Engineered glucocerebrosidase variants

By genetically engineering the amino acid sequence of β-glucocerebrosidase, an engineered GCase peptide was developed, which solved the problems of poor efficacy of existing treatments for central nervous system symptoms and immune responses, and achieved a more efficient and stable treatment for Gaucher disease.

JP2026506873APending Publication Date: 2026-02-27エンボールド セラピューティクス インコーポレイテッド
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Patent Information

Application Number
JP2025545135
Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-05-12
Filing Date
2024-02-01
Publication Date
2026-02-27

AI Technical Summary

Technical Problem

Existing treatments for Gaucher disease, such as enzyme replacement therapy and substrate reduction therapy, have limited effectiveness in treating central nervous system-related symptoms. Furthermore, some patients develop an immune response to recombinant enzymes, leading to reduced treatment efficacy. There is a need to develop more effective enzyme replacement therapies to increase the availability of functional GCases in target organs and reduce the need for frequent intravenous injections.

Method used

An engineered β-glucocerebrosidase (GCase) peptide was developed, and its amino acid sequence was genetically engineered to enhance its activity and stability in target organs, including mutations at specific amino acid positions, to enhance its function in patients with Gaucher disease.

Benefits of technology

It improves the activity and stability of engineered GCase in target organs, reduces the immune response to enzyme replacement therapy, and provides a more effective treatment for Gaucher disease, particularly for the improvement of central nervous system symptoms.

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Abstract

The present disclosure provides engineered glucocerebrosidases, polynucleotides encoding the engineered glucocerebrosidases, and methods of treating deficiencies in glucocerebroside activity using the engineered glucocerebrosidases or recombinant polynucleotides encoding the engineered glucocerebrosidases. The present disclosure further provides methods or uses of the engineered glucocerebrosidases or recombinant polynucleotides to treat conditions or diseases associated with deficiencies in glucocerebrosidase activity, such as Gaucher's disease.
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Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 482,947, filed February 2, 2023, and U.S. Provisional Patent Application No. 63 / 502,058, filed May 12, 2023, the entire contents of which are incorporated herein by reference.

[0002] Reference to a sequence listing, table, or computer program The Sequence Listing, created on January 13, 2024, having a file size of 3,846,697 bytes, and submitted concurrently herewith under file name CX7-244WO3_ST26, is a part of the present specification and is incorporated herein by reference.

[0003] Technical Field The present disclosure relates to the use of engineered β-glucocerebrosidase (GCase) polypeptides, polynucleotides encoding engineered β-glucocerebrosidase polypeptides, and engineered polypeptides or recombinant polynucleotides, or compositions thereof, in therapeutic applications. [Background technology]

[0004] background Gaucher disease (GD) is a lipid storage disorder characterized by the accumulation of glucocerebroside within the cells of patients, including the macrophage-monocyte system. GD is caused by mutations that result in a deficiency in the activity of the enzyme β-glucocerebrosidase (GCase), an enzyme with glucosylceramidase activity (EC 3 2 1.45) that hydrolyzes the beta-glycosidic bond of glucocerebroside (glucosylceramide or GL1) to glucose and ceramide. The monocyte-macrophage system is particularly affected in the disease due to the role of macrophages in removing dead and dying red blood cells and white blood cells, whose membranes are rich in GL1. This leads to the accumulation of GL1 in these macrophages, also known as Gaucher cells. These Gaucher cells infiltrate the bone marrow, spleen, liver, and other organs, causing organomegaly in addition to increasing the inflammatory state of the macrophage-monocyte system. GCase deficiency is generally caused by mutations in the GBA1 gene that impair the activity and / or production of the enzyme. GD is a recessive genetic disorder.

[0005] GD generally manifests in three primary forms. GD type 1 (GD1), also known as non-neuropathic GD, does not involve the central nervous system and is the most common form. Individuals with GD type 1 are prone to bruising, exhibit chronic fatigue, enlarged liver or spleen, and bone pain or degeneration. Some of the symptoms are due to low levels of platelets (thrombocytopenia) and low levels of circulating blood cells (anemia).

[0006] GD2 type (GD2), also known as acute neuropathic GD, appears in newborns and infants and is characterized by neurological complications due to the abnormal accumulation of glucocerebroside and its deacylation product, glucosphingosine, in the central nervous system. Affected infants exhibit loss of motor skills, decreased muscle tone, and involuntary muscle spasms, which can cause slow and rigid limb movements and strabismus. Patients with GD2 type may also have anemia and thrombocytopenia. GD2 type often progresses to life-threatening complications, including respiratory distress. Severely affected newborns exhibit skin abnormalities and may die within the first few weeks of life. Children with GD2 type have a shortened lifespan of approximately 1 to 3 years.

[0007] Perinatal lethal or fetal / neonatal GD is a rare form of GD type 2, occurring in fewer than 5% of patients. This form of GD is severe and associated with death before 3 months of age or in utero. The fetus / newborn may present with widespread swelling of the skin (edema or anasarca) leading to fluid accumulation in the heart, skin, or lungs (hydrops fetalis). Other symptoms include bleeding within the skull (intracranial hemorrhage), scaling of the skin with a reddish appearance (nonbullous ichthyosiform erythroderma), and joints becoming fixed and contracted in a bent position (arthrogryposis multiplex congenita).

[0008] Type 3 Gaucher disease (GD3), also known as chronic neuropathic GD, occurs during the first decade of life. In addition to blood and bone abnormalities, affected individuals develop neurological complications that develop later in onset and progression than those in type 2 Gaucher disease. These include mental deterioration, involuntary movements (ataxia), and muscle spasms of the limbs or the whole body (myoclonic seizures). A significant number of patients also develop pulmonary disease, including interstitial lung disease. There can be wide variability in the presentation and clinical course among patients with type 3 Gaucher disease. Some affected patients live into their teens or early twenties, while others live much longer (in their thirties or forties). With increasing difficulty, affected individuals may require assistance with daily living tasks (e.g., eating, bathing, and walking).

[0009] Gaucher disease type 1 (GD1) can be treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). ERT uses a recombinant form of human β-glucocerebrosidase, typically administered by intravenous infusion every two weeks. ERTs approved for treating GD include Cerezyme® (imiglucerase), VPRIV® (velaglucerase alfa), and Elelyso® (taliglucerase alfa). The amino acid composition of imiglucerase and taliglucerase alfa differs from human GCase, but velaglucerase has the same amino acid sequence as found in humans. Taliglucerase alfa also differs from velaglucerase alfa and imiglucerase in its glycosylation due to its production in plants. While ERT provides clinically significant relief of GD symptoms, some patients develop antibodies against the recombinant enzyme, thereby reducing its effectiveness.

[0010] SRTs reduce the accumulation of toxic GL1 by targeting the inhibition of UDP-glucose ceramide glucosyltransferase, the first enzyme in the pathway that glycosylates sphingolipids. This enzyme is involved in the synthesis of GL1 and other glycosphingolipids. Approved SRTs include miglustat (Zavesca®) and eliglustat (Cerdelga®). Miglustat is primarily used as a second-line treatment for patients who cannot receive or tolerate ERT.

[0011] ERT is effective in treating mild forms of GD and improving quality of life by alleviating the visceral and hematologic aspects of the disease. However, immune responses to the administered enzymes can reduce its effectiveness, and progression of the disease involving the central nervous system is not treated by ERT.

[0012] Therefore, there is a need for improved, effective therapies for the treatment of GD that increase the availability of functional GCase to affected target organs, avoiding the need for frequent intravenous injections of GCase preparations approved to treat GD. Summary of the Invention

[0013] overview The present disclosure provides engineered glucocerebrosidase, recombinant polynucleotides encoding the engineered glucocerebrosidase, and pharmaceutical compositions comprising the engineered glucocerebrosidase or recombinant polynucleotide. The present disclosure further provides methods or uses of the engineered glucocerebrosidase or recombinant polynucleotide for treating conditions or diseases associated with a deficiency of glucocerebrosidase activity, such as Gaucher's disease.

[0014] In one aspect, the engineered glucocerebrosidase, or functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:2-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:2-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2, 54, 220, 984, or 1150, or relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2, 54, 220, 984, or 1150.

[0015] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150, wherein the amino acid sequence comprises one or more substitutions.

[0016] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0017] In some embodiments, the engineered glucocerebrosidase comprises a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to an even-numbered SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions.

[0019] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 16, 117, 133, 134, 137, 138, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 3 01, 314, 315, 316, 319, 325, 329, 332, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 4 or 536, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0020] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0021] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 265 / 372 / 489, 243, 23, 109, 265, 109 / 195 / 243, 23 / 70 / 207 / 214 / 265, 23 / 70, 23 / 214 / 243 / 265, 65 / 70 / 109 / 214 / 319 / 372 / 481, 23 / 265 / 319, 23 / 65 / 70 / 141 / 319 / 481, 23 / 265, 372, 214, 23 / 65 / 70 / 109 / 141 / 214, 319, 65 / 372, 109 / 195, 23 / 65, 20 7 / 265 / 319, 109 / 207 / 214 / 372, 23 / 319 / 372 / 489, 65 / 109 / 214 / 243, 23 / 141 / 195, 70 / 207 / 214 / 489, 65 / 70 / 265 / 319 / 372, 214 / 265, 265 / 319, 65 / 243, 6 5 / 70 / 195, 489, 23 / 65 / 141, 23 / 372, 70 / 141, 23 / 195, 214 / 319, 23 / 207 / 214 / 243 / 265 / 481, 65 / 214 / 243 / 265 / 319, 214 / 243, 243 / 265, 265 / 489, 214 / 24 3 / 265, 207 / 243 / 372, 195 / 243, 65 / 481, 207 / 214 / 243 / 319, 70 / 141 / 195 / 265, 23 / 265 / 372, 46 / 99 / 134 / 230 / 373 / 449, 68 / 211 / 230, 373 / 449, 46 / 68 / 3 36, 68 / 230 / 314 / 373, 68, 68 / 336 / 373, 99 / 314, 46, 46 / 230 / 261, 373, 230 / 314, 68 / 211, 68 / 336 / 449, 46 / 336, 336 / 449, 46 / 68 / 336 / 373, 46 / 99, 99 / 261 , 99, 68 / 134, 46 / 68 / 134 / 230 / 336 / 373, 46 / 68, 68 / 99 / 211 / 230, 99 / 134, 68 / 99 / 336, 449, 68 / 99, 46 / 230 / 336, 68 / 154 / 449, 336 / 373 / 449, 134 / 336, 68 / 99 / 230, 46 / 68 / 134 / 154, 68 / 99 / 230 / 373 / 449, 134, 46 / 68 / 154 / 211 / 230 / 261 / 336, 68 / 99 / 154 / 261 / 336, 230 / 449, 46 / 68 / 314, 154, 68 / 449, 99 / 449,At least one substitution or set of substitutions at 68 / 154 / 230 / 314 / 336 / 449, 46 / 68 / 154 / 314 / 336 / 373, 46 / 230, 336, or 46 / 99 / 230 / 373, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0022] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions at amino acid position(s), and ... 30 / 319 / 337 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 154 / 195 / 214 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 374 / 408, 65 / 68 / 154 / 195 / 207 / 214 / 230 / 263 / 3 19 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 374 / 408 / 471, 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 408, 65 / 68 / 70 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 214 / 230 / 263 / 337 / 374 / 408, 214 / 230 / 263 / 374 / 4 08, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 230 / 408, 65 / 68 / 70 / 99 / 154 / 207 / 230 / 319 / 408, 65 / 68 / 70 / 214 / 230 / 408, 65 / 68 / 70 / 195 / 214 / 230 / 263 / 361 / 471, 19 5 / 207 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 214 / 230 / 408 / 489, 65 / 68 / 70 / 195 / 207 / 230 / 408, 207 / 230 / 319 / 374 / 408, 65 / 68 / 70 / 99 / 195 / 207 / 230 / 408,195 / 214 / 230 / 263 / 408, 195 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 214 / 408, 207 / 214 / 230 / 319 / 374 / 408, 207 / 214 / 230 / 374 / 408, 154 / 207 / 230 / 374 / 408 / 489, 207 / 230 / 263 / 408, 207 / 230 / 408 / 471, 207 / 230 / 374 / 408, 214 / 230 / 319 / 408, 214 / 230 / 374 / 408 / 489, 207 / 230 / 408, 65 / 68 / 70 / 154 / 20 7 / 408, 207 / 230 / 263 / 337 / 408, 195 / 214 / 230 / 408, 207 / 230 / 408 / 489, 214 / 230 / 408, 230 / 361 / 408, 207 / 214 / 408, 214 / 263 / 408, 230 / 374 / 408, 207 / 408, 195 / 214 / 263, 374 / 408, or 408, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0023] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or relative to a reference sequence corresponding to SEQ ID NO:2.

[0024] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase variant described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0025] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising one substitution or set of substitutions of an engineered glucocerebrosidase variant set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to the reference sequence corresponding to SEQ ID NO:2.

[0026] In some embodiments, the engineered glucocerebrosidase comprises a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150.

[0027] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648.

[0028] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence comprises one or more substitutions.

[0029] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150, or relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150.

[0030] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133 , 134, 137, 138, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 3 25, 329, 332, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 504 , 505, 506, 507, 508, 509, 510, 512, 514, 515, 516, 518, 519, 520, 522, 524, 525, 529, 532, 533, 534, or 536, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0031] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0032] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54.

[0033] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions.

[0034] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 68 / 336, 65 / 99 / 319, 65 / 68 / 319, 68 / 319, 68, 65 / 319, 195, 65 / 154 / 319, 65 / 68 / 230, 65, 65 / 68 / 99 / 154 / 230 / 319, 68 / 230, 195 / 230, 65 / 230, 141 / 319, 154 / 336, 68 / 154, 68 / 195 / 230 / 319 / 336, 68 / 99 / 195 / 230 / 319, 68 / 195, 65 / 68, 68 / 141 / 230 / 319, 65 / 68 / 99 / 230 / 319, 68 / 99 / 141 / 319, 68 / 154 / 319, 141 / 154, 68 / 99, 99 / 230 / 319, 99, 230, 68 / 230 / 319,154 / 319, 65 / 68 / 154 / 319, 141, 336, 141 / 230, 68 / 154 / 336, 65 / 68 / 99 / 319, 154, 319, 230 / 319, 68 / 154, 99 / 141 / 319, 509, 108, 108 / 117 / 509, 519, 108 / 519, 88, 117 / 41 3 / 509, 509 / 519, 413, 108 / 117 / 315, 117 / 271, 117 / 413 / 509 / 529, 413 / 509, 469, 524, 507, 525, 489, 104, 95, 359, 374, 107, 89, 260, 110, 186, 370, 191, 333, 189, 468, 473, 115, 53, 449, 518, 236, 481, 59 / 220, 258, 467, 51, 415, 103, 493, 78, 335, 520, 101, 341, 471, 211, 206, 93, 490, 71, 79, 180, 172, 207, 63, 142, 529, 77, 212, 474, 106, 386 / 507, 117, 373, 337, 107 / 246, 516, 233, 91, 194, 84, 361, 171, 536, 50, 116, 239, 44, 40, 113, 243, 169, 504, 505, 109, 479, 477, 97, 182, 111, 508, 314, 102, 338, 96, 138, 42, 263, 222, 522, 265, 514, 515, 246, 46, 133, 332, 506, 512, 137, 190,At least one substitution or set of substitutions at amino acid positions 105, 301, 329, 339, or 151, and the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, or relative to a reference sequence corresponding to SEQ ID NO: 54.

[0035] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:220.

[0036] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more substitutions.

[0037] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 65 / 195 / 361, 514, 79 / 195 / 263 / 361, 65 / 374, 79 / 195, 65 / 230, 65 / 233 / 263 / 337 / 359 / 361 / 374, 79 / 536, 79 / 230 / 233 / 239 / 359 / 361 / 471, 65 / 471, 374, 65 / 195 / 230 / 233 / 337 / 374 / 514, 65 / 195 / 233 / 359 / 536, 230, 65 / 195 / 263 / 276 / 359 / 361 / 374 / 471, 79, 195 / 230 / 233 / 337 / 374 / 536, 195 / 230, 195 / 230 / 337 / 359 / 361, 230 / 263, 65 / 230 / 233, 263, 195 / 230 / 337 / 361 / 374 / 471, 65 / 195, 374 / 471, 374 / 536, 239, 65, 230 / 233 / 536, 230 / 263 / 374, 65 / 195 / 230 / 263 / 337 / 361 / 374 / 471, 79 / 374, 65 / 361 / 374 / 471 / 536, 195, 233, 79 / 230 / 359 / 374, 536, 230 / 359 / 361 / 374 / 514, 65 / 79 / 230, 195 / 239 / 263 / 361 / 374 / 514, 195 / 263 / 374, and at least one substitution or set of substitutions at 195 / 374, 65 / 230 / 233 / 361 / 536, 230 / 233 / 374 / 471, 65 / 233 / 359 / 361 / 471, 65 / 359 / 471, 65 / 79, or 65 / 359, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:220.

[0038] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984.

[0039] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984, wherein the amino acid sequence comprises one or more substitutions.

[0040] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises the amino acid sequence at amino acid position(s) 359, 79 / 359, 79 / 97 / 182, 97 / 359, 97, 79 / 182, 201, 404, 484, 446, 494 / 495, 495, 412, 385, 100, 184, 82, 75, 71, 41, 99, 94, 92, 98, 114, 95, 102, 96, 105, 113, 325, 336, 262, 386, 360, 400, 342, 390, 264, 298, at least one substitution or set of substitutions at 382, ​​408, 449, 459, 434, 476, 502, 29 / 534, 497, 485, 533, 536, 510, 532, 489, 534, 494, 182, 137, 204, 194, 202, 144, 142, 175, 220, 150, or 206, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:984 or are relative to a reference sequence corresponding to SEQ ID NO:984.

[0041] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or to a reference sequence corresponding to SEQ ID NO: 1150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150.

[0042] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:1218-1554, or a reference sequence corresponding to SEQ ID NO:1218-1554, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:1150, or relative to the reference sequence corresponding to SEQ ID NO:1150.

[0043] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 96 / 194 / 382 / 484, 359 / 382 / 412 / 494 / 495, 92 / 96 / 175 / 182 / 382, 96 / 359 / 382 / 484 / 494 / 495, 92 / 175 / 194 / 359 / 382, 359 / 382 / 400 / 412, 92 / 96 / 182 / 382 / 400 / 484, 92 / 96 / 382 / 494 / 495, 92 / 96 / 175 / 382 / 494 / 495, 96 / 182 / 359 / 382 / 484, 92 / 96 / 3 82, 92 / 382 / 494 / 495 / 534, 92 / 96 / 382 / 400 / 484, 92 / 96 / 175 / 382, 92 / 175 / 382 / 400 / 484, 92 / 96 / 175 / 182 / 382 / 412 / 484, 92 / 96 / 201 / 382 / 484, 92 / 182 / 194 / 382, 92 / 96 / 175 / 194 / 359 / 382 / 400, 92 / 359 / 382, 96 / 382, 92 / 175 / 382 / 484 / 494 / 495, 96 / 201 / 359 / 382, 92, 194, 201, 359, 96, 92 / 96 / 382 / 400 / 4 84 / 495, 382 / 495, 382, ​​92 / 382, 71 / 175 / 325 / 385, 325 / 385, 71 / 325 / 502, 71 / 175 / 325 / 385 / 502, 202 / 325 / 385, 97 / 175 / 385 / 510, 325 / 385 / 510, 100 / 1 75 / 325, 71 / 175 / 325 / 360 / 385, 97 / 175 / 510, 71 / 97 / 100 / 175 / 325 / 385, 71 / 175 / 510, 71 / 97 / 325 / 502, 71 / 100 / 325 / 385, 202 / 385 / 489, 325 / 385 / 502, 7 1 / 100 / 325 / 385 / 510, 71 / 100 / 175 / 325 / 385, 385 / 510, 97 / 325 / 385, 71, 97 / 202 / 325 / 360 / 385 / 510, 502, 71 / 97 / 103 / 325, 100 / 325 / 385 / 502 / 510, 71 / 137 / 175 / 325 / 360 / 385 / 502, 71 / 276 / 325 / 385 / 510, 71 / 175 / 360 / 385 / 502, 100 / 175 / 325 / 385 / 489 / 502, 71 / 175 / 360 / 385 / 485 / 502, 95 / 113, 494 / 536,95 / 99 / 113 / 182 / 184 / 264 / 386, 184 / 536, 95 / 142 / 144 / 182 / 184 / 386 / 449, 144 / 204 / 386 / 400 / 449, 113 / 449 / 536, 536, 95 / 386 / 536, 264, 99 / 113 / 386 / 449, 95 / 386, 95 / 99 / 204 / 386, 113 / 142 / 386 / 449, 182 / 184 / 264 / 494, 113 / 142 / 182 / 494, 113, 99 / 264 / 449, 95, 95 / 99, 99 / 113 / 449, 95 / 113 / 449 / 5 36, 95 / 99 / 113, 386 / 536, 99 / 182 / 184, 142 / 386 / 400 / 536, 35 / 264 / 449 / 536, 142 / 144 / 220, 99 / 142, 29 / 102 / 182 / 276 / 336 / 390 / 459 / 502 / 532, 41 / 262 / 336 / 502, 29 / 97 / 150 / 262 / 336 / 502 / 532, 29, 532, 102 / 390 / 459 / 502 / 532, 29 / 262 / 276 / 336 / 495, 29 / 182 / 262 / 459 / 532, 92 / 99 / 102 / 502, 29 / 97 / 1 02 / 182 / 502 / 532, 336 / 459 / 495 / 502 / 532, 150 / 182 / 336 / 390, 150 / 262 / 495, 29 / 495, 29 / 102 / 182 / 262 / 502, 29 / 336 / 459 / 502 / 532, 182 / 495 / 502 / 532, 102 / 336, 150 / 182 / 262 / 495, 459 / 495 / 502 / 532, 41 / 182 / 390 / 495, 29 / 41 / 182 / 336 / 495, 97 / 102 / 495, 182 / 262 / 495, 29 / 262 / 502, 29 / 102 / 502, 1 or at least one substitution or set of substitutions at 82 / 336 / 495, 29 / 150 / 182 / 262 / 459 / 495, 502 / 532, 386, 497, 99, 512, 69, 507, 445, 75, 487, 184, 90, 235, 434, 489, 86, 182, 446, 490, 299, 102, 230, 68, 141, 97, 316, 522, 224, 478, 480, 368, 412, 449, 265, or 494, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150;or relative to the reference sequence corresponding to SEQ ID NO: 1150.

[0044] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0045] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0046] In some embodiments, the engineered glucocerebrosidase has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1691, 1692, 1693, 1694, 1695, 1696 98%, 99%, or more sequence identity, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0047] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises residues 40-536 of an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, or an amino acid sequence comprising an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1.

[0048] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence comprising residues 40-536 of an even-numbered SEQ ID NO:4-1648, or an even-numbered SEQ ID NO:4-1648, optionally, the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0049] In some embodiments, the engineered glucocerebrosidase has glucocerebrosidase activity, hi some embodiments, the engineered glucocerebrosidase has glucocerebrosidase activity and one or more improved properties compared to a reference glucocerebrosidase.

[0050] In some embodiments, the engineered glucocerebrosidase has glucocerebrosidase activity and improved properties selected from: i) increased enzymatic activity, ii) increased expression or expression efficiency, iii) increased stability at neutral or basic pH, iv) increased stability at acidic pH, v) increased stability in serum or plasma, vi) increased thermostability, vii) increased uptake and / or intracellular stability in cells such as macrophages, and viii) reduced immunogenicity, compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO:2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO:2, 54, 220, 984, or 1150. In some embodiments, the improved properties are compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2.

[0051] In a further aspect, the present disclosure provides a recombinant polynucleotide encoding an engineered glucocerebrosidase, hi some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase of the present disclosure.

[0052] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 118-1608 of SEQ ID NO: 53, 219, 983, or 1149, or a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 53, 219, 983, or 1149, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

[0053] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 118-1608 of an odd-numbered SEQ ID NO:3-1647, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:3-1647, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

[0054] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence that is codon-optimized for expression of the encoded glucocerebrosidase.

[0055] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising residues 118-1608 of an odd-numbered SEQ ID NO: 3-1647, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 3-1647.

[0056] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence that includes nucleotide residues 118 to 1608 of SEQ ID NO: 53, 219, 983, or 1149, or that includes SEQ ID NO: 53, 219, 983, or 1149.

[0057] In a further aspect, the recombinant polynucleotide is provided in an expression vector. In some embodiments, the expression vector comprises a recombinant polynucleotide encoding an engineered glucocerebrosidase described herein.

[0058] In some embodiments, the recombinant polynucleotide in the expression vector is operably linked to a control sequence. In some embodiments, the control sequence includes at least a promoter, particularly a heterologous promoter. In some embodiments, the expression vector includes a viral vector, particularly for introducing the recombinant polynucleotide encoding the engineered glucocerebrosidase into a cell or for administration to a subject.

[0059] In another aspect, the present disclosure provides a host cell comprising an expression vector comprising a recombinant polynucleotide encoding the engineered glucocerebrosidase disclosed herein. In some embodiments, the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell, particularly a human cell.

[0060] In another aspect, the present disclosure further provides methods for producing an engineered glucocerebrosidase, the method comprising culturing a host cell under suitable culture conditions such that the engineered glucocerebrosidase is expressed or produced. In some embodiments, the method further comprises recovering the engineered glucocerebrosidase from the culture medium and / or the host cell. In some embodiments, the method further comprises purifying the engineered glucocerebrosidase.

[0061] In another aspect, the engineered glucocerebrosidase or recombinant polynucleotide is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the engineered glucocerebrosidase or a recombinant polynucleotide encoding the engineered glucocerebrosidase, or an expression vector thereof, and a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion into humans.

[0062] In some embodiments, the pharmaceutical composition is a gene therapy composition comprising a recombinant polynucleotide encoding the engineered glucocerebrosidase. In some embodiments, the gene therapy composition comprises a viral vector comprising a recombinant polynucleotide encoding the engineered glucocerebrosidase. In some embodiments, the viral vector is an adenovirus, adeno-associated virus, lentivirus, retrovirus, or herpesvirus vector.

[0063] In some embodiments, the gene therapy composition comprises a recombinant polynucleotide encoding an engineered glucocerebrosidase formulated with a liposome, a cationic polymer, a dendrimer, or conjugated to a cell-penetrating peptide.

[0064] In some embodiments, the engineered glucocerebrosidase or a recombinant polynucleotide encoding the engineered glucocerebrosidase is used to treat and / or prevent symptoms of a deficiency in glucocerebrosidase activity in a subject. In some embodiments, the method or use for treating a deficiency in glucocerebrosidase activity in a subject comprises administering to a subject in need thereof an effective amount of the engineered glucocerebrosidase, recombinant polynucleotide encoding the engineered glucocerebrosidase, or gene therapy composition comprising a recombinant polynucleotide encoding the engineered glucocerebrosidase described herein.

[0065] In some embodiments, the use of the engineered glucocerebrosidase, or a recombinant polynucleotide encoding the engineered glucocerebrosidase, or a gene therapy composition thereof, is for the preparation of a medicament for treating a deficiency in glucocerebrosidase activity in a subject.

[0066] In some embodiments, the subject having a deficiency in glucocerebrosidase activity suffers from Gaucher disease or Parkinson's disease. [Brief explanation of the drawings]

[0067] [Figure 1] Graphs are provided showing expression of five GCase variants as determined by Western blot and GCase activity (4-MU-βGLU hydrolysis) in the supernatants of cultures of Expi293F (Panels A and B), ExpiCHO (Panels C and D), and HepG2 (Panels E and F).

[0068] [Figure 2] Graphs are provided showing the residual GCase activity (4-MU-βGLU hydrolysis) of the supernatants of 11 GCase variants after incubation in neutral pH solution. The GCase material used in the experiments was derived from high-throughput expression cultures of Expi293F transfected with DNA for the 11 GCase variants.

[0069] [Figure 3] 1 provides a graph showing the residual GCase activity (4-MU-βGLU hydrolysis) of the supernatants of 11 GCase variants after 1 hour of incubation at 47° C. The GCase material used in the experiments was derived from a high-throughput expression culture of Expi293F transfected with the DNA of the 11 GCase variants.

[0070] [Figure 4] A graph showing the residual GCase activity (4-MU-βGLU hydrolysis) of the supernatants of 11 GCase variants after incubation with human serum for 1 hour at 37° C. The GCase material used in the experiments was derived from a high-throughput expression culture of Expi293F transfected with the DNA of the 11 GCase variants.

[0071] [Figure 5]

[0023] Figure 1 provides graphs showing the cross-correcting ability of GCase variants in which HepG2 cells were transfected with DNA encoding GCase SEQ ID NO: 2, 54, 220, or 984 and co-cultured with macrophages in a transwell setting. 4-MU-βGLU hydrolytic activity was assessed in each compartment (Panel A - HepG2 cell lysate; Panel B - secreted protein from HepG2 in conditioned medium; and Panel C - evidence of uptake in macrophage lysate) and normalized to the activity of GCase SEQ ID NO: 2.

[0072] [Figure 6] This figure provides graphs showing the cross-correcting ability of GCase variants when delivered as vectored AAV9 constructs. HepG2 cells were transduced with AAV-packaged DNA of SEQ ID NO: 1 or 983, and macrophages were co-cultured in a transwell environment. 4-MU-βGLU hydrolysis activity was assessed in each compartment (Panel A - HepG2 cell lysate; Panel B - secreted protein from HepG2 in conditioned medium; and Panel C - evidence of uptake in macrophage lysate). GCase-induced clearance of GL1 (Panel D) and lyso-GL1 (Panel E) was determined in macrophages.

[0073] [Figure 7] 1 provides graphs showing the stability and residual activity of two GCase variants when challenged with (Panel A) incubation in serum, (Panel B) incubation in artificial CSF, and (Panel C) incubation over a thermal gradient for 1 hour, as described in Example 11.

[0074] [Figure 8]HepG2 cells (Panel A - secreted protein; Panel B - lysate), Expi293F (Panel C - secreted protein; Panel D - lysate), and RAW264.7 (Panel E - secreted protein; Panel F - lysate) were transduced with DNA of SEQ ID NO: 1 or 983 in the presence of etoposide, and the resulting GCase activity was measured using standard 4-MU-βGLU hydrolysis activity in the conditioned medium (Panels A, C, E) or lysate (Panels B, D, F) of each cell line. Graphs are provided showing the transduction ability of GCase variants when delivered as vectored AAV9 constructs.

[0075] [Figure 9] Graphs are provided showing tissue GCase activity in WT or D409V / D409V mice after dosing with vehicle or AAV9 packaged SEQ ID NO:1 or SEQ ID NO:983. GCase activity in plasma (Panel A - pre-dosing; Panel B - day 14; and Panel C - day 28), liver (Panel D), spleen (Panel E), and brain (Panel F) are described in Example 8. Data are presented as mean + / - standard deviation. Data are presented as mean + / - standard deviation. Treatment groups were analyzed by one-way ANOVA multiple comparisons, and significant differences were indicated by *: P<0.05, **: P<0.01, and ***: P<0.005. *: P<0.05; **: P<0.01; ***: P<0.005.

[0076] [Figure 10]

[0023] Figure 1 provides graphs showing GL1 abundance in tissues of WT or D409V / D409V mice after dosing with vehicle or AAV9 packaged SEQ ID NO:1 or SEQ ID NO:983. GL1 was measured in the liver (Panel A), spleen (Panel B), and brain (Panel C) as described in Example 8. GL1 is the sum of the 16:0, 18:0, and 24:1 isoforms analyzed. Data are presented as mean + / - standard deviation. Treatment groups were analyzed by one-way ANOVA multiple comparisons, and significant differences were indicated by *: P<0.05, **: P<0.01, and ****: P<0.001. *: P<0.05; **: P<0.01; ****: P<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0077] Detailed Description The present disclosure provides engineered glucocerebrosidase variants. In some embodiments, the engineered glucocerebrosidase variants have glucocerebrosidase activity and are characterized by improved enzymatic properties, including, but not limited to, increased enzymatic activity, increased expression efficiency, increased stability at neutral or basic pH, increased stability at acidic pH, increased serum stability, enhanced uptake into cells such as macrophages, and / or reduced immunogenicity. Additionally, recombinant polynucleotides encoding the engineered glucocerebrosidases for treating glucocerebrosidase deficiencies, such as Gaucher disease, and uses of the engineered glucocerebrosidases, e.g., as enzyme replacement therapy, or e.g., as gene therapy, are provided.

[0078] Abbreviations and definitions Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art.

[0079] It is understood that the present invention is not limited to the particular methodology, protocols, and reagents described, which may vary depending on the circumstances under which they are used by those skilled in the art. Accordingly, the terms defined immediately below are more fully described by reference to this application as a whole.

[0080] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0081] Additionally, the term "including" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0082] It should be further understood that where the description of embodiments uses the term "comprising" and its cognates, the embodiments may also be described using the phrase "consisting essentially of" or "consisting of."

[0083] Numerical ranges are intended to be inclusive of the numbers defining the range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were all expressly written herein.

[0084] The term "about" refers to an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value. In some instances, "about" encompasses values ​​within 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value.

[0085] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be had by reference to the entire application. Accordingly, the terms defined immediately below are more fully defined by reference to the entire application. Nevertheless, to facilitate understanding of the invention, certain terms are defined below.

[0086] The "EC" numbers refer to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0087] "ATCC" refers to the American Type Culture Collection, whose biorepository collection contains genes and strains.

[0088] "NCBI" refers to the National Center for Bioinformation and the sequence databases contained therein.

[0089] "Gaucher disease" or "GD" refers to a metabolic disorder associated with a deficiency of the enzyme glucocerebrosidase, which leads to the accumulation of certain lipids, particularly the glycolipid glucocerebroside, in cells and certain tissues and organs.

[0090] "Glucocerebrosidase," "β-glucocerebrosidase," "acid β-glucosidase," and "GCase" are used interchangeably herein to refer to a glycoside hydrolase with glucosylceramidase activity that cleaves the β-glycosyl bond of glucocerebroside to produce glucose and ceramide. In humans, GCase is encoded by the GBA1 gene. In lysosomes, wild-type GCase is membrane-associated and interacts with the activating protein saposin C (SapC). The GCase enzyme is classified as EC 3.2.1.45.

[0091] "Protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0092] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine ​​(Cys or C), glycine (Gly or G), glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When three-letter abbreviations are used, amino acids may be in either the L- or D-configuration about the α-carbon (Cα) unless clearly preceded by "L" or "D" or otherwise apparent from the context in which the abbreviation is used. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, whereas "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When one-letter abbreviations are used, an uppercase letter designates an amino acid in the L-configuration about the α-carbon, and a lowercase letter designates an amino acid in the D-configuration about the α-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When polypeptide sequences are represented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is represented in the amino (N) to carboxy (C) direction, in accordance with common convention.

[0093] "Fusion protein," and "chimeric protein," and "chimera" refer to a hybrid protein created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant techniques (e.g., molecular biology techniques that are readily known in the art).

[0094] "Mature protein" or "mature polypeptide" refers to the final processed biological protein or polypeptide or product.

[0095] A "proprotein," "propolypeptide," or "propeptide" refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification to form a biologically active protein, polypeptide, or peptide. In some embodiments, the post-translational modification is a cleavage reaction to form the protein, polypeptide, or peptide. A "proenzyme" refers to a precursor polypeptide that is processed by post-translational modification, particularly a cleavage reaction, to form an active enzyme.

[0096] A "preproprotein," "prepropolypeptide," or "prepropeptide" refers to a precursor protein, polypeptide, or peptide that includes a signal sequence or signal peptide and can be post-translationally modified, particularly a cleavage reaction, to produce a proprotein, propolypeptide, or propeptide. Generally, the cleavage reaction removes the signal sequence to produce the proprotein, propolypeptide, or propeptide. A "preproenzyme" refers to a precursor protein, polypeptide, or peptide that is processed post-translationally, particularly a cleavage reaction, to remove the signal sequence and form a proenzyme.

[0097] "Full-length" in the context of a protein or polypeptide refers to a protein or polypeptide that has not been treated to alter the amino acid sequence of the entire protein or polypeptide. For example, a full-length protein is the entire protein encoded by the corresponding mRNA.

[0098] "Polynucleotide" is used herein to refer to a polymer containing at least two nucleotides, where the nucleotides are either deoxyribonucleotides or ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless specifically noted, abbreviated nucleosides can be either ribonucleosides or 2'-deoxyribonucleosides. Nucleosides, individually or collectively, can be designated as either ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is represented as a string of single-letter abbreviations, the sequence is represented in the 5' to 3' direction, according to common convention, and phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid.

[0099] "Engineered," "recombinant," "non-naturally occurring," and "variant," when used in reference to a cell, polynucleotide, or polypeptide, refer to a material or materials that do not otherwise occur in nature or are not identical thereto, but that correspond to native or naturally occurring forms of the material, produced or derived from synthetic materials and / or modified by manipulation using recombinant techniques.

[0100] "Wild-type" and "naturally-occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that can be isolated from a natural source and is present in an organism that has not been intentionally modified by human manipulation.

[0101] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a polypeptide or protein.

[0102] "Percent sequence identity (%)" is used herein to refer to a comparison between a polynucleotide and a polypeptide and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by aligning the nucleic acid base or amino acid residue where either the same nucleic acid base or amino acid residue occurs in both sequences, or where the nucleic acid base or amino acid residue contains a gap, to determine the number of positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection as known in the art.Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (See Altschul et al., J. Mol. Biol., 1990, 215:403-410, and Altschul et al., 1977, Nucleic Acids Res., 1977, 3389-3402, respectively). Software for performing BLAST analyses is publicly available via the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (See Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped if the cumulative alignment score deviates from its maximum actual value by an amount X, if the cumulative score falls below zero due to the accumulation of one or more negatively scored residue alignments, or if the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary sequence alignments and determination of percent sequence identity can utilize the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0103] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or at least 100 residues in length, across the length or full length of the nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) similar sequences (i.e., portions of the complete sequence) between the two sequences and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" is based on a primary amino acid sequence, and the reference sequence may have one or more changes in the primary sequence. For example, the phrase "a reference sequence corresponding to SEQ ID NO: 2 with a proline at the residue corresponding to X51" (or "a reference sequence corresponding to SEQ ID NO: 2 with a proline at the residue corresponding to position 51") refers to a reference sequence in which the residue corresponding to position X51 in SEQ ID NO: 2 (e.g., serine) has been changed to a proline.

[0104] A "comparison window" refers to a conceptual segment of contiguous nucleotide positions or amino acid residues over which a sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15-20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window may be longer than 15-20 contiguous residues, optionally including a window of 30, 40, 50, 100, or longer.

[0105] When used in the context of the numbering of a given amino acid or polynucleotide sequence, "corresponding to," "reference," or "relative to" refers to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified relative to the reference sequence, rather than the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as an engineered glucocerebrosidase, can be aligned with a reference sequence by introducing gaps to optimize residue identity between the two sequences. In these cases, despite the presence of gaps, the residues in the given amino acid or polynucleotide sequence are numbered relative to the reference sequence to which it is aligned.

[0106] "Mutation" refers to an alteration of a nucleic acid sequence. In some embodiments, a mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, a mutation comprises an addition, such that an amino acid is added (e.g., inserted) to the original polypeptide sequence. In some further embodiments, a mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations can be present in a given sequence. In some embodiments, a "substitution" comprises a deletion of an amino acid, which can be represented by a "-" symbol.

[0107] "Amino acid difference" and "residue difference" refer to the difference in an amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, a "residue difference at position X51 compared to SEQ ID NO:2" (or "residue difference at position 51 compared to SEQ ID NO:2") refers to the difference in the amino acid residue at the polypeptide position corresponding to position 51 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a serine at position 51, then a "residue difference at position X51 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 51 of SEQ ID NO:2. In some instances herein, a particular amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding residue and position in the reference polypeptide (as described above), and "Y" is a single-letter identifier for the amino acid found in the engineered polypeptide (i.e., the residue that differs from the reference polypeptide). In some instances (e.g., in the Tables in the Examples), the disclosure also provides specific amino acid differences, designated with the conventional designation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, an amino acid difference, e.g., a substitution, is designated with the abbreviation "nB," without an identifier for the residue in the reference sequence. In some embodiments, the phrase "amino acid residue nB" indicates the presence of an amino acid residue in the engineered polypeptide, which may or may not be a substitution in the context of the reference sequence.

[0108] In some cases, polypeptides of the present disclosure can contain one or more amino acid residue differences relative to a reference sequence, as indicated by a list of the specific positions at which the residue difference relative to the reference sequence occurs. In some embodiments, when more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X46M / X46R, X46M / R, or 46M / R). The present disclosure includes engineered polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0109] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a substitution set includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions present in any of the engineered glucocerebrosidase polypeptides, such as those listed in any of the tables in the Examples. In these substitution sets, individual substitutions are separated by a semicolon (";"; e.g., K46R;P68V or abbreviated 46R;68V) or a slash (" / "; e.g., K46R / P68V or abbreviated 46R / 68V).

[0110] "Conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid with a hydroxyl side chain with another amino acid with a hydroxyl side chain (e.g., serine and threonine), an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid with a basic side chain with another amino acid with a basic side chain (e.g., lysine and arginine), an amino acid with an acidic side chain with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid with another hydrophobic or hydrophilic amino acid, respectively.

[0111] A "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, defined groups and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., glycine for proline), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions may be an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0112] "Deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids comprising the reference enzyme, while retaining enzymatic activity and / or improving the properties of the engineered enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous.

[0113] "Insertion" refers to a modification to a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion can be in the internal portion of the polypeptide, or at the carboxy- or amino-terminus. As used herein, insertion includes fusion proteins known in the art. The insertion can be a continuous segment of amino acids or can be separated by one or more amino acids in a naturally occurring polypeptide.

[0114] As used interchangeably herein, a "functional fragment" or a "biologically active fragment" refers to a polypeptide that has amino-terminal and / or carboxy-terminal deletion(s), and / or internal deletion(s), but whose remaining amino acid sequence is identical to corresponding positions in a sequence to which it is compared (e.g., a full-length engineered glucocerebrosidase of the present disclosure), and that retains substantially all of the activity of the full-length polypeptide.

[0115] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Engineered glucocerebrosidase polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, an engineered glucocerebrosidase polypeptide may be an isolated polypeptide.

[0116] A "substantially pure" or "purified" polypeptide or protein refers to a composition in which the polypeptide species is present as the predominant species (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition). Generally, a substantially purified composition is one in which the species of interest comprises at least about 50 percent of the macromolecular species present in the composition, either molar or weight percent. Generally, a substantially pure glucocerebrosidase composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more by weight of all macromolecular species present in the composition. In some embodiments, the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated engineered glucocerebrosidase polypeptide is a substantially pure polypeptide composition.

[0117] "Improved enzymatic properties" refers to engineered glucocerebrosidase polypeptides that exhibit any improved enzymatic properties compared to a reference glucocerebrosidase polypeptide and / or compared to a wild-type glucocerebrosidase polypeptide or another engineered glucocerebrosidase polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermal activity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved solvent stability, increased tolerance to acidic, neutral, or basic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, increased lysosomal stability, and the like.

[0118] "Increased enzymatic activity" or "enhanced catalytic activity" refers to improved properties of an engineered glucocerebrosidase polypeptide, which can be expressed by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the rate of substrate-to-product conversion (e.g., the rate of substrate-to-product conversion starting with a specific amount of glucocerebrosidase within a specific period of time) compared to a reference glucocerebrosidase enzyme. Exemplary methods for determining enzymatic activity are provided in the Examples. Any property associated with enzymatic activity can be affected, including the classical enzyme properties of Km, Vmax, or kcat, and changes thereto can result in increased enzymatic activity.

[0119] "Improved tolerance to acidic pH" refers to an engineered glucocerebrosidase that has increased stability (higher retained activity after exposure to an acidic pH, e.g., pH 2-6.6, for a specified period of time, e.g., 1 hour, up to 24 hours) compared to a reference glucocerebrosidase or another enzyme.

[0120] "Improved tolerance to neutral or basic pH" means an engineered glucocerebrosidase that has increased stability (higher retained activity after exposure to pH 7 or above for a specified period of time (e.g., 1 hour, up to 24 hours)) compared to a reference glucocerebrosidase or another enzyme.

[0121] "Improved cellular uptake" means that the engineered glucocerebrosidases provided herein exhibit increased uptake into cells (e.g., via endocytosis or transport) compared to a reference glucocerebrosidase (including wild-type glucocerebrosidase) or another enzyme. In some embodiments, the cells are cultured GD patient cells (higher retained intracellular activity after incubation with the cultured cells for a particular period of time compared to a reference glucocerebrosidase or another enzyme). In some additional embodiments, the engineered glucocerebrosidases provided herein exhibit greater retained intracellular activity in cultured cells for a particular period of time compared to a reference glucocerebrosidase (including wild-type glucocerebrosidase) or another enzyme. In some additional embodiments, the period is about 4 hours, while in some other embodiments, the period is less than 4 hours (e.g., 1, 2, or 3 hours), and in some alternative embodiments, the period is 4 hours or more (e.g., 5, 6, 7, 8, or more).

[0122] "Reduced immunogenicity" and "reduced immunogenicity" refer to an engineered glucocerebrosidase that induces a reduced immune response compared to a wild-type or another reference glucocerebrosidase.

[0123] As used herein, "deimmunized" refers to engineering a protein sequence to create a variant that is predicted to be less immunogenic than a wild-type or reference protein. In some embodiments, the predicted deimmunization is complete, in that the variant protein is predicted not to stimulate an immune response in patients to whom the variant protein is administered. This response can be measured by various methods, including, but not limited to, the presence or abundance of anti-drug antibodies, the presence or abundance of neutralizing antibodies, the presence of an anaphylactic response, peptide presentation on major histocompatibility complex II (MHC-II) proteins, or the frequency or intensity of cytokine release upon administration of the protein. In some embodiments, the variant protein is less immunogenic than the wild-type or reference protein. In some embodiments, deimmunization involves modifications to subsequences of the protein (e.g., epitopes) recognized by human leukocyte antigen (HLA) receptors. In some embodiments, these epitopes are removed by altering their amino acid sequence to produce a deimmunized variant protein in which such subsequences are no longer recognized by HLA receptors. In some other embodiments, these epitopes retain binding affinity to HLA receptors but are not presented. In some embodiments, the deimmunized proteins exhibit lower levels of response in biochemical and cell biological predictors of human immunological responses, including dendritic cell T cell activation assays or (HLA) peptide binding assays. In some embodiments, these epitopes are removed by altering their amino acid sequence to produce deimmunized variant proteins in which the epitopes are no longer recognized by T cell receptors. In still other embodiments, the deimmunized proteins induce anergy in their corresponding T cells, activate T regulatory cells, or result in clonal deletion of recognizing B cells. In some embodiments, the Total Immunogenicity Score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity).

[0124] As used herein, "physiological pH" means the pH range typically found in the blood of a subject (e.g., a human).

[0125] "Basic pH" (eg, used in reference to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH > 7, eg, a pH range > 7-11.

[0126] "Acidic pH" (eg, used in reference to improved stability to or increased tolerance to acidic pH conditions) means a pH <7, particularly a pH range of about 2 to <7.

[0127] " Hybridization stringency " refers to the hybridization conditions, such as washing conditions, in the hybridization of nucleic acid. Generally, hybridization reaction is carried out under the condition of lower stringency, followed by washing under various but higher stringency (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term "moderately stringent hybridization" refers to the conditions that allow target DNA to bind with the complementary nucleic acid that has about 90% identity with target polynucleotide, and has about 60% identity with target DNA, preferably about 75% identity, about 85% identity. Exemplary moderately stringent conditions are those equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to conditions that are about 10°C or less from the thermal melting temperature, Tm, determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization at 42°C under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 65°C in 0.1x SSPE and 0.1% SDS.Another high stringency condition is hybridization in 5×SSC containing 0.1% (w:v) SDS at 65° C., followed by a wash in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, and moderately stringent conditions, are described in the references cited above.

[0128] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those that are preferentially used in a particular organism so that the encoded protein can be expressed more efficiently in the target organism. Although the genetic code is degenerate in that most amino acids are represented by multiple codons, called "synonyms" or "synonymous" codons, it is well known that the codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for certain genes, genes with common functions or ancestral origins, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding an engineered glucocerebrosidase polypeptide can be codon-optimized for optimal production from the host organism selected for expression.

[0129] The term "control sequences," as used herein, includes all components necessary or advantageous for the expression of polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. In some embodiments, at a minimum, control sequences include a promoter, and transcriptional and translational stop signals.

[0130] "Operably linked" or "operatively linked" is defined herein as a configuration in which control sequences are appropriately positioned (i.e., in functional relationship) with a polynucleotide of interest such that the control sequences direct or regulate expression of the polynucleotide, and, as appropriate, direct or regulate expression of the encoded polypeptide of interest.

[0131] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. Promoter sequences include transcriptional control sequences that mediate expression of the polynucleotide of interest. Promoters may be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0132] "Suitable reaction conditions" refer to conditions in an enzymatic conversion reaction solution (e.g., ranges of enzyme load, substrate load, temperature, pH, buffer, cosolvent, etc.) that allow the glucocerebrosidase polypeptide of the present application to convert a substrate into a desired product. Exemplary "suitable reaction conditions" are provided herein and illustrated by the Examples. "Substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by a glucocerebrosidase polypeptide. "Product" in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of a glucocerebrosidase polypeptide on a substrate.

[0133] "Culturing" refers to the growth of a population of cells under any suitable conditions (eg, using liquid, gel, or solid medium).

[0134] "Vector" refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector operably linked to suitable control sequences capable of affecting expression of the polynucleotide in a suitable host and, if relevant, expression of the polypeptide encoded by the polynucleotide sequence. In some embodiments, an "expression vector" has a promoter sequence operably linked to the polynucleotide sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0135] "Gene therapy vector" refers to a vehicle or carrier suitable for delivering a polynucleotide sequence to a cell for therapeutic purposes. In some embodiments, the vector encapsulates a gene (e.g., a transgene) or polynucleotide sequence for delivery to a cell or tissue, including, but not limited to, non-viral vectors such as adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and liposomes. It is not intended that the present disclosure be limited to any particular gene therapy vector, as any vehicle suitable for a given setting may find use herein. Gene therapy vectors may be designed to deliver genes to a specific species or host or cell, or may find more general applicability.

[0136] "Expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0137] "Production" refers to the production of proteins and / or other compounds by a cell. The term is intended to encompass any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0138] "Heterologous" refers to the relationship between two or more nucleic acid or protein sequences (e.g., promoter sequences, signal peptides, transcription terminator sequences, etc.) that are derived from different sources and are not related in nature.

[0139] "Host cell" and "host strain" refer to a suitable host for an expression vector containing a recombinant polynucleotide provided herein (e.g., a polynucleotide encoding a glucocerebrosidase variant). In some embodiments, a host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known to those skilled in the art.

[0140] "Therapeutic agent" refers to a compound administered to a subject exhibiting signs or symptoms of pathology that has a beneficial or desired medical effect.

[0141] "Gene therapy" refers to the delivery of a gene, polydeoxyribonucleotide, or polynucleotide sequence(s) to cells or tissues using a gene therapy vector or formulation to modify those cells or tissues for the treatment or prevention of disease. In some embodiments, gene therapy can be transient, for example, by introducing a gene, polydeoxyribonucleotide, or polynucleotide sequence(s) into a cell, which results in the transient presence of the gene, polydeoxyribonucleotide, or polynucleotide. In some embodiments, gene therapy can involve replacing a disease-causing mutant gene with a healthy copy of the gene, or inactivating or "knockout" an improperly functioning mutant gene, or providing a sufficient functional copy of the gene to treat and / or prevent a disease or condition. In some embodiments, gene therapy is used to treat a disease in a patient.

[0142] "mRNA therapy" refers to the delivery of mRNA polyribonucleotides to cells or tissues to modify those cells or tissues in order to treat or prevent disease. In some embodiments, the mRNA polynucleotides for delivery to cells or tissues are formulated, for example, but not limited to, in liposomes. In some embodiments, mRNA therapy is used to treat a patient's disease.

[0143] "Cell therapy" refers to the delivery of exogenously modified living cells to a patient to provide a defective gene to treat or prevent disease. The modified cells are then reintroduced into the body.

[0144] By "effective amount" is meant an amount sufficient to bring about a desired result. One of ordinary skill in the art can determine the effective amount by using routine experimentation.

[0145] "Subject" includes mammals such as humans, non-human primates, livestock, companion animals (e.g., cats, dogs, rodents), and laboratory animals (e.g., rodents and lagomorphs). "Patient" means any subject being evaluated for, treated for, or experiencing a disease.

[0146] "Composition" and "formulation" encompass products comprising at least one engineered glucocerebrosidase of the present disclosure or a recombinant polynucleotide encoding an engineered glucocerebrosidase intended for any suitable use (e.g., pharmaceutical composition, etc.).

[0147] "Administration" and "administering" an engineered glucocerebrosidase, or a composition thereof, means providing an engineered glucocerebrosidase of the present disclosure, or a composition thereof, to a subject (e.g., a subject affected by Gaucher disease).

[0148] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., a human) comprising a pharmaceutically effective amount of an engineered glucocerebrosidase polypeptide disclosed herein and an acceptable carrier and / or excipient.

[0149] "Pharmaceutically acceptable" means a substance that may be administered to a subject without causing any undesired biological effects or interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0150] When used in reference to pharmaceutical compositions, "carrier" means any of the standard pharmaceutical carriers, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0151] "Excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API; e.g., an engineered glucocerebrosidase polypeptide of the disclosure). Excipients are typically included for formulation and / or administration purposes.

[0152] A "therapeutically effective amount," when used in reference to symptoms of a disease / condition, refers to an amount and / or concentration of a compound (e.g., an engineered glucocerebrosidase polypeptide) that ameliorates, attenuates, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of a symptom(s). A "therapeutically effective amount," when used in reference to a disease / condition, refers to an amount and / or concentration of a composition (e.g., an engineered glucocerebrosidase polypeptide) that ameliorates, attenuates, or eliminates the disease / condition. In some embodiments, the term is used in reference to the amount of a composition that elicits a biological (e.g., medical) response by a tissue, system, or animal subject as desired by a researcher, physician, veterinarian, or other clinician.

[0153] "Treating" a disease, disorder, or syndrome or "treatment," as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., preventing the development of clinical symptoms of the disease, disorder, or syndrome in an animal that may be exposed to or susceptible to the disease, disorder, or syndrome, but that does not also experience or display symptoms of the disease, disorder, or syndrome; (ii) arresting the disease, disorder, or syndrome, i.e., halting its development; and (iii) alleviating the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. Thus, the terms "treating," "treat," and "treatment" encompass preventative (e.g., prophylactic) treatment and palliative treatment. As known in the art, adjustments may be necessary for systemic versus local delivery, age, weight, overall health, sex, diet, duration of administration, drug interactions, and severity of the condition, and can be ascertained through routine experimentation by one of ordinary skill in the art.

[0154] Engineered glucocerebrosidase polypeptides In one aspect, the present disclosure provides engineered glucocerebrosidases with improved properties. As noted above, in some embodiments, the engineered glucocerebrosidases are characterized by, among other things, improved activity, improved expression efficiency, increased stability at neutral and acidic pH, increased serum stability, enhanced cellular uptake, and / or reduced immunogenicity.

[0155] In some embodiments, the engineered glucocerebrosidase, or biologically active fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NOs: 2-1648, or a reference sequence corresponding to SEQ ID NOs: 2-1648, wherein the amino acid sequence contains one or more substitutions relative to a reference sequence corresponding to residues 40-536 of SEQ ID NOs: 2, 54, 220, 984, or 1150, or a reference sequence corresponding to SEQ ID NOs: 2, 54, 220, 984, or 1150.

[0156] In some embodiments, the engineered glucocerebrosidase, or biological fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150, wherein the amino acid sequence contains one or more substitutions.

[0157] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0158] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions.

[0159] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to an even-numbered SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions.

[0160] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to an even-numbered SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions.

[0161] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 16, 117, 133, 134, 137, 138, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 3 01, 314, 315, 316, 319, 325, 329, 332, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 4 or 536, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0162] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one of the following substitutions: 23A, 29I, 35T, 40H / S, 41H, 42A, 44V, 46M / R, 50A, 51P, 53L, 59N, 63L, 65L / M / S / V, 68F / L / Q / T / V / W / Y, 69D, 70L, 71F / G / H / L / M / R / S / T / Y, 75A / E / H / N, 77G / H / I / N / R / T, 78L / M, 79I / R / V, 82S, 84A / G / K, 86E / H, 88L, 89C / D, 90A / M, 91I / T, 92A / E / G / H / K / Q, 93S, 94E / N, 95G / H / L / T / W, 96D / E / G / I / M / N / R / S / V, 97E / I / K / M / N / Q / R, 98P, 99E / F / G / K / L / R / S / V / W, 100N / R, 101L / Q / R / S / T / V / W, 102A / E / N / P / S, 103A / L / P / S, 104M / V, 105I / M / T, 106W, 107A / C / D / E / M / Q / S, 108I, 109D / G / L / M / R, 1 10A / H / Q, 111F / H / L, 113G / I / N / P / Q / R, 114Y, 115M / R / V, 116E / M / Q / R / V / Y, 11 7I / T, 133M, 134S, 137Q / V, 138S, 141D / E, 142A / M / N / Y, 144M, 150T, 151L, 15 4E, 169V, 171C / P / V, 172S, 175D, 180C, 182D / E / G / H / M / S / T, 184I / Q / V, 186G, 189R / S, 190W, 191N / R, 194H / L / Q, 195M / W, 201Q / R / T, 202Q, 204Q, 206F / I / R , 207S / D, 211I / L / N, 212A / N, 214F, 220A / G, 222I, 224M, 230F / L / Y, 233A / G / Q / R, 235T, 236M, 239I / R / T, 243K / L / M / R / V, 246K / R, 258T, 260R, 261K, 262Q, 263G, 264I, 265N / R / S / T, 271T, 276T, 298W, 299V, 301K, 314D / G / R, 315T, 316 H, 319F / I / V, 325V, 329L, 332R, 333C / L / T, 335M, 336A / E / G / R, 337N, 338R, 3 39A, 341R / S, 342Q, 359E / P, 360E, 361L / V / W / Y, 368D, 370R, 372D, 373A / V / Y,374T / V, 382A, 385G / H / N / Q / R, 386I / L / P, 390A, 400E / Q / T, 404Q, 408D / E, 412N / W, 413W / Y, 4 15C, 434G / Q, 445G / N, 446H / N, 449E / K / L / M / R / S / V, 459M, 467H / L / M / N / R, 468G, 469E, 471E / F / L / R / S / T / V, 473C / I / L, 474A / D / S, 476A, 477G / H / R / S / V, 478N, 479A / L / R, 480G, 481H / K / T , 484E, 485M / S / T, 487S, 489A / E / G / L / N / Q / T, 490G / I / N / Q / R / S / T / V, 493T, 494A / T, 495G / S, 4 97A / M, 502K / Q, 504G / P / T / W, 505I / L / R, 506T, 507A / I / L / M / R / T / Y, 508L / Q / R / S / T, 509F / G / I / R / V / W, 510S, 512H / L / Q, 514E, 515G / T, 516L, 518E / L / S / V, 519I, 520D / K / R / S / V, 522S / T / V , 524K / S, 525C / Q, 529M / Q, 532V, 533Y, 534K, or 536A / C / D / H / I / L / M / P / R / T / W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0163] It's a nice little snack and it's a nice smile on your face Material keys include and 1 of grades G23A, L29I, S35T. A40H / S, R41H, P42A, I44V, K46M / R, Y50A, S51P, V53L, A59N, D63L, F65L / M / S / V、P68F / L / Q / V / T / W / Y、T69D、F70L、P71F / G / H / L / M / R / S / T / Y、T75A / E / H / N、 S77G / H / I / N / R / T、R78L / M、Y79I / R / V,T82S、S84A / G / K、R86E / H、M88L、E89C / D L90A / M, S91I / T, M92A / E / G / H / K / Q, G93S, P94E / N, I95G / H / L / T / W, Q96D / E / G / I / M / N / R / S / V、A97E / I / K / M / N / Q / R、N98P、H99E / F / G / K / L / R / S / V / W、T100N / R、G101L / Q / R / S / T / V / W、T102A / E / N / P / S、G103A / L / P / S、L104M / V、L105I / M / T、L106W、T107A / C / D / E / M / Q / S、L108I、Q109D / G / L / M / R、P110A / H / Q、E111F / H / L、K113G / I / N / P / Q / R、F114Y、Q115M / R / V、K116E / M / Q / R / V / Y、V117I / T、L1 33M, A134S, P137Q / V, P138S, N141D / E, L142A / M / N / Y, L144M, E150T, E151L. G154E, I169V, T171C / P / V, Y172S, A175D, D180C, Q182D / E / G / H / M / S / T, H184 I / Q / V、F186G、P189R / S、E190W、E191N / R、K194H / L / Q、L195M / W、H201Q / R / T、R 202Q、L204Q、L206F / I / R、A207D / S、V211I / L / N、S212A / N、L214F、S220A / G、T 222I、L224M、V230F / L / Y、K233A / G / Q / R、S235T、L236M、Q239I / R / T、I243K / L / M / R / V、Q246K / R、A258T、A260R、E261K、H262Q、K263G、L264I、Q265N / R / S / T A271T, S276T, F298W, I299V, R301K, N314D / G / R, V315T, R316H, M319F / I / V.L325V, H329L, K332R, V333C / L / T, L335M, T336A / E / G / R, D337N, P338R, E339A, A341R / S, K342Q, A359E / P, K360E, A361L / V / W / Y, R368D, F370R, N372D, T373A / V / Y, M374T / V, V382A, K385G / H / N / Q / R, F386I / L / P, S390A, M400E / Q / T, H404Q, T408D / E, Y412N / W, H413W / Y, V415C, R434G / Q, I445G / N, T446H / N, T449E / K / L / M / R / S / V, L459M, P467H / L / M / N / R, E468G, G469E, Q 471E / F / L / R / S / T / V, V473C / I / L, G474A / D / S, V476A, A477G / H / R / S / V, S478N, Q479A / L / R, K480G, N481H / K / T, D484E, A485M / S / T, A487S, M489A / E / G / L / N / Q / T, H490G / I / N / Q / R / S / T / V, G493T, S494A / T, A495G / S, V497A / M, R502K / Q, S504G / P / T / W, K505I / L / R, D506T, V507A / I / L / M / R / T / Y, P508L / Q / R / S / T, L509F / G / I / R / V / W, T510S, K512H / L / Q, P514E, A515G / T, V516L, F518E / L / S / V, L519I, E520D / K / R / S / V, I522S / T / V, P524K / S, G525C / Q, H529M / Q, L532V, W533Y, R534K, or Q536A / C / D / H / I / L / M / P / R / T / W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0164] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0165] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase includes at least one substitution 65L / M / S / V, 68F / L / Q / T / V / W / Y, 70L, 99E / F / G / K / L / R / S / V / W, 154E, 195M / W, 207S / D, 214F, 230F / L / Y, 263M, 319F / I / V, 337N, 361L / V / W / Y, 374T / V, 408D / E, 471E / F / L / R / S / T / V, or 489A / E / G / L / N / Q / T, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0166] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase includes at least one substitution 665L / V, 68L, 70L, 99R, 154E, 195W, 207S, 214F, 230L / Y, 263G, 319I, 337N, 361W, 374T, 408E, 471T, or 489L, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0167] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 70, 207, 214, or 489, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 70L, 207S / D, 214F, or 489A / E / G / L / N / Q / T, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 70L, 207S, 214F, or 489L, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set at amino acid positions 70 / 207 / 214 / 489, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set 70L / 207S / 214F / 489L, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0168] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 99, 154, 230, or 319, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 65L / M / S / V, 68F / L / Q / T / V / W / Y, 99E / F / G / K / L / R / S / V / W, 154E, 230F / L / Y, or 319F / I / V, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 65L, 68L, 99R, 154E, 230L, or 319I, or a combination thereof, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set at amino acid positions 65 / 68 / 99 / 154 / 230 / 319, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set 65L / 68L / 99R / 154E / 230L / 319I, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0169] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 195, 230, 263, 337, 361, 374, or 471, or a combination thereof, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 65L / M / S / V, 195M / W, 230F / L / Y, 263G, 337N, 361L / V / W / Y, 374T / V, or 471E / F / L / R / S / T / V, or a combination thereof, where the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 65V, 195W, 230Y, 263G, 337N, 361W, 374T, or 471T, or a combination thereof, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set at amino acid positions 65, 195, 230, 263, 337, 361, 374, or 471, where the amino acid position is relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set 65V / 195W / 230Y / 263G / 337N / 361W / 374T / 471T, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0170] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 408, the amino acid position relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 408D / E, the amino acid position relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0171] In some embodiments, the amino acid sequence of the engineered glucocerebroside has amino acid position(s) 265 / 372 / 489, 243, 23, 109, 265, 109 / 195 / 243, 23 / 70 / 207 / 214 / 265, 23 / 70, 23 / 214 / 243 / 265, 65 / 70 / 109 / 214 / 319 / 372 / 481, 23 / 265 / 319, 23 / 65 / 70 / 141 / 319 / 481, 23 / 265, 372, 214, 23 / 65 / 70 / 109 / 141 / 214, 319, 65 / 372, 109 / 195, 23 / 65, 207 / 265 / 319, 109 / 207 / 214 / 372, 23 / 319 / 372 / 489, 65 / 109 / 214 / 243, 23 / 141 / 195, 70 / 207 / 214 / 489, 65 / 70 / 265 / 319 / 372, 214 / 265, 265 / 319, 65 / 243, 65 / 70 / 195, 489, 23 / 65 / 141, 23 / 372, 70 / 141, 23 / 195, 214 / 319, 23 / 207 / 214 / 243 / 265 / 481, 65 / 214 / 243 / 265 / 319, 214 / 243, 243 / 265, 265 / 489, 214 / 243 / 265, 207 / 243 / 372, 195 / 243, 65 / 481, 207 / 214 / 243 / 319, 70 / 141 / 195 / 265, 23 / 265 / 372, 46 / 99 / 134 / 230 / 373 / 449, 68 / 211 / 230, 373 / 449, 46 / 68 / 33 6, 68 / 230 / 314 / 373, 68, 68 / 336 / 373, 99 / 314, 46, 46 / 230 / 261, 373, 230 / 314, 68 / 211, 68 / 336 / 449, 46 / 336, 336 / 449, 46 / 68 / 336 / 373, 46 / 99, 99 / 261, 99, 68 / 134, 46 / 68 / 134 / 230 / 336 / 373, 46 / 68, 68 / 99 / 211 / 230, 99 / 134, 68 / 99 / 336, 449, 68 / 99, 46 / 230 / 336, 68 / 154 / 449, 336 / 373 / 449, 134 / 336, 68 / 99 / 230, 46 / 68 / 134 / 154, 68 / 99 / 230 / 373 / 449, 134, 46 / 68 / 154 / 211 / 230 / 261 / 336, 68 / 99 / 154 / 261 / 336, 230 / 449, 46 / 68 / 314, 154, 68 / 449, 99 / 449,At least one substitution or set of substitutions at 68 / 154 / 230 / 314 / 336 / 449, 46 / 68 / 154 / 314 / 336 / 373, 46 / 230, 336, or 46 / 99 / 230 / 373, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0172] In some embodiments, the amino acid sequence of the engineered glucocerebroside has at least one substitution or set of substitutions 265T / 372D / 489L, 243K, 23A, 109D, 265T, 109D / 195M / 243K, 23A / 70L / 207S / 214F / 265T, 23A / 70L, 23A / 214F / 243K / 265T, 65L / 70L / 109D / 214F / 319I / 372D / 481T, 23A / 265T / 319I, 23A / 65L / 70L / 141D / 319I / 481T, 23A / 265T, 372D, 214F, 265T ... 3A / 65L / 70L / 109D / 141D / 214F, 319I, 65L / 372D, 109D / 195M, 23A / 65L, 207 S / 265T / 319I, 109D / 207S / 214F / 372D, 23A / 319I / 372D / 489L, 65L / 109D / 21 4F / 243K, 23A / 141D / 195M, 70L / 207S / 214F / 489L, 65L / 70L / 265T / 319I / 37 2D, 214F / 265T, 265T / 319I, 65L / 243K, 65L / 70L / 195M, 489L, 23A / 65L / 141D , 23A / 372D, 70L / 141D, 23A / 195M, 214F / 319I, 23A / 207S / 214F / 243K / 265T / 481T, 65L / 214F / 243K / 265T / 319I, 214F / 243K, 243K / 265T, 265T / 489L, 21 4F / 243K / 265T, 207S / 243K / 372D, 195M / 243K, 65L / 481T, 207S / 214F / 243K / 319I, 70L / 141D / 195M / 265T, 23A / 265T / 372D, 46R / 99R / 134S / 230L / 373Y / 449V, 68L / 211L / 230L, 373Y / 449V, 46R / 68V / 336A, 68V / 230L / 314D / 373Y, 68V, 68L / 336A / 373Y, 99R / 314D, 46R, 46R / 230L / 261K, 373Y, 230L / 314D, 68 L / 211L, 68V / 336A / 449V, 46R / 336A, 336A / 449V, 46R / 68V / 336A / 373Y, 46R / 99R, 99R / 261K, 99R, 68V / 134S, 46R / 68V / 134S / 230L / 336A / 373Y, 46R / 68V,68L / 99R / 211L / 230L, 99R / 134S, 68V / 99R / 336A, 449V, 68V / 99R, 46R / 230L / 336A, 68V / 154E / 449V, 336A / 373Y / 449V, 134S / 336A, 68V / 99R / 230L, 68L, 46R / 68V / 134S / 154E, 68V / 99R / 230L / 373Y / 449V, 134S, 46R / 68V / 154E / 211L / 230L / 261K / 336A, 68V / 99R / 154E / 261 K / 336A, 230L / 449V, 46R / 68L / 314D, 154E, 68L / 449V, 99R / 449V, 68V / 154E / 230L / 314D / 336A / 449V, 46R / 68V / 154E / 314D / 336A / 373Y, 46R / 230L, 336A, or 46R / 99R / 230L / 373Y, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0173] In some embodiments, the amino acid sequence of the engineered glucocerebroside contains at least one substitution or set of substitutions Q265T / N372D / M489L, I243K, G23A, Q109D, Q265T, Q109D / L195M / I243K, G23A / F70L / A207S / L214F / Q265T, G23A / F70L, G23A / L214F / I243K / Q265T, F65L / F70L / Q109D / L214F / M319I / N372D / N481T, G23A / Q265T / M319I, G23A / F65L / F70L / N14 1D / M319I / N481T, G23A / Q265T, N372D, L214F, G23A / F65L / F70L / Q109D / N14 1D / L214F, M319I, F65L / N372D, Q109D / L195M, G23A / F65L, A207S / Q265T / M3 19I, Q109D / A207S / L214F / N372D, G23A / M319I / N372D / M489L, F65L / Q109D / L214F / I243K, G23A / N141D / L195M, F70L / A207S / L214F / M489L, F65L / F70L / Q 265T / M319I / N372D, L214F / Q265T, Q265T / M319I, F65L / I243K, F65L / F70L / L195M, M489L, G23A / F65L / N141D, G23A / N372D, F70L / N141D, G23A / L195M, L 214F / M319I, G23A / A207S / L214F / I243K / Q265T / N481T, F65L / L214F / I243K / Q265T / M319I, L214F / I243K, I243K / Q265T, Q265T / M489L, L214F / I243K / Q2 65T, A207S / I243K / N372D, L195M / I243K, F65L / N481T, A207S / L214F / I243K / M319I, F70L / N141D / L195M / Q265T, G23A / Q265T / N372D, K46R / H99R / A134S / V230L / T373Y / T449V, P68L / V211L / V230L, T373Y / T449V, K46R / P68V / T336 A, P68V / V230L / N314D / T373Y, P68V, P68L / T336A / T373Y, H99R / N314D, K46R,K46R / V230L / E261K, T373Y, V230L / N314D, P68L / V211L, P68V / T336A / T449V, K46R / T336A, T336A / T449V, K 46R / P68V / T336A / T373Y, K46R / H99R, H99R / E261K, H99R, P68V / A134S, K46R / P68V / A134S / V230L / T336A / T3 73Y, K46R / P68V, P68L / H99R / V211L / V230L, H99R / A134S, P68V / H99R / T336A, T449V, P68V / H99R, K46R / V230 L / T336A, P68V / G154E / T449V, T336A / T373Y / T449V, A134S / T336A, P68V / H99R / V230L, P68L, K46R / P68V / A1 34S / G154E, P68V / H99R / V230L / T373Y / T449V, A134S, K46R / P68V / G154E / V211L / V230L / E261K / T336A, P68V / H99R / G154E / E261K / T336A, V230L / T449V, K46R / P68L / N314D, G154E, P68L / T449V, H99R / T449V, P68V / G15 4E / V230L / N314D / T336A / T449V, K46R / P68V / G154E / N314D / T336A / T373Y, K46R / V230L, T336A, or K46R / H99R / V230L / T373Y, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0174] In some embodiments, the amino acid sequence of the engineered glucocerebroside comprises amino acid position(s) 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408, 65 / 68 / 154 / 195 / 214 / 230 / 319 / 337 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 154 / 195 / 214 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 374 / 408, 65 / 68 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 31 9 / 337 / 361 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 374 / 408 / 471, 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 408, 65 / 68 / 70 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 214 / 230 / 263 / 337 / 374 / 408, 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 230 / 408, 65 / 68 / 70 / 99 / 154 / 207 / 230 / 319 / 408 , 65 / 68 / 70 / 214 / 230 / 408, 65 / 68 / 70 / 195 / 214 / 230 / 263 / 361 / 471, 195 / 207 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 214 / 230 / 408 / 489, 65 / 68 / 70 / 195 / 207 / 230 / 408, 207 / 230 / 319 / 374 / 408, 65 / 68 / 70 / 99 / 195 / 207 / 230 / 408, 195 / 214 / 230 / 263 / 408, 195 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 214 / 408,207 / 214 / 230 / 319 / 374 / 408, 207 / 214 / 230 / 374 / 408, 154 / 207 / 230 / 374 / 408 / 489, 207 / 230 / 263 / 408, 207 / 230 / 408 / 471, 207 / 230 / 374 / 408, 214 / 230 / 319 / 408, 214 / 230 / 374 / 408 / 489, 207 / 230 / 408, 65 / 68 / 70 / 154 / 207 / 408, 207 / 230 / 263 / 337 / 408, 195 / 214 / At least one substitution or set of substitutions at 230 / 408, 207 / 230 / 408 / 489, 214 / 230 / 408, 230 / 361 / 408, 207 / 214 / 408, 214 / 263 / 408, 230 / 374 / 408, 207 / 408, 195 / 214 / 263, 374 / 408, or 408, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0175] In some embodiments, the amino acid sequence of the engineered glucocerebroside has at least one substitution or set of substitutions 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E, 65V / 68L / 154E / 195W / 214F / 230Y / 319I / 337N / 374T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 374 T / 408E, 65V / 68L / 70L / 154E / 195W / 214F / 230Y / 319I / 374T / 408E / 489L, 65 V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 337N / 361W / 374T / 4 08E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 3 19I / 337N / 374T / 408E, 65V / 68L / 154E / 195W / 207S / 214F / 230Y / 263G / 319 I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207 S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 489L, 65V / 68L / 70L / 9 9R / 154E / 195W / 207S / 214F / 230Y / 374T / 408E / 471T, 68L / 70L / 99R / 154E / 1 95W / 207S / 214F / 230Y / 263G / 319I / 408E, 65V / 68L / 70L / 207S / 230Y / 374T / 408E, 65V / 68L / 70L / 214F / 230Y / 263G / 337N / 374T / 408E, 214F / 230Y / 263 G / 374T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 230Y / 408E, 65V / 68L / 70L / 99R / 154E / 207S / 230Y / 319I / 408E, 65V / 68L / 70L / 214F / 230Y / 408E,65V / 68L / 70L / 195W / 214F / 230Y / 263G / 361W / 471T, 195W / 207S / 230Y / 319I / 374T / 408E / 489L, 65V / 68L / 70L / 214F / 230 Y / 408E / 489L, 65V / 68L / 70L / 195W / 207S / 230Y / 408E, 207S / 230Y / 319I / 374T / 408E, 65V / 68L / 70L / 99R / 195W / 207S / 23 0Y / 408E, 195W / 214F / 230Y / 263G / 408E, 195W / 207S / 230Y / 374T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 214F / 408E, 207 S / 214F / 230Y / 319I / 374T / 408E, 207S / 214F / 230Y / 374T / 408E, 154E / 207S / 230Y / 374T / 408E / 489L, 207S / 230Y / 263G / 408E, 207S / 230Y / 408E / 471T, 207S / 230Y / 374T / 408E, 214F / 230Y / 319I / 408E, 214F / 230Y / 374T / 408E / 489L, 207S / 23 0Y / 408E, 65V / 68L / 70L / 154E / 207S / 408E, 207S / 230Y / 263G / 337N / 408E, 195W / 214F / 230Y / 408E, 207S / 230Y / 408E / 48 9L, 214F / 230Y / 408E, 230Y / 361W / 408E, 207S / 214F / 408E, 214F / 263G / 408E, 230Y / 374T / 408E, 207S / 408E, 195W / 214F / 263G, 374T / 408E, or 408E, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0176] In some embodiments, the amino acid sequence of the engineered glucocerebroside comprises at least one substitution or set of substitutions F65V / P68L / F70L / H99R / G154E / L195W / A207S / L214F / V230Y / K263G / M319I / D337N / A361W / M374T / T408E / Q471T / M489L, F65V / P68L / F70L / H99R / G154E / L195W / A207S / L214F / V230Y / K263G / M319I / D337N / A361W / M374T / T408E, F65V / P68L / G1 54E / L195W / L214F / V230Y / M319I / D337N / M374T / T408E, F65V / P68L / F70L / H 99R / G154E / L195W / A207S / L214F / V230Y / K263G / M374T / T408E, F65V / P68L / F 70L / G154E / L195W / L214F / V230Y / M319I / M374T / T408E / M489L, F65V / P68L / F70L / H99R / G154E / L195W / A207S / L214F / V230Y / M319I / D337N / A361W / M374T / T408E / Q471T / M489L, F65V / P68L / F70L / H99R / G154E / L195W / A207S / L214F / V230Y / K263G / M319I / D337N / M374T / T408E, F65V / P68L / G154E / L195W / A207 S / L214F / V230Y / K263G / M319I / D337N / A361W / M374T / T408E / Q471T / M489L, F65V / P68L / F70L / H99R / G154E / L195W / A207S / L214F / V230Y / K263G / M319I / D 337N / A361W / M374T / T408E / M489L, F65V / P68L / F70L / H99R / G154E / L195W / A 207S / L214F / V230Y / M374T / T408E / Q471T, P68L / F70L / H99R / G154E / L195W / A 207S / L214F / V230Y / K263G / M319I / T408E, F65V / P68L / F70L / A207S / V230Y / M374T / T408E, F65V / P68L / F70L / L214F / V230Y / K263G / D337N / M374T / T408E,L214F / V230Y / K263G / M374T / T408E、F65V / P68L / F70L / H99R / G154E / L195W / A207S / V230Y / T408E、F65V / P68L / F70L / H99R / G154E / A207S / V230Y / M319I / T408E、F65V / P68L / F70L / L214F / V230Y / T408E、F65V / P68L / F70L / L195W / L214F / V230Y / K263G / A361W / Q471T、L195W / A207S / V230Y / M319I / M374T / T408E / M489L、F65V / P68L / F70L / L214F / V230Y / T408E / M489L、F65V / P68L / F70L / L195W / A207S / V230Y / T408E、A207S / V230Y / M319I / M374T / T408E、F65V / P68L / F70L / H99R / L195W / A207S / V230Y / T408E、L195W / L214F / V230Y / K263G / T408E、L195W / A207S / V230Y / M374T / T408E、F65V / P68L / F70L / H99R / G154E / L195W / L214F / T408E、A207S / L214F / V230Y / M319I / M374T / T408E、A207S / L214F / V230Y / M374T / T408E、G154E / A207S / V230Y / M374T / T408E / M489L、A207S / V230Y / K263G / T408E、A207S / V230Y / T408E / Q471T、A207S / V230Y / M374T / T408E、L214F / V230Y / M319I / T408E、L214F / V230Y / M374T / T408E / M489L、A207S / V230Y / T408E、F65V / P68L / F70L / G154E / A207S / T408E、A207S / V230Y / K263G / D337N / T408E、L195W / L214F / V230Y / T408E、A207S / V230Y / T408E / M489L、L214F / V230Y / T408E、V230Y / A361W / T408E、A207S / L214F / T408E、L214F / K263G / T408E、V230Y / M374T / T408E、A207S / T408E、L195W / L214F / K263G、M374T / T408E、or T408E, and the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 2, or are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0177] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase has amino acid position(s) 68 / 70 / 207 / 214 / 336 / 489, 65 / 70 / 99 / 207 / 214 / 319 / 489, 65 / 68 / 70 / 207 / 214 / 319 / 489, 68 / 70 / 207 / 214 / 319 / 489, 68 / 70 / 207 / 214 / 489, 65 / 70 / 207 / 214 / 319 / 489, 70 / 195 / 207 / 214 / 489, 65 / 154 / 70 / 207 / 214 / 319 / 489, 70 / 207 / 214 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 489, 68 / 70 / 207 / 214 / 230 / 489, 70 / 195 / 207 / 214 / 230 / 489, 65 / 70 / 207 / 214 / 230 / 489, 70 / 141 / 207 / 214 / 319 / 489, 70 / 154 / 207 / 214 / 336 / 489, 68 / 70 / 154 / 207 / 214 / 489, 68 / 70 / 195 / 207 / 214 / 230 / 319 / 336 / 489, 68 / 70 / 99 / 195 / 207 / 214 / 230 / 319 / 489, 68 / 70 / 195 / 207 / 214 / 48 9, 65 / 68 / 70 / 207 / 214 / 489, 68 / 70 / 141 / 207 / 214 / 230 / 319 / 489, 65 / 68 / 70 / 99 / 207 / 214 / 230 / 319 / 489, 68 / 70 / 99 / 141 / 207 / 214 / 319 / 489, 68 / 70 / 154 / 2 07 / 214 / 319 / 489, 70 / 141 / 154 / 207 / 214 / 489, 68 / 70 / 99 / 207 / 214 / 489, 70 / 99 / 207 / 214 / 230 / 319 / 489, 70 / 99 / 207 / 214 / 489, 70 / 207 / 214 / 230 / 489, 68 / 70 / 207 / 214 / 230 / 319 / 489, 70 / 154 / 207 / 214 / 319 / 489, 65 / 68 / 70 / 154 / 207 / 214 / 319 / 489, 70 / 141 / 207 / 214 / 489, 70 / 207 / 214 / 336 / 489, 70 / 141 / 207 / 2 14 / 230 / 489, 68 / 70 / 154 / 207 / 214 / 336 / 489, 65 / 68 / 70 / 99 / 207 / 214 / 319 / 489, 70 / 154 / 207 / 214 / 489, 70 / 207 / 214 / 319 / 489, 70 / 207 / 214 / 230 / 319 / 489,70 / 99 / 141 / 207 / 214 / 319 / 489、70 / 207 / 214 / 489 / 509、70 / 108 / 207 / 214 / 489、70 / 108 / 117 / 207 / 214 / 489 / 509、70 / 207 / 214 / 489 / 519、70 / 108 / 207 / 214 / 489 / 519、70 / 88 / 207 / 214 / 489、70 / 117 / 207 / 214 / 413 / 489 / 509、70 / 207 / 214 / 489 / 509 / 519、70 / 207 / 214 / 413 / 489、70 / 108 / 117 / 207 / 214 / 315 / 489、70 / 117 / 207 / 214 / 271 / 489、70 / 117 / 207 / 214 / 413 / 489 / 509 / 529、70 / 207 / 214 / 413 / 489 / 509、70 / 207 / 214 / 489 / 469、70 / 207 / 214 / 489 / 524、70 / 207 / 214 / 489 / 507、70 / 207 / 214 / 489 / 525、70 / 104 / 207 / 214 / 489、70 / 95 / 207 / 214 / 489、70 / 207 / 214 / 359 / 489、70 / 207 / 214 / 374 / 489、70 / 107 / 207 / 214 / 489、70 / 89 / 207 / 214 / 489、70 / 207 / 214 / 260 / 489、70 / 110 / 207 / 214 / 489、70 / 186 / 207 / 214 / 489、70 / 207 / 214 / 370 / 489、70 / 191 / 207 / 214 / 489、70 / 207 / 214 / 333 / 489、70 / 189 / 207 / 214 / 489、70 / 207 / 214 / 468 / 489、70 / 207 / 214 / 473 / 489、70 / 115 / 207 / 214 / 489、53 / 70 / 207 / 214 / 489、70 / 207 / 214 / 449 / 489、70 / 207 / 214 / 489 / 518、70 / 207 / 214 / 236 / 489、70 / 207 / 214 / 481 / 489、59 / 70 / 207 / 214 / 220 / 489、70 / 207 / 214 / 258 / 489、70 / 207 / 214 / 467 / 489、51 / 70 / 207 / 214 / 489、70 / 207 / 214 / 415 / 489、70 / 103 / 207 / 214 / 489、70 / 207 / 214 / 489 / 493、70 / 78 / 207 / 214 / 489、65 / 70 / 207 / 214 / 489、70 / 207 / 214 / 335 / 489、70 / 207 / 214 / 489 / 520、70 / 101 / 207 / 214 / 489、70 / 207 / 214 / 341 / 489、70 / 207 / 214 / 471 / 489、70 / 207 / 211 / 214 / 489、70 / 206 / 207 / 214 / 489、70 / 93 / 207 / 214 / 489、70 / 207 / 214 / 489 / 490、70 / 71 / 207 / 214 / 489、70 / 79 / 207 / 214 / 489、70 / 180 / 207 / 214 / 489、70 / 172 / 207 / 214 / 489、70 / 142 / 207 / 214 / 489、70 / 207 / 214 / 489 / 529、70 / 77 / 207 / 214 / 489、70 / 207 / 212 / 214 / 489、70 / 207 / 214 / 474 / 489、70 / 106 / 207 / 214 / 489、70 / 207 / 214 / 386 / 489 / 507、70 / 117 / 207 / 214 / 489、70 / 207 / 214 / 373 / 489、70 / 207 / 214 / 337 / 489、70 / 107 / 207 / 214 / 246 / 489、70 / 207 / 214 / 489 / 516、70 / 207 / 214 / 233 / 489、70 / 91 / 207 / 214 / 489、70 / 194 / 207 / 214 / 489、70 / 84 / 207 / 214 / 489、70 / 207 / 214 / 361 / 489、70 / 171 / 207 / 214 / 489、70 / 207 / 214 / 489 / 536、50 / 70 / 207 / 214 / 489、70 / 116 / 207 / 214 / 489、70 / 207 / 214 / 239 / 489、44 / 70 / 207 / 214 / 489、40 / 70 / 207 / 214 / 489、70 / 113 / 207 / 214 / 489、70 / 207 / 214 / 243 / 489、70 / 169 / 207 / 214 / 489、70 / 207 / 214 / 489 / 504、70 / 207 / 214 / 489 / 505、70 / 109 / 207 / 214 / 489、70 / 207 / 214 / 479 / 489、70 / 207 / 214 / 477 / 489、70 / 97 / 207 / 214 / 489、70 / 182 / 207 / 214 / 489、70 / 111 / 207 / 214 / 489、70 / 207 / 214 / 489 / 508、70 / 207 / 214 / 314 / 489、70 / 102 / 207 / 214 / 489、70 / 207 / 214 / 338 / 489、70 / 96 / 207 / 214 / 489、70 / 138 / 207 / 214 / 489, 42 / 70 / 207 / 214 / 489, 70 / 207 / 214 / 263 / 489, 70 / 207 / 214 / 222 / 489, 70 / 207 / 214 / 489 / 522, 70 / 207 / 214 / 265 / 489, 70 / 207 / 214 / 48 9 / 514, 70 / 207 / 214 / 489 / 515, 70 / 207 / 214 / 246 / 489, 70 / 207 / 214, 46 / 70 / 207 / 214 / 489, 70 / 133 / 207 / 214 / 489, 70 / 207 / 214 / 332 / 489, 70 / 207 / 214 / 489 / 506, 70 / 207 / 214 / 489 / 512, 70 / 137 / 207 / 214 / 489, 70 / 190 / 207 / 214 / 489, 70 / 105 / 207 / 214 / 489, 70 / 207 / 214 / 301 / 489, 70 / 207 / 214 / 329 / 489, 70 / 207 / 214 / 339 / 489, or 70 / 151 / 207 / 214 / 489, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0178] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 68L / 70L / 207S / 214F / 336A / 489L, 65L / 70L / 99R / 207S / 214F / 319I / 489L, 65L / 68L / 70L / 207S / 214F / 319I / 489L, 68V / 70L / 207S / 214F / 319I / 489L, 68V / 70L / 207S / 214F / 489L, 65L / 70L / 207S / 214F / 319I / 489L, 70L / 195M / 207S / 214F / 489L , 65L / 154E / 70L / 207S / 214F / 319I / 489L, 70L / 207S / 214F / 489L, 70L / 207S / 214F / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 489L, 68L / 70L / 207S / 214F / 230L / 489L, 65L / 68V / 70L / 207S / 214F / 319I / 489L, 70L / 195M / 207S / 214F / 230L / 489L, 65L / 70L / 207S / 214F / 230L / 489L, 70L / 141D / 207S / 2 14F / 319I / 489L, 70L / 154E / 207S / 214F / 336A / 489L, 68L / 70L / 207S / 214F / 3 19I / 489L, 68V / 70L / 154E / 207S / 214F / 489L, 68V / 70L / 195M / 207S / 214F / 230 L / 319I / 336A / 489L, 68V / 70L / 99R / 195M / 207S / 214F / 230L / 319I / 489L, 68V / 70L / 195M / 207S / 214F / 489L, 65L / 68V / 70L / 207S / 214F / 489L, 68L / 70L / 141 D / 207S / 214F / 230L / 319I / 489L, 65L / 68V / 70L / 99R / 207S / 214F / 230L / 319I / 489L, 68L / 70L / 99R / 141D / 207S / 214F / 319I / 489L, 68V / 70L / 154E / 207S / 21 4F / 319I / 489L, 70L / 141D / 154E / 207S / 214F / 489L, 68V / 70L / 99R / 207S / 214 F / 489L, 70L / 99R / 207S / 214F / 230L / 319I / 489L, 70L / 99R / 207S / 214F / 489L,70L / 207S / 214F / 230L / 489L, 68V / 70L / 207S / 214F / 230L / 319I / 489L, 68L / 70L / 207S / 214F / 489L, 70L / 154E / 207S / 214F / 319I / 489L, 65L / 68L / 70L / 15 4E / 207S / 214F / 319I / 489L、70L / 141D / 207S / 214F / 489L、68V / 70L / 207S / 21 4F / 230L / 489L、70L / 207S / 214F / 336A / 489L、70L / 141D / 207S / 214F / 230L / 4 89L、68V / 70L / 154E / 207S / 214F / 336A / 489L、65L / 68L / 70L / 207S / 214F / 489 L、65L / 68V / 70L / 99R / 207S / 214F / 319I / 489L、70L / 154E / 207S / 214F / 489L、 70L / 207S / 214F / 319I / 489L, 70L / 207S / 214F / 230L / 319I / 489L, 68L / 70L / 154E / 207S / 214F / 489L, 70L / 99R / 141D / 207S / 214F / 319I / 489L, 68L / 70L / 15 4E / 207S / 214F / 319I / 489L、70L / 207S / 214F / 489L / 509F、70L / 108I / 207S / 214F / 489L、70L / 108I / 117I / 207S / 214F / 489L / 509F、70L / 207S / 214F / 489L / 519I、70L / 108I / 207S / 214F / 489L / 519I、70L / 88L / 207S / 214F / 489L、70L / 117I / 207S / 214F / 413Y / 489L / 509F、70L / 207S / 214F / 489L / 509F / 519I、70L / 207S / 214F / 413Y / 489L、70L / 108I / 117I / 207S / 214F / 315T / 489L、70L / 117 I / 207S / 214F / 271T / 489L、70L / 117I / 207S / 214F / 413Y / 489L / 509F / 529Q、7 0L / 207S / 214F / 413Y / 489L / 509F、70L / 207S / 214F / 489L / 469E、70L / 207S / 2 14F / 489L / 524K、70L / 207S / 214F / 489L / 507M、70L / 207S / 214F / 489L / 509I、70L / 207S / 214F / 489L / 525Q、70L / 207S / 214F / 489A、70L / 104M / 207S / 214F / 489L、70L / 207S / 214F / 489L / 507A、70L / 95G / 207S / 214F / 489L、70L / 207S / 214F / 359E / 489L、70L / 207S / 214F / 374V / 489L、70L / 207S / 214F / 489L / 509W、70L / 107D / 207S / 214F / 489L、70L / 89C / 207S / 214F / 489L、70L / 207S / 214F / 260R / 489L、70L / 110Q / 207S / 214F / 489L、70L / 186G / 207S / 214F / 489L、70L / 207S / 214F / 370R / 489L、70L / 207S / 214F / 489L / 509R、70L / 191R / 207S / 214F / 489L、70L / 207S / 214F / 333L / 489L、70L / 189R / 207S / 214F / 489L、70L / 207S / 214F / 468G / 489L、70L / 207S / 214F / 473I / 489L、70L / 115M / 207S / 214F / 489L、70L / 191N / 207S / 214F / 489L、53L / 70L / 207S / 214F / 489L、70L / 207S / 214F / 489L / 524S、70L / 207S / 214F / 449L / 489L、70L / 207S / 214F / 489L / 518E、70L / 207S / 214F / 236M / 489L、70L / 207S / 214F / 481H / 489L、70L / 115R / 207S / 214F / 489L、59N / 70L / 207S / 214F / 220G / 489L、70L / 207S / 214F / 489G、70L / 207S / 214F / 258T / 489L、70L / 207S / 214F / 467N / 489L、51P / 70L / 207S / 214F / 489L、70L / 207S / 214F / 415C / 489L、70L / 103S / 207S / 214F / 489L、70L / 207S / 214F / 489L / 493T、70L / 207S / 214F / 489T、70L / 207S / 214F / 489L / 518V、70L / 78L / 207S / 214F / 489L、70L / 195W / 207S / 214F / 489L、65M / 70L / 207S / 214F / 489L、70L / 207S / 214F / 413W / 489L、70L / 207S / 214F / 335M / 489L、70L / 207S / 214F / 489L / 520K、70L / 107E / 207S / 214F / 489L、70L / 101Q / 207S / 214F / 489L、70L / 207S / 214F / 449K / 489L、70L / 207S / 214F / 481K / 489L、70L / 104V / 207S / 214F / 489L、70L / 207S / 214F / 341R / 489L、70L / 78M / 207S / 214F / 489L、70L / 207S / 214F / 489L / 507L、70L / 207S / 214F / 449M / 489L、70L / 207S / 214F / 471L / 489L、70L / 207S / 211L / 214F / 489L、70L / 207S / 214F / 489L / 520D、70L / 103P / 207S / 214F / 489L、70L / 206F / 207S / 214F / 489L、70L / 93S / 207S / 214F / 489L、70L / 207S / 214F / 489L / 490S、70L / 207S / 214F / 473C / 489L、70L / 207S / 214F / 489L / 507R、70L / 207S / 211I / 214F / 489L、70L / 71S / 207S / 214F / 489L、70L / 79R / 207S / 214F / 489L、70L / 180C / 207S / 214F / 489L、70L / 207S / 214F / 489L / 509G、70L / 206R / 207S / 214F / 489L、70L / 207S / 211N / 214F / 489L、70L / 172S / 207S / 214F / 489L、70L / 207S / 214F / 489L / 490R、70L / 207D / 214F / 489L、70L / 115V / 207S / 214F / 489L、63L / 70L / 207S / 214F / 489L、70L / 142A / 207S / 214F / 489L、70L / 207S / 214F / 489L / 529M、70L / 71R / 207S / 214F / 489L、70L / 207S / 214F / 473L / 489L、70L / 95T / 207S / 214F / 489L、70L / 107A / 207S / 214F / 489L、70L / 207S / 214F / 489L / 520V、70L / 101L / 207S / 214F / 489L、70L / 207S / 214F / 489L / 520S、70L / 71H / 207S / 214F / 489L、70L / 89D / 207S / 214F / 489L、70L / 77I / 207S / 214F / 489L、70L / 207S / 214F / 489L / 507Y、70L / 107C / 207S / 214F / 489L、70L / 207S / 214F / 489L / 518S、70L / 207S / 212A / 214F / 489L、70L / 71F / 207S / 214F / 489L、70L / 207S / 214F / 489L / 525C、70L / 207S / 214F / 449R / 489L、70L / 107M / 207S / 214F / 489L、70L / 207S / 214F / 467H / 489L、70L / 207S / 214F / 474D / 489L、70L / 101S / 207S / 214F / 489L、70L / 106W / 207S / 214F / 489L、70L / 207S / 214F / 386L / 489L / 507M、70L / 207S / 214F / 471S / 489L、70L / 107Q / 207S / 214F / 489L、68Q / 70L / 207S / 214F / 489L、70L / 79I / 207S / 214F / 489L、70L / 207S / 214F / 489L / 518L、70L / 207S / 214F / 474A / 489L、70L / 107S / 207S / 214F / 489L、70L / 117T / 207S / 214F / 489L、70L / 207S / 214F / 467M / 489L、70L / 207S / 214F / 471R / 489L、70L / 207S / 214F / 341S / 489L、70L / 207S / 214F / 489L / 490G、70L / 207S / 214F / 471F / 489L、70L / 207S / 214F / 489L / 490T、70L / 207S / 214F / 333T / 489L、70L / 101R / 207S / 214F / 489L、70L / 207S / 214F / 373V / 489L、70L / 101V / 207S / 214F / 489L、70L / 101W / 207S / 214F / 489L、65S / 70L / 207S / 214F / 489L、70L / 207S / 214F / 489L / 490V、70L / 207S / 214F / 337N / 489L、70L / 207S / 214F / 374T / 489L、68Y / 70L / 207S / 214F / 489L、70L / 95L / 207S / 214F / 489L、70L / 71G / 207S / 214F / 489L、70L / 207S / 214F / 471V / 489L、70L / 107M / 207S / 214F / 246R / 489L、70L / 110A / 207S / 214F / 489L、70L / 142Y / 207S / 214F / 489L、65V / 70L / 207S / 214F / 489L、70L / 71M / 207S / 214F、 / 489L、70L / 77G / 207S / 214F / 489L、70L / 77R / 207S / 214F / 489L、70L / 207S / 214F / 230Y / 489L、70L / 207S / 214F / 489L / 490Q、70L / 101T / 207S / 214F / 489 L, 70L / 103A / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 507T, 70L / 207S / 214F / 471T / 489L, 70L / 207S / 214F / 489L / 509V, 70L / 207S / 214F / 489L / 520R 70L / 207S / 214F / 319F / 489L, 70L / 142N / 207S / 214F / 489L, 70L / 79V / 207S / 214F / 489L, 70L / 207S / 214F / 467L / 489L, 70L / 77N / 207S / 214F / 489L, 70L / 71L / 207S / 214F / 489L, 68W / 70L / 207S / 214F / 489L, 70L / 189S / 207S / 214F / 489L, 70L / 207S / 214F / 359P / 489L, 70L / 71Y / 207S / 214F / 489L, 70L / 77T / 20 7S / 214F / 489L, 70L / 142M / 207S / 214F / 489L, 70L / 207S / 214F / 373A / 489L, 70L / 207S / 214F / 319V / 489L, 70L / 207S / 212N / 214F / 489L, 70L / 110H / 207S / 214F / 489L、70L / 207S / 214F / 333C / 489L、70L / 207S / 214F / 489L / 490I、68F / 70L / 207S / 214F / 489L、70L / 207S / 214F / 474S / 489L、70L / 103L / 207S / 21 4F / 489L, 70L / 77H / 207S / 214F / 489L, 70L / 207S / 214F / 471E / 489L, 70L / 207S / 214F / 489L / 516L, 70L / 207S / 214F / 467R / 489L, 70L / 207S / 214F / 233R / 489L, 70L / 91T / 207S / 214F / 489L, 70L / 194L / 207S / 214F / 489L, 70L / 84G / 207S / 214F / 489L, 70L / 207S / 214F / 361V / 489L, 70L / 207S / 214F / 233A / 489L70L / 171V / 207S / 214F / 489L、70L / 207S / 214F / 489L / 536L、50A / 70L / 207S / 2 14F / 489L、70L / 116R / 207S / 214F / 489L、70L / 207S / 214F / 239T / 489L、44V / 7 0L / 207S / 214F / 489L、40S / 70L / 207S / 214F / 489L、70L / 113N / 207S / 214F / 48 9L、70L / 91I / 207S / 214F / 489L、70L / 99F / 207S / 214F / 489L、70L / 207S / 214F / 243L / 489L、70L / 169V / 207S / 214F / 489L、70L / 207S / 214F / 489L / 504P、70L / 207S / 214F / 489L / 505R、70L / 109R / 207S / 214F / 489L、70L / 207S / 214F / 489L / 536T、70L / 207S / 214F / 479A / 489L、70L / 207S / 214F / 479L / 489L、70L / 207S / 214F / 477H / 489L、70L / 84K / 207S / 214F / 489L、70L / 97N / 207S / 214F / 489L、7 0L / 97K / 207S / 214F / 489L, 70L / 182M / 207S / 214F / 489L, 70L / 111H / 207S / 214F / 489L, 70L / 207S / 214F / 361Y / 489L, 70L / 207S / 214F / 489L / 508R, 70L / 20 7S / 214F / 314R / 489L, 70L / 207S / 214F / 336G / 489L, 70L / 102E / 207S / 214F / 489L, 70L / 207S / 214F / 338R / 489L, 70L / 207S / 214F / 489L / 505L, 70L / 99L / 207 S / 214F / 489L、70L / 96S / 207S / 214F / 489L、70L / 138S / 207S / 214F / 489L、70L / 207S / 214F / 489L / 508T、70L / 96I / 207S / 214F / 489L、70L / 207S / 214F / 489L / 508Q、70L / 207S / 214F / 243M / 489L、70L / 111L / 207S / 214F / 489L、70L / 207S / 214F / 489L / 508S、70L / 207S / 214F / 479R / 489L、42A / 70L / 207S / 214F / 489L、70L / 207S / 214F / 239I / 489L, 70L / 97I / 207S / 214F / 489L, 70L / 116E / 207S / 214F / 489L, 70L / 113I / 207S / 214F / 489L, 70L / 207S / 214F / 477V / 489L, 70L / 2 07S / 214F / 489L / 536I, 70L / 207S / 214F / 263G / 489L, 70L / 99S / 207S / 214F / 489L, 70L / 207S / 214F / 222I / 489L, 70L / 207S / 214F / 489L / 536D, 70L / 207S / 2 14F / 477G / 489L, 70L / 207S / 214F / 489L / 536W, 70L / 207S / 214F / 489L / 536C, 70L / 207S / 214F / 336R / 489L, 70L / 207S / 214F / 243R / 489L, 70L / 116V / 207S / 214F / 489L、70L / 207S / 214F / 489L / 536R、70L / 207S / 214F / 489L / 522V、70L / 102S / 207S / 214F / 489L、70L / 97R / 207S / 214F / 489L、40H / 70L / 207S / 214F / 4 89L、70L / 207S / 214F / 243V / 489L、70L / 207S / 214F / 265S / 489L、70L / 116Q / 2 07S / 214F / 489L、70L / 207S / 214F / 489L / 514E、70L / 207S / 214F / 489L / 508L、 70L / 207S / 214F / 489L / 515T, 70L / 207S / 214F / 246R / 489L, 70L / 207S / 214F / 361W / 489L, 70L / 111F / 207S / 214F / 489L, 70L / 207S / 214F, 70L / 207S / 214F / 477R / 489L, 70L / 116Y / 207S / 214F / 489L, 46R / 70L / 207S / 214F / 489L, 70L / 133M / 207S / 214F / 489L, 70L / 207S / 214F / 332R / 489L, 70L / 207S / 214F / 239R / 489L、70L / 109G / 207S / 214F / 489L、70L / 207S / 214F / 489L / 506T、70L / 207S / 214F / 489L / 504G、70L / 207S / 214F / 233Q / 489L、70L / 96R / 207S / 214F / 489L、70L / 99G / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 504T, 70L / 96G / 207S / 2 14F / 489L, 70L / 97M / 207S / 214F / 489L, 70L / 109L / 207S / 214F / 489L, 70L / 2 07S / 214F / 489L / 515G, 70L / 207S / 214F / 233G / 489L, 70L / 84A / 207S / 214F / 489L, 70L / 102N / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 504W, 70L / 207S / 214F / 265R / 489L, 70L / 99V / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 512 L, 70L / 137V / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 536A, 70L / 113R / 20 7S / 214F / 489L, 70L / 171P / 207S / 214F / 489L, 70L / 190W / 207S / 214F / 489L, 70L / 207S / 214F / 314G / 489L, 46M / 70L / 207S / 214F / 489L, 70L / 182H / 207S / 214F / 489L, 70L / 207S / 214F / 246K / 489L, 70L / 105T / 207S / 214F / 489L, 70L / 207S / 214F / 301K / 489L, 70L / 207S / 214F / 489L / 522T, 70L / 207S / 214F / 32 9L / 489L, 70L / 96V / 207S / 214F / 489L, 70L / 207S / 214F / 339A / 489L, 70L / 20 7S / 214F / 477S / 489L, 70L / 113G / 207S / 214F / 489L, 70L / 207S / 214F / 361L / 489L, 70L / 207S / 214F / 489L / 505I, 70L / 105M / 207S / 214F / 489L, 70L / 116M / 207S / 214F / 489L, 70L / 96M / 207S / 214F / 489L, 70L / 109M / 207S / 214F / 489L, 70L / 171C / 207S / 214F / 489L, 70L / 207S / 214F / 489L / 536M, or 70L / 151L / 207S / 214F / 489L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2;or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0179] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase includes at least one substitution or set of substitutions of an engineered glucocerebrosidase described in Tables 4.1, 4.2, 4.3, and 4.4, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0180] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 361 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 319 / 489 / 514, 65 / 68 / 70 / 79 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 361 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 374 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 263 / 319 / 337 / 359 / 361 / 374 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 207 / 214 / 230 / 319 / 489 / 536, 65 / 68 / 70 / 79 / 99 / 154 / 207 / 214 / 230 / 233 / 239 / 319 / 359 / 361 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 233 / 319 / 337 / 374 / 489 / 514, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 233 / 319 / 359 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 276 / 319 / 359 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 207 / 214 / 230 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 31 9 / 233 / 337 / 374 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 359 / 361 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 263 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 361 / 374 / 471 / 489,65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 374 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 239 / 319 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 319 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 263 / 374 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 207 / 214 / 230 / 319 / 374 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 361 / 374 / 471 / 489 / 536, 65 / 68 / 70 / 79 / 99 / 154 / 207 / 214 / 230 / 319 / 359 / 374 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 359 / 3 61 / 374 / 489 / 514, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 239 / 263 / 361 / 374 / 319 / 489 / 514, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 374 / 489, 6 5 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 374 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 319 / 361 / 489 / 536, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 3 At least one substitution, or set of substitutions, at 19 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 233 / 319 / 359 / 361 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 359 / 471 / 489, or 65 / 68 / 70 / 99 / 154 / 207 / 214 / 230 / 319 / 359 / 489, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0181] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 319I / 361W / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 361W / 489L, 65L / 68L / 70L / 79V / 99R / 154E / 195W / 207S / 214F / 230L / 263G ...99R / 154E / 207S / 214F / 230L / 319I / 361W / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 361W / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 2 7S / 214F / 230L / 319I / 374T / 489L, 65L / 68L / 70L / 79V / 99R / 154E / 195W / 207S / 214F / 230L / 319I / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 319I / 4 89L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 233A / 263G / 319I / 337N / 35 9P / 361W / 374T / 489L, 65L / 68L / 70L / 79V / 99R / 154E / 207S / 214F / 230L / 319I / 489L / 536D, 65L / 68L / 70L / 79V / 99R / 154E / 207S / 214F / 230Y / 233A / 239R / 31 9I / 359P / 361W / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 471T / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 374T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 233A / 319I / 337N / 374T / 48 9L / 514E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 233A / 319I / 359 P / 489L / 536D, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 319I / 489L, 65V / 6 8L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 263G / 276T / 319I / 359P / 361W / 37 4T / 471T / 489L, 65L / 68L / 70L / 79V / 99R / 154E / 207S / 214F / 230L / 319I / 489L,65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 233A / 337N / 374T / 489L / 536D、65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 489L、 65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 337N / 359P / 361W / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 263G / 319I / 489L, 65V / 68 L / 70L / 99R / 154E / 207S / 214F / 230Y / 233G / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 263G / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 2 14F / 230L / 319I / 374T / 471T / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 374T / 489L / 536D, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 2 39R / 319I / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 233G / 319I / 489L / 536L, 65L / 68L 70L / 99R / 154E / 207S / 214F / 230Y / 319I / 263G / 374T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489 L, 65L / 68L / 70L / 79V / 99R / 154E / 207S / 214F / 230L / 319I / 374T / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 361W / 374T / 471T / 489L / 536D,65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 233G / 319I / 489L, 65L / 68L / 70L / 79V / 99R / 15 4E / 207S / 214F / 230Y / 319I / 359P / 374T / 489L, 65L / 68L / 70L / 99R / 154E / 207 S / 214F / 230L / 319I / 489L / 536D, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 319I / 359P / 361W / 374T / 489L / 514E, 65V / 68L / 70L / 79V / 99R / 154E / 207S / 2 14F / 230Y / 319I / 489L, 65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 23 9R / 263G / 361W / 374T / 319I / 489L / 514E, 65L / 68L / 70L / 99R / 154E / 195W / 207 S / 214F / 230L / 263G / 319I / 374T / 489L, 65L / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230L / 319I / 374T / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 2 33A / 319I / 361W / 489L / 536L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230Y / 23 3A / 319I / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 233 A / 319I / 359P / 361W / 471T / 489L, 65L / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 489L / 536L, 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 359P / 471T / 489L, 65V / 68L / 70L / 79V / 99R / 154E / 207S / 214F / 230L / 319I / 489L, or 65V / 68L / 70L / 99R / 154E / 207S / 214F / 230L / 319I / 359P / 489L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0182] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase described in Table 5.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0183] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 79 / 97 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 97 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 97 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 79 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 201 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 404 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 484 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 446 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 494 / 495, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 495, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 412 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 385 / 471 / 489,65 / 68 / 70 / 99 / 100 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 184 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 385 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 82 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 75 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、41 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 94 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 98 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 114 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 95 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 105 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 360 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 400 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 342 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 390 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 264 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 298 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 449 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 459 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 434 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 502、29 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 534、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 497、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 533、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 536、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 485 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 510、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 532、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 534、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489 / 494、65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 204 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489、65 / 68 / 70 / 99 / 154 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 202 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 144 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 36 1 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 220 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, 65 / 68 / 70 / 99 / 150 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 471 / 489, or 65 / 68 2, or a set of substitutions at amino acid positions 40-536 of SEQ ID NO:2, or a reference sequence corresponding to SEQ ID NO:2.

[0184] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 79V / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 79V / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 471T / 489L, 99R / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 4 71T / 489L, 65V / 68L / 70L / 97R / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 3 19I / 337N / 359P / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 97R / 99R / 154E / 1 95W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 79V / 99R / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 36 1W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 201T / 207S / 214F / 2 30Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 404Q / 471T / 489L, 6 5V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 484E / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 201R / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 446N / 471T / 489L,65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 494T / 495G, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 495G、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 412W / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 385N / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F 230Y / 263G / 319I / 337N / 361W / 374T / 385G / 471T / 489L, 65V / 68L / 70L / 99R / 100N / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 99R / 154E / 184V / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 99R / 154E / 385H / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 82S / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 4 89L, 65V / 68L / 70L / 75A / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 71T / 99R / 154E / 195W / 207S / 214 F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 41H / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L,65V / 68L / 70L / A97K / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99E / 154E / 195W / 207S / 214F / 23 0Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 97Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65 V / 68L / 70L / P94N / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 3 61W / 374T / 471T / 489L、65V / 68L / 70L / 99R / 100R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 92A / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L 65V / 68L / 70L / 98P / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99W / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 92G / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 99R / 114Y / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / I95H / 99R / 154E / 195W / 207S / 214F / 23 0Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 99R / T102A / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / M92K / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 96D / 99R / 154E / 195W / 207S / 214F 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 94E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L 65V / 68L / 70L / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / I95W / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 105I / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L 65V / 68L / 70L / 99R / 113Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 92H / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 92Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 6 5V / 68L / 70L / 92E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 2 63G / 319I / L325V / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 336E / 337N / 361W / 374T / 471T / 489L65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 385Q / 471T / 489L / 262Q / 263G / 319I / 337N / 361W / 374T / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 471T / 489L、 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 360E / 361W / 374T / 471T / 489L / 263G / 319I / 337N / 361W / 374T / 400T / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 342Q / 471T / 489L、 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 400E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 400Q / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 390A / 471T / 489L、 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 264I / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y 263G / 298W / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 471T / 489L65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408D / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 449E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 459M / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / , 263G / 319I / 337N / 361W / 374T / 434G / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 7 0L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 33 7N / 361W / 374T / 471T / 489L / 502K, 29I / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 534K, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 502Q, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 497A, 65V / 68L / 70L / 99R / 154E / 195 W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 533Y, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 47 1T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337 N / 361W / 374T / 471T / 489L / 536P、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 536H, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 485M / 489L65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 510S 2V, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 534K, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L / 494T 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 182G / 195W / 207S / 214F / 230 Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 204Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 194H / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 184Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 202Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L65V / 68L / 70L / 99R / 154E / 182S / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230 Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 144M / 15 4E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65 V / 68L / 70L / 99R / 154E / 195W / H201Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 182D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154 E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L , 65V / 68L / 70L / 99R / 154E / 182T / 195W / 207S / 214F / 230Y / 263G / 319I / 337 N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 194Q / 195W / 207S / 21 4F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, 65V / 68L / 70L / 99R / 1 54E / 195W / 207S / 214F / 220A / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 48 9L, 65V / 68L / 70L / 99R / 150T / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, or 65V / 68L / 70L / 99R / 154E / 195W / 206I / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 471T / 489L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0185] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase described in Tables 6.1 and 6.2, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0186] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 65 / 68 / 70 / 96 / 99 / 154 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 484 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 412 / 471 / 489 / 494 / 495, 65 / 68 / 70 / 92 / 96 / 99 / 154 / 175 / 1 82 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489, 65 / 68 / 70 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 471 / 484 / 489 / 494 / 495, 65 / 68 / 70 / 92 / 99 / 154 / 175 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 15 4 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 400 / 408 / 412 / 471 / 489, 65 / 68 / 70 / 92 / 96 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 400 / 408 / 471 / 484 / 489, 65 / 68 / 70 / 92 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489 / 494 / 495, 65 / 6 8 / 70 / 92 / 96 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489 / 494 / 495, 65 / 68 / 70 / 96 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 471 / 484 / 489, 65 / 68 / 70 / 92 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489,65 / 68 / 70 / 92 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489 / 494 / 495 / 534、65 / 68 / 70 / 92 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 400 / 408 / 471 / 484 / 489、65 / 68 / 70 / 92 / 96 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 400 / 408 / 471 / 484 / 489、65 / 68 / 70 / 92 / 96 / 99 / 154 / 175 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 412 / 471 / 484 / 489、65 / 68 / 70 / 92 / 96 / 99 / 154 / 195 / 201 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 484 / 489、65 / 68 / 70 / 92 / 99 / 154 / 182 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 92 / 96 / 99 / 154 / 175 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 400 / 408 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 484 / 489 / 494 / 495、65 / 68 / 70 / 96 / 99 / 154 / 195 / 201 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 194 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 201 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 359 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 92 / 96 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 400 / 408 / 471 / 484 / 489 / 495、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489 / 495、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 92 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 382 / 408 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 502、65 / 68 / 70 / 99 / 154 / 195 / 202 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 97 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 99 / 100 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 360 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 97 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 510、65 / 68 / 70 / 71 / 97 / 99 / 100 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 510、65 / 68 / 70 / 71 / 97 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 99 / 100 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 202 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 99 / 100 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 71 / 99 / 100 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 97 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489、65 / 68 / 70 / 71 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 97 / 99 / 154 / 195 / 202 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 360 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 97 / 99 / 103 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 100 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 502 / 510、65 / 68 / 70 / 71 / 99 / 137 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 360 / 361 / 374 / 385 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 276 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 510、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 360 / 361 / 374 / 385 / 408 / 471 / 489 / 502、65 / 68 / 70 / 99 / 100 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 325 / 337 / 361 / 374 / 385 / 408 / 471 / 489 / 502、65 / 68 / 70 / 71 / 99 / 154 / 175 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 360 / 361 / 374 / 385 / 408 / 471 / 485 / 489 / 502、65 / 68 / 70 / 95 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 494 / 536、65 / 68 / 70 / 95 / 99 / 113 / 154 / 182 / 184 / 195 / 207 / 21、 4 / 230 / 263 / 264 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 184 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 536、65 / 68 / 70 / 95 / 99 / 142 / 144 / 154 / 182 / 184 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 449 / 471 / 489、65 / 68 / 70 / 99 / 144 / 154 / 195 / 204 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 400 / 408 / 449 / 471 / 489、65 / 68 / 70 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 449 / 471 / 489 / 536、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 536、65 / 68 / 70 / 95 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489 / 536、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 264 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 449 / 471 / 489、65 / 68 / 70 / 95 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489、65 / 68 / 70 / 95 / 99 / 154 / 195 / 204 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489、65 / 68 / 70 / 99 / 113 / 142 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 449 / 471 / 489、65 / 68 / 70 / 99 / 154 / 182 / 184 / 195 / 207 / 214 / 230 / 263 / 264 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 494、65 / 68 / 70 / 99 / 113 / 142 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 494、65 / 68 / 70 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 449 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 264 / 319 / 337 / 361 / 374 / 408 / 449471 / 489、65 / 68 / 70 / 95 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 95 / 99 / 113 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 449 / 471 / 489 / 536、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489 / 536、65 / 68 / 70 / 99 / 154 / 182 / 184 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 142 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 400 / 408 / 471 / 489 / 536、35 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 264 / 319 / 337 / 361 / 374 / 408 / 471 / 449 / 489 / 536、65 / 68 / 70 / 99 / 142 / 144 / 154 / 195 / 207 / 214 / 220 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 142 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、29 / 65 / 68 / 70 / 99 / 102 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 276 / 319 / 336 / 337 / 361 / 374 / 390 / 408 / 459 / 471 / 489 / 502 / 532、41 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489 / 502、29 / 65 / 68 / 70 / 97 / 99 / 150 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489 / 502 / 53229 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 532、65 / 68 / 70 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 390 / 408 / 459 / 471 / 489 / 502 / 532、29 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 276 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489 / 495、29 / 65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 459 / 471 / 489 / 532、65 / 68 / 70 / 92 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502、29 / 65 / 68 / 70 / 97 / 99 / 102 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502 / 532、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 459 / 471 / 489 / 495 / 502 / 532、65 / 68 / 70 / 99 / 150 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 390 / 408 / 471 / 489、65 / 68 / 70 / 99 / 150 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495、29 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495、29 / 65 / 68 / 70 / 99 / 102 / 154 / 182 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502、29 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 459 / 471 / 489 / 502 / 532、65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495 / 502 / 532、65 / 68 / 70 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 150 / 154 / 182 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 459 / 471 / 489 / 495 / 502 / 532、41 / 65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 390 / 408 / 471 / 489 / 495、29 / 41 / 65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489 / 495、65 / 68 / 70 / 97 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495、65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 495、29 / 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502、29 / 65 / 68 / 70 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502、65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 336 / 337 / 361 / 374 / 408 / 471 / 489 / 495、29 / 65 / 68 / 70 / 99 / 150 / 154 / 182 / 195 / 207 / 214 / 230 / 262 / 263 / 319 / 337 / 361 / 374 / 408 / 459 / 471 / 489 / 495、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 502 / 532、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 386 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 497、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 512、65 / 68 / 69 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 507、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 445 / 471 / 489、65 / 68 / 70 / 75 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 487 / 489、65 / 68 / 70 / 99 / 154 / 184 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 90 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 235 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 434 / 471 / 489、65 / 68 / 70 / 86 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 182 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489、65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 、 263 / 319 / 337 / 361 / 374 / 408 / 446 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 490, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 299 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 102 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 141 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 97 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 , 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 522, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 224 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 478 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 3 61 / 374 / 408 / 471 / 480 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 412 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 26 At least one substitution in 3 / 265 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489 / 494, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 316 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 368 / 374 / 408 / 471 / 489,or a set of substitutions, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 2, or are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0187] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase contains at least one substitution or set of substitutions 65V / 68L / 70L / 96E / 99R / 154E / 194Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 484E / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 484E / 489L, 4T / 382A / 408E / 412W / 471T / 489L / 494T / 495G, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 175D / 182S / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 382A / 40 8E / 471T / 484E / 489L / 494T / 495G, 65V / 68L / 70L / 92E / 99R / 154E / 175D / 194Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374 T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 382A / 400E / 408E / 412W / 471T / 489L, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 182S / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 400E / 408E / 471T / 48 4E / 489L, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L / 494T / 495G,65V / 68L / 70L / 92Q / 96E / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L / 494T / 495G R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 92Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L / 494T / 495G / 534K, 65V / 68L / 70L / 92E / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 400Q / 408E / 471T / 484E / 489L, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 92Q / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 400Q / 408E / 47 1T / 484E / 489L, 65V / 68L / 70L / 92E / 96E / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L / 494T / 495G, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 175D / 182S / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 412W / 471T / 484E / 489L65V / 68L / 70L / 92Q / 96E / 99R / 154E / 195W / 201Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 484E / 489L, 65V / 68L / 70L / 92E / 9 9R / 154E / 182S / 194Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 92Q / 96E / 99R / 154E / 175D / 194Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 382A / 400E / 408E / 471T / 489L、65V / 68L / 70L / 92Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 359P / 361W / 374T / 382A / 408E / 471T / 489L, 65V / 68L / 70L / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 3 82A / 408E / 471T / 489L, 65V / 68L / 70L / 92Q / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 484E / 489L / 49 4T / 495G、65V / 68L / 70L / 96E / 99R / 154E / 195W / 201Q / 207S / 214F / 230Y / 26 3G / 319I / 337N / 359P / 361W / 374T / 382A / 408E / 471T / 489L、65V / 68L / 70L / 9 2E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 92Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 194Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 201Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L 7N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 92E / 96E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 400E / 408E / 471T / 484E / 489L / 495G, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489L / 495G, 65 V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361 W / 374T / 382A / 408E / 471T / 489L、65V / 68L / 70L / 92E / 99R / 154E / 195W / 2 07S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 382A / 408E / 471T / 489 L、65V / 68L / 70L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 3 19I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L, 65V / 68L / 70L / 71T / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 408E / 471T / 489L / 502Q65V / 68L / 70L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L / 502Q, 65V / 68L / 70L / 99R / 154 E / 195W / 202Q / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385N / 408E / 471T / 489L, 65V / 68L / 70L / 97Q / 99R / 154E / 175D / 195W / 207S / 214F / 230 Y / 263G / 319I / 337N / 361W / 374T / 385H / 408E / 471T / 489L / 510S, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 3 85Q / 408E / 471T / 489L / 510S、65V / 68L / 70L / 99R / 100R / 154E / 175D / 195W / 20 7S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 408E / 471T / 489L、65V / 6 8L / 70L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 360E / 361W / 374T / 385N / 408E / 471T / 489L, 65V / 68L / 70L / 97Q / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 4 89L / 510S、65V / 68L / 70L / 71T / 97Q / 99R / 100R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L, 65V / 68L / 70L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W 374T / 408E / 471T / 489L / 510S, 65V / 68L / 70L / 71T / 97Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 408E / 471T / 489L / 502Q65V / 68L / 70L / 71T / 99R / 100R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385Q / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 202Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 385N / 408E / 471T / 489L / 489E, 65V 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385N / 408E / 471T / 489L / 502Q 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L / 502Q 65V / 68L / 70 L / 71T / 99R / 100R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L / 510S, 65V / 68L / 70L / 71T / 99R / 100R / 15 4E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385 N / 408E / 471T / 489L、65V / 68L / 70L / 71T / 99R / 100R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 385N / 408E / 471T / 489L / 510S, 65V / 68L / 70L / 97Q / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385N / 408E / 471T / 48 9L, 65V / 68L / 70L / 71T / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 97Q / 99R / 154E / 195W / 202Q / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 360E / 361W / 374T / 385G / 408E / 471T / 489L / 510S、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502Q, 65V / 68L / 70L / 71T / 97Q / 99R / 103S / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374 T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 100R / 154E / 195W / 207S / 214F / 230 Y / 263G / 319I / 325V / 337N / 361W / 374T / 385H / 408E / 471T / 489L / 502Q / 510S、65V / 68L / 70L / 71T / 99R / 137Q / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 360E / 361W / 374T / 385H / 408E / 471T / 489L / 502Q, 65V / 68L / 70L / 71T / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 276T / 319I / 325V / 337N / 361W / 374T / 385R / 408E / 471T / 489L / 510S, 65V / 68L / 70 L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 360E / 361W / 374T / 385N / 408E / 471T / 489L / 502Q、65V / 68L / 70L / 99R / 100R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 325V / 337N / 361W / 374T / 385N / 408E / 471T / 489E / 502Q、65V / 68L / 70L / 71T / 99R / 154E / 175D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 360E / 361W / 374T / 385H / 408E / 471T / 485S / 489L / 502Q, 65V / 68L / 70L / 95W / 99R / 113Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 494T / 536P, 65V / 68L / 70L / 95W / 99W / 113Q / 154E / 182D / 184V / 195W / 207S / 214F / 230Y / 263G / 264I / 319I / 337N / 361W / 374T / 386I / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 184Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 536P65V / 68L / 70L / 95W / 99R / 142N / 144M / 154E / 182G / 184Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 449E / 471T / 489L、65V / 6 8L / 70L / 99R / 144M / 154E / 195W / 204Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 400T / 408E / 449E / 471T / 489L, 65V / 68L / 70L / 99R / 113Q 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 449E / 471T / 489L / 536P 65V / 68L / 70L / 99R 471T / 489L / 536P, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 264I / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99W / 113 Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 449E / 471T / 489L, 65V / 68L / 70L / 95W / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 471T / 489L, 65V / 68L / 70L / 95W / 99W / 154E / 195W / 204Q / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 471T / 489L、65V / 68L / 70L / 99R / 113Q / 142N / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 449E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 182G / 184Q / 195W / 207S / 214F / 230Y / 263G / 2 64I / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 494T、65V / 68L / 70L / 99 R / 113Q / 142N / 154E / 182D / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 494T、65V / 68L / 70L / 99R / 113Q / 154E / 19 5W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99W / 154E / 195W / 207S / 214F / 230Y / 263G / 264I / 319I / 337N / 361W / 374T / 408E / 449E / 471T / 489L, 65V / 68L / 70L / 95W / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 4 89L, 65V / 68L / 70L / 95W / 99W / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99W / 113Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 449E / 471T / 489L, 65V / 68L / 70L / 95W / 99R / 113Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 449E / 471T / 489L / 536P, 65V / 68L / 70L / 95W / 99W / 113Q / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 408E / 471T / 489L / 536P,65V / 68L / 70L / 99W / 154E / 182G / 184Q / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 142N / 154E / 195W / 2 07S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386I / 400T / 408E / 471T / 489L / 536P、35T / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 264I / 319I / 337N / 361W / 374T / 408E / 471T / 449E / 489L / 536P、65V / 68L / 70L / 99R / 142N / 144M / 154E / 195W / 207S / 214F / 220A / 230Y / 263G / 319I / 337N / 361W / 3 74T / 408E / 471T / 489L, 65V / 68L / 70L / 99W / 142N / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 29I / 65V / 68L / 70L / 99 R / 102A / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 276T / 319I / 336E / 337N / 361W / 374T / 390A / 408E / 459M / 471T / 489L / 502K / 532V、41H / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 336E / 337N / 361W / 374T / 408E / 471T / 489L / 502K, 29I / 65V / 68L / 70L / 97R / 99R / 150T / 154E / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 336E / 337N / 361W / 374T / 408E / 471T / 489L / 502K / 532V, 29I / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263 G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 532V,65V / 68L / 70L / 99R / 102A / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 390A / 408E / 459M / 4 71T / 489L / 502K / 532V、29I / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 262Q / 263G / 276T / 319I / 336E / 337N / 361W / 374T / 408E / 471T / 489L / 495G、29I / 65V / 68L / 70L / 99R / 154E / 182T / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 459M / 471T / 489L / 532V、65V / 68L / 70L / 92K / 99G / 102A / 154E / 195W、 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K, 29I / 65V / 68L / 70L / 97R / 99R / 102A / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K / 532V, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 336E / 337N / 361W / 374T / 408E / 459M / 471T / 489L / 495S / 502K / 532V, 65V / 68L / 70L / 99R / 150T / 154E / 182T / 195W / 207S / 214F / 230Y / 263G / 319I / 336E / 337N / 361W / 374T / 390A / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 150T / 154E / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / M374T / 408E / 471T / 489L / 495G, 29I / 65V / 6 8L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 495S, 29I / 65V / 68L / 70L / 99R / 102A / 154E / 182T / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K, 29I / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 336E / 337N / 361W / 374T / 408E / 459M / 471T / 489L / 502K / 532V, 65V / 68L / 70L / 99R / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 495G / 502K / 532V, 65V / 68L / 70L / 99R / 102A / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 336E / 337N / 361W / 374T / 408E / 471T / 489L65V / 68L / 70L / 99R / 150T / 154E / 182M / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 495S、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 459M / 471T / 489L / 495S / 502K / 532V、41H / 65V / 68L / 70L / 99R / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 390A / 408E / 471T / 489L / 495G, 29I / 41H / 65V / 68L / 70L / 99R / 154E / 182T / 195W / 207S / 214F / 230Y / 263G / 31 9I / 336E / 337N / 361W / 374T / 408E / 471T / 489L / 495S、65V / 68L / 70L / 97R / 9 9R / 102A / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408 E / 471T / 489L / 495S, 65V / 68L / 70L / 99R / 154E / 182T / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 495S, 29I / 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K, 29I / 65V / 68L / 70L / 99R / 102A / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K、65V / 68L / 70L / 99R / 154E / 182M / 195W / 207S / 214F / 230Y / 263G / 319I / 336E 337N / 361W / 374T / 408E / 471T / 489L / 495S, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K29I / 65V / 68L / 70L / 99R / 150T / 154E / 182T / 195W / 207S / 214F / 230Y / 262Q / 263G / 319I / 337N / 361W / 374T / 408E / 459M / 471T / 489L / 495G, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 502K / 532V, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386P / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T 489L / 497M, 65V / 68L / 70L / 99K / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207 S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 512H、65V / 68L / 69D / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 507I, 65V / 68L / 70L / 99 R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 445 N / 471T / 489L、65V / 68L / 70L / 75N / 99R / 154E / 195W / 207S / 214F / 230Y / 263G 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 75H / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 512Q, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 487S / 489L, 65V / 68L / 70L / 99R / 113P / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 3 61W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 184I / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 90M / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y 235T / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 434Q / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 445G / 471T / 489L, 65V / 68L / 70L / 99R / 1 54E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489N, 65V / 68L / 70L / 90A / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 386L / 408E / 471T / 489L65V / 68L / 70L / 75E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 86E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489Q, 65V / 68L / 70L / 99R / 154E / 182E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 446H / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230 Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 490N, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 299V / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 1 02P / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230F / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68T / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L65V / 68L / 70L / 99R / 141E / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 97E / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 522S、65V / 68L / 70L / 99R / 15、 4E / 195W / 207S / 214F / 224M / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 478N / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 480G / 489L、65V / 68L / 70L / 86H / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337 N / 361W / 374T / 408E / 412N / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 449S / 471T / 48 9L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 265N / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L / 494A、65V / 68L / 70L / 96N / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 20 7S / 214F / 230Y / 263G / 316H / 319I / 337N / 361W / 374T / 408E / 471T / 489L、65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 368D / 374T / 408E / 471T / 489L, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0188] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase described in Tables 12.1 and 12.2, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0189] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at an amino acid position set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid position is relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or is relative to a reference sequence corresponding to SEQ ID NO:2.

[0190] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or relative to a reference sequence corresponding to SEQ ID NO:2.

[0191] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions at an amino acid position(s) set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0192] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase variant described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0193] In some embodiments, the engineered glucocerebrosidase comprises a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150.

[0194] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648.

[0195] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence comprises one or more substitutions.

[0196] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150, or relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150.

[0197] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133 , 134, 137, 138, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 3 25, 329, 332, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 504 , 505, 506, 507, 508, 509, 510, 512, 514, 515, 516, 518, 519, 520, 522, 524, 525, 529, 532, 533, 534, or 536, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0198] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase has at least one substitution or deletion of amino acid residues 23A, 29I, 35T, 40H / S, 41H, 42A, 44V, 46M / R, 50A, 51P, 53L, 59N, 63L, 65L / M / S / V, 68F / L / Q / T / V / W / Y, 69D, 70L, 71F / G / H / L / M / R / S / T / Y, 75A / E / H / N, 77G / H / I / N / R / T, 78L / M, 79I / R / V, 82S, 84A / G / K, 86E / H, 88L, 89C / D, 90A / M, 91I / T, 92A / 2E / G / H / K / Q, 93S, 94E / N, 95G / H / L / T / W, 96D / E / G / I / M / N / R / S / V, 97E / I / K / M / N / Q / R, 98P, 99E / F / G / K / L / R / S / V / W, 100N / R, 101L / Q / R / S / T / V / W, 102A / E / N / P / S, 103A / L / P / S, 104M / V, 105I / M / T, 106W, 107A / C / D / E / M / Q / S, 108I, 109D / G / L / M / R, 110A / H / Q, 111F / H / L, 113G / I / N / P / Q / R, 114Y, 115M / R / V, 116E / M / Q / R / V / Y, 117I / T, 133M, 134S, 137Q / V, 138S, 141D / E, 142A / M / N / Y, 144M, 150 T, 151L, 154E, 169V, 171C / P / V, 172S, 175D, 180C, 182D / E / G / H / M / S / T, 184I / Q / V, 186G, 189R / S, 190W, 191N / R, 194H / L / Q, 195M / W, 201Q / R / T, 202Q, 204Q , 206F / I / R, 207S / D, 211I / L / N, 212A / N, 214F, 220A / G, 222I, 224M, 230F / L / Y , 233A / G / Q / R, 235T, 236M, 239I / R / T, 243K / L / M / R / V, 246K / R, 258T, 260R, 2 61K, 262Q, 263G, 264I, 265N / R / S / T, 271T, 276T, 298W, 299V, 301K, 314D / G / R , 315T, 316H, 319F / I / V, 325V, 329L, 332R, 333C / L / T, 335M, 336A / E / G / R, 33 7N, 338R, 339A, 341R / S, 342Q, 359E / P, 360E, 361L / V / W / Y, 368D, 370R, 372D,373A / V / Y, 374T / V, 382A, 385G / H / N / Q / R, 386I / L / P, 390A, 400E / Q / T, 404Q, 408D / E, 412N / W, 41 3W / Y, 415C, 434G / Q, 445G / N, 446H / N, 449E / K / L / M / R / S / V, 459M, 467H / L / M / N / R, 468G, 469E, 471 E / F / L / R / S / T / V, 473C / I / L, 474A / D / S, 476A, 477G / H / R / S / V, 478N, 479A / L / R, 480G, 481H / K / T, 484E, 485M / S / T, 487S, 489A / E / G / L / M / N / Q / T, 490G / I / N / Q / R / S / T / V, 493T, 494A / T, 495G / S, 497 A / M, 502K / Q, 504G / P / T / W, 505I / L / R, 506T, 507A / I / L / M / R / T / Y, 508L / Q / R / S / T, 509F / G / I / R / V / W, 510S, 512H / L / Q, 514E, 515G / T, 516L, 518E / L / S / V, 519I, 520D / K / R / S / V, 522S / T / V, 524K / S, 525C / Q, 529M / Q, 532V, 533Y, or 534K, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0199] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0200] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase has at least one substitution or amino acid residues 65L / M / S / V, 68F / L / Q / T / V / W / Y, 70L, 99E / F / G / K / L / R / S / V / W, 154E, 195M / W, 207S / D, 214F, 230F / L / Y, 263M, 319F / I / V, 337N, 361L / V / W / Y, 374T / V, 408D / E, 471E / F / L / R / S / T / V, or 489A / E / G / L / N / Q / T, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0201] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or amino acid residues 665L / V, 68L, 70L, 99R, 154E, 195W, 207S, 214F, 230L / Y, 263G, 319I, 337N, 361W, 374T, 408E, 471T, or 489L, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0202] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54.

[0203] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions.

[0204] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 68 / 336, 65 / 99 / 319, 65 / 68 / 319, 68 / 319, 68, 65 / 319, 195, 65 / 154 / 319, 65 / 68 / 230, 65, 65 / 68 / 99 / 154 / 230 / 319, 68 / 230, 195 / 230, 65 / 230, 141 / 319, 154 / 336, 68 / 154, 68 / 195 / 230 / 319 / 336, 68 / 99 / 195 / 230 / 319, 68 / 195, 65 / 68, 68 / 141 / 230 / 319, 65 / 68 / 99 / 230 / 319, 68 / 99 / 141 / 319, 68 / 154 / 319, 141 / 154, 68 / 99, 99 / 230 / 319, 99, 230, 68 / 230 / 319,154 / 319, 65 / 68 / 154 / 319, 141, 336, 141 / 230, 68 / 154 / 336, 65 / 68 / 99 / 319, 154, 319, 230 / 319, 68 / 154, 99 / 141 / 319, 509, 108, 108 / 117 / 509, 519, 108 / 519, 88, 117 / 41 3 / 509, 509 / 519, 413, 108 / 117 / 315, 117 / 271, 117 / 413 / 509 / 529, 413 / 509, 469, 524, 507, 525, 489, 104, 95, 359, 374, 107, 89, 260, 110, 186, 370, 191, 333, 189, 468, 473, 115, 53, 449, 518, 236, 481, 59 / 220, 258, 467, 51, 415, 103, 493, 78, 335, 520, 101, 341, 471, 211, 206, 93, 490, 71, 79, 180, 172, 207, 63, 142, 529, 77, 212, 474, 106, 386 / 507, 117, 373, 337, 107 / 246, 516, 233, 91, 194, 84, 361, 171, 536, 50, 116, 239, 44, 40, 113, 243, 169, 504, 505, 109, 479, 477, 97, 182, 111, 508, 314, 102, 338, 96, 138, 42, 263, 222, 522, 265, 514, 515, 246, 46, 133, 332, 506, 512, 137, 190,At least one substitution or set of substitutions at amino acid positions 105, 301, 329, 339, or 151, and the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, or relative to a reference sequence corresponding to SEQ ID NO: 54.

[0205] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 68L / 336A, 65L / 99R / 319I, 65L / 68L / 319I, 68V / 319I, 68V, 65L / 319I, 195M, 65L / 154E / 319I, 65L / 68L / 230L, 65L, 65L / 68L / 99R / 154E / 230L / 319I, 68L / 230L, 65L / 68V / 319I, 195M / 230L, 65L / 230L, 141D / 319I, 154E / 336A, 68L / 319I, 68V / 1 54E, 68V / 195M / 230L / 319I / 336A, 68V / 99R / 195M / 230L / 319I, 68V / 195M, 65 L / 68V, 68L / 141D / 230L / 319I, 65L / 68V / 99R / 230L / 319I, 68L / 99R / 141D / 319 I, 68V / 154E / 319I, 141D / 154E, 68V / 99R, 99R / 230L / 319I, 99R, 230L, 68V / 2 30L / 319I, 68L, 154E / 319I, 65L / 68L / 154E / 319I, 141D, 68V / 230L, 336A, 141 D / 230L, 68V / 154E / 336A, 65L / 68L, 65L / 68V / 99R / 319I, 154E, 319I, 230L / 3 19I, 68L / 154E, 99R / 141D / 319I, 68L / 154E / 319I, 509F, 108I, 108I / 117I / 50 9F, 519I, 108I / 519I, 88L, 117I / 413Y / 509F, 509F / 519I, 413Y, 108I / 117I / 315T, 117I / 271T, 117I / 413Y / 509F / 529Q, 413Y / 509F, 469E, 524K, 507M, 509 I, 525Q, 489A, 104M, 507A, 95G, 359E, 374V, 509W, 107D, 89C, 260R, 110Q, 18 6G, 370R, 509R, 191R, 333L, 189R, 468G, 473I, 115M, 191N, 53L, 524S, 449L, 5 18E, 236M, 481H, 115R, 59N / 220G, 489G, 258T, 467N, 51P, 415C, 103S, 493T, 4 89T, 518V, 78L, 195W, F65M, 413W, 335M, 520K, 107E, 101Q, 449K, 481K, 104V,341R、78M、507L、449M、471L、211L、520D、103P、206F、93S、490S、473C、507R 、211I、71S、79R、180C、509G、206R、211N、172S、490R、207D、115V、63L、142A、 529M、71R、473L、95T、107A、520V、101L、520S、71H、89D、77I、507Y、107C、51 8S、212A、71F、525C、449R、107M、467H、474D、101S、106W、386L / 507M、471S、1 07Q、68Q、79I、518L、474A、107S、117T、467M、471R、341S、490G、471F、490T、 333T、101R、373V、101V、101W、65S、490V、337N、374T、68Y、95L、71G、471V、10 7M / 246R、110A、142Y、65V、71M、77G、77R、230Y、490Q、101T、103A、507T、471 T、509V、520R、319F、142N、79V、467L、77N、71L、68W、189S、359P、71Y、77T、14 2M, 373A, 319V, 212N, 110H, 333C, 490I, 68F, 474S, 103L, 77H, 471E, 516L, 467R, 233R, 91T, 194L, 84G, 361V, 233A, 171V, 536L, 50A, 116R, 239T, 44V, 40S 113N, 91I, 99F, 243L, 169V, 504P, 505R, 109R, 536T, 479A, 479L, 477H, 84K, 97N, 97K, 182M, 111H, 361Y, 508R, 314R, 336G, 102E, 338R, 505L, 99L, 96S, 1 38S, 508T, 96I, 508Q, 243M, 111L, 508S, 479R, 42A, 239I, 97I, 116E, 113I, 477V, 536I, 263G, 99S, 222I, 536D, 477G, 536W, 536C, 336R, 243R, 116V, 536R 522V、102S、97R、40H、243V、265S、116Q、514E、508L、515T、246R、361W、111F 、489M、477R、116Y、46R、133M、332R、239R、109G、506T、504G、233Q、96R、99G、504T, 96G, 97M, 109L, 515G, 233G, 84A, 102N, 504W, 265R, 99V, 512L, 137V, 536A, 113R, 171P, 190W, 314G, 46M, 182H, 246K, 105T, 301K, 522T, 329L, 96V, 339A, 477S, 113G, 361L, 505I, 105M, 116M, 96M, 109M, 171C, 536M, or 151L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, or are relative to a reference sequence corresponding to SEQ ID NO: 54.

[0206] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions P68L / T336A, F65L / H99R / M319I, F65L / P68L / M319I, P68V / M319I, P68V, F65L / M319I, L195M, F65L / G154E / M319I, F65L / P68L / V230L, F65L, F65L / P68L / H99R / G154E / V230L / M319I, P68L / V230L, F65L / P68V / M319I, L195M ... 0L, N141D / M319I, G154E / T336A, P68L / M319I, P68V / G154E, P68V / L195M / V2 30L / M319I / T336A, P68V / H99R / L195M / V230L / M319I, P68V / L195M, F65L / P6 8V, P68L / N141D / V230L / M319I, F65L / P68V / H99R / V230L / M319I, P68L / H99R / N141D / M319I, P68V / G154E / M319I, N141D / G154E, P68V / H99R, H99R / V230L / M319I, H99R, V230L, P68V / V230L / M319I, P68L, G154E / M319I, F65L / P68L / G 154E / M319I, N141D, P68V / V230L, T336A, N141D / V230L, P68V / G154E / T336A , F65L / P68L, F65L / P68V / H99R / M319I, G154E, M319I, V230L / M319I, P68L / G 154E, H99R / N141D / M319I, P68L / G154E / M319I, L509F, L108I, L108I / V117I / L509F, L519I, L108I / L519I, M88L, V117I / H413Y / L509F, L509F / L519I, H41 3Y, L108I / V117I / V315T, V117I / A271T, V117I / H413Y / L509F / H529Q, H413Y / L509F, G469E, P524K, V507M, L509I, G525Q, L489A, L104M, V507A, I95G, A3 59E, M374V, L509W, T107D, E89C, A260R, P110Q, F186G, F370R, L509R, E191R,V333L、P189R、E468G、V473I、Q115M、E191N、V53L、P524S、T449L、F518E、L236M、N481H、Q115R、A59N / S220G、L489G、A258T、P467N、S51P、V415C、G103S、G493T、L489T、F518V、R78L、L195W、F65M、H413W、L335M、E520K、T107E、G101Q、T449K、N481K、L104V、A341R、R78M、V507L、T449M、Q471L、V211L、E520D、G103P、L206F、G93S、H490S、V473C、V507R、V211I、P71S、Y79R、D180C、L509G、L206R、V211N、Y172S、H490R、S207D、Q115V、D63L、L142A、H529M、P71R、V473L、I95T、T107A、E520V、G101L、E520S、P71H、E89D、S77I、V507Y、T107C、F518S、S212A、P71F、G525C、T449R、T107M、P467H、G474D、G101S、L106W、F386L / V507M、Q471S、T107Q、P68Q、Y79I、F518L、G474A、T107S、V117T、P467M、Q471R、A341S、H490G、Q471F、H490T、V333T、G101R、T373V、G101V、G101W、F65S、H490V、D337N、M374T、P68Y、I95L、P71G、Q471V、T107M / Q246R、P110A、L142Y、F65V、P71M、S77G、S77R、V230Y、H490Q、G101T、G103A、V507T、Q471T、L509V、E520R、M319F、L142N、Y79V、P467L、S77N、P71L、P68W、P189S、A359P、P71Y、S77T、L142M、T373A、M319V、S212N、P110H、V333C、H490I、P68F、G474S、G103L、S77H、Q471E、V516L、P467R、K233R、S91T、K194L、S84G、A361V、K233A、T171V、Q536L、Y50A、K116R、Q239T、I44V、A40S、K113N、S91I、H99F、I243L、I169V、S504P、K505R, Q109R, Q536T, Q479A, Q479L, A477H, S84K, A97N, A97K, Q182M, E111H, A361Y, P508R, N314R, T336G, T1 02E, P338R, K505L, H99L, Q96S, P138S, P508T, Q96I, P508Q, I243M, E111L, P508S, Q479R, P42A, Q239I, A97I, K 116E, K113I, A477V, Q536I, K263G, H99S, T222I, Q536D, A477G, Q536W, Q536C, T336R, I243R, K116V, Q536R, I 522V, T102S, A97R, A40H, I243V, Q265S, K116Q, P514E, P508L, A515T, Q246R, A361W, E111F, L489M, A477R, K11 6Y, K46R, L133M, K332R, Q239R, Q109G, D506T, S504G, K233Q, Q96R, H99G, S504T, Q96G, A97M, Q109L, A515G, K 233G, S84A, T102N, S504W, Q265R, H99V, K512L, P137V, Q536A, K113R, T171P, E190W, N314G, K46M, Q182H, Q246 K, L105T, R301K, I522T, H329L, Q96V, E339A, A477S, K113G, A361L, K505I, L105M, K116M, Q96M, Q109M, T171C, Q536M, or E151L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, or are relative to a reference sequence corresponding to SEQ ID NO: 54.

[0207] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:220.

[0208] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more substitutions.

[0209] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid position(s) 65 / 195 / 361, 514, 79 / 195 / 263 / 361, 65 / 374, 79 / 195, 65 / 230, 65 / 233 / 263 / 337 / 359 / 361 / 374, 79 / 536, 79 / 230 / 233 / 239 / 359 / 361 / 471, 65 / 471, 374, 65 / 195 / 230 / 233 / 337 / 374 / 514, 65 / 195 / 233 / 359 / 536, 230, 65 / 195 / 263 / 276 / 359 / 361 / 374 / 471, 79, 195 / 230 / 233 / 337 / 374 / 536, 195 / 230, 195 / 230 / 337 / 359 / 361, 230 / 263, 65 / 230 / 233, 263, 195 / 230 / 337 / 361 / 374 / 471, 65 / 195, 374 / 471, 374 / 536, 239, 65, 230 / 233 / 536, 230 / 263 / 374, 65 / 195 / 230 / 263 / 337 / 361 / 374 / 471, 79 / 374, 65 / 361 / 374 / 471 / 536, 195, 233, 79 / 230 / 359 / 374, 536, 230 / 359 / 361 / 374 / 514, 65 / 79 / 230, 195 / 239 / 263 / 361 / 374 / 514, 195 / 263 / 374, and at least one substitution or set of substitutions at 195 / 374, 65 / 230 / 233 / 361 / 536, 230 / 233 / 374 / 471, 65 / 233 / 359 / 361 / 471, 65 / 359 / 471, 65 / 79, or 65 / 359, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:220.

[0210] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 65V / 195W / 361W, 514E, 79V / 195W / 263G / 361W, 65V / 374T, 79V / 195W, 65V / 230Y, 65V / 233A / 263G / 337N / 359P / 361W / 374T, 79V / 536D, 79V / 230Y / 233A / 239R / 359P / 361W / 471T, 65V / 471T, 374T, 65V / 195W / 230Y Y / 233A / 337N / 374T / 514E, 65V / 195W / 233A / 359P / 536D, 230Y, 65V / 195W / 263G / 276T / 359P / 361W / 374T / 471T, 79V, 195W / 230Y / 233A / 33 7N / 374T / 536D, 195W / 230Y, 195W / 230Y / 337N / 359P / 361W, 230Y / 263G, 65V / 230Y / 233G, 263G, 195W / 230Y / 337N / 361W / 374T / 471T, 65V / 1 95W, 374T / 471T, 374T / 536D, 239R, 65V, 230Y / 233G / 536L, 230Y / 263G / 374T, 65V / 195W / 230Y / 263G / 337N / 361W / 374T / 471T, 79V / 374T, 65V / 361W / 374T / 471T / 536D, 195W, 233G, 79V / 230Y / 359P / 374T, 536D, 230Y / 359P / 361W / 374T / 514E, 65V / 79V / 230Y, 195W / 239R / 263G / 3 and 61W / 374T / 514E, 195W / 263G / 374T, 195W / 374T, 65V / 230Y / 233A / 361W / 536L, 230Y / 233A / 374T / 471T, 65V / 233A / 359P / 361W / 471T, 536L, 65V / 359P / 471T, 65V / 79V, or 65V / 359P, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:220.

[0211] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions L65V / L195W / A361W, P514E, Y79V / L195W / K263G / A361W, L65V / M374T, Y79V / L195W, L65V / L230Y, L65V / K233A / K263G / D337N / A359P / A361W / M374T, Y79V / Q536D, Y79V / L230Y / K233A / Q239R / A359P / A361W / Q471T, L65V / Q471T, M374T, L 65V / L195W / L230Y / K233A / D337N / M374T / P514E, L65V / L195W / K233A / A359 P / Q536D, L230Y, L65V / L195W / K263G / S276T / A359P / A361W / M374T / Q471T, Y79V, L195W / L230Y / K233A / D337N / M374T / Q536D, L195W / L230Y, L195W / L2 30Y / D337N / A359P / A361W, L230Y / K263G, L65V / L230Y / K233G, K263G, L195W / L230Y / D337N / A361W / M374T / Q471T, L65V / L195W, M374T / Q471T, M374T / Q 536D, Q239R, L65V, L230Y / K233G / Q536L, L230Y / K263G / M374T, L65V / L195 W / L230Y / K263G / D337N / A361W / M374T / Q471T, Y79V / M374T, L65V / A361W / M 374T / Q471T / Q536D, L195W, K233G, Y79V / L230Y / A359P / M374T, Q536D, L230 Y / A359P / A361W / M374T / P514E, L65V / Y79V / L230Y, L195W / Q239R / K263G / A361W / M374T / P514E, L195W / K263G / M374T, L195W / M374T, L65V / L230Y / K233A / A361W / Q536L, L230Y / K233A / M374T / Q471T, L65V / K233A / A359P / A361W / Q471T, Q536L, L65V / A359P / Q471T, L65V / Y79V, L65V / A359P,It is relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 220, or it is relative to a reference sequence corresponding to SEQ ID NO: 220.

[0212] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984.

[0213] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984, wherein the amino acid sequence comprises one or more substitutions.

[0214] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises the amino acid sequence at amino acid position(s) 359, 79 / 359, 79 / 97 / 182, 97 / 359, 97, 79 / 182, 201, 404, 484, 446, 494 / 495, 495, 412, 385, 100, 184, 82, 75, 71, 41, 99, 94, 92, 98, 114, 95, 102, 96, 105, 113, 325, 336, 262, 386, 360, 400, 342, 390, 264, 298, at least one substitution or set of substitutions at 382, ​​408, 449, 459, 434, 476, 502, 29 / 534, 497, 485, 533, 536, 510, 532, 489, 534, 494, 182, 137, 204, 194, 202, 144, 142, 175, 220, 150, or 206, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:984 or are relative to a reference sequence corresponding to SEQ ID NO:984.

[0215] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions 359P, 79V / 359P, 79V / 97R / 182M, 97R / 359P, 97R, 79V / 182M, 201T, 404Q, 484E, 201R, 446N, 494T / 495G, 495G, 412W, 385N, 385G , 100N, 184V, 385H, 82S, 75A, 71T, 41H, 97K, 99E, 97Q, 94N, 100R, 92A, 98P, 99W, 92G, 114Y, 95H, 1 02A, 92K, 96D, 94E, 96E, 95W, 105I, 113Q, 92H, 92Q, 92E, 325V, 336E, 385Q, 262Q, 386I, 360E, 400 T, 342Q, 400E, 400Q, 390A, 264I, 298W, 382A, 408D, 449E, 459M, 434G, 476A, 408E, 502K, 29I / 534 K, 502Q, 497A, 485S, 533Y, 495S, 536P, 536H, 485M, 510S, 485T, 532V, 489E, 534K, 494T, 182G, 13 7Q, L204Q, 194H, 184Q, 202Q, 182S, 144M, 142N, 201Q, 182D, 175D, 182T, 194Q, 220A, 150T, or 206I, and the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 984, or are relative to a reference sequence corresponding to SEQ ID NO: 984.

[0216] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions A359P, Y79V / A359P, Y79V / A97R / Q182M, A97R / A359P, A97R, Y79V / Q182M, H201T, H404Q, D484E, H201R, T446N, S494T / A495G, A495G, Y412W, K385N, K385G, T10 0N, H184V, K385H, T82S, T75A, P71T, R41H, A97K, R99E, A97Q, P94N, T100R, M92A, N98P, R99W, M92G, F114Y, I95H, T102A, M92K, Q96D, P94E, Q96E, I95W, L105I, K113Q, M92H, M92Q, M92E, L325V, T336E, K385Q, H262Q, F386I, K360 E, M400T, K342Q, M400E, M400Q, S390A, L264I, F298W, V382A, T408D, T449E, L459M, R434G, V476A, T408E, R502K, L29I / R534K, R502Q, V497A, A485S, W533Y, A495S, Q536P, Q536H, A485M, T510S, A485T, L532V, L489E, R534K, S49 4T, Q182G, P137Q, L204Q, K194H, H184Q, R202Q, Q182S, L144M, L142N, H201Q, Q182D, A175D, Q182T, K194Q, S220A, E150T, or L206I, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 984, or are relative to a reference sequence corresponding to SEQ ID NO: 984.

[0217] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or to a reference sequence corresponding to SEQ ID NO: 1150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150.

[0218] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:1218-1554, or a reference sequence corresponding to SEQ ID NO:1218-1554, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:1150, or relative to the reference sequence corresponding to SEQ ID NO:1150.

[0219] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises amino acid positions 96 / 194 / 382 / 484, 359 / 382 / 412 / 494 / 495, 92 / 96 / 175 / 182 / 382, 96 / 359 / 382 / 484 / 494 / 495, 92 / 175 / 194 / 359 / 382, 359 / 382 / 400 / 412, 92 / 96 / 182 / 382 / 400 / 484, 92 / 96 / 382 / 494 / 495, 92 / 96 / 175 / 382 / 494 / 495, 96 / 182 / 359 / 382 / 484, 92 / 96 / 3 82, 92 / 382 / 494 / 495 / 534, 92 / 96 / 382 / 400 / 484, 92 / 96 / 175 / 382, 92 / 175 / 382 / 400 / 484, 92 / 96 / 175 / 182 / 382 / 412 / 484, 92 / 96 / 201 / 382 / 484, 92 / 182 / 194 / 382, 92 / 96 / 175 / 194 / 359 / 382 / 400, 92 / 359 / 382, 96 / 382, 92 / 175 / 382 / 484 / 494 / 495, 96 / 201 / 359 / 382, 92, 194, 201, 359, 96, 92 / 96 / 382 / 400 / 4 84 / 495, 382 / 495, 382, ​​92 / 382, 71 / 175 / 325 / 385, 325 / 385, 71 / 325 / 502, 71 / 175 / 325 / 385 / 502, 202 / 325 / 385, 97 / 175 / 385 / 510, 325 / 385 / 510, 100 / 1 75 / 325, 71 / 175 / 325 / 360 / 385, 97 / 175 / 510, 71 / 97 / 100 / 175 / 325 / 385, 71 / 175 / 510, 71 / 97 / 325 / 502, 71 / 100 / 325 / 385, 202 / 385 / 489, 325 / 385 / 502, 7 1 / 100 / 325 / 385 / 510, 71 / 100 / 175 / 325 / 385, 385 / 510, 97 / 325 / 385, 71, 97 / 202 / 325 / 360 / 385 / 510, 502, 71 / 97 / 103 / 325, 100 / 325 / 385 / 502 / 510, 71 / 137 / 175 / 325 / 360 / 385 / 502, 71 / 276 / 325 / 385 / 510, 71 / 175 / 360 / 385 / 502, 100 / 175 / 325 / 385 / 489 / 502, 71 / 175 / 360 / 385 / 485 / 502, 95 / 113, 494 / 536,95 / 99 / 113 / 182 / 184 / 264 / 386, 184 / 536, 95 / 142 / 144 / 182 / 184 / 386 / 449, 144 / 204 / 386 / 400 / 449, 113 / 449 / 536, 536, 95 / 386 / 536, 264, 99 / 113 / 386 / 449, 95 / 386, 95 / 99 / 204 / 386, 113 / 142 / 386 / 449, 182 / 184 / 264 / 494, 113 / 142 / 182 / 494, 113, 99 / 264 / 449, 95, 95 / 99, 99 / 113 / 449, 95 / 113 / 449 / 5 36, 95 / 99 / 113, 386 / 536, 99 / 182 / 184, 142 / 386 / 400 / 536, 35 / 264 / 449 / 536, 142 / 144 / 220, 99 / 142, 29 / 102 / 182 / 276 / 336 / 390 / 459 / 502 / 532, 41 / 262 / 336 / 502, 29 / 97 / 150 / 262 / 336 / 502 / 532, 29, 532, 102 / 390 / 459 / 502 / 532, 29 / 262 / 276 / 336 / 495, 29 / 182 / 262 / 459 / 532, 92 / 99 / 102 / 502, 29 / 97 / 1 02 / 182 / 502 / 532, 336 / 459 / 495 / 502 / 532, 150 / 182 / 336 / 390, 150 / 262 / 495, 29 / 495, 29 / 102 / 182 / 262 / 502, 29 / 336 / 459 / 502 / 532, 182 / 495 / 502 / 532, 102 / 336, 150 / 182 / 262 / 495, 459 / 495 / 502 / 532, 41 / 182 / 390 / 495, 29 / 41 / 182 / 336 / 495, 97 / 102 / 495, 182 / 262 / 495, 29 / 262 / 502, 29 / 102 / 502, 1 or at least one substitution or set of substitutions at 82 / 336 / 495, 29 / 150 / 182 / 262 / 459 / 495, 502 / 532, 386, 497, 99, 512, 69, 507, 445, 75, 487, 184, 90, 235, 434, 489, 86, 182, 446, 490, 299, 102, 230, 68, 141, 97, 316, 522, 224, 478, 480, 368, 412, 449, 265, or 494, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150;or relative to the reference sequence corresponding to SEQ ID NO: 1150.

[0220] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase has at least one substitution or set of substitutions 96E / 194Q / 382A / 484E, 359P / 382A / 412W / 494T / 495G, 92Q / 96E / 175D / 182S / 382A, 96E / 359P / 382A / 484E / 494T / 495G, 92E / 175D / 194Q / 359P / 382A, 359P / 382A / 400E / 412W, 92Q / 96E / 182S / 382A / 400E / 484E, 92Q / 96E / 382A / 494T / 495 G, 92Q / 96E / 175D / 382A / 494T / 495G, 96E / 182S / 359P / 382A / 484E, 92E / 96E / 382A, 92Q / 382A / 494T / 495G / 534K, 92E / 96E / 382A / 400Q / 484E, 92Q / 96E / 175D / 382A, 92Q / 175D / 382A / 400Q / 484E, 92E / 96E / 175D / 382A / 494T / 495G , 92Q / 96E / 175D / 182S / 382A / 412W / 484E, 92Q / 96E / 201Q / 382A / 484E, 92E / 1 82S / 194Q / 382A, 92Q / 96E / 175D / 194Q / 359P / 382A / 400E, 92Q / 359P / 382A, 96E / 382A, 92Q / 175D / 382A / 484E / 494T / 495G, 96E / 201Q / 359P / 382A, 92E, 92Q, 194Q, 201Q, 359P, 96E, 92E / 96E / 382A / 400E / 484E / 495G, 382A / 495G, 382A, 92E / 382A, 71T / 175D / 325V / 385H, 325V / 385H, 71T / 325V / 502Q, 71T / 1 75D / 325V / 385H / 502Q, 202Q / 325V / 385N, 97Q / 175D / 385H / 510S, 325V / 385 Q / 510S, 100R / 175D / 325V, 71T / 175D / 325V / 360E / 385N, 97Q / 175D / 510S, 7 1T / 97Q / 100R / 175D / 325V / 385H, 71T / 175D / 510S, 71T / 97Q / 325V / 502Q, 71 T / 100R / 325V / 385Q, 202Q / 385N / 489E, 325V / 385N / 502Q, 325V / 385H / 502Q,71T / 100R / 325V / 385H / 510S、71T / 100R / 175D / 325V / 385N、71T / 100R / 175D / 325V / 385H、385N / 510S、97Q / 325V / 385N、71T、97Q / 202Q / 325V / 360E / 385G / 510S、502Q、71T / 97Q / 103S / 325V、100R / 325V / 385H / 502Q / 510S、71T / 137Q / 175D / 325V / 360E / 385H / 502Q、71T / 276T / 325V / 385R / 510S、71T / 175D / 360E / 385N / 502Q、100R / 175D / 325V / 385N / 489E / 502Q、71T / 175D / 360E / 385H / 485 S / 502Q、95W / 113Q、494T / 536P、95W / 99W / 113Q / 182D / 184V / 264I / 386I、184 Q / 536P、95W / 142N / 144M / 182G / 184Q / 386I / 449E、144M / 204Q / 386I / 400T / 4 49E、113Q / 449E / 536P、536P、95W / 386I / 536P、264I、99W / 113Q / 386I / 449E、9 5W / 386I、95W / 99W / 204Q / 386I、113Q / 142N / 386I / 449E、182G / 184Q / 264I / 4 94T、113Q / 142N / 182D / 494T、113Q、99W / 264I / 449E、95W、95W / 99W、99W / 113 Q / 449E、95W / 113Q / 449E / 536P、95W / 99W / 113Q、386I / 536P、99W / 182G / 184Q 、142N / 386I / 400T / 536P、35T / 264I / 449E / 536P、142N / 144M / 220A、99W / 142N 、29I / 102A / 182M / 276T / 336E / 390A / 459M / 502K / 532V、41H / 262Q / 336E / 502 K、29I / 97R / 150T / 262Q / 336E / 502K / 532V、29I、532V、102A / 390A / 459M / 502K / 532V、29I / 262Q / 276T / 336E / 495G、29I / 182T / 262Q / 459M / 532V、92K / 99G / 102A / 502K、29I / 97R / 102A / 182M / 502K / 532V、336E / 459M / 495S / 502K / 532V、150T / 182T / 336E / 390A, 150T / 262Q / 495G, 29I / 495S, 29I / 102A / 182T / 262Q / 502K, 29I / 336E / 459M / 502K / 532V, 182M / 495G / 502K / 532V, 102A / 336E, 150T / 182M / 262Q / 495S, 459M / 495S / 502K / 532V, 41H / 182M / 390A / 495G, 29I / 41H / 182T / 336E / 495S, 97R / 102A / 495S, 182T / 262Q / 495S, 29I / 262Q / 502K, 29I / 102A / 502K, 182M / 336E / 495S, 502K, 29I / 150T / 182T / 262Q / 459M / 495G, 502K / 532V, 386P, 497M, 99K, 512H, 69D, 507I, 445N, 75N, 75H, 512Q, 487S, 113P, 184I, 90M, 235T, 434Q, 445G, 489N, 90A, 386L, 75E, 86E, 489Q, 182E, 446H, 490N, 299V, 96N, 102P, 2 30F, 68T, 141E, 97E, 316H, 522S, 224M, 478N, 480G, 368D, 86H, 412N, 449S, 265N, or 494A, and the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 1150.

[0221] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions Q96E / K194Q / V382A / D484E, A359P / V382A / Y412W / S494T / A495G, M92Q / Q96E / A175D / Q182S / V382A, Q96E / A359P / V382A / D484E / S494T / A495G, M92E / A175D / K194Q / A359P / V382A, A359P / V382A / M400E / Y412W, M92Q / Q96E / Q182S / V382A A / M400E / D484E, M92Q / Q96E / V382A / S494T / A495G, M92Q / Q96E / A175D / V38 2A / S494T / A495G, Q96E / Q182S / A359P / V382A / D484E, M92E / Q96E / V382A, M 92Q / V382A / S494T / A495G / R534K, M92E / Q96E / V382A / M400Q / D484E, M92Q / Q96E / A175D / V382A, M92Q / A175D / V382A / M400Q / D484E, M92E / Q96E / A175D / V382A / S494T / A495G, M92Q / Q96E / A175D / Q182S / V382A / Y412W / D484E, M92 Q / Q96E / H201Q / V382A / D484E, M92E / Q182S / K194Q / V382A, M92Q / Q96E / A17 5D / K194Q / A359P / V382A / M400E, M92Q / A359P / V382A, Q96E / V382A, M92Q / A 175D / V382A / D484E / S494T / A495G, Q96E / H201Q / A359P / V382A, M92E, M92Q , K194Q, H201Q, A359P, Q96E, M92E / Q96E / V382A / M400E / D484E / A495G, V38 2A / A495G, V382A, M92E / V382A, P71T / A175D / L325V / K385H, L325V / K385H, P71T / L325V / R502Q, P71T / A175D / L325V / K385H / R502Q, R202Q / L325V / K38 5N, A97Q / A175D / K385H / T510S, L325V / K385Q / T510S, T100R / A175D / L325V,P71T / A175D / L325V / K360E / K385N, A97Q / A175D / T510S, P71T / A97Q / T100R / A175D / L325V / K385H, P71T / A175D / T510S, P71T / A97Q / L325V / R502Q, P71T / T100R / L325V / K385Q, R202Q / K385N / L489E, L325V / K385N / R502Q, L325V / K385H / R502Q, P71T / T100R / L325V / K385H / T510S, P71T / T100R / A175D / L325V / K385N, P71T / T100R / A175D / L325V / K385H, K385N / T510S, A97Q / L325V / K385N, P71T, A97Q / R202Q / L325V / K360E / K385G / T510S, R502Q, P71T / A97Q / G103 S / L325V、T100R / L325V / K385H / R502Q / T510S、P71T / P137Q / A175D / L325V / K 360E / K385H / R502Q、P71T / S276T / L325V / K385R / T510S、P71T / A175D / K360E / K385N / R502Q, T100R / A175D / L325V / K385N / L489E / R502Q, P71T / A175D / K360E / K385H / A485S / R502Q, I95W / K113Q, S494T / Q536P, I95W / R99W / K113Q / Q1 82D / H184V / L264I / F386I、H184Q / Q536P、I95W / L142N / L144M / Q182G / H184Q / F386I / T449E、L144M / L204Q / F386I / M400T / T449E、K113Q / T449E / Q536P、Q5 36P、I95W / F386I / Q536P、L264I、R99W / K113Q / F386I / T449E、I95W / F386I、I 95W / R99W / L204Q / F386I、K113Q / L142N / F386I / T449E、Q182G / H184Q / L264I / S494T、K113Q / L142N / Q182D / S494T、K113Q、R99W / L264I / T449E、I95W、I95W / R99W、R99W / K113Q / T449E、I95W / K113Q / T449E / Q536P、I95W / R99W / K113Q、F386I / Q536P, R99W / Q182G / H184Q, L142N / F386I / M400T / Q536P, S35T / L264I / T449E / Q536P, L142N / L144M / S220A, R99W / L142N, L29I / T102A / Q182M / S2 76T / T336E / S390A / L459M / R502K / L532V、R41H / H262Q / T336E / R502K、L29I / A97R / E150T / H262Q / T336E / R502K / L532V、L29I、L532V、T102A / S390A / L459M / R502K / L532V、L29I / H262Q / S276T / T336E / A495G、L29I / Q182T / H262Q / L459M / L532V、M92K / R99G / T102A / R502K、L29I / A97R / T102A / Q182M / R502K / L53 2V, T336E / L459M / A495S / R502K / L532V, E150T / Q182T / T336E / S390A, E150T / H262Q / A495G, L29I / A495S, L29I / T102A / Q182T / H262Q / R502K, L29I / T336E / L459M / R502K / L532V、Q182M / A495G / R502K / L532V、T102A / T336E、E150T / Q 182M / H262Q / A495S、L459M / A495S / R502K / L532V、R41H / Q182M / S390A / A495 G, L29I / R41H / Q182T / T336E / A495S, A97R / T102A / A495S, Q182T / H262Q / A495S, L29I / H262Q / R502K, L29I / T102A / R502K, Q182M / T336E / A495S, R502K, L2 9I / E150T / Q182T / H262Q / L459M / A495G、R502K / L532V、F386P、V497M、R99K、 K512H、T69D、V507I、I445N、T75N、T75H、K512Q、A487S、K113P、H184I、L90M、S 235T、R434Q、I445G、L489N、L90A、F386L、T75E、R86E、L489Q、Q182E、T446H、 H490N、I299V、Q96N、T102P、Y230F、L68T、N141E、A97E、R316H、I522S、L224M、S478N, K480G, R368D, R86H, Y412N, T449S, Q265N, or S494A, and the amino acid positions are relative to the reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 1150, or relative to the reference sequence corresponding to SEQ ID NO: 1150.

[0222] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at an amino acid position set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid position is relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0223] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0224] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions at an amino acid position(s) set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0225] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0226] In some embodiments, the engineered glucocerebrosidase has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 The amino acid sequences include those having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0227] In some embodiments, the amino acid sequence of the engineered glucocerebrosidase comprises residues 40-536 of an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and 6.2, or an amino acid sequence comprising an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1.

[0228] In some embodiments, the engineered glucocerebrosidase is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 ,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 , 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1026、1028、1030、1032、1034、1036、1038、1040、1042、1044、1046、1048、1050、1052、1054、1056、1058、1060、1062、1064、1066、1068、1070、1072、1074、1076、1078、1080、1082、1084、1086、1088、1090、1092、1094、1096、1098、1100、1102、1104、1106、1108、1110、1112、1114、1116、1118、1120、1122、1124、1126、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1218、1220、1222、1224、1226、1228、1230、1232、1234、1236、1238、1240、1242、1244、1246、1248、1250、1252、1254、1256、1258、1260、1262、1264、1266、1268、1270、1272、1274、1276、1278、1280、1282、1284、1286、1288、1290、1292、1294、1296、1298、1300、1302、1304、1306、1308、1310、1312、1314、1316、1318、1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1 416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1511 2, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608 , 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, or 1648.

[0229] In some embodiments, the engineered glucocerebrosidase is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 ,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 , 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1026、1028、1030、1032、1034、1036、1038、1040、1042、1044、1046、1048、1050、1052、1054、1056、1058、1060、1062、1064、1066、1068、1070、1072、1074、1076、1078、1080、1082、1084、1086、1088、1090、1092、1094、1096、1098、1100、1102、1104、1106、1108、1110、1112、1114、1116、1118、1120、1122、1124、1126、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1218、1220、1222、1224、1226、1228、1230、1232、1234、1236、1238、1240、1242、1244、1246、1248、1250、1252、1254、1256、1258、1260、1262、1264、1266、1268、1270、1272、1274、1276、1278、1280、1282、1284、1286、1288、1290、1292、1294、1296、1298、1300、1302、1304、1306、1308、1310、1312、1314、1316、1318、1320、1322、1324、1326、1328、1330、1332、1334、1336、1338、1340、1342、1344、1346、1348、1350、1352、1354、1356、1358、1360、1362、1364、1366、1368、1370、1372、1374、1376、1378、1380、1382、1384、1386、1388、1390、1392、1394、1396、1398、1400、1402、1404、1406、1408、1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1481 2, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1555 556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628 , 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, or 1648.

[0230] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence comprising residues 40-536 of an even-numbered SEQ ID NO:4-1648, or comprises an even-numbered SEQ ID NO:4-1648. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, or 5 substitutions. In some embodiments, the amino acid substitutions comprise non-conservative and / or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

[0231] In some embodiments, the engineered glucocerebrosidase is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 ,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 , 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1026、1028、1030、1032、1034、1036、1038、1040、1042、1044、1046、1048、1050、1052、1054、1056、1058、1060、1062、1064、1066、1068、1070、1072、1074、1076、1078、1080、1082、1084、1086、1088、1090、1092、1094、1096、1098、1100、1102、1104、1106、1108、1110、1112、1114、1116、1118、1120、1122、1124、1126、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1218、1220、1222、1224、1226、1228、1230、1232、1234、1236、1238、1240、1242、1244、1246、1248、1250、1252、1254、1256、1258、1260、1262、1264、1266、1268、1270、1272、1274、1276、1278、1280、1282、1284、1286、1288、1290、1292、1294、1296、1298、1300、1302、1304、1306、1308、1310、1312、1314、1316、1318、1320、1322、1324、1326、1328、1330、1332、1334、1336、1338、1340、1342、1344、1346、1348、1350、1352、1354、1356、1358、1360、1362、1364、1366、1368、1370、1372、1374、1376、1378、1380、1382、1384、1386、1388、1390、1392、1394、1396、1398、1400、1402、1404、1406、1408、1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1 472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1533 34, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1595 In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises residues 40-536 of 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, or 1648. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, or 5 substitutions. In some embodiments, the amino acid substitutions include non-conservative and / or conservative substitutions. In some embodiments, the substitutions include conservative substitutions.

[0232] In some embodiments, the engineered glucocerebrosidase is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 ,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 24, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 38 6, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448 , 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1026、1028、1030、1032、1034、1036、1038、1040、1042、1044、1046、1048、1050、1052、1054、1056、1058、1060、1062、1064、1066、1068、1070、1072、1074、1076、1078、1080、1082、1084、1086、1088、1090、1092、1094、1096、1098、1100、1102、1104、1106、1108、1110、1112、1114、1116、1118、1120、1122、1124、1126、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1218、1220、1222、1224、1226、1228、1230、1232、1234、1236、1238、1240、1242、1244、1246、1248、1250、1252、1254、1256、1258、1260、1262、1264、1266、1268、1270、1272、1274、1276、1278、1280、1282、1284、1286、1288、1290、1292、1294、1296、1298、1300、1302、1304、1306、1308、1310、1312、1314、1316、1318、1320、1322、1324、1326、1328、1330、1332、1334、1336、1338、1340、1342、1344、1346、1348、1350、1352、1354、1356、1358、1360、1362、1364、1366、1368、1370、1372、1374、1376、1378、1380、1382、1384、1386、1388、1390、1392、1394、1396、1398、1400、1402、1404、1406、1408、1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 147 0, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1531 32, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, or 1648. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally includes 1, 2, 3, 4, or 5 substitutions. In some embodiments, the amino acid substitutions include non-conservative and / or conservative substitutions. In some embodiments, the substitutions include conservative substitutions.

[0233] In some embodiments, the engineered glucocerebrosidase comprises an amino acid sequence comprising residues 40-536 of, or comprising, SEQ ID NO: 54, 220, 984, or 1150. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered glucocerebrosidase optionally comprises 1, 2, 3, 4, or 5 substitutions. In some embodiments, the amino acid substitutions comprise non-conservative and / or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

[0234] In some embodiments, the engineered glucocerebrosidase described herein has glycoside hydrolase activity. In some embodiments, the engineered glucocerebrosidase has glucosylceramidase activity, which can cleave the β-glycosyl bond of glucocerebroside. In some embodiments, the engineered glucocerebrosidase has glucocerebrosidase activity and one or more improved properties compared to a reference glucocerebrosidase. In some embodiments, exemplary improved properties are provided in the Examples.

[0235] In some embodiments, the engineered glucocerebrosidase has increased enzymatic activity compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150. In some embodiments, the engineered glucocerebrosidase has at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more, activity compared to the reference glucocerebrosidase. In some embodiments, the increased enzymatic activity is based on the assays described in the Examples.

[0236] In some embodiments, the engineered glucocerebrosidase has increased expression or expression efficiency compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150. In some embodiments, the increased expression or expression efficiency is in the liver, or liver cells, or macrophages.

[0237] In some embodiments, the engineered glucocerebrosidase has increased stability at neutral or basic pH compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

[0238] In some embodiments, the engineered glucocerebrosidase has increased stability at acidic pH compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

[0239] In some embodiments, the engineered glucocerebrosidase has increased stability with respect to exposure to serum and / or plasma compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

[0240] In some embodiments, the engineered glucocerebrosidase has increased thermal stability compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150. In some embodiments, the increased stability is at 44°C to 47°C, or 30°C to 55°C, for a defined period of time, e.g., 1 hour.

[0241] In some embodiments, the engineered glucocerebrosidase is characterized by increased expression and / or secretion from cells, such as macrophages, compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

[0242] In some embodiments, the engineered glucocerebrosidase is characterized by increased uptake and / or intracellular stability in cells, such as macrophages, compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

[0243] In some embodiments, the engineered glucocerebrosidase has reduced immunogenicity compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, or 984, or a sequence corresponding to SEQ ID NO: 2, 54, 220, or 984.

[0244] In some of the foregoing embodiments, the improved properties are compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2.

[0245] In some embodiments, a glucocerebrosidase of the disclosure exhibits at least one improved property selected from: i) increased enzymatic activity, ii) increased expression or expression efficiency, iii) increased stability at neutral or basic pH, iv) increased stability at acidic pH, v) increased stability in serum or plasma, vi) increased thermostability, vii) increased uptake and / or intracellular stability in cells such as macrophages, and viii) reduced immunogenicity, or any combination of i), ii), iii), iv), v), vi), vii), and viii), compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2. In some embodiments, the reference glucocerebrosidase has a sequence corresponding to residues 40-536 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2.

[0246] In some embodiments, the engineered glucocerebrosidase is a "deimmunized" glucocerebrosidase (e.g., as shown in the Examples in Table 14.1). In some embodiments, the engineered glucocerebrosidase is characterized by a reduced total immunogenicity score (TIS) and / or immunogenic hits (IHC) compared to a reference glucocerebrosidase, the reference glucocerebrosidase having a sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, as provided in the Examples in Table 14.1.

[0247] In some embodiments, the "deimmunized" engineered glucocerebrosidase is selected from the group consisting of 4, 12, 22, 30, 32, 38, 44, 46, 50, 54, 56, 58, 60, 64, 72, 76, 80, 82, 84, 86, 88, 90, 92, 96, 98, 200, 208, 212, 216, 218, 222, 226, 228, 232, 236, 242, 244, 254, 256, 260, 262, 266, 272, 274, 276, 278, 280, 282, 286, 292, 298, 300, 302, 304, 306, 308, 312, 314, 316, 318, 320, 322, 326, 328, 330, 332, 336, 338, 340, 342, 344, 346, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 18, 320, 322, 324, 326, 330, 334, 336, 338, 340, 342, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 382, ​​384, 386, 388, 39 0, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 422, 424, 426, 428, 430, 432, 434, 436, 438, 444, 446, 448, 450, 452, 454, 456, 458 , 460, 462, 464, 466, 468, 470, 472, 476, 478, 480, 482, 484, 490, 492, 496, 500, 502, 508, 510, 512, 514, 516, 518, 524, 526, 528, 532, 534, 536, 538, 540, 542, 544, 546, 552, 554, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 588, 590, 592, 594, 596, 600, 602, 604, 606, 608, 610, 612, 6 18, 620, 626, 628, 630, 632, 634, 636, 638, 640, 644, 646, 648, 652, 654, 656, 660, 662, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 69 2, 694, 696, 698, 700, 706, 708, 710, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 740, 742, 746, 748, 750, 752, 756, 760, 762, 766, 768, 770,772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 794, 796, 798, 800, 802, 804, 806, 808, 810, 814, 816, 818, 822, 828, 830, 834, 836, 842, 844, 846, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 52, 854, 856, 858, 860, 862, 864, 866, 868, 870, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 910, 914, 916, 918, 92 0, 924, 928, 932, 936, 940, 946, 948, 952, 956, 958, 960, 964, 968, 970, 980, 984, 988, 990, 994, 998, 1002, 1004, 1006, 1008, 1012, 1016, 1020, 1022, 102 4, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1073 4, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 112 4, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1173 an amino acid sequence comprising residues 40 to 536 of SEQ ID NO: 4, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, or 1216, or SEQ ID NO: 4, 12, 22, 30, 32, 38, 44, 46, 50, 54, 56, 58, 60, 64, 72, 76, 80, 82, 84, 86, 88, 90, 92, 96, 98, 200, 208, 212, 216, 218, 222, 226, 228, 232, 236,242、244、254、256、260、262、266、272、274、276、278、280、282、286、292、298、300、302、304、306、308、312、314、316、318、320、322、324、326、330、334、336、338、340、342、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、382、384、386、388、390、392、394、396、398、400、402、404、406、408、410、412、414、416、418、422、424、426、428、430、432、434、436、438、444、446、448、450、452、454、456、458、460、462、464、466、468、470、472、476、478、480、482、484、490、492、496、500、502、508、510、512、514、516、518、524、526、528、532、534、536、538、540、542、544、546、552、554、558、560、562、564、566、568、570、572、574、576、578、580、582、584、588、590、592、594、596、600、602、604、606、608、610、612、618、620、626、628、630、632、634、636、638、640、644、646、648、652、654、656、660、662、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、706、708、710、714、716、718、720、722、724、726、728、730、732、734、736、740、742、746、748、750、752、756、760、762、766、768、770、772、774、776、778、780、782、784、786、788、790、794、796、798、800、802、804、806、808、810、814、816、818、822、828、830、834、836、842、844、846、850、852、854、856、858、860、862、864、866, 868, 870, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 910, 914, 916, 918, 920, 924, 928, 932, 936, 940, 946, 948, 952, 956, 958, 960, 964, 968, 970, 980, 984, 988, 990, 994, 998, 1002, 1004, 1006, 1008, 1012, 1016, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, or 1216.

[0248] In some embodiments, the engineered glucocerebrosidase comprises any full-length engineered glucocerebrosidase described herein. In some embodiments, the glucocerebrosidase comprises the "preproprotein" or "prepropolypeptide" of any of the engineered glucocerebrosidases described herein. In some embodiments, the signal sequence of the full-length glucocerebrosidase or its "preproprotein" or "prepropolypeptide" can comprise any compatible signal sequence or signal peptide, including synthetic, mouse (e.g., IgG), or human signal sequences or signal peptides. In some embodiments, the signal sequence of an exemplary glucocerebrosidase herein is replaced with a compatible signal sequence or signal peptide, e.g., synthetic or mouse. In some embodiments, the signal sequence comprises the signal sequence of a naturally occurring full-length human glucocerebrosidase.

[0249] In some embodiments, the engineered glucocerebrosidase is processed or cleaved, for example, to a propolypeptide or proprotein form lacking a signal sequence. In some embodiments, the engineered glucocerebrosidase is processed or cleaved to a mature form of the engineered glucocerebrosidase, as described herein. In some embodiments, the mature form of the engineered glucocerebrosidase comprises an active form of the engineered glucocerebrosidase.

[0250] Thus, for each and every engineered glucocerebrosidase described herein, the present disclosure provides a prepropolypeptide, propolypeptide, or mature biologically active form of the engineered glucocerebrosidase, including each engineered glucocerebrosidase disclosed in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1. In some embodiments, the engineered glucocerebrosidase propolypeptide comprises the amino acid sequence from amino acid residue 21 of the engineered glucocerebrosidase polypeptide, or the amino acid sequence from amino acid residue 40 to the carboxy terminus, e.g., amino acid residue 536.

[0251] In some embodiments, the engineered glucocerebrosidase is expressed as a fusion protein. In some embodiments, the engineered glucocerebrosidase described herein can be fused to various polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification. In some embodiments, the engineered glucocerebrosidase fusion protein comprises a glycine-histidine or histidine tag (His-tag). In some embodiments, the engineered glucocerebrosidase fusion protein comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the engineered glucocerebrosidase fusion protein comprises a GST, SUMO, STREP, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) terminus of the engineered glucocerebrosidase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the engineered glucocerebrosidase polypeptide.

[0252] In some embodiments, for any of the engineered glucocerebrosidases disclosed herein, the engineered glucocerebrosidase is purified or is a purified preparation or composition. In some embodiments, the engineered glucocerebrosidase is provided in solution, as a lyophilizate, or immobilized on a solid substrate, porous substrate, membrane, filter, or particle.

[0253] In some embodiments, the present disclosure further provides functional or biologically active fragments of the engineered glucocerebrosidases described herein. In some embodiments, the functional or biologically active fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the glucocerebrosidase from which it is derived (i.e., the parent engineered glucocerebrosidase). In some embodiments, a functional or biologically active fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of glucocerebrosidase. In some embodiments, functional fragments are truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, and fewer than 50 amino acids.

[0254] In some embodiments, functional or biologically active fragments of the engineered glucocerebrosidases described herein comprise at least one mutation or set of mutations in the amino acid sequence of the engineered glucocerebrosidases described herein. Thus, in some embodiments, functional or biologically active fragments of the engineered glucocerebrosidases exhibit enhanced or improved properties relative to a mutation or set of mutations in the parent engineered glucocerebrosidase.

[0255] In some further embodiments, as discussed herein, functional or biologically active fragments include biologically processed polypeptides of engineered glucocerebrosidase, such as functional or biologically active fragments produced upon expression in a mammalian cell or patient, or when the engineered glucocerebrosidase is administered to a patient, particularly a human patient. In some embodiments, functional or biologically active fragments are derived from engineered glucocerebrosidases shown in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and in the Sequence Listing.

[0256] Polynucleotides encoding engineered polypeptides, expression vectors, and host cells In another aspect, the present disclosure provides a polynucleotide that encodes the engineered glucocerebrosidase described herein.In some embodiments, the polynucleotide is operably linked with one or more heterologous or homologous regulatory sequences, which control gene expression to produce a recombinant polynucleotide that can express engineered glucocerebrosidase.The expression construct that includes the heterologous polynucleotide that encodes engineered glucocerebrosidase can be introduced into suitable host cells to express the corresponding engineered glucocerebrosidase polypeptide.

[0257] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provide a description of all polynucleotides capable of encoding a polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of a vast number of nucleic acids, all of which encode engineered glucocerebrosidases. Thus, a person skilled in the art with knowledge of a particular amino acid sequence can create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides capable of encoding the engineered glucocerebrosidases described herein by selecting combinations based on possible codon choices, and all such variations should be considered specifically disclosed for any polypeptide described herein, including the engineered glucocerebrosidase variants provided in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and the Sequence Listing.

[0258] As noted above, it should be understood that the present disclosure provides recombinant polynucleotides encoding each and every engineered glucocerebrosidase described herein, or biologically or functionally active fragments thereof, or prepropolypeptides, propolypeptides, or mature polypeptides.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:2-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:2-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2, 54, 220, or 1150, or relative to the reference sequence corresponding to SEQ ID NO:2, 54, 220, or 1150.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, or 1150, or to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 1150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, or 1150.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions.

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or to a reference sequence corresponding to an even-numbered SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2.

[0264] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that at amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 38, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 325, 329, 3 32, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 5 and a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at residues 40-536 of SEQ ID NO:2, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at amino acid positions 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at amino acid position 70, 207, 214, or 489, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at amino acid positions 65, 68, 99, 154, 230, or 319, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at amino acid positions 65, 195, 230, 263, 337, 361, 374, or 471, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0269] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 265 / 372 / 489, 243, 23, 109, 265, 109 / 195 / 243, 23 / 70 / 207 / 214 / 265, 23 / 70, 23 / 214 / 243 / 265, 65 / 70 / 109 / 214 / 319 / 372 / 481, 23 / 265 / 319, 23 / 65 / 70 / 141 / 319 / 481, 23 / 265, 372, 214, 23 / 65 / 70 / 109 / 141 / 214, 319, 65 / 372, 109 / 195, 23 / 65, 207 / 265 / 319, 109 / 2 07 / 214 / 372, 23 / 319 / 372 / 489, 65 / 109 / 214 / 243, 23 / 141 / 195, 70 / 207 / 214 / 489, 65 / 70 / 265 / 319 / 372, 214 / 265, 265 / 319, 65 / 243, 65 / 70 / 195, 489, 23 / 65 / 141, 23 / 372, 70 / 141, 23 / 195, 214 / 319, 23 / 207 / 214 / 243 / 265 / 481, 65 / 214 / 243 / 265 / 319, 214 / 243, 243 / 265, 265 / 489, 214 / 243 / 265, 207 / 243 / 372 , 195 / 243, 65 / 481, 207 / 214 / 243 / 319, 70 / 141 / 195 / 265, 23 / 265 / 372, 46 / 99 / 134 / 230 / 373 / 449, 68 / 211 / 230, 373 / 449, 46 / 68 / 336, 68 / 230 / 314 / 373, 6 8, 68 / 336 / 373, 99 / 314, 46, 46 / 230 / 261, 373, 230 / 314, 68 / 211, 68 / 336 / 449, 46 / 336, 336 / 449, 46 / 68 / 336 / 373, 46 / 99, 99 / 261, 99, 68 / 134, 46 / 68 / 134 / 230 / 336 / 373, 46 / 68, 68 / 99 / 211 / 230, 99 / 134, 68 / 99 / 336, 449, 68 / 99, 46 / 230 / 336, 68 / 154 / 449, 336 / 373 / 449, 134 / 336, 68 / 99 / 230, 46 / 68 / 134 / 154 , 68 / 99 / 230 / 373 / 449, 134, 46 / 68 / 154 / 211 / 230 / 261 / 336, 68 / 99 / 154 / 261 / 336, 230 / 449, 46 / 68 / 314, 154, 68 / 449, 99 / 449, 68 / 154 / 230 / 314 / 336 / 449,The present invention also includes a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence containing at least one substitution set at 46 / 68 / 154 / 314 / 336 / 373, 46 / 230, 336, or 46 / 99 / 230 / 373, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0270] In some embodiments, the recombinant polynucleotide comprises a sequence encoding the amino acid sequence at amino acid position(s) 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408, 65 / 68 / 154 / 195 / 214 / 230 / 319 / 337 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 154 / 195 / 214 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 23 0 / 263 / 319 / 337 / 374 / 408, 65 / 68 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 374 / 408 / 471, 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 408, 65 / 68 / 70 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 214 / 230 / 263 / 337 / 374 / 408, 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 230 / 408, 65 / 68 / 70 / 99 / 154 / 207 / 230 / 319 / 408, 65 / 68 / 70 / 214 / 230 / 408, 65 / 68 / 70 / 195 / 214 / 230 / 263 / 361 / 471, 195 / 207 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 214 / 230 / 408 / 489, 65 / 68 / 70 / 195 / 207 / 2 30 / 408, 207 / 230 / 319 / 374 / 408, 65 / 68 / 70 / 99 / 195 / 207 / 230 / 408, 195 / 214 / 230 / 263 / 408, 195 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 214 / 408,207 / 214 / 230 / 319 / 374 / 408, 207 / 214 / 230 / 374 / 408, 154 / 207 / 230 / 374 / 408 / 489, 207 / 230 / 263 / 408, 207 / 230 / 408 / 471, 207 / 230 / 374 / 408, 214 / 230 / 319 / 408, 214 / 230 / 374 / 408 / 489, 207 / 230 / 408, 65 / 68 / 70 / 154 / 207 / 408, 207 / 230 / 263 / 337 / 408, 195 / 214 / 230 / 408, 207 / 230 / 408 and a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence containing at least one substitution set at residues 40-536 of SEQ ID NO:2, 214 / 230 / 408, 230 / 361 / 408, 207 / 214 / 408, 214 / 263 / 408, 230 / 374 / 408, 207 / 408, 195 / 214 / 263, 374 / 408, or 408, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at an amino acid position set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:2.

[0273] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions at an amino acid position(s) set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions of an engineered glucocerebrosidase variant set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:2.

[0275] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150.

[0276] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or to a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648.

[0277] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150.

[0278] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150, or relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150.

[0279] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that at amino acid positions 23, 29, 35, 40, 41, 42, 44, 46, 50, 51, 53, 59, 63, 65, 68, 69, 70, 71, 75, 77, 78, 79, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 137, 138, 141, 142 , 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 325, 329, 332, 333, 335, 336, 337, 3 38, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 504, 505, 506, 507, 508, 509, 510, 512, 514, 51 The present invention also includes a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at residues 5, 516, 518, 519, 520, 522, 524, 525, 529, 532, 533, 534, or 536, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0280] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to the reference sequence corresponding to SEQ ID NO:54.

[0281] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence comprises one or more substitutions.

[0282] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 68 / 336, 65 / 99 / 319, 65 / 68 / 319, 68 / 319, 68, 65 / 319, 195, 65 / 154 / 319, 65 / 68 / 230, 65, 65 / 68 / 99 / 154 / 230 / 319, 68 / 230, 195 / 230, 65 / 230, 141 / 319, 154 / 336, 68 / 154, 68 / 195 / 230 / 319 / 336, 68 / 99 / 195 / 230 / 319, 68 / 195, 65 / 68, 68 / 141 / 23 0 / 319, 65 / 68 / 99 / 230 / 319, 68 / 99 / 141 / 319, 68 / 154 / 319, 141 / 154, 68 / 99, 99 / 230 / 319, 99, 230, 68 / 230 / 319,154 / 319, 65 / 68 / 154 / 319, 141, 336, 141 / 230, 68 / 154 / 336, 65 / 68 / 99 / 319, 154, 319, 230 / 319, 68 / 154, 99 / 141 / 319, 509, 108, 108 / 117 / 509, 519, 108 / 519, 88, 117 / 413 / 509, 509 / 519, 413, 108 / 117 / 315, 117 / 271, 117 / 413 / 509 / 529, 413 / 509, 469, 524, 507, 525, 489, 104, 95, 359, 374, 107, 89, 260, 110, 186, 370, 191, 333, 189, 468, 473, 115, 53, 449, 518, 236, 481, 59 / 220, 258, 467, 51, 415, 103, 493, 78, 335, 520, 101, 341, 471, 211, 206, 93, 490, 71, 79, 180 , 172, 207, 63, 142, 529, 77, 212, 474, 106, 386 / 507, 117, 373, 337, 107 / 246, 516, 233, 91, 194, 84, 361, 171, 536, 50, 116, 239, 44, 40, 113, 243, 169, 504, 505, 109, 479, 477, 97, 182, 111, 508, 314, 102, 338, 96, 138, 42, 263, 222, 522, 265, 514, 515, 246, 46, 133, 332, 506, 512, 137, 190,The invention also includes a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence including at least one substitution, or set of substitutions, at 105, 301, 329, 339, or 151, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, or are relative to a reference sequence corresponding to SEQ ID NO: 54.

[0283] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:220.

[0284] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 220.

[0285] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 65 / 195 / 361, 514, 79 / 195 / 263 / 361, 65 / 374, 79 / 195, 65 / 230, 65 / 233 / 263 / 337 / 359 / 361 / 374, 79 / 536, 79 / 230 / 233 / 239 / 359 / 361 / 471, 65 / 471, 374, 65 / 195 / 230 / 233 / 337 / 374 / 514, 65 / 195 / 2 33 / 359 / 536, 230, 65 / 195 / 263 / 276 / 359 / 361 / 374 / 471, 79, 195 / 230 / 233 / 337 / 374 / 536, 195 / 230, 195 / 230 / 337 / 359 / 361, 230 / 263, 65 / 230 / 233, 263, 195 / 230 / 337 / 361 / 374 / 471, 65 / 195, 374 / 471, 374 / 536, 239, 65, 230 / 233 / 536, 230 / 263 / 374, 65 / 195 / 230 / 263 / 337 / 361 / 374 / 471, 79 / 374, 65 / 361 / 374 / 471 / 536, 195, 233, 79 / 230 / 359 / 374, 536, 230 / 359 / 361 / 374 / 514, 65 / 79 / 230, 195 / 239 / 263 / 361 / 374 / 514, 195 / 263 / 374, 195 / 374, 65 / 230 / 233 / 361 / 536, 230 / 233 / 3 The present invention also includes a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution, or set of substitutions, at 74 / 471, 65 / 233 / 359 / 361 / 471, 65 / 359 / 471, 65 / 79, or 65 / 359, where the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:220.

[0286] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or to a reference sequence corresponding to SEQ ID NO: 984, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 984 or to the reference sequence corresponding to SEQ ID NO: 984.

[0287] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1022-1216, or a reference sequence corresponding to SEQ ID NO: 1022-1216, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or relative to the reference sequence corresponding to SEQ ID NO: 984.

[0288] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that of an amino acid at amino acid position(s) 359, 79 / 359, 79 / 97 / 182, 97 / 359, 97, 79 / 182, 201, 404, 484, 446, 494 / 495, 495, 412, 385, 100, 184, 82, 75, 71, 41, 99, 94, 92, 98, 114, 95, 102, 96, 105, 113, 325, 336, 262, 386, 360, 400, 342, 390, 264, 298, 382, ​​408, 449, 459, 434, 476, 502, The present invention also includes a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions at 29 / 534, 497, 485, 533, 536, 510, 532, 489, 534, 494, 182, 137, 204, 194, 202, 144, 142, 175, 220, 150, or 206, where the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:984 or are relative to a reference sequence corresponding to SEQ ID NO:984.

[0289] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or to a reference sequence corresponding to SEQ ID NO: 1150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150.

[0290] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:1218-1554, or a reference sequence corresponding to SEQ ID NO:1218-1554, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:1150, or relative to the reference sequence corresponding to SEQ ID NO:1150.

[0291] In some embodiments, the recombinant polynucleotide comprises a sequence encoding the amino acid sequence at amino acid position(s) 96 / 194 / 382 / 484, 359 / 382 / 412 / 494 / 495, 92 / 96 / 175 / 182 / 382, 96 / 359 / 382 / 484 / 494 / 495, 92 / 175 / 194 / 359 / 382, 359 / 382 / 400 / 412, 92 / 96 / 182 / 382 / 400 / 484, 92 / 96 / 382 / 494 / 495, 92 / 96 / 175 / 382 / 494 / 495, 96 / 182 / 359 / 382 / 484, 92 / 96 / 382, 92 / 382 / 494 / 495 / 534, 92 / 96 / 382 / 400 / 484, 92 / 96 / 175 / 382, 92 / 175 / 382 / 400 / 484, 92 / 96 / 175 / 182 / 382 / 412 / 484, 92 / 96 / 201 / 382 / 484, 92 / 182 / 194 / 382, 92 / 96 / 175 / 194 / 359 / 382 / 400, 92 / 359 / 382, 96 / 382, 92 / 175 / 382 / 484 / 494 / 495, 96 / 201 / 359 / 382, 92, 194, 201, 359, 96, 92 / 96 / 382 / 400 / 484 / 495 , 382 / 495, 382, ​​92 / 382, 71 / 175 / 325 / 385, 325 / 385, 71 / 325 / 502, 71 / 175 / 325 / 385 / 502, 202 / 325 / 385, 97 / 175 / 385 / 510, 325 / 385 / 510, 100 / 175 / 325, 71 / 175 / 325 / 360 / 385, 97 / 175 / 510, 71 / 97 / 100 / 175 / 325 / 385, 71 / 175 / 510, 71 / 97 / 325 / 502, 71 / 100 / 325 / 385, 202 / 385 / 489, 325 / 385 / 502, 71 / 1 00 / 325 / 385 / 510, 71 / 100 / 175 / 325 / 385, 385 / 510, 97 / 325 / 385, 71, 97 / 202 / 325 / 360 / 385 / 510, 502, 71 / 97 / 103 / 325, 100 / 325 / 385 / 502 / 510, 71 / 137 / 175 / 325 / 360 / 385 / 502, 71 / 276 / 325 / 385 / 510, 71 / 175 / 360 / 385 / 502, 100 / 175 / 325 / 385 / 489 / 502, 71 / 175 / 360 / 385 / 485 / 502, 95 / 113, 494 / 536,95 / 99 / 113 / 182 / 184 / 264 / 386, 184 / 536, 95 / 142 / 144 / 182 / 184 / 386 / 449, 144 / 204 / 386 / 400 / 449, 113 / 449 / 536, 536, 95 / 386 / 536, 264, 99 / 113 / 386 / 449, 95 / 386, 95 / 99 / 204 / 386, 113 / 142 / 386 / 449, 182 / 184 / 264 / 494, 113 / 142 / 182 / 494, 113, 99 / 264 / 449, 95, 95 / 99, 99 / 113 / 449, 95 / 113 / 449 / 536 , 95 / 99 / 113, 386 / 536, 99 / 182 / 184, 142 / 386 / 400 / 536, 35 / 264 / 449 / 536, 142 / 144 / 220, 99 / 142, 29 / 102 / 182 / 276 / 336 / 390 / 459 / 502 / 532, 41 / 262 / 336 / 502, 29 / 97 / 150 / 262 / 336 / 502 / 532, 29, 532, 102 / 390 / 459 / 502 / 532, 29 / 262 / 276 / 336 / 495, 29 / 182 / 262 / 459 / 532, 92 / 99 / 102 / 502, 29 / 97 / 102 / 182 / 502 / 532, 336 / 459 / 495 / 502 / 532, 150 / 182 / 336 / 390, 150 / 262 / 495, 29 / 495, 29 / 102 / 182 / 262 / 502, 29 / 336 / 459 / 502 / 532, 182 / 495 / 502 / 532, 1 02 / 336, 150 / 182 / 262 / 495, 459 / 495 / 502 / 532, 41 / 182 / 390 / 495, 29 / 41 / 182 / 336 / 495, 97 / 102 / 495, 182 / 262 / 495, 29 / 262 / 502, 29 / 102 / 502, 182 / 33 and a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions at amino acid positions 6 / 495, 29 / 150 / 182 / 262 / 459 / 495, 502 / 532, 386, 497, 99, 512, 69, 507, 445, 75, 487, 184, 90, 235, 434, 489, 86, 182, 446, 490, 299, 102, 230, 68, 141, 97, 316, 522, 224, 478, 480, 368, 412, 449, 265, or 494, wherein the amino acid positions are:It is relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 1150, or it is relative to a reference sequence corresponding to SEQ ID NO: 1150.

[0292] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution at an amino acid position set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or is relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0293] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0294] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions at an amino acid position(s) set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0295] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising at least one substitution or set of substitutions of an engineered glucocerebrosidase set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, or 984, or 1150.

[0296] In some embodiments, the recombinant polynucleotide has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

[0297] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising residues 40-536 of an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, and 6.2, or an amino acid sequence comprising an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, and 6.2.

[0298] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising, or including, residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150.

[0299] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered glucocerebrosidase comprising an amino acid sequence comprising residues 40-536 of an even-numbered SEQ ID NO:4-1648, or an even-numbered SEQ ID NO:4-1648.

[0300] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 118-1608 of an odd-numbered SEQ ID NO:3-1647, or a reference sequence corresponding to an odd-numbered SEQ ID NO:3-1647, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

[0301] In some embodiments, the recombinant polynucleotide is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 21 9, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 27, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 38 9, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513,515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、872、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、219、923、925、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987、989、991、993、995、997、999、1001、1003、1005、1007、1009、1011、1013、1015、1017、1019、1021、1023、1025、1027、1029、1031、1033、1035、1037、1039、1041、1043、1045、1047、1049、1051、1053、1055、1057、1059、1061、1063、1065、1067、1069、1071、1073、1075、1077、1079、1081、1083、1085、1087、1089、1091、1093、1095、1097、1099、1101、1103、1105、1107、1109、1111、1113、1115、1117、1119、1121、1123、1125、1127、1129、1131、1133、1135、1137、1139、1141、1143、1145、1147、1149、1151、1153、1155、1157、1159、1161、1163、1165、1167、1169、1171、1173、1175、1177、1179、1181、1183、1185、1187、1189、1191、1193、1195、1197、1199、1201、1203、1205、1207、1209、1211、1213、1215、1217、1219、1221、1223、1225、1227、1229、1231、1233、1235、1237、1239、1241、1243、1245、1247、1249、1251、1253、1255、1257、1259、1261、1263、1265、1267、1269、1271、1273、1275、1277、1279、1281、1283、1285、1287、1289、1291、1293、1295、1297、1299、1301、1303、1305、1307、1309、1311、1313、1315、1317、1319、1321、1323、1325、1327、1329、1331、1333、1335、1337、1339、1341、1343、1345、1347、1349、1351、1353、1355、1357、1359、1361、1363、1365、1367、1369、1371、1373、1375、1377、1379、1381、1383、1385、1387、1389、1391、1393、1395、1397、1399、1401、1403、1405、1407、1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1486 7, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1 567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645 or a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 118 to 1608 of 1647, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

[0302] In some embodiments, the engineered glucocerebrosidase is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 2 37, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 19 9, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 399, 400, 401, 402, 403, 404, 87, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 44 9, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511,513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、872、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、219、923、925、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987、989、991、993、995、997、999、1001、1003、1005、1007、1009、1011、1013、1015、1017、1019、1021、1023、1025、1027、1029、1031、1033、1035、1037、1039、1041、1043、1045、1047、1049、1051、1053、1055、1057、1059、1061、1063、1065、1067、1069、1071、1073、1075、1077、1079、1081、1083、1085、1087...

Claims

1. 1. An engineered glucocerebrosidase, or a biologically active fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NOs: 2-1648, or to a reference sequence corresponding to SEQ ID NOs: 2-1648, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NOs: 2, 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NOs: 2, 54, 220, 984, or 1150.

2. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150.

3. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:

2.

4. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:4-1648, or to a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 137, 138, 141, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 325 , 329, 332, 333, 335, 336, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 50 6. The engineered glucocerebrosidase of any one of claims 1 to 5, comprising at least one substitution at amino acid position 4, 505, 506, 507, 508, 509, 510, 512, 514, 515, 516, 518, 519, 520, 522, 524, 525, 529, 532, 533, 534, or 536, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

7. Preface to the operation of the されたグルコセレブロシダーゼのPreface to the アミノacid preparation sequence が, less なくとも1つの replaces 23A, 29I, 35T, 40H / S, 41H, 4 2A, 44V, 46M / R, 50A, 51P, 53L, 59N, 63 L, 65L / M / S / V, 68F / L / Q / T / V / W / Y, 69D , 70L, 71F / G / H / L / M / R / S / T / Y, 75A / E / H / N, 77G / H / I / N / R / T, 78L / M, 79I / R / V , 82S, 84A / G / K, 86E / H, 88L, 89C / D, 90 A / M, 91I / T, 92A / E / G / H / K / Q, 93S, 94E / N、95G / H / L / T / W、96D / E / G / I / M / N / R / S / V, 97E / I / K / M / N / Q / R, 98P, 99E / F / G / K / L / R / S / V / W, 100N / R, 101L / Q / R / S / T / V / W, 102A / E / N / P / S, 103A / L / P / S, 104 M / V, 105I / M / T, 106W, 107A / C / D / E / M / Q / S, 108I, 109D / G / L / M / R, 110A / H / Q, 111F / H / L, 113G / I / N / P / Q / R, 114Y, 11 5M / R / V, 116E / M / Q / R / V / Y, 117I / T, 133 M, 134S, 137Q / V, 138S, 141D / E, 142A / M / N / Y, 144M, 150T, 151L, 154E, 169V, 171C / P / V, 172S, 175D, 180C, 182D / E / G / H / M / S / T, 184I / Q / V, 186G, 189R / S, 190W, 191N / R, 194H / L / Q, 195M / W, 201 Q / R / T, 202Q, 204Q, 206F / I / R, 207S / D , 211I / L / N, 212A / N, 214F, 220A / G, 222I, 224M, 230F / L / Y, 233A / G / Q / R, 235T , 236M, 239I / R / T, 243K / L / M / R / V, 246 K / R, 258T, 260R, 261K, 262Q, 263G, 26 4I, 265N / R / S / T, 271T, 276T, 298W, 29 9V, 301K, 314D / G / R, 315T, 316H, 319F / I / V, 325V, 329L, 332R, 333C / L / T, 335 M, 336A / E / G / R, 337N, 338R, 339A, 341 R / S, 342Q, 359E / P, 360E, 361L / V / W / Y , 368D, 370R, 372D, 373A / V / Y, 374T / V,382A, 385G / H / N / Q / R, 386I / L / P, 390A, 400E / Q / T, 404Q, 408D / E, 412N / W, 413W / Y, 415C, 434G / Q, 445G / N, 446H / N, 449E / K / L / M / R / S / V, 459M, 467H / L / M / N / R, 468G, 469E, 471E / F / L / R / S / T / V, 473 C / I / L, 474A / D / S, 476A, 477G / H / R / S / V, 478N, 479A / L / R, 480G, 481H / K / T, 484E, 485M / S / T, 487S , 489A / E / G / L / N / Q / T, 490G / I / N / Q / R / S / T / V, 493T, 494A / T, 495G / S, 497A / M, 502K / Q, 504G / P / T / W, 505I / L / R, 506T, 507A / I / L / M / R / T / Y, 508L / Q / R / S / T, 509F / G / I / R / V / W, 510S, 512H / L / Q, 514E , 515G / T, 516L, 518E / L / S / V, 519I, 520D / K / R / S / V, 522S / T / V, 524K / S, 525C / Q, 529M / Q, 532V, 53 7. The engineered glucocerebrosidase of any one of claims 1 to 6, comprising 3Y, 534K, or 536A / C / D / H / I / L / M / P / R / T / W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

8. 6. The engineered glucocerebrosidase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

9. 9. The engineered glucocerebrosidase of any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 65L / M / S / V, 68F / L / Q / T / V / W / Y, 70L, 99E / F / G / K / L / R / S / V / W, 154E, 195M / W, 207S / D, 214F, 230F / L / Y, 263M, 319F / I / V, 337N, 361L / V / W / Y, 374T / V, 408D / E, 471E / F / L / R / S / T / V, or 489A / E / G / L / N / Q / T, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:

2.

10. 9. The engineered glucocerebrosidase of any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution 665L / V, 68L, 70L, 99R, 154E, 195W, 207S, 214F, 230L / Y, 263G, 319I, 337N, 361W, 374T, 408E, 471T, or 489L, or a combination thereof, and wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:

2.

11. The amino acid sequence of the engineered glucocerebrosidase may comprise at amino acid position(s) 265 / 372 / 489, 243, 23, 109, 265, 109 / 195 / 243, 23 / 70 / 207 / 214 / 265, 23 / 70, 23 / 214 / 243 / 265, 65 / 70 / 109 / 214 / 319 / 372 / 481, 23 / 265 / 319, 23 / 65 / 70 / 141 / 319 / 481, 23 / 265, 372, 214, 23 / 65 / 70 / 109 / 141 / 214, 319, 65 / 372, 109 / 195, 23 / 65, 207 / 265 / 319, 109 / 207 / 214 / 372, 23 / 319 / 372 / 489, 65 / 109 / 214 / 243, 23 / 141 / 195, 70 / 207 / 214 / 489, 65 / 70 / 265 / 319 / 372, 214 / 265, 265 / 319, 65 / 243, 65 / 70 / 195, 489, 23 / 65 / 141, 23 / 372, 70 / 141, 23 / 195, 214 / 319, 23 / 207 / 214 / 243 / 265 / 481, 65 / 214 / 243 / 265 / 319, 214 / 243, 243 / 265, 265 / 489, 214 / 243 / 26 5, 207 / 243 / 372, 195 / 243, 65 / 481, 207 / 214 / 243 / 319, 70 / 141 / 195 / 265, 23 / 265 / 372, 46 / 99 / 134 / 230 / 373 / 449, 68 / 211 / 230, 373 / 449, 46 / 68 / 336, 68 / 230 / 314 / 373, 68, 68 / 336 / 373, 99 / 314, 46, 46 / 230 / 261, 373, 230 / 314, 68 / 211, 68 / 336 / 449, 46 / 336, 336 / 449, 46 / 68 / 336 / 373, 46 / 99, 99 / 261, 9 9, 68 / 134, 46 / 68 / 134 / 230 / 336 / 373, 46 / 68, 68 / 99 / 211 / 230, 99 / 134, 68 / 99 / 336, 449, 68 / 99, 46 / 230 / 336, 68 / 154 / 449, 336 / 373 / 449, 134 / 336, 68 / 99 / 230, 46 / 68 / 134 / 154, 68 / 99 / 230 / 373 / 449, 134, 46 / 68 / 154 / 211 / 230 / 261 / 336, 68 / 99 / 154 / 261 / 336, 230 / 449, 46 / 68 / 314, 154, 68 / 449, 99 / 449,7. The engineered glucocerebrosidase of any one of claims 1 to 6, comprising at least one substitution set at 68 / 154 / 230 / 314 / 336 / 449, 46 / 68 / 154 / 314 / 336 / 373, 46 / 230, 336, or 46 / 99 / 230 / 373, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

12. The amino acid sequence of the engineered glucocerebrosidase may contain at least one of the following substitution sets: 265T / 372D / 489L, 243K, 23A, 109D, 265T, 109D / 195M / 243K, 23A / 70L / 207S / 214F / 265T, 23A / 70L, 23A / 214F / 243K / 265T, 65L / 70L / 109D / 214F / 319I / 372D / 481T, 23A / 265T / 319I, 23A / 65L / 70L / 141D / 319I / 481T, 23A / 265T, 372D, 214F, 23A / 65L / 70L / 109D / 141D / 214F, 319I, 65L / 372D, 109D / 195M, 23A / 65L, 207S / 265T / 319I, 109D / 207S / 214F / 372D, 23A / 319I / 372D / 489L, 65L / 109D / 214F / 243K, 23A / 141D / 195M, 70L / 207S / 214F / 489L, 65L / 70L / 265T / 319I / 372D, 214F / 265T, 265T / 319I, 65L / 243K, 65L / 70L / 195M, 489L, 23A / 65L / 141D, 23A / 372D, 70L / 141D, 23A / 195M, 214F / 319I, 23A / 207S / 214F / 243K / 265T / 481T, 65L / 214F / 243K / 265T / 319I, 214F / 243K, 243K / 265T, 265T / 489L, 214F / 243K / 265T, 207S / 243 K / 372D, 195M / 243K, 65L / 481T, 207S / 214F / 243K / 319I, 70L / 141D / 195M / 26 5T, 23A / 265T / 372D, 46R / 99R / 134S / 230L / 373Y / 449V, 68L / 211L / 230L, 373Y / 449V, 46R / 68V / 336A, 68V / 230L / 314D / 373Y, 68V, 68L / 336A / 373Y, 99R / 31 4D, 46R, 46R / 230L / 261K, 373Y, 230L / 314D, 68L / 211L, 68V / 336A / 449V, 46R / 336A, 336A / 449V, 46R / 68V / 336A / 373Y, 46R / 99R, 99R / 261K, 99R, 68V / 134S, 46R / 68V / 134S / 230L / 336A / 373Y, 46R / 68V, 68L / 99R / 211L / 230L, 99R / 134S,68V / 99R / 336A, 449V, 68V / 99R, 46R / 230L / 336A, 68V / 154E / 449V, 336A / 373Y / 449V, 134S / 336A, 68V / 99R / 230L, 68L, 46R / 68V / 134S / 154 E.

12. The engineered glucocerebrosidase of any one of claims 1 to 6, comprising the amino acid sequences 46R / 68V / 154E / 230L / 314D / 336A / 449V, 46R / 68V / 154E / 314D / 336A / 373Y, 46R / 230L, 336A, or 46R / 99R / 230L / 373Y, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

13. The amino acid sequence of the engineered glucocerebrosidase may comprise at amino acid position(s) 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408, 65 / 68 / 154 / 195 / 214 / 230 / 319 / 337 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 154 / 195 / 214 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 374 / 408, 65 / 68 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 337 / 361 / 374 / 408 / 471 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 3 37 / 361 / 374 / 408 / 489, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 374 / 408 / 471, 68 / 70 / 99 / 154 / 195 / 207 / 214 / 230 / 263 / 319 / 408, 65 / 68 / 70 / 207 / 230 / 37 4 / 408, 65 / 68 / 70 / 214 / 230 / 263 / 337 / 374 / 408, 214 / 230 / 263 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 207 / 230 / 408, 65 / 68 / 70 / 99 / 154 / 207 / 230 / 319 / 408, 6 5 / 68 / 70 / 214 / 230 / 408, 65 / 68 / 70 / 195 / 214 / 230 / 263 / 361 / 471, 195 / 207 / 230 / 319 / 374 / 408 / 489, 65 / 68 / 70 / 214 / 230 / 408 / 489, 65 / 68 / 70 / 195 / 207 / 230 / 408, 207 / 230 / 319 / 374 / 408, 65 / 68 / 70 / 99 / 195 / 207 / 230 / 408, 195 / 214 / 230 / 263 / 408, 195 / 207 / 230 / 374 / 408, 65 / 68 / 70 / 99 / 154 / 195 / 214 / 408,207 / 214 / 230 / 319 / 374 / 408, 207 / 214 / 230 / 374 / 408, 154 / 207 / 230 / 374 / 408 / 489, 207 / 230 / 263 / 408, 207 / 230 / 408 / 471, 207 / 230 / 374 / 408, 214 / 230 / 319 / 408, 214 / 230 / 374 / 408 / 489, 207 / 230 / 408, 65 / 68 / 70 / 154 / 207 / 408, 207 / 230 / 263 / 337 / 408, 195 / 214 / 230 / 408, 207 / 230 / 40 7. The engineered glucocerebrosidase of any one of claims 1 to 6, comprising at least one substitution, or set of substitutions, at amino acid positions 8 / 489, 214 / 230 / 408, 230 / 361 / 408, 207 / 214 / 408, 214 / 263 / 408, 230 / 374 / 408, 207 / 408, 195 / 214 / 263, 374 / 408, or 408, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

14. The amino acid sequence of the engineered glucocerebrosidase may contain at least one substitution or set of substitutions 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E, 65V / 68L / 154E / 195W / 214F / 230Y / 319I / 337N / 374T / 408E, T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 374T / 40 8E, 65V / 68L / 70L / 154E / 195W / 214F / 230Y / 319I / 374T / 408E / 489L, 65V / 6 8L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 319I / 337N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 374T / 408E, 65V / 68L / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 3 37N / 361W / 374T / 408E / 471T / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 263G / 319I / 337N / 361W / 374T / 408E / 489L, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 214F / 230Y / 374T / 408E / 471T, 68L / 70L / 99R / 154E / 19 5W / 207S / 214F / 230Y / 263G / 319I / 408E, 65V / 68L / 70L / 207S / 230Y / 374T / 408E, 65V / 68L / 70L / 214F / 230Y / 263G / 337N / 374T / 408E, 214F / 230Y / 263 G / 374T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 207S / 230Y / 408E, 65V / 68L / 70L / 99R / 154E / 207S / 230Y / 319I / 408E, 65V / 68L / 70L / 214F / 230Y / 408E,65V / 68L / 70L / 195W / 214F / 230Y / 263G / 361W / 471T, 195W / 207S / 230Y / 319I / 374T / 408E / 489L, 65V / 68L / 70L / 214F / 230Y / 40 8E / 489L, 65V / 68L / 70L / 195W / 207S / 230Y / 408E, 207S / 230Y / 319I / 374T / 408E, 65V / 68L / 70L / 99R / 195W / 207S / 230Y / 408E, 1 95W / 214F / 230Y / 263G / 408E, 195W / 207S / 230Y / 374T / 408E, 65V / 68L / 70L / 99R / 154E / 195W / 214F / 408E, 207S / 214F / 230Y / 3 19I / 374T / 408E, 207S / 214F / 230Y / 374T / 408E, 154E / 207S / 230Y / 374T / 408E / 489L, 207S / 230Y / 263G / 408E, 207S / 230Y / 408 E / 471T, 207S / 230Y / 374T / 408E, 214F / 230Y / 319I / 408E, 214F / 230Y / 374T / 408E / 489L, 207S / 230Y / 408E, 65V / 68L / 70L / 15 4E / 207S / 408E, 207S / 230Y / 263G / 337N / 408E, 195W / 214F / 230Y / 408E, 207S / 230Y / 408E / 489L, 214F / 230Y / 408E, 230Y / 361W 14. The engineered glucocerebrosidase of any one of claims 1 to 6 and 13, comprising the amino acid positions 207S / 214F / 408E, 214F / 263G / 408E, 230Y / 374T / 408E, 207S / 408E, 195W / 214F / 263G, 374T / 408E, 408E, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:2 or relative to a reference sequence corresponding to SEQ ID NO:

2.

15. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to a reference sequence corresponding to SEQ ID NO:

2.

16. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase variant set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:2, or are relative to a reference sequence corresponding to SEQ ID NO:

2.

17. 10. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising one substitution or set of substitutions of an engineered glucocerebrosidase variant set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:2 or are relative to the reference sequence corresponding to SEQ ID NO:

2.

18. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150.

19. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or to a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648.

20. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or to a reference sequence corresponding to SEQ ID NO: 54, 220, 984, or 1150, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150.

21. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of an even-numbered SEQ ID NO:4-1648, or a reference sequence corresponding to an even-numbered SEQ ID NO:4-1648, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, 220, 984, or 1150, or relative to the reference sequence corresponding to SEQ ID NO:54, 220, or 984, or 1150.

22. 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 133, 134, 137, 138, 1, 142, 144, 150, 151, 154, 169, 171, 172, 175, 180, 182, 184, 186, 189, 190, 191, 194, 195, 201, 202, 204, 206, 207, 211, 212, 214, 220, 222, 224, 230, 233, 235, 236, 239, 243, 246, 258, 260, 261, 262, 263, 264, 265, 271, 276, 298, 299, 301, 314, 315, 316, 319, 325, 329, 332, 333, 335, 33 6, 337, 338, 339, 341, 342, 359, 360, 361, 368, 370, 372, 373, 374, 382, ​​385, 386, 390, 400, 404, 408, 412, 413, 415, 434, 445, 446, 449, 459, 467, 468, 469, 471, 473, 474, 476, 477, 478, 479, 480, 481, 484, 485, 487, 489, 490, 493, 494, 495, 497, 502, 504, 505, 506, 507, 508, 509, 510, 51 22. The engineered glucocerebrosidase of claim 20 or 21, comprising at least one substitution at amino acid position 2, 514, 515, 516, 518, 519, 520, 522, 524, 525, 529, 532, 533, 534, or 536, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

23. Preface to the operation of the されたグルコセレブロシダーゼのPreface to the アミノacid preparation sequence が, less なThe acid residues 23A, 29I, 35T, and 40 are replaced by くとも1つの. H / S, 41H, 42A, 44V, 46M / R, 50A, 51P, 5 3L, 59N, 63L, 65L / M / S / V, 68F / L / Q / T / V / W / Y、69D、70L、71F / G / H / L / M / R / S / T / Y, 75A / E / H / N, 77G / H / I / N / R / T, 78L / M , 79I / R / V, 82S, 84A / G / K, 86E / H, 88L, 89C / D, 90A / M, 91I / T, 92A / 2E / G / H / K / Q , 93S, 94E / N, 95G / H / L / T / W, 96D / E / G / I / M / N / R / S / V, 97E / I / K / M / N / Q / R, 98P , 99E / F / G / K / L / R / S / V / W, 100N / R, 101 L / Q / R / S / T / V / W, 102A / E / N / P / S, 103A / L / P / S, 104M / V, 105I / M / T, 106W, 107A / C / D / E / M / Q / S, 108I, 109D / G / L / M / R, 110A / H / Q, 111F / H / L, 113G / I / N / P / Q / R, 114Y, 115M / R / V, 116E / M / Q / R / V / Y, 1 17I / T, 133M, 134S, 137Q / V, 138S, 141 D / E, 142A / M / N / Y, 144M, 150T, 151L, 1 54E, 169V, 171C / P / V, 172S, 175D, 180 C, 182D / E / G / H / M / S / T, 184I / Q / V, 186G , 189R / S, 190W, 191N / R, 194H / L / Q, 19 5M / W, 201Q / R / T, 202Q, 204Q, 206F / I / R, 207S / D, 211I / L / N, 212A / N, 214F, 220A / G, 222I, 224M, 230F / L / Y, 233A / G / Q / R, 235T, 236M, 239I / R / T, 243K / L / M / R / V, 246K / R, 258T, 260R, 261K, 262Q , 263G, 264I, 265N / R / S / T, 271T, 276T , 298W, 299V, 301K, 314D / G / R, 315T, 31 6H, 319F / I / V, 325V, 329L, 332R, 333C / L / T, 335M, 336A / E / G / R, 337N, 338R, 3 39A, 341R / S, 342Q, 359E / P, 360E, 361 L / V / W / Y, 368D, 370R, 372D, 373A / V / Y,374T / V, 382A, 385G / H / N / Q / R, 386I / L / P, 390A, 400E / Q / T, 404Q, 408D / E, 412N / W, 413W / Y, 415C, 434 G / Q, 445G / N, 446H / N, 449E / K / L / M / R / S / V, 459M, 467H / L / M / N / R, 468G, 469E, 471E / F / L / R / S / T / V, 47 3C / I / L, 474A / D / S, 476A, 477G / H / R / S / V, 478N, 479A / L / R, 480G, 481H / K / T, 484E, 485M / S / T, 487S, 4 89A / E / G / L / M / N / Q / T, 490G / I / N / Q / R / S / T / V, 493T, 494A / T, 495G / S, 497A / M, 502K / Q, 504G / P / T / W, 50 5I / L / R, 506T, 507A / I / L / M / R / T / Y, 508L / Q / R / S / T, 509F / G / I / R / V / W, 510S, 512H / L / Q, 514E, 515G / T , 516L, 518E / L / S / V, 519I, 520D / K / R / S / V, 522S / T / V, 524K / S, 525C / Q, 529M / Q, 532V, 533Y, or 534 K, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

24. 23. The engineered glucocerebrosidase of any one of claims 20-22, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution at amino acid position 65, 68, 70, 99, 154, 195, 207, 214, 230, 263, 319, 337, 361, 374, 408, 471, or 489, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

25. The amino acid sequence of the engineered glucocerebrosidase may have at least one substitution or amino acid residues 65L / M / S / V, 68F / L / Q / T / V / W / Y, 70L, 99E / F / G / K / L / R / S / V / W, 154E, 195M / W, 207S / D, 214F, 230F / L / Y, 263M, 319F / I / V, 337N, 361L / V / W / Y, 374T / V, 408D / E, 471E / F / L / R / S / T / W, 472E / F / L / R / S / T / W, 471F ...

25. The engineered glucocerebrosidase of any one of claims 20-22 and 24, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

26. 26. The engineered glucocerebrosidase of any one of claims 20-22, 24, and 25, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or amino acid residues 665L / V, 68L, 70L, 99R, 154E, 195W, 207S, 214F, 230L / Y, 263G, 319I, 337N, 361W, 374T, 408E, 471T, or 489L, or a combination thereof, and wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

27. 21. The engineered glucocerebrosidase of claim 20, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or a reference sequence corresponding to SEQ ID NO:54, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or to the reference sequence corresponding to SEQ ID NO:

54.

28. 22. The engineered glucocerebrosidase of claim 21, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54, or a reference sequence corresponding to SEQ ID NO:

54.

29. The amino acid sequence of the engineered glucocerebrosidase may comprise at amino acid position(s) 68 / 336, 65 / 99 / 319, 65 / 68 / 319, 68 / 319, 68, 65 / 319, 195, 65 / 154 / 319, 65 / 68 / 230, 65, 65 / 68 / 99 / 154 / 230 / 319, 68 / 230, 195 / 230, 65 / 230, 141 / 319, 154 / 336, 68 / 154, 68 / 195 / 230 / 319 / 336, 68 / 99 / 195 / 230 / 319, 68 / 195, 65 / 68, 68 / 141 / 230 / 319, 65 / 68 / 99 / 230 / 319, 68 / 99 / 141 / 319, 68 / 154 / 319, 141 / 154, 68 / 99, 99 / 230 / 319, 99, 230, 68 / 230 / 319, 154 / 319, 65 / 68 / 154 / 319, 141, 336, 141 / 230, 68 / 154 / 336, 65 / 68 / 99 / 319, 154, 319, 230 / 319, 68 / 154, 99 / 141 / 319, 509, 108, 108 / 117 / 509, 519, 108 / 519, 88, 117 / 413 / 50 9, 509 / 519, 413, 108 / 117 / 315, 117 / 271, 117 / 413 / 509 / 529, 413 / 509, 469, 524, 507, 525, 489, 104, 95, 359, 374, 107, 89, 260, 110, 186, 370, 191, 333, 189, 468, 473, 115, 53, 449, 518, 236, 481, 59 / 220, 258, 467, 51, 415, 103, 493, 78, 335, 520, 101, 341, 471, 211, 206, 93, 490, 71, 79, 18 0, 172, 207, 63, 142, 529, 77, 212, 474, 106, 386 / 507, 117, 373, 337, 107 / 246, 516, 233, 91, 194, 84, 361, 171, 536, 50, 116, 239, 44, 40, 113, 243, 169, 504, 505, 109, 479, 477, 97, 182, 111, 508, 314, 102, 338, 96, 138, 42, 263, 222, 522, 265, 514, 515, 246, 46, 133, 332, 506, 512, 137, 190,29. The engineered glucocerebrosidase of claim 27 or 28, comprising at least one substitution, or set of substitutions, at amino acid positions 105, 301, 329, 339, or 151, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40 to 536 of SEQ ID NO:54, or relative to a reference sequence corresponding to SEQ ID NO:

54.

30. The amino acid sequence of the engineered glucocerebrosidase may contain at least one substitution or set of substitutions: 68L / 336A, 65L / 99R / 319I, 65L / 68L / 319I, 68V / 319I, 68V, 65L / 319I, 195M, 65L / 154E / 319I, 65L / 68L / 230L, 65L, 65L / 68L / 99R / 154E / 230L / 319I, 68L / 230L, 65L / 68V / 319I, 195M / 230L, 65L / 230L, 141D / 319I, 154E / 336A, 68L / 319I, 68V / 154E, 68V / 195M / 230L / 319I / 336A, 68V / 99R / 195M / 230L / 319I, 68V / 195M, 65L / 68V, 6 8L / 141D / 230L / 319I, 65L / 68V / 99R / 230L / 319I, 68L / 99R / 141D / 319I, 68V / 1 54E / 319I, 141D / 154E, 68V / 99R, 99R / 230L / 319I, 99R, 230L, 68V / 230L / 319 I, 68L, 154E / 319I, 65L / 68L / 154E / 319I, 141D, 68V / 230L, 336A, 141D / 230L, 68V / 154E / 336A, 65L / 68L, 65L / 68V / 99R / 319I, 154E, 319I, 230L / 319I, 68L / 154E, 99R / 141D / 319I, 68L / 154E / 319I, 509F, 108I, 108I / 117I / 509F, 519 I, 108I / 519I, 88L, 117I / 413Y / 509F, 509F / 519I, 413Y, 108I / 117I / 315T, 1 17I / 271T, 117I / 413Y / 509F / 529Q, 413Y / 509F, 469E, 524K, 507M, 509I, 525Q , 489A, 104M, 507A, 95G, 359E, 374V, 509W, 107D, 89C, 260R, 110Q, 186G, 370 R, 509R, 191R, 333L, 189R, 468G, 473I, 115M, 191N, 53L, 524S, 449L, 518E, 23 6M, 481H, 115R, 59N / 220G, 489G, 258T, 467N, 51P, 415C, 103S, 493T, 489T, 5 18V, 78L, 195W, F65M, 413W, 335M, 520K, 107E, 101Q, 449K, 481K, 104V, 341R,78M、507L、449M、471L、211L、520D、10 3P、206F、93S、490S、473C、507R、211I 、71S、79R、180C、509G、206R、211N、17 2S、490R、207D、115V、63L、142A、529M、 71R, 473L, 95T, 107A, 520V, 101L, 520S, 71H, 89D, 77I, 507Y, 107C, 518S, 21 2A, 71F, 525C, 449R, 107M, 467H, 474D, 101S, 106W, 386L / 507M, 471S, 107Q, 6 8Q、79I、518L、474A、107S、117T、467M 、471R、341S、490G、471F、490T、333T、 101R、373V、101V、101W、65S、490V、33 7N、374T、68Y、95L、71G、471V、107M / 24 6R、110A、142Y、65V、71M、77G、77R、23 0Y、490Q、101T、103A、507T、471T、509 V、520R、319F、142N、79V、467L、77N、7 1L、68W、189S、359P、71Y、77T、142M、37 3A, 319V, 212N, 110H, 333C, 490I, 68F, 474S, 103L, 77H, 471E, 516L, 467R, 2 33R、91T、194L、84G、361V、233A、171V 、536L、50A、116R、239T、44V、40S、113N 、91I、99F、243L、169V、504P、505R、10 9R、536T、479A、479L、477H、84K、97N、 97K, 182M, 111H, 361Y, 508R, 314R, 336G, 102E, 338R, 505L, 9L, 96S, 138S, 5 08T, 96I, 508Q, 243M, 111L, 508S, 479R, 42A, 239I, 97I, 116E, 113I, 477V, 5 36i, 263g, 99s, 222i, 536d, 477g, 536w, 536c, 336r, 243r, 116v, 536r, 522v 102S、97R、40H、243V、265S、116Q、514 E、508L、515T、246R、361W、111F、489M 、477R、116Y、46R、133M、332R、239R、1 09G、506T、504G、233Q、96R、99G、504T、30. The engineered glucocerebrosidase of any one of claims 27-29, comprising 96G, 97M, 109L, 515G, 233G, 84A, 102N, 504W, 265R, 99V, 512L, 137V, 536A, 113R, 171P, 190W, 314G, 46M, 182H, 246K, 105T, 301K, 522T, 329L, 96V, 339A, 477S, 113G, 361L, 505I, 105M, 116M, 96M, 109M, 171C, 536M, or 151L, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:54 or are relative to a reference sequence corresponding to SEQ ID NO:

54.

31. 21. The engineered glucocerebrosidase of claim 20, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or to the reference sequence corresponding to SEQ ID NO:

220.

32. 22. The engineered glucocerebrosidase of claim 21, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 924-1020, or a reference sequence corresponding to SEQ ID NO: 924-1020, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 220, or relative to the reference sequence corresponding to SEQ ID NO:

220.

33. The amino acid sequence of the engineered glucocerebrosidase may comprise at amino acid position(s) 65 / 195 / 361, 514, 79 / 195 / 263 / 361, 65 / 374, 79 / 195, 65 / 230, 65 / 233 / 263 / 337 / 359 / 361 / 374, 79 / 536, 79 / 230 / 233 / 239 / 359 / 361 / 471, 65 / 471, 374, 65 / 195 / 230 / 233 / 337 / 374 / 514, 65 / 195 / 233 / 359 / 536, 230, 65 / 195 / 263 / 276 / 359 / 361 / 374 / 471, 79, 195 / 230 / 233 / 337 / 374 / 536, 195 / 230, 195 / 230 / 337 / 359 / 361, 230 / 263, 65 / 230 / 233, 263, 195 / 230 / 337 / 361 / 374 / 471, 65 / 195, 374 / 471, 374 / 536, 239, 65, 230 / 233 / 5 36, 230 / 263 / 374, 65 / 195 / 230 / 263 / 337 / 361 / 374 / 471, 79 / 374, 65 / 361 / 374 / 471 / 536, 195, 233, 79 / 230 / 359 / 374, 536, 230 / 359 / 361 / 374 / 514, 65 / 79 / 230, 195 / 239 / 263 / 361 / 374 / 514, 195 / 263 / 374, 195 / 374, 65 / 230 / 233 / 361 / 536, 33. The engineered glucocerebrosidase of claim 31 or 32, comprising at least one substitution, or set of substitutions, at 230 / 233 / 374 / 471, 65 / 233 / 359 / 361 / 471, 65 / 359 / 471, 65 / 79, or 65 / 359, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:

220.

34. The amino acid sequence of the engineered glucocerebrosidase contains at least one substitution or set of substitutions 65V / 195W / 361W, 514E, 79V / 195W / 263G / 361W, 65V / 374T, 79V / 195W, 65V / 230Y, 65V / 233A / 263G / 337N / 359P / 361W / 374T, 79V / 536D, 79V / 230Y / 233A / 239R / 359P / 361W / 471T, 65V / 471T, 374T, 65V / 195W / 230Y / 233A / 337N / 374 T / 514E, 65V / 195W / 233A / 359P / 536D, 230Y, 65V / 195W / 263G / 276T / 359P / 361W / 374T / 471T, 79V, 195W / 230Y / 233A / 337N / 374T / 536D, 195W / 2 30Y, 195W / 230Y / 337N / 359P / 361W, 230Y / 263G, 65V / 230Y / 233G, 263G, 195W / 230Y / 337N / 361W / 374T / 471T, 65V / 195W, 374T / 471T, 374T / 536D , 239R, 65V, 230Y / 233G / 536L, 230Y / 263G / 374T, 65V / 195W / 230Y / 263G / 337N / 361W / 374T / 471T, 79V / 374T, 65V / 361W / 374T / 471T / 536D, 195 W, 233G, 79V / 230Y / 359P / 374T, 536D, 230Y / 359P / 361W / 374T / 514E, 65V / 79V / 230Y, 195W / 239R / 263G / 361W / 374T / 514E, 195W / 263G / 374T, 1 34. The engineered glucocerebrosidase of any one of claims 31-33, comprising 95W / 374T, 65V / 230Y / 233A / 361W / 536L, 230Y / 233A / 374T / 471T, 65V / 233A / 359P / 361W / 471T, 536L, 65V / 359P / 471T, 65V / 79V, or 65V / 359P, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:220, or are relative to a reference sequence corresponding to SEQ ID NO:

220.

35. 21. The engineered glucocerebrosidase of claim 20, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO:984, or a reference sequence corresponding to SEQ ID NO:984, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO:984, or relative to the reference sequence corresponding to SEQ ID NO:

984.

36. 22. The engineered glucocerebrosidase of claim 21, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1022-1216, or a reference sequence corresponding to SEQ ID NO: 1022-1216, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 984, or relative to the reference sequence corresponding to SEQ ID NO:

984.

37. 102, 96, 105, 113, 325, 336, 262, 386, 360, 400, 342, 390, 264, 298, 382, ​​408, 449, 459, 434, 37. The engineered glucocerebrosidase of claim 35 or 36, comprising at least one substitution or set of substitutions at 476, 502, 29 / 534, 497, 485, 533, 536, 510, 532, 489, 534, 494, 182, 137, 204, 194, 202, 144, 142, 175, 220, 150, or 206, wherein said amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:984, or are relative to a reference sequence corresponding to SEQ ID NO:

984.

38. 10. The amino acid sequence of the engineered glucocerebrosidase contains at least one substitution or set of substitutions: 359P, 79V / 359P, 79V / 97R / 182M, 97R / 359P, 97R, 79V / 182M, 201T, 404Q, 484E, 201R, 446N, 494T / 495G, 495G, 412W, 385N, 385G, 100N, 184V, 385H, 82S, 75A, 71T, 41H, 97K, 99E, 97Q, 94N, 100R, 92A, 98P, 99W, 92G, 114Y, 95H, 102A, 92K, 96D, 94E, 96H, 104Y ...

38. The engineered glucocerebrosidase of any one of claims 35-37, comprising: E, 95W, 105I, 113Q, 92H, 92Q, 92E, 325V, 336E, 385Q, 262Q, 386I, 360E, 400T, 342Q, 400E, 400Q, 390A, 264I, 298W, 382A, 408D, 449E, 459M, 434G, 476A, 408E, 502K, wherein the amino acid sequence contains one or more substitutions relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO:984, or relative to a reference sequence corresponding to SEQ ID NO:

984.

39. 21. The engineered glucocerebrosidase of claim 20, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or to a reference sequence corresponding to SEQ ID NO: 1150, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150.

40. 22. The engineered glucocerebrosidase of claim 21, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1218-1554, or a reference sequence corresponding to SEQ ID NO: 1218-1554, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or relative to the reference sequence corresponding to SEQ ID NO: 1150.

41. The amino acid sequence may comprise amino acid position(s) 96 / 194 / 382 / 484, 359 / 382 / 412 / 494 / 495, 92 / 96 / 175 / 182 / 382, 96 / 359 / 382 / 484 / 494 / 495, 92 / 175 / 194 / 359 / 382, 359 / 382 / 400 / 412, 92 / 96 / 182 / 382 / 400 / 484, 92 / 96 / 382 / 494 / 495, 92 / 96 / 175 / 382 / 494 / 495, 96 / 182 / 359 / 382 / 484, 92 / 96 / 382, 92 / 382 / 494 / 495 / 534, 92 / 96 / 382 / 400 / 484, 92 / 96 / 175 / 382, 92 / 175 / 382 / 400 / 484, 92 / 96 / 175 / 182 / 382 / 412 / 484, 92 / 96 / 201 / 382 / 484, 92 / 182 / 194 / 382, 92 / 96 / 175 / 194 / 359 / 38 2 / 400, 92 / 359 / 382, 96 / 382, 92 / 175 / 382 / 484 / 494 / 495, 96 / 201 / 359 / 382, 92, 194, 201, 359, 96, 92 / 96 / 382 / 400 / 484 / 495, 382 / 495, 382, ​​92 / 382, 71 / 1 75 / 325 / 385, 325 / 385, 71 / 325 / 502, 71 / 175 / 325 / 385 / 502, 202 / 325 / 385, 97 / 175 / 385 / 510, 325 / 385 / 510, 100 / 175 / 325, 71 / 175 / 325 / 360 / 385, 97 / 17 5 / 510, 71 / 97 / 100 / 175 / 325 / 385, 71 / 175 / 510, 71 / 97 / 325 / 502, 71 / 100 / 325 / 385, 202 / 385 / 489, 325 / 385 / 502, 71 / 100 / 325 / 385 / 510, 71 / 100 / 175 / 325 / 385, 385 / 510, 97 / 325 / 385, 71, 97 / 202 / 325 / 360 / 385 / 510, 502, 71 / 97 / 103 / 325, 100 / 325 / 385 / 502 / 510, 71 / 137 / 175 / 325 / 360 / 385 / 502, 71 / 276 / 32 5 / 385 / 510, 71 / 175 / 360 / 385 / 502, 100 / 175 / 325 / 385 / 489 / 502, 71 / 175 / 360 / 385 / 485 / 502, 95 / 113, 494 / 536, 95 / 99 / 113 / 182 / 184 / 264 / 386, 184 / 536,95 / 142 / 144 / 182 / 184 / 386 / 449, 144 / 204 / 386 / 400 / 449, 113 / 449 / 536, 536, 95 / 386 / 536, 264, 99 / 113 / 386 / 449, 95 / 386, 95 / 99 / 204 / 386, 113 / 142 / 386 / 449, 182 / 184 / 264 / 494, 113 / 142 / 182 / 494, 113, 99 / 264 / 449, 95, 95 / 99, 99 / 113 / 449, 95 / 113 / 449 / 536, 95 / 99 / 113, 386 / 536, 99 / 182 / 184, 14 2 / 386 / 400 / 536, 35 / 264 / 449 / 536, 142 / 144 / 220, 99 / 142, 29 / 102 / 182 / 276 / 336 / 390 / 459 / 502 / 532, 41 / 262 / 336 / 502, 29 / 97 / 150 / 262 / 336 / 502 / 532, 29, 532, 102 / 390 / 459 / 502 / 532, 29 / 262 / 276 / 336 / 495, 29 / 182 / 262 / 459 / 532, 92 / 99 / 102 / 502, 29 / 97 / 102 / 182 / 502 / 532, 336 / 459 / 495 / 502 / 53 2, 150 / 182 / 336 / 390, 150 / 262 / 495, 29 / 495, 29 / 102 / 182 / 262 / 502, 29 / 336 / 459 / 502 / 532, 182 / 495 / 502 / 532, 102 / 336, 150 / 182 / 262 / 495, 459 / 495 / 502 / 532, 41 / 182 / 390 / 495, 29 / 41 / 182 / 336 / 495, 97 / 102 / 495, 182 / 262 / 495, 29 / 262 / 502, 29 / 102 / 502, 182 / 336 / 495, 29 / 150 / 182 / 262 / 459 / 495 , 502 / 532, 386, 497, 99, 512, 69, 507, 445, 75, 487, 184, 90, 235, 434, 489, 86, 182, 446, 490, 299, 102, 230, 68, 141, 97, 316, 522, 224, 478, 480, 368, 412, 449, 265, or 494, wherein said amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 1150.The engineered glucocerebrosidase of claim 39 or 40.

42. 10. The amino acid sequence of claim 1, wherein the amino acid sequence has at least one substitution or set of substitutions 96E / 194Q / 382A / 484E, 359P / 382A / 412W / 494T / 495G, 92Q / 96E / 175D / 182S / 382A, 96E / 359P / 382A / 484E / 494T / 495G, 92E / 175D / 194Q / 359P / 382A, 359P / 382A / 400E / 412W, 92Q / 96E / 182S / 382A / 400E / 484E, 92Q / 96E / 382A / 494T / 495G, 92Q / 96E / 175D / 382A / 494T / 495G, 96E / 182S / 359P / 382A / 484E, 92E / 96E / 382A, 92Q / 382A / 494T / 495G / 534K, 92E / 96E / 382A / 400Q / 484E, 92Q / 96E / 175D / 382A, 92Q / 175D / 382A / 400Q / 484E, 92 E / 96E / 175D / 382A / 494T / 495G, 92Q / 96E / 175D / 182S / 382A / 412W / 484E, 92Q / 96E / 201Q / 382A / 484E, 92E / 182S / 194Q / 382A, 92Q / 96E / 175D / 194Q / 359P / 3 82A / 400E, 92Q / 359P / 382A, 96E / 382A, 92Q / 175D / 382A / 484E / 494T / 495G, 9 6E / 201Q / 359P / 382A, 92E, 92Q, 194Q, 201Q, 359P, 96E, 92E / 96E / 382A / 400E / 484E / 495G, 382A / 495G, 382A, 92E / 382A, 71T / 175D / 325V / 385H, 325V / 385H , 71T / 325V / 502Q, 71T / 175D / 325V / 385H / 502Q, 202Q / 325V / 385N, 97Q / 175D / 385H / 510S, 325V / 385Q / 510S, 100R / 175D / 325V, 71T / 175D / 325V / 360E / 385 N, 97Q / 175D / 510S, 71T / 97Q / 100R / 175D / 325V / 385H, 71T / 175D / 510S, 71T / 9 7Q / 325V / 502Q, 71T / 100R / 325V / 385Q, 202Q / 385N / 489E, 325V / 385N / 502Q, 325V / 385H / 502Q, 71T / 100R / 325V / 385H / 510S, 71T / 100R / 175D / 325V / 385N,71T / 100R / 175D / 325V / 38H、381N / 510S、97Q / 3257 / 301N、71T、971 / 2021 / 3 - 510S、71T / 137Q / 175D / 325V / 360E / 385H / 502Q、71T / 276T / 325T / 385R / 510S / D / 360E9385H / 485S / 502Q、95W / 113Q、494T / 536P、95W / 99W / 111Q / 182 / 184 V / 264I / 386I、181Q / 535P、95W / 142N / 14M / 182G / 181Q / 386I / 49E112M / 2 04Q / 386I / 400T / 449E、11Q / 499E / 535P、536P、95W / 386I / 535P255I999G / The 182G / 184Q9264I / 494T、113Q / 142N1182 / / 99T、113Q99W / 261I / / 49E99W 、95W / 99W、99W / 113Q / 119E、95W / 1111 / 119G / 535、95W / 99W / 113Q、381I / 53 6P、99W / 182G9181Q、142N / 386I / 400T / 536P、35T / 264I / 449E / 535、142 / / 1 44M / 220A、99W / 142N、29I / 102A / 182M / 276T / 336E / 390A / 459M / 5025 / 2322 41H / 262Q / 336E / 502K、29I / 97R150T / 262Q / 336E / 502K / 5327 02A / 390A / 459M / 502K / 532V、29I / 262Q / 276T / 336E / 495G、29I / 182T / 262G / 459M / 532V、92K / 99G / 10A / 502K、29I / 97R / 102A / 182M / 5025 / 5325、336E / 4 59M / 495S / 502K / 532V、11T / 182T / 336E / 390A、11T / 262Q / 49G、29I / 295S29I / 102A / 182T / 262Q / 502K, 29I / 336E / 459M / 502K / 532V, 182M / 495G / 502K / 532V, 102A / 33 6E, 150T / 182M / 262Q / 495S, 459M / 495S / 502K / 532V, 41H / 182M / 390A / 495G, 29I / 41H / 182T / 3 36E / 495S, 97R / 102A / 495S, 182T / 262Q / 495S, 29I / 262Q / 502K, 29I / 102A / 502K, 182M / 336E / 495S, 502K, 29I / 150T / 182T / 262Q / 459M / 495G, 502K / 532V, 386P, 497M, 99K, 512H, 69D, 507 42. The engineered glucocerebrosidase of any one of claims 39-41, comprising: I, 445N, 75N, 75H, 512Q, 487S, 113P, 184I, 90M, 235T, 434Q, 445G, 489N, 90A, 386L, 75E, 86E, 489Q, 182E, 446H, 490N, 299V, 96N, 102P, 230F, 68T, 141E, 97E, 316H, 522S, 224M, 478N, 480G, 368D, 86H, 412N, 449S, 265N, or 494A, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 1150.

43. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

44. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises at least one substitution or set of substitutions of an engineered glucocerebrosidase set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1, and wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

45. and a reference sequence containing one substitution or set of substitutions of an engineered glucocerebrosidase as set forth in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, 6.2, 12.1, 12.2, and 13.1 that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1301, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1401, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1501, 1510, 1520, 1530, 1540, 1550, 1560, 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence having 6%, 97%, 98%, 99% or more sequence identity, wherein the amino acid positions are relative to a reference sequence corresponding to residues 40-536 of SEQ ID NO: 54, 220, 984, or 1150, or are relative to a reference sequence corresponding to SEQ ID NO: 54, 220, or 984, or 1150.

46. 2. The engineered glucocerebrosidase of claim 1, comprising an amino acid sequence comprising residues 40-536 of an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, and 6.2, or an amino acid sequence comprising an engineered glucocerebrosidase described in Tables 3.1, 3.2, 4.1, 4.2, 4.3, 4.4, 5.1, 6.1, and 6.

2.

47. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises residues 40-536 of an even-numbered SEQ ID NO:4-1648, or comprises an even-numbered SEQ ID NO:4-1648, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

48. 2. The engineered glucocerebrosidase of claim 1, wherein the amino acid sequence of the engineered glucocerebrosidase comprises residues 40 to 536 of SEQ ID NO: 54, 220, 984, or 1150, or comprises SEQ ID NO: 54, 220, 984, or 1150, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

49. 49. The engineered glucocerebrosidase of any one of claims 1 to 48, wherein the engineered glucocerebrosidase has glucocerebrosidase and one or more improved properties.

50. 50. The engineered glucocerebrosidase of any one of claims 1-49, wherein the engineered glucocerebrosidase has glucocerebrosidase activity and improved properties selected from: i) increased enzymatic activity; ii) increased expression or expression efficiency; iii) increased stability at neutral or basic pH; iv) increased stability at acidic pH; v) increased stability in serum or plasma; vi) increased thermal stability; vii) increased uptake and / or intracellular stability in cells such as macrophages; and viii) reduced immunogenicity, or any combination of i), ii), iii), iv), v), vi), vii), and vii), and viii), compared to a reference glucocerebrosidase having a sequence corresponding to residues 40-536 of SEQ ID NO: 2, 54, 220, 984, or 1150, or a sequence corresponding to SEQ ID NO: 2, 54, 220, 984, or 1150.

51. 51. The engineered glucocerebrosidase of claim 49 or 50, wherein the reference glucocerebrosidase has a sequence corresponding to residues 40 to 536 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:

2.

52. 52. The engineered glucocerebrosidase of claims 1-51, wherein the engineered glucocerebrosidase is a purified preparation.

53. 52. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered glucocerebrosidase of any one of claims 1 to 51.

54. 53. The recombinant polynucleotide of claim 52, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 118-1608 of SEQ ID NO: 53, 219, 983, or 1149, or a reference polynucleotide sequence corresponding to SEQ ID NO: 53, 219, 983, or 1149, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

55. 53. The recombinant polynucleotide of claim 52, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 118-1608 of an odd-numbered SEQ ID NO:3-1647, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:3-1647, wherein the recombinant polynucleotide encodes a glucocerebrosidase.

56. 56. The recombinant polynucleotide of any one of claims 53 to 55, wherein the polynucleotide sequence is codon-optimized for expression of the encoded glucocerebrosidase.

57. 54. The recombinant polynucleotide of claim 53, comprising a polynucleotide sequence comprising residues 118 to 1608 of an odd-numbered SEQ ID NO: 3-1647, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 3-1647.

58. 54. The recombinant polynucleotide of claim 53, comprising a polynucleotide sequence comprising residues 118 to 1608 of, or comprising, SEQ ID NO: 53, 219, 983, or 1149.

59. An expression vector comprising the recombinant polynucleotide of any one of claims 53 to 58.

60. 60. The expression vector of claim 59, wherein the recombinant polynucleotide is operably linked to a control sequence.

61. 61. The expression vector of claim 60, wherein the control sequence comprises at least a promoter.

62. 62. The expression vector of any one of claims 59 to 61, wherein the expression vector comprises a viral vector.

63. A host cell comprising the expression vector of any one of claims 59 to 62.

64. 64. The host cell of claim 63, comprising a mammalian cell.

65. 65. The host cell of claim 64, comprising a human cell.

66. 66. A method for producing an engineered glucocerebrosidase in a host cell, the method comprising culturing the host cell of any one of claims 63 to 65 under suitable culture conditions such that the engineered glucocerebrosidase is expressed.

67. 67. The method of claim 66, further comprising recovering the engineered glucocerebrosidase from the culture and / or host cell.

68. 68. The method of claim 66 or 67, further comprising purifying the engineered glucocerebrosidase.

69. 62. A pharmaceutical composition comprising the engineered glucocerebrosidase of any one of claims 1 to 52, the recombinant polynucleotide of any one of claims 53 to 58, or the expression vector of any one of claims 59 to 62.

70. 70. The pharmaceutical composition of claim 69, further comprising a pharmaceutically acceptable carrier and / or excipient.

71. 71. The pharmaceutical composition of claim 69 or 70, wherein the composition is suitable for parenteral injection or infusion into humans.

72. A gene therapy composition comprising the recombinant polynucleotide of any one of claims 53 to 58.

73. 73. The gene therapy composition of claim 72, wherein the gene therapy composition comprises a viral vector containing the recombinant polynucleotide.

74. 74. The gene therapy composition of claim 73, wherein the viral vector is an adenovirus, adeno-associated virus, lentivirus, retrovirus, or herpesvirus vector.

75. 73. The gene therapy composition of claim 72, wherein the recombinant polynucleotide is formulated in a liposome, cationic polymer, dendrimer, or conjugated to a cell-penetrating peptide.

76. 10. A method for treating and / or preventing symptoms of a deficiency in glucocerebrosidase activity in a subject, the method comprising administering to a subject having a deficiency in glucocerebrosidase activity an effective amount of an engineered glucocerebrosidase of any one of claims 1-52, a pharmaceutical composition of any one of claims 69-71, or a gene therapy composition of any one of claims 72-75.

77. 77. The method of claim 76, wherein the subject has Gaucher disease.

78. 78. The method of claim 76 or 77, wherein the symptoms of a deficiency in glucocerebrosidase activity are ameliorated.

79. 10. Use of an engineered glucocerebrosidase according to any one of claims 1 to 52, a pharmaceutical composition according to any one of claims 69 to 71, or a gene therapy composition according to any one of claims 72 to 75 for the treatment of a deficiency in glucocerebrosidase activity in a subject.

80. 8. Use of the engineered glucocerebrosidase of any one of claims 1 to 52 or the gene therapy composition of any one of claims 72 to 75 in the preparation of a medicament for treating a deficiency in glucocerebrosidase activity in a subject.

81. 81. The use of claim 79 or 80, wherein the deficiency in glucocerebrosidase activity is Gaucher disease.