Engineered lipase variants

Abstract

To provide engineered lipase polypeptides and compositions thereof.SOLUTION: Engineered lipase polypeptides according to the invention have been optimized to provide improved thermostability, protease stability, and stability under a wide range of pH conditions, including acidic (pH<7) and basic (pH>7) conditions. The invention also relates to use of compositions comprising the engineered lipase polypeptides for therapeutic and / or nutritional purposes. Further provided are polynucleotides encoding the engineered lipase polypeptides, as well as methods for producing the engineered polynucleotides and lipase polypeptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 894,019, filed August 30, 2019, the entire contents of which are incorporated herein by reference for all purposes.

[0002] FIELD OF THE INVENTION The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides are optimized to provide improved thermostability, protease stability, and stability under a wide range of pH conditions, including acidic (pH<7) conditions. The present invention also relates to the use of compositions comprising the engineered lipase polypeptides for therapeutic and / or nutritional purposes. The present invention also provides polynucleotides encoding the engineered lipase polypeptides, as well as methods for making the engineered polynucleotides and lipase polypeptides.

[0003] Reference to a sequence listing, table or computer program An official copy of the Sequence Listing is submitted contemporaneously herewith as an ASCII text file via EFS-Web with the filename "CX7-187WO2_ST25.txt," a creation date of August 27, 2020, and a size of 4.30 megabytes. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Pancreatic enzyme replacement therapy (PERT) has found use in the treatment of pancreatic enzyme insufficiency (PEI). Various disorders, including pancreatitis, cystic fibrosis, celiac disease, inflammatory bowel disease, and pancreatic cancer, can result in PEI as a result of reduced secretion of pancreatic enzymes into the duodenum. This can lead to poor food digestion and inadequate intestinal absorption of fat, protein, carbohydrates, and vitamins, leading to malnutrition. Orally administered PERT treatments are currently available, but some individuals may not experience relief due to insufficient activity of PERT in the gastrointestinal tract and / or poor patient compliance due to the significant medication burden associated with current treatment protocols. In some cases, the coefficient of fat absorption (CFA) and / or coefficient of nitrogen absorption (CNA) are inferior to those of healthy patients, leading to weight loss and other health concerns. Therefore, there remains a need in the art for improved PERT treatments.

[0005] Summary of the Invention The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides are optimized to provide improved thermostability, protease stability, and stability under a wide range of pH conditions, including acidic (pH<7) conditions. The present invention also relates to the use of compositions comprising the engineered lipase polypeptides for therapeutic and / or nutritional purposes. The present invention also provides polynucleotides encoding the engineered lipase polypeptides, as well as methods for making the engineered polynucleotides and lipase polypeptides.

[0006] The present invention provides recombinant lipases and / or biologically active recombinant lipase fragments comprising an amino acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant lipase and / or biologically active recombinant lipase fragment comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some additional embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 2, 3, 4, 22, 27 / 46 / 97 / 136 / 149 / 385, 27 / 46 / 385, 27 / 70 / 136 / 231 / 385, 27 / 136 / 323 / 385, 27 / 149, 27 / 149 / 385, 27 / 385, 34, 38, 41, 44, 46 / 70, 46 / 149 / 183 / 231 / 385, 48, 70, 73, 73 / 305, 82, 82 / 94 / 101 / 194 / 199, 82 / 94 / 199, 83, 85, 87, 89, 92, 94, 96, 97 / 149 / 385, 135, 136 / 385, 140, 141, 142, 144, 146, 149, 149 / 231 / 385, 151, 174, 175, 178, 181, 183 / 385, 189, 194, 194 / 199, 195, 195 / 231 / 385, 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 219, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379,and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some further embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, wherein the recombinant lipase is selected from the group consisting of 2H, 2M, 2T, 3A, 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97A / 136V / 149E / 385P, 27V / 46T / 385P, 27V / 70 N / 136V / 231E / 385P, 27V / 136V / 323I / 385P, 27V / 149E, 27V / 149E / 385P, 27V / 385P, 34D, 38A, 38H, 38L, 41V, 44E, 46T / 70N, 46T / 149E / 183L / 2 31E / 385P, 48V, 70N, 73C / 305I, 73I, 73R, 82C, 82E, 82E / 94S / 101S / 194N / 199L, 82E / 94S / 199L, 82F, 82G, 82L, 83G, 83K, 85I, 85W, 87R, 89A, 8 9T, 89V, 89W, 92A, 94S, 96E, 96N, 96S, 97A / 149E / 385P, 135G, 135Q, 135S, 135T, 135V, 136V / 385P, 140T, 141A, 141F, 141L, 141S, 142A, 142F, 142I, 142L, 144M, 144R, 144W, 146A, 146F, 146S, 146Y, 149E, 149E / 231E / 385P, 149K, 149R, 151A, 151I, 151L, 174H, 174R, 175G, 175L, 175R, 178R, 178T, 181H, 183L / 385P, 189A, 189E, 189W, 194N, 194N / 199L, 194T, 195D / 231E / 385P, 195V, 199L, 199L / 213A, 210A, 210V, 212C, 212G, 212R, 212T, 213A / 330T, 213H, 216R, 216T, 216W, 218D, 218G, 218I, 218M, 218T, 219C, 219G, 219K, 219R, 226R, 231E / 385P, 238S, 247R, 250T,and at least one substitution or set of substitutions selected from 270V, 274D, 281K, 281R, 292A, 292C, 292L, 292V, 296M, 296R, 300A, 300D, 300T, 308A, 330Y, 338N, 379T, 385A, 385C, 385D, 385P, 385R, and 385T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of E2H, E2M, E2T, T3A, T3G, T3R, T3S, S4M, S4W, S4Y, G22N, Y27V / R46T / Y97A / Y136V / T149E / F385P, Y27V / R46T / F385P, Y27V / K70N / Y136V / Q231E / F385P, Y27V / Y136V / K323I / F385P, Y27V / T149E, Y 27V / T149E / F385P, Y27V / F385P, K34D, F38A, F38H, F38L, N41V, G44E, R46T / K70N, R46T / T149E / F183L / Q231E / F385P, Y48V, K 70N, T73C / V305I, T73I, T73R, K82C, K82E, K82E / P94S / D101S / K194N / I199L, K82E / P94S / I199L, K82F, K82G, K82L, E83G, E83 K, G85I, G85W, A87R, F89A, F89T, F89V, F89W, T92A, P94S, I96E, I96N, I96S, Y97A / T149E / F385P, Y135G, Y135Q, Y135S, Y135T , Y135V, Y136V / F385P, P140T, E141A, E141F, E141L, E141S, E142A, E142F, E142I, E142L, I144M, I144R, I144W, P146A, P146F , P146S, P146Y, T149E, T149E / Q231E / F385P, T149K, T149R, G151A, G151I, G151L, E174H, E174R, Q175G, Q175L, Q175R, S178R,S178T, K181H, F183L / F385P, S189A, S189E, S189W, K194N, K194N / I199L , K194T, Q195D / Q231E / F385P, Q195V, I199L, I199L / P213A, K210A, K210 V, Q212C, Q212G, Q212R, Q212T, P213A / N330T, P213H, S216R, S216T, S21 6W, H218D, H218G, H218I, H218M, H218T, A219C, A219G, A219K, A219R, T2 26R, Q231E / F385P, Y238S, E247R, Q250T, R270V, T274D, G281K, G281R, M292A, M292C, M292L, M292V, S296M, S296R, Q300A, Q300D, Q300T, R308A, N330Y, K338N, N379T, F385A, F385C, F385D, F385P, F385R, and F385T, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.

[0007] In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 231 / 296 / 300, 4 / 96 / 149 / 292 / 296 / 300, 4 / 96 / 219 / 296, 4 / 96 / 231 / 296 / 300, 4 / 96 / 292 / 296 / 300, 4 / 149 / 174, 4 / 174 / 219 / 292 / 296, 4 / 231 / 296 / 300, 27, 27 / 34 / 82, 27 / 73 / 82 / 218, 27 / 89, 27 / 89 / 178, 27 / 89 / 218, 27 / 218, 34 / 73 / 218, 34 / 82, 34 / 218, 73 / 82, 73 / 82 / 183, 94 / 146 / 175, 96 / 149 / 174 / 292 / 296, 96 / 149 / 231 / 292 / 296, 96 / 149 / 292 / 296, 96 / 174 / 219 / 231 / 292 / 296, 96 / 231 / 296, 146 / 175 / 189 / 281, 149 / 174 / 292 / 296, 149 / 174 / 300, 149 / 219 / 231 / 292 / 296 / 300, 149 / 231 / 292 / 296, 149 / 231 / 292 / and 296 / 300, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 3S / 4W / 96E / 296R / 300D, ...E / 296R / 300D, 3S / 4W / 96E / 296E / 296R / 300D, 3S / 4W / 96E / 296E / 296R / 300D, 3S / 4W / 96E / 296E / 296R / 300D, 3S / 4W / 96E / 296E / 296R / 300D, 3S / 4W / 96E / 296E / 296E / 23S / 292L / 296R, 4W, 4W / 11A / 149E / 292L / 296R / 300D, 4W / 96E / 149E / 231E / 296R / 300D, 4W / 96E / 149E / 292L / 296R / 300D, 4W / 96E / 219K / 296R, 4W / 96E / 231E / 296R / 300D, 4W / 96E / 292L / 296R / 300D, 4W / 149E / 174R, 4W / 174H / 219K / 292L / 296R, 4W / 231E / 296R / 300D, 27V, 27V / 34D / 82E, 27V / 73I / 82L / 218I, 27V / 73I / 82L / 218M, 27V / 89T, 27V / 89T / 178P, 27V / 89T / 218I, 27V / 218I, 34D / 73I / 218I, 34D / 82C, 34D / 218M, 73I / 82C, 73I / 82L / 183L, 94S / 146F / 175G, 96E / 149E / 174H / 292L / 296R, 96E / 149E / 231E / 292L / 296R, 96E / 149E / 292L / 296R, 96E / 174H / 219K / 231E / 292L / 296R, 96E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174H / 292L / 2 96R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 231E / 292L / 296R, 149E / 231E / 292L / 296R / 300D, 149E / 296R , 149E / 296R / 300D, 174H / 296R, 174R / 231E, 218I, 231E, 231E / 292L / 296R, 231E / 292L / 296R / 300D, 231E / 296R, 231E / 300D, 292L / 296R, 292L / 296R / 300D, 296R, and 296R / 300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of T3S / S4W / I96E / S296R / Q300D, ...T3S / M292L / S296R, S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S296R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S29 6R / Q300D, S4W / T149E / E174R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73 I / K82L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H2 18I, K34D / T73I / H218I, K34D / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F 183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / Q231E / M292L / S296R, I96E / T149E / M292L / S296R, I96E / E174H / A219K / Q23 1E / M292L / S296R, I96E / Q231E / S296R, P146F / Q175G / S189A / G281K, T149E / E174H / M292L / S296R, T149E / E174H / Q300D, T149E / A219K / Q231E / M292L / S2 96R / Q300D, T149E / Q231E / M292L / S296R, T149E / Q231E / M292L / S296R / Q300 and at least one substitution or set of substitutions selected from: D, T149E / S296R, T149E / S296R / Q300D, E174H / S296R, E174R / Q231E, H218I, Q231E, Q231E / M292L / S296R, Q231E / M292L / S296R / Q300D, Q231E / S296R, Q231E / Q300D, M292L / S296R, M292L / S296R / Q300D, S296R, and S296R / Q300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94.

[0008] In some additional embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350 or a functional fragment thereof, and the recombinant lipase is , 4 / 137, 4 / 175 / 189 / 218, 4 / 175 / 189 / 300, 4 / 175 / 218 / 224, 4 / 175 / 218 / 373, 4 / 185, 4 / 189 / 218 / 373, 4 / 193, 4 / 218 / 300 / 369 / 373, 4 / 228, 4 / 233, 4 / 243, 4 / 271, 4 / 303, 4 / 334, 4 / 336, 4 / 375, 25, 27 / 82 / 174 / 175 / 189 / 218 / 300 / 369 / 373, 27 / 82 / 174 / 175 / 218 / 300 / 369, 27 / 82 / 174 / 175 / 218 / 300 / 373 / 382, 27 / 82 / 174 / 218 / 300 / 373, 27 / 189 / 218 / 373, 28, 33, 99, 102, 104, 134, 153, 174 / 175 / 189, 174 / 373, 175 / 189 / 218 / 300 / 373, and at least one substitution or set of substitutions at one or more positions selected from 175 / 189 / 373, 175 / 218, 175 / 218 / 382, 189 / 218 / 300 / 373, 191, 193, 231, 233, 243, 293, 303, 331, 336, 339, 368, and 373, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 189A / 218M ... , 4W / 175G / 189A / 300D, 4W / 175G / 218M / 224A, 4W / 175G / 218M / 373F, 4W / 185L, 4W / 189A / 218M / 373F, 4W / 193T, 4W / 218M / 300D / 369 N / 373F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 175G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 382M, 27V / 82C / 174H / 2 18M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218M / 3 and 373F, 175G / 218M, 175G / 218M / 382M, 175L / 189A / 373F, 189A / 218M / 300D / 373F, 191L, 193L, 231Q, 233G, 233S, 233W, 243R, 293R, 303P, 331R, 336R, 339K, 368A, 368T, and 373F, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S189A / Q3 00D, S4W / Q175G / H218M / V224A, S4W / Q175G / H218M / Y373F, S4W / I185L, S4W / S189A / H218M / Y373F, S4W / Q193T, S4W / H218M / Q300D / S369N / Y373F, S4W / P228E, S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S4W / A 336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218 M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S369N, Y 27V / K82C / E174H / Q175G / H218M / Q300D / Y373F / H382M, Y27V / K82C / E174H / H218M / Q300D / Y373F, Y27V / S189A / H218M / Y373F, R28N, L33Y, Q99D, E102L , N104H, E134R, N153G, E174H / Q175L / S189A, E174H / Y373F, Q175G / S189A / H and at least one substitution or set of substitutions selected from 218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T, and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.

[0009] In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, and the recombinant lipase comprises 6, 33, 33 / 174 / 193 / 243, 33 / 174 / 334, 33 / 175 / 218 / 303, 33 / 175 / 218 / 334 / 339, 33 / 193, 34, 38, 46, 48, 70 / 271 / 293 / 334, 144, 174, 174 / 175 / 193, 174 / 175 / 218 / 339, 174 / 193 / 303 / 375, 17 4 / 193 / 375, 174 / 218 / 233 / 271 / 293 / 303, 174 / 218 / 271 / 303, 175 / 193 / 218 / 233 / 243 / 375, 175 / 193 / 218 / 243, 175 / 218 / 375, 181, 189, 193, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 303 , 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281, and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 26A, 26R, 26S, 33Y, 33Y / 174H / 193L / 243R ... 3Y / 174H / 334T, 33Y / 175G / 218M / 303P, 33Y / 175G / 218M / 334T / 339K, 33Y / 193L, 34L, 38A, 38G, 46P, 48L, 70H / 271D / 293R / 334T, 144L, 174H, 174H / 175G / 193L, 174H / 175G / 218M / 339K, 174H / 193L / 303P / 375A, 174H / 193L / 375A, 174 H / 218M / 233R / 271D / 293R / 303P, 174H / 218M / 271D / 303P, 174R, 175G / 193L / 218M / 233R / 243R / 375A, 175G / 193L / 2 18M / 243R, 175G / 218M / 375A, 181Q, 189H, 193L, 193L / 218M / 243R, 193L / 271D / 303P / 334T, 193L / 293R / 303P, 194T, and at least one substitution or set of substitutions selected from 195I, 195L, 195Y, 199H, 210V, 218C, 218D, 218M, 218M / 243R, 218M / 243R / 303P / 334T, 218M / 303P, 218P, 225L, 238W, 274D, 281K, 281P, 330F, and 330H, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0010] In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / L243R, L33Y / E174H / D334T ... Y / L175G / H218M / A303P, L33Y / L175G / H218M / D334T / Q339K, L33Y / Q193L, K34L, F38A, F38G, R46P, Y48L, K70H / G271D / Q293R / D334T , I144L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E174H / H218 M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N375A, L175G / Q193L / H21 8M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303P / D334T, Q193L / Q293R / A303P, K1 and at least one substitution or set of substitutions selected from: 94T, Q195I, Q195L, Q195Y, I199H, K210V, H218C, H218D, H218M, H218M / L243R, H218M / L243R / A303P / D334T, H218M / A303P, H218P, M225L, Y238W, T274D, G281K, G281P, N330F, and N330H, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0011] In some additional embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174, 34 / 174 / 193 / 195 / 243 / 281, ... 81, 34 / 174 / 193 / 195 / 303 / 330, 34 / 193 / 195 / 225 / 243, 34 / 193 / 243 / 303 / 330, 34 / 281 / 330, 38, 38 / 174 / 193 / 195 / 243 / 330, 38 / 174 / 193 / 225 / 274 / 283 / 303 / 330, 38 / 174 / 193 / 303, 38 / 174 / 195 / 281 / 330, 38 / 174 / 225 / 243 / 281 / 330, 38 / 174 / 281, 38 / 174 / 281 / 303, 38 / 174 / 281 / 303 / 345, 38 / 193 / 195 / 22 5 / 303 / 345, 38 / 195 / 243 / 303, 38 / 195 / 281 / 303 / 330, 38 / 195 / 281 / 330, 38 / 195 / 303, 49, 49 / 51 / 98 / 252 / 311, 49 / 51 / 123 / 252 / 344, 49 / 98 / 120 / 252 / 3 44, 49 / 120 / 252 / 344, 49 / 123 / 252 / 344, 49 / 123 / 264 / 344, 49 / 123 / 311, 49 / 252 / 344, 49 / 311 / 344, 49 / 344, 51, 51 / 252 / 344, 51 / 344, 98, 98 / 344, 123 / 2 and at least one substitution or set of substitutions at one or more positions selected from: 52 / 344, 129, 160, 161, 174 / 193 / 195 / 225, 174 / 193 / 303 / 330, 174 / 195 / 225 / 281 / 303 / 330 / 345, 174 / 195 / 243 / 281 / 345, 174 / 195 / 281 / 303, 174 / 225 / 243 / 281 / 303 / 345, 174 / 281 / 330, 174 / 303, 195 / 225 / 303 / 330, 252, 268, and 344, wherein the amino acid positions of the polypeptide sequence are:The recombinant lipase is numbered with reference to SEQ ID NO: 540. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38A ... 4L / 174G, 34L / 174G / 193L / 195L / 243R / 281P, 34L / 174H / 193L / 195I / 303P / 330F, 34L / 193L / 195I / 225L / 243R, 34L / 193L / 243R / 303P / 330F, 34L / 28 1P / 330H, 38A / 174H / 193L / 225L / 274D / 283I / 303P / 330F, 38A / 174R / 281K / 303P / 345I, 38A / 193L / 195L / 225L / 303P / 345I, 38A / 195I / 303P, 38A / 195 Y / 281K / 303P / 330F, 38G, 38G / 174G / 193L / 303P, 38G / 174G / 281K / 303P, 3 8G / 174H / 195L / 281P / 330F, 38G / 174H / 225L / 243R / 281K / 330H, 38G / 174H / 281K, 38G / 174R / 193L / 195L / 243R / 330H, 38G / 195I / 281P / 330F, 38G / 19 5Y / 243R / 303P, 49S, 49T, 49T / 51A / 98P / 252V / 311W, 49T / 51A / 123Q / 252V / 344H, 49T / 98P / 120T / 252V / 344H, 49T / 120T / 252V / 344H, 49T / 123Q / 252V / 344H, 49T / 123Q / 264S / 344H, 49T / 123Q / 311W, 49T / 252V / 344H, 49T / 311 W / 344H, 49T / 344H, 51A / 252V / 344H, 51A / 344H, 51V, 98P / 344H, 98R, 123Q / 252V / 344H, 129F, 160T, 161I, 174G / 195I / 225L / 281K / 303P / 330F / 345I,and at least one substitution or set of substitutions selected from 174G / 225L / 243R / 281P / 303P / 345I, 174H / 195I / 243R / 281K / 345I, 174H / 281P / 330H, 174R / 193L / 195I / 225L, 174R / 193L / 303P / 330H, 174R / 195I / 281K / 303P, 174R / 303P, 195Y / 225L / 303P / 330H, 252V, 268T, 344I, 344V, and 344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:540. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38A / E174R / A303P / F345I, K34L / F38G / E174R / M225L / A303P, K34L / E174G ...G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38A / E174G / Q19 <h2 style=";text-align:left;direction:ltr">43R / G281P, K34L / E174H / Q193L / Q195I / A303P / N330F, K34L / Q193L / Q195I / M225L / L243R, K34L / Q193L / L243R / A303P / N330F, K34L / G281P / N330H, F38A / E174H / Q193L / M225L / T274D / M283I / A303P / N330F、F38A / E174R / G281K / A303P / F345I、F38A / Q193L / Q195L / M225L / A303P / F345I、F38A / Q195I / A303P、F 38A / Q195Y / G281K / A303P / N330F、F38G、F38G / E174G / Q193L / A303P、F38G / E 174G / G281K / A303P、F38G / E174H / Q195L / G281P / N330F、F38G / E174H / M225L / L243R / G281K / N330H,F38G / E174H / G281K,F38G / E174R,Q193L / Q195L,L243R / N330H,F38G / Q195I / G281P / N330F,F38G / Q195Y / L243R / A303P,V49S,V49 T、V49T / T51A / G98P / M252V / L311W、V49T / T51A / E123Q / M252V / S344H、V49T / G98P / M120T / M252V / S344H、V49T / M120T / M252V / S344H、V49T / E123Q / M252V / S344H、V49T / E123Q / T264S / S344H、V49T / E123Q / L311W、V49T / M252V / S344 H、V49T / L311W / S344H、V49T / S344H、T51A / M252V / S344H、T51A / S344H、T51V、 G98P / S344H、G98R、E123Q / M252V / S344H、S129F、S160T、L161I、E174G / Q195 I / M225L / G281K / A303P / N330F / F345I、E174G / M225L / L243R / G281P / A303P / F 345I, E174H / Q195I / L243R / G281K / F345I, E174H / G281P / N330H, E174R / Q193L / Q195I / M225L, E174R / Q193L / A303P / N330H, E174R / Q195I / G281K / A303Pand at least one substitution or set of substitutions selected from E174R / A303P, Q195Y / M225L / A303P / N330H, M252V, S268T, S344I, S344V, and S344W, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 540.

[0012] In some additional embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 237 / 287 / 291, 1 6 / 168 / 177, 24, 24 / 278, 34 / 49 / 161 / 193 / 243 / 344, 34 / 49 / 161 / 193 / 344, 34 / 49 / 161 / 243 / 344, 34 / 49 / 161 / 252 / 268 / 344, 34 / 49 / 193 / 243 / 252 / 344, 34 / 49 / 193 / 344, 34 / 49 / 243 / 252, 34 / 49 / 252 / 268 / 344, 34 / 161 / 193 / 243 / 252 / 268, 34 / 161 / 193 / 243 / 268 / 344, 34 / 161 / 193 / 243 / 344, 34 / 161 / 193 / 252 / 268 / 344, 34 / 161 / 193 / 252 / 344, 34 / 161 / 193 / 268 / 344, 34 / 161 / 243 / 281 / 344, 34 / 161 / 344, 34 / 193 / 243 / 252 / 344, 34 / 193 / 252 / 268 / 344, 34 / 193 / 268 / 281, 34 / 252 / 268 / 281 / 344, 34 / 252 / 344, 49 / 161 / 193 / 243 / 252 / 344, 49 / 161 / 193 / 252 / 281 / 344, 49 / 161 / 243 / 252 / 344, 49 / 193 / 197 / 243 / 252 / 281 / 3 44, 49 / 193 / 243 / 252 / 344, 49 / 193 / 243 / 344, 49 / 243 / 344, 67 / 154 / 237 / 287, 67 / 168 / 237, 67 / 168 / 237 / 287, 67 / 177 / 237, 154 / 237 / 286 / 287, 154 / 286 / 287, 161 / 193 / 252 / 344, 161 / 193 / 344, 161 / 344, 168 / 237, 186 / 187 / 278, 193 / 243 / 281 / 344, 193 / 252 / 268 / 344, 193 / 252 / 281 / 344, 237, 237 / 287 / 291,and 281 / 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646, wherein the recombinant lipase is selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 287L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 16F / 67A / 237Q / 287L / 291A, 16F / 168T / 177L, 24M, 24M / 278L, 34K / 49T / 161I / 193L / 243R / 344H, 34K / 49T / 161I / 193L / 243R / 344V, 34K / 49T / 161I / 193L / 344W, 34K / 49T / 161I / 243R / 344H, 34K / 49T / 161I / 252V / 268T / 344V, 34K / 49T / 193L / 2 43R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 2 52V / 268T, 34K / 161I / 193L / 243R / 268T / 344V, 34K / 161I / 193L / 243R / 344W, 34K / 161I / 193L / 252V / 268T / 344H, 34K / 161I / 193L / 252V / 26 8T / 344V, 34K / 161I / 193L / 252V / 344H, 34K / 161I / 193L / 252V / 344V, 34K / 161I / 193L / 252V / 344W, 34K / 161I / 193L / 268T / 344H, 34K / 161 I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,34K / 252V / 268T / 281P / 344V, 34K / 252V / 344V, 49T / 161I / 193L / 243R / 252V / 344W, 49T / 161I / 193L / 252V / 281P / 344H, 49T / 161I / 243R / 252V / 344V, 49T / 193L / 197G / 243R / 252V / 281P / 344H, 49T / 193L / 243R / 252V / 344V, 49T / 193L / 243R / 344W, 49T / 243R / 344V, 67A / 154Y / 237Q / 287L, 67A / 168T / 237Q, 67A / 168T / 237Q / 287L, 67A / 17 and at least one substitution or set of substitutions selected from 7L / 237Q, 154Y / 237Q / 286V / 287L, 154Y / 286V / 287L, 161I / 193L / 252V / 344V, 161I / 193L / 344W, 161I / 344W, 168T / 237Q, 186T / 187A / 278L, 193L / 243R / 281P / 344W, 193L / 252V / 268T / 344V, 193L / 252V / 281P / 344H, 237Q, 237Q / 287L / 291A, and 281P / 344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:646. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, V11I / Y67A / S168T / F287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y67A / A237, <h2 style=";text-align:left;direction:ltr">Q / F287L / S291A、L16F / S168T / V177L、F24M、F24M / Y278L、L34K / V49T / L161I / Q193L / L243R / S344H、L34K / V49T / L161I / Q193L / L243R / S344V、L34K / V49T / L161I / Q193L / S344W、L34K / V49T / L161I / L243R / S344H、L34K / V49T / L161I / M252V / S268T / S344V、L34K / V49T / Q193L / L243R / M252V / S344W、L34K / V49T / Q 193L / S344H, L34K / V49T / Q193L / S344W, L34K / V49T / L243R / M252V, L34K / V49T / M252V / S268T / S344V, L34K / L161I / Q193L / L243R / M252V / S268T, L34K / L1 61I / Q193L / L243R / S268T / S344V、L34K / L161I / Q193L / L243R / S344W、L34K / L161I / Q193L / M252V / S268T / S344H、L34K / L161I / Q193L / M252V / S268T / S344 V、L34K / L161I / Q193L / M252V / S344H、L34K / L161I / Q193L / M252V / S344V、L3 4K / L161I / Q193L / M252V / S344W、L34K / L161I / Q193L / S268T / S344H、L34K / L1 61I / L243R / G281P / S344V、L34K / L161I / S344V、L34K / L161I / S344W、L34K / Q193L / L243R / M252V / S344W、L34K / Q193L / M252V / S268T / S344H、L34K / Q193L / S268T / G281P、L34K / M252V / S268T / G281P / S344V、L34K / M252V / S344V、V49T / L161I / Q193L / L243R / M252V / S344W、V49T / L161I / Q193L / M252V / G281P / S34 4H、V49T / L161I / L243R / M252V / S344V、V49T / Q193L / S197G / L243R / M252V / G 281P / S344H、V49T / Q193L / L243R / M252V / S344V、V49T / Q193L / L243R / S344W、V49T / L243R / S344V, Y67A / W154Y / A237Q / F287L, Y67A / S168T / A237Q, Y67A / S168T / A237Q / F287L, Y67A / V177L / A237Q, W1 54Y / A237Q / L286V / F287L, W154Y / L286V / F287L, L161I / Q193L / M252V / S344V, L161I / Q193L / S344W, L161I / S344W, S168T / and at least one substitution or set of substitutions selected from A237Q, H186T / L187A / Y278L, Q193L / L243R / G281P / S344W, Q193L / M252V / S268T / S344V, Q193L / M252V / G281P / S344H, A237Q, A237Q / F287L / S291A, and G281P / S344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:758 or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24M / 168T / 252V / 281P, 24M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S and 281P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 758. In some further embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758, wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from F24M / S168T / M252V / G281P, F24M / A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S, and G281P, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758.

[0013] In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / 252 / 260 and 385, 38, 49, 70, 149, 161, 174, 175, 189, 193, 225, 231, 243, 252, 271, 287, 292, 293, 296, 303, 334, 344, 373, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70K / 149T / 161L / 174E / 175Q / 189S / 193Q / 225M / 231Q / 243L / 252M / 271G / 287F / 292M / 293Q / 296S / 303A / 334D / 344S / 373Y / 385F, 24G, 24G, 24F ... H, 24I, 24K, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24W, 24Y, 38A, 38C, 38D, 38E, 38F, 38H , 38I, 38K, 38L, 38M, 38N, 38P, 38Q, 38R, 38S, 38T, 38V, 38W, 38Y, 49A, 49C, 49D, 49E, 49F, 49G, 49H, 49I, 49K, 49L, 49M, 49N, 49P, 49Q, 49R, 49S, 49V, 49W, 49Y, 70A, 70C, 70D, 70E, 7 0F, 70G, 70I, 70K, 70L, 70M, 70N, 70P, 70Q, 70R, 70S, 70T, 70V, 70W, 70Y, 149A, 149C, 149D,149F、149G、149H、149I、149K、149L、149M、149N、149P、149Q、149R、149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M、271N、271P、271Q、271R, 271S, 271T, 271V, 271W, 271Y, 287A, 287C, 287D, 287E, 287F, 287G , 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 287S, 287T, 287V, 287W , 287Y, 292A, 292C, 292D, 292E, 292F, 292G, 292H, 292I, 292K, 292M, 292 N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293 E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293P, 293Q, 293S, 29 3T, 293V, 293W, 293Y, 296A, 296C, 296D, 296E, 296F, 296G, 296H, 296I, 29 6K, 296L, 296M, 296N, 296P, 296Q, 296S, 296T, 296V, 296W, 296Y, 303A, 3 03C, 303D, 303E, 303F, 303G, 303H, 303I, 303K, 303L, 303M, 303N, 303Q, 3 03R, 303S, 303T, 303V, 303W, 303Y, 334A, 334C, 334D, 334E, 334F, 334G, 334H, 334I, 334K, 334L, 334M, 334N, 334P, 334Q, 334R, 334S, 334V, 334W, 334Y, 344A, 344C, 344D, 344E, 344F, 344G, 344I, 344K, 344L, 344M, 344N , 344P, 344Q, 344R, 344S, 344T, 344V, 344W, 344Y, 373A, 373C, 373D, 373E , 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373P, 373Q, 373R, 373S, 373T, 373V, 373W, 373Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W, and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase has at least 85% amino acid sequence identity with SEQ ID NO: 868.and a polypeptide sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the recombinant lipase, or a functional fragment thereof, including any of M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / L287F / L292M / R293Q / R296S / P303A / T334. D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M2 4R, M24S, M24T, M24V, M24W, M24Y, G38A, G38C, G38D, G38E, G38F, G38H, G38I , G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T 49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P , T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H 70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70 Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I16 1C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I161N, I161P , I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C, R174D, R 174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R174P, R17 4Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L175E, L175F,<h2 style=";text-align:left;direction:ltr">L175G、L175H、L175I、L175K、L175M、L175N、L175P、L175Q、L175R、L175S、L1 75T、L175V、L175W、L175Y、A189C、A189D、A189E、A189F、A189G、A189H、A189 I、A189K、A189L、A189M、A189N、A189P、A189Q、A189R、A189S、A189T、A189V、 A189W、A189Y、L193A、L193C、L193D、L193E、L193F、L193G、L193H、L193I、L19 3K, L193M, L193N, L193P, L193Q, L193R, L193S, L193T, L193V, L193W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, L225H, L225I, L225K, L225M, L 225N、L225P、L225Q、L225R、L225S、L225T、L225V、L225W、L225Y、E231A、E23 1C、E231D、E231F、E231G、E231H、E231I、E231K、E231L、E231M、E231N、E231P、 E231Q, E231R, E231S, E231T, E231V, E231W, E231Y, R243A, R243C, R243D, R243E, R243F, R243G, R243H, R243I, R243K, R243L, R243M, R243N, R243P, R243 Q、R243S、R243T、R243V、R243W、R243Y、V252A、V252C、V252D、V252E、V252F、 V252G、V252H、V252I、V252K、V252L、V252M、V252N、V252P、V252Q、V252R、V25 2S、V252T、V252W、V252Y、D271A、D271C、D271E、D271F、D271G、D271H、D271I 、D271K、D271L、D271M、D271N、D271P、D271Q、D271R、D271S、D271T、D271V、D 271W, D271Y, L287A, L287C, L287D, L287E, L287F, L287G, L287H, L287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287YL292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292K, L292M, L292 N, L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R2, 93D, R293E, R293F, R293G, R293H, R293I, R293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R296C, R296D, R296E, R296F, R296G, R296H, R296I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R296T, R296V, R296W, R296 Y, P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303S, P303T, P303V, P3 03W, P303Y, T334A, T334C, T334D, T334E, T334F, T334G, T334H, T334I, T334K, T334L, T334M, T334N, T334P, T334Q, T334R, T334S, T 334V, T334W, T334Y, H344A, H344C, H344D, H344E, H344F, H344G, H344I, H344K, H344L, H344M, H344N, H344P, H344Q, H344R, H344S , H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F373Q, F373 and at least one substitution or set of substitutions selected from: R, F373S, F373T, F373V, F373W, F373Y, P385A, P385C, P385D, P385E, P385F, P385G, P385H, P385I, P385K, P385L, P385M, P385N, P385Q, P385R, P385S, P385T, P385V, P385W, and P385Y, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868.

[0014] In some additional embodiments, the recombinant lipase comprises at least one mutation at at least one position set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1, wherein said positions are numbered with reference to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to at least one sequence set forth in the even-numbered sequences of SEQ ID NOs: 14-1796. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one sequence set forth in the even-numbered sequences of SEQ ID NOs: 14-1796. In some additional embodiments, the recombinant lipase comprises at least one sequence set forth in the even-numbered sequences of SEQ ID NOs: 14-1796.

[0015] In some additional embodiments, the recombinant lipase is more thermostable than the lipase of SEQ ID NO: 2. In some further embodiments, the recombinant lipase is more thermostable than the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase retains enzymatic activity after exposure to high and / or low temperatures. In some additional embodiments, the recombinant lipase retains greater enzymatic activity after exposure to high and / or low temperatures compared to the reference sequence. In some embodiments, the reference sequence is a wild-type lipase, while in some other embodiments, the reference sequence is another recombinant lipase.

[0016] In some embodiments, the recombinant lipase is stable and / or active in a low pH environment, while in other embodiments, the recombinant lipase is stable and / or active in a high pH environment, and in still further embodiments, the recombinant lipase is stable and / or active in a neutral pH environment. In some embodiments, the recombinant lipase is stable and / or active in low and high pH environments, and in some additional embodiments, the lipase is stable and / or active in low, neutral, and high pH environments. In some embodiments, the recombinant lipase retains enzymatic activity after exposure to low, high, and / or neutral pH environments. In some additional embodiments, the recombinant lipase is more stable and / or active in high, neutral, and / or low pH environments compared to the reference sequence. In some embodiments, the reference sequence is a wild-type lipase, while in other embodiments, the reference sequence is another engineered lipase. In some further embodiments, the recombinant lipase is more stable and / or active at a pH below 7 (i.e., under acidic pH conditions or levels) than the lipase of SEQ ID NO:2. In some additional embodiments, the recombinant lipase is more stable and / or active at a pH below 7 (i.e., under acidic pH conditions or levels) than the lipase of SEQ ID NO:94, 350, 442, 540, 646, 758, and / or 868. In some further embodiments, the recombinant lipase is more stable and / or active at a pH below 5 than the lipase of SEQ ID NO:2. In some additional embodiments, the recombinant lipase is more stable and / or active at pH 5 than the lipase of SEQ ID NO:94, 350, 442, 540, 646, 758, and / or 688. In some further embodiments, the recombinant lipase is more stable and / or active at pH 3.5 than the lipase of SEQ ID NO:2.In some further embodiments, the recombinant lipase is more stable and / or active at pH 3.5 than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some further embodiments, the recombinant lipase is more stable and / or active at pH 3 than the lipase of SEQ ID NO: 2. In some further embodiments, the recombinant lipase is more stable and / or active at pH 3 than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase is more resistant to proteolysis than the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase is more resistant to proteolysis than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some additional embodiments, the recombinant lipase is resistant to proteolysis by trypsin. In some additional embodiments, the recombinant lipase is more resistant to proteolysis by trypsin than the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase is more resistant to proteolysis by trypsin than the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase is resistant to proteolysis by chymotrypsin. In some additional embodiments, the recombinant lipase is more resistant to proteolysis by chymotrypsin than the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase is more resistant to proteolysis by chymotrypsin than the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some further embodiments, the recombinant lipase is resistant to proteolysis by pepsin, trypsin, and / or chymotrypsin. In some additional embodiments, the recombinant lipase is more resistant to proteolysis by pepsin, trypsin, and / or chymotrypsin than the lipase of SEQ ID NO:2.In some embodiments, the recombinant lipase is more resistant to proteolysis by pepsin, trypsin, and / or chymotrypsin than the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868.

[0017] In some additional embodiments, the recombinant lipase is more active in the presence of at least one bile salt than the lipase of SEQ ID NO: 2. In some additional embodiments, the recombinant lipase is more active in the presence of at least one bile salt than the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some additional embodiments, the recombinant lipase retains enzymatic activity after exposure to bile salts. In some additional embodiments, the recombinant lipase retains greater enzymatic activity after exposure to bile salts compared to the reference sequence. In some embodiments, the reference sequence is the lipase of SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868. In some additional embodiments, the bile salt is taurocholate.

[0018] In yet some additional embodiments, the recombinant lipase is more stable and / or active at acidic pH, more thermostable, more resistant to proteolysis, and / or more active in the presence of at least one bile salt than the lipase of SEQ ID NO: 2. In yet some further embodiments, the lipase is stable in foods and / or beverages. In yet some additional embodiments, the recombinant lipase is more stable and / or active at acidic pH, more thermostable, more resistant to proteolysis, and / or more active in the presence of at least one bile salt than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least one improved property selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits at least one improved property selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least two improved properties selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 2.In some embodiments, the recombinant lipase exhibits at least two improved properties selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least three improved properties selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits at least three improved properties selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits improved properties of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits improved properties of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least one further improved property in addition to at least one improved property selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt compared to the lipase of SEQ ID NO: 2.In some embodiments, a recombinant lipase. exhibits at least one improved property selected from improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as well as at least one further improved property, compared to the lipase of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to acidic pH; iii) increased tolerance to pH 3.5; iv) increased tolerance to pH 3; v) increased tolerance to at least one protease; vi) increased tolerance to at least one bile salt; vii) increased thermostability; or any combination of i), ii), iii), iv), v), vi), and vii), compared to the reference sequence. In some embodiments, the reference sequence is SEQ ID NO:2, while in some alternative embodiments, the reference sequence is selected from SEQ ID NOs:94, 350, 442, 540, 646, 758, and 868. In some embodiments, the recombinant lipase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to acid pH; iii) increased tolerance to pH 3.5; iv) increased tolerance to pH 3; v) increased tolerance to at least one protease; vi) increased tolerance to at least one bile salt; vii) increased thermostability; or any combination of i), ii), iii), iv), v), vi), and vii), compared to at least one reference sequence. In some embodiments, the reference sequence is SEQ ID NO:2, while in some alternative embodiments, the reference sequence is selected from SEQ ID NOs:94, 350, 442, 540, 646, 758, and 868.In some embodiments, the recombinant lipase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to acid pH; iii) increased tolerance to pH 3.5; iv) increased tolerance to pH 3; v) increased tolerance to at least one protease; vi) increased tolerance to at least one bile salt; vii) increased thermostability; or any combination of i), ii), iii), iv), v), vi), and vii), compared to at least two or more reference sequences. In some embodiments, the protease is selected from pepsin, trypsin, and / or chymotrypsin. In some embodiments, the reference sequence is selected from SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and 868. In some further embodiments, the recombinant lipase is purified.

[0019] The present invention also provides recombinant polynucleotide sequences encoding at least one recombinant lipase provided herein. In some embodiments, the recombinant polynucleotide sequences are codon-optimized. In some further embodiments, the recombinant polynucleotide comprises a sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to at least one sequence set forth in the odd-numbered sequences of SEQ ID NOS: 13-1795. In some further embodiments, the polynucleotide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one sequence set forth in an odd-numbered sequence of SEQ ID NOs: 13-1795. In some additional embodiments, the invention provides at least one sequence set forth in an odd-numbered sequence of SEQ ID NOs: 13-1795. In some additional embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences of SEQ ID NOs: 13-1795. In some embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences of SEQ ID NOs: 13-1795, wherein the sequence encodes a recombinant polypeptide provided herein. In some embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences of SEQ ID NOs: 13-1795. The recombinant polynucleotides provided herein comprise sequences selected from the odd-numbered sequences of SEQ ID NOs: 3-1795, which encode recombinant polypeptides provided in the even-numbered sequences provided in SEQ ID NOs: 14-1796. In some further embodiments, the recombinant polynucleotides encoding the recombinant lipases provided herein comprise sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to at least one sequence set forth in the odd-numbered sequences of SEQ ID NOs: 13-1795. In some further embodiments, a recombinant polynucleotide encoding a recombinant lipase provided herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one sequence set forth in the odd-numbered sequences of SEQ ID NOs: 13-1795. In some further embodiments, a recombinant polynucleotide encoding a recombinant lipase provided herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one sequence set forth in the odd-numbered sequences of SEQ ID NOs: 13-1795, and the recombinant lipase comprises a polypeptide sequence comprising an even-numbered sequence as provided in SEQ ID NOs: 14-1796.

[0020] The present invention also provides an expression vector comprising at least one recombinant polynucleotide sequence provided herein. The present invention also provides an expression vector comprising at least one recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. In some additional embodiments, the recombinant polynucleotide sequence is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence is a promoter. In some further embodiments, the promoter is a heterologous promoter.

[0021] The present invention also provides a host cell comprising at least one expression vector provided herein. The present invention also provides a host cell comprising at least one expression vector comprising at least one recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. The present invention also provides a host cell comprising an expression vector comprising at least one recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. The present invention also provides a host cell comprising an expression vector comprising at least one recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. In some embodiments, the host cell is eukaryotic, while in some alternative embodiments, the host cell is prokaryotic. In some embodiments, the host cell is Escherichia coli. In some alternative embodiments, the host cell is Saccharomyces cerevisiae.

[0022] The present invention also provides methods for producing at least one recombinant lipase, comprising culturing at least one host cell provided herein under conditions such that the recombinant lipase encoded by the recombinant polynucleotide is produced. In some embodiments, the methods further comprise recovering the lipase. In yet some additional embodiments, the methods further comprise purifying the lipase.

[0023] The present invention also provides compositions comprising at least one recombinant lipase provided herein. In some embodiments, the composition comprising at least one recombinant lipase comprises a pharmaceutical composition. In some additional embodiments, the pharmaceutical composition is suitable for treating pancreatic insufficiency. In some additional embodiments, the pharmaceutical composition is suitable for treating pancreatic insufficiency. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion into humans. In some additional embodiments, the pharmaceutical composition is suitable for oral administration to humans. In some further embodiments, the pharmaceutical composition is suitable for other modes of administration to humans. The present invention also provides compositions comprising at least one recombinant lipase provided herein, wherein the compositions are suitable for other uses.

[0024] The present invention also provides methods for treating and / or preventing symptoms of pancreatic insufficiency in a subject, comprising providing a subject with pancreatic insufficiency and providing the subject with a pharmaceutical composition provided herein. In some embodiments, administering a composition comprising at least one recombinant lipase to the subject improves the symptoms of pancreatic insufficiency in the subject. In some additional embodiments, the pharmaceutical composition further comprises at least one protease. In some further embodiments, the pharmaceutical composition further comprises at least one amylase. In yet some additional embodiments, the pharmaceutical composition further comprises at least one protease and at least one amylase.

[0025] In some further embodiments, the subject can eat a diet whose lipid content is less restricted than that required by a subject exhibiting symptoms of pancreatic insufficiency. In some additional embodiments, the subject is an infant, while in some other embodiments, the subject is a child. In yet some further embodiments, the subject is an adult, while in some alternative embodiments, the subject is a young adult. The present invention further provides a pharmaceutical comprising at least one recombinant lipase provided herein.

[0026] In some embodiments, the present invention provides a method for breaking down fats and / or lipids, comprising providing at least one fat and / or lipid and at least one engineered lipase of the present invention, and exposing the fat and / or lipid to at least one engineered amylase under conditions whereby the fat and / or lipid is broken down. In some additional embodiments, the present invention provides a method for breaking down fats and / or lipids, comprising providing a composition comprising at least one fat and / or lipid and at least one engineered lipase of the present invention, and exposing the fat and / or lipid to a composition comprising at least one engineered amylase under conditions whereby the fat and / or lipid is broken down. The present invention also provides uses of compositions comprising at least one recombinant lipase provided herein. The present invention provides a method for fat and / or lipid hydrolysis, comprising providing a fat and / or lipid and at least one engineered lipase provided herein, and exposing the fat and / or lipid to the engineered lipase under conditions whereby the fat and / or lipid is hydrolyzed by the engineered amylase. In some further embodiments, the present invention provides methods for fat and / or lipid hydrolysis comprising providing a composition comprising fat and / or lipid and at least one engineered lipase provided herein, and exposing the fat and / or lipid to the composition under conditions such that the fat and / or lipid is hydrolyzed by the engineered lipase of the composition. DETAILED DESCRIPTION OF THE INVENTION

[0027] Description of the Invention The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides are optimized to provide improved thermostability, protease stability, and stability under a wide range of pH conditions, including acidic (pH<7) conditions. The present invention also relates to the use of compositions comprising the engineered lipase polypeptides for therapeutic purposes. In some embodiments, the lipase variants of the present invention find use in the PERT treatment of PEI conditions. In some additional embodiments, the lipase is administered in a manner that does not require an enteric coating and / or a proton pump inhibitor (PPI). The present invention also provides polynucleotides encoding the engineered lipase polypeptides, as well as methods for making the engineered polynucleotides and lipase polypeptides.

[0028] Abbreviations and definitions: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and nucleic acid chemistry described below are well known and commonly used in the art. Such techniques are well known and described in many textbooks and references well known to those skilled in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All patents, patent applications, papers and publications mentioned herein, both above and below, are expressly incorporated herein by reference.

[0029] Although any suitable methods and materials similar or equivalent to those described herein can be used to practice the present invention, some methods and materials are described herein. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described, as the specific methodologies, protocols, and reagents may vary depending on the circumstances used by those skilled in the art. Therefore, the terms defined immediately below are more fully explained by reference to this application as a whole. All patents, patent applications, papers, and publications mentioned in this specification, both above and below, are expressly incorporated herein by reference.

[0030] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] Numerical ranges are 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 all narrower numerical ranges were expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if all lower (or higher) numerical limitations were expressly written herein.

[0032] The term "about" refers to an acceptable error for a particular value. In some instances, "about" refers to within 0.05%, 0.5%, 1.0%, or 2.0% of a given value's range. In some instances, "about" refers to within 1, 2, 3, or 4 standard deviations of a given value.

[0033] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention that may be had by reference to this application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to this application as a whole. Nevertheless, to facilitate understanding of the invention, certain terms are defined below.

[0034] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0035] As used herein, the term "comprising" and its cognates are used in an inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0036] As used herein, "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0037] As used herein, "ATCC" refers to the American Type Culture Collection, whose biorepository collection contains genes and strains.

[0038] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0039] As used herein, "lipase" refers to any enzyme commonly referred to as "lipase" that catalyzes the hydrolysis of fats by hydrolyzing the ester bonds of triglycerides. Pancreatic lipase is important in the breakdown of fats into fatty acids, glycerol and other alcohols. Lipase is essential for the digestion, transport and processing of dietary lipids in most organisms.

[0040] As used herein, the term "lipid" refers to a class of water-insoluble macromolecules including fatty acids and their esters, sterols, prenols, certain sparingly soluble vitamins, and other related compounds. "Fats" are a subset of lipids composed of fatty acid esters (e.g., triglycerides made from glycerol and three fatty acids). It is not intended that the present invention be limited to any particular lipid and / or fat. Given the context, the terms "fats" and "lipids" are used interchangeably herein.

[0041] As used herein, "protease" (as well as "proteinase" and "peptidase") refers to a number of enzymes that hydrolyze proteins. There are many proteases involved in the breakdown of proteins into smaller polypeptide units or single amino acids. Proteases are important for many biological functions, including the digestion of ingested protein, protein catabolism, and cell signaling.

[0042] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0043] "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. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0044] The terms "engineered," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to material that has been modified in an otherwise non-naturally occurring manner, or that is identical to the natural or native form of the material, but that has been produced or derived from synthetic material and / or by manipulation using recombinant techniques, or material that corresponds to the natural or native form of the material.

[0045] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host, such as E. coli or S. cerevisiae. Variants of recombinant polypeptides can be made by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (region-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: U.S. Patent Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393; 7,783,428; and 8,383,346. After variants are made, they can be screened for any desired properties (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability and / or acidic pH stability, etc.). In some embodiments, "recombinant lipase polypeptides" (also referred to herein as "engineered lipase polypeptides," "variant lipase enzymes," and "lipase variants") are used.

[0046] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0047] As used herein, "coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0048] The term "percent (%) sequence identity" is used herein to refer to a comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window. 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 in both sequences where the same nucleic acid base or amino acid residue exists 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 determining the number of positions in both sequences where the same nucleic acid base or amino acid residue exists, or where the nucleic acid base or amino acid residue is aligned with a gap, 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. Those skilled in the art will understand 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., 2:482

[1981] ), by the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as is 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., 215:403-410

[1990] ; and Altschul et al., Nucleic Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a 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. These word hits are then extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. 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 when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls to 0 or below due to the accumulation of 1 or more negative-scoring residue alignments; or when 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 as defaults 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 as defaults 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 89:10915

[1989] ). Exemplary sequence alignments and determinations of percent sequence identity can use the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0049] As used herein, 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, such as 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, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each contain (1) similar sequences (i.e., a portion of the complete sequence) between the two sequences and (2) additional sequences that differ between the two polynucleotides, 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" can be based on a primary amino acid sequence, which may have one or more changes in the primary sequence. A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window can contain 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, including windows of 30, 40, 50, 100, or longer, as desired.

[0050] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference to," 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, residue numbers or residue positions in a given polymer are specified with respect to the reference sequence, not by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered lipase, can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence is done with respect to the reference sequence to which the given amino acid or polynucleotide sequence is aligned.

[0051] As used herein, "mutation" refers to any change in a polypeptide or polynucleotide sequence. It is intended to encompass any number (i.e., one or more) of substitutions, insertions, deletions, and / or rearrangements present in the sequence (i.e., compared to the starting or reference sequence). Thus, mutations in the sequence result in the production of variant polypeptides (e.g., variant or recombinant lipases) as provided herein.

[0052] As used herein, an "amino acid difference" or "residue difference" refers 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 X92 compared to SEQ ID NO:2" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 92 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a threonine at position 92, then a "residue difference at position X92 compared to SEQ ID NO:2" refers to the presence of an amino acid residue other than threonine at the position in the polypeptide corresponding to position 92 of SEQ ID NO:2 (e.g., T92A). In most examples herein, a specific amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding position 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 residue in the reference polypeptide). In some instances (e.g., in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1), the disclosure also provides specific amino acid differences, designated by the conventional notation "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 instances, the polypeptides of the disclosure can contain one or more amino acid residue differences relative to the reference sequence, as indicated by a list of designated positions where the residue difference occurs relative to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position in the polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X2H / X2M / X2T or X2H / M / T or E2H / M / T). In some embodiments, the enzyme variant contains more than one substitution. These substitutions are separated by a slash for ease of reading (e.g., Y27V / F385P).The present application includes engineered polypeptide sequences that contain one or more amino acid differences, including one or both conservative and non-conservative amino acid substitutions.

[0053] As used herein, " conservative amino acid substitution " refers to the substitution of a residue with a different residue that has a similar side chain, and therefore typically includes the substitution of the amino acid in a polypeptide with the amino acid of the same or similar defined class of amino acid.For example, but not limited to, the amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid (such as alanine, valine, leucine and isoleucine); the amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain (such as serine and threonine); the amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain (such as phenylalanine, tyrosine, tryptophan and histidine); the amino acid with a basic side chain can be substituted with another amino acid with a basic side chain (such as lysine and arginine); the amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain (such as aspartic acid or glutamic acid); and / or the hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0054] As used herein, "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 defined groups rather than within a defined group, and affect (a) the structure of the peptide backbone in the area of substitution (e.g., proline instead of glycine), (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.

[0055] As used herein, "deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing 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 that make up the reference enzyme, while retaining the 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 continuous segments or can be discontinuous.

[0056] As used herein, "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.

[0057] As used herein, an asterisk (*) used in the context of a polynucleotide sequence indicates the presence of a stop codon within the polynucleotide sequence. In some embodiments, the variant protease is truncated relative to the starting or reference sequence due to the presence of the stop codon.

[0058] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion (e.g., the sequence is truncated), but the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered lipase of the invention), and that retains substantially all of the activity of the full-length polypeptide.

[0059] As used herein, an "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany the polypeptide. The term encompasses polypeptides that have been removed or purified from the polypeptide's naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant lipase 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, a recombinant lipase polypeptide may be an isolated polypeptide.

[0060] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component that naturally accompanies the molecule. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0061] As used herein, "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant than any other individual macromolecular species in the composition, on a molar or weight basis). A substantially purified composition is generally achieved when the target species constitutes at least about 50% of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure lipase 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 of all macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the target species is purified from a starting preparation to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), and the composition essentially consists 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 recombinant lipase polypeptide is a substantially pure polypeptide composition. In some embodiments, the isolated recombinant lipase polypeptide is a substantially pure polypeptide composition. In some embodiments, the substantially pure recombinant lipase polypeptide preparation is added to a formulation suitable for use in the present invention (e.g., polysaccharides, surfactants, etc.).

[0062] As used herein, the terms "improved enzymatic property" and "improved property" refer to a property of an engineered lipase polypeptide, including any improved enzymatic property compared to a reference lipase polypeptide and / or a wild-type lipase polypeptide or another engineered lipase polypeptide. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermoactivity, 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 chemoselectivity, improved solvent stability, increased tolerance to acidic or basic pH, increased tolerance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), and altered temperature profile.

[0063] As used herein, "increased enzymatic activity" or "enhanced catalytic activity" refers to an improved property of an engineered lipase polypeptide, which can be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product over a specified period of time using a specified amount of lipase) compared to a reference lipase enzyme. Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max or k cat Any property associated with enzyme activity can be affected, including classical enzyme properties of, and the change can result in increased enzyme activity. The improvement in enzyme activity can be from about 1.1-fold the enzyme activity of the corresponding wild-type enzyme to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzyme activity over a naturally occurring lipase or another engineered lipase from which the lipase polypeptide is derived.

[0064] Lipase activity can be measured by any suitable method known in the art (e.g., standard assays such as monitoring changes in the spectrophotometric properties of reactants or products). In some embodiments, the amount of product produced can be measured by high-performance liquid chromatography (HPLC) separation combined with UV absorbance. In some embodiments, the amount of product produced can be measured using a RAPIDFIRE® mass spectrometer, while in some other embodiments, the product can be measured using alternative methods known in the art. Enzyme activity comparisons are performed using defined preparations of enzymes, defined assays under set conditions, and one or more defined substrates, as described in further detail herein.

[0065] As used herein, the terms "protease stable" and "proteolytic stability" refer to the ability of a protein (e.g., a recombinant lipase of the invention) to function and withstand proteolysis mediated by any proteolytic enzyme or other proteolytic compound or agent, and retain its function after exposure to the protease. The terms are not intended to be limited to the use of any particular protease to assess protein stability. Indeed, the engineered lipases of the invention are stable and retain enzymatic activity in the presence of or after exposure to various proteases. In some embodiments, the engineered lipases are stable in the presence of trypsin, chymotrypsin, and / or pepsin. However, it is not intended that the invention be limited to any particular protease or to any particular method of assessing proteolytic stability.

[0066] As used herein, the term "pH stability" refers to the ability of a protein (e.g., a recombinant lipase of the invention) to function after incubation at a particular pH. In some embodiments, the invention provides recombinant lipases that are stable over a range of pH, including, but not limited to, a range of pH from pH 2 to pH 7. In some embodiments, the recombinant lipase is stable over different pH ranges, as shown in the examples provided herein. It is not intended that the invention be limited to any pH stability level or pH range.

[0067] As used herein, the term "improved tolerance to acidic pH" means that a recombinant lipase according to the present invention has increased stability (higher retained activity at about pH 7, 6, 5, 4, 3, 2, or even lower pH after exposure to acidic pH for a specified period of time [e.g., 1 hour, up to 24 hours, etc.]) compared to a reference lipase or another enzyme.

[0068] As used herein, "physiological pH" refers to the pH range typically found in a subject's (eg, human) blood (eg, pH 7.2-7.4).

[0069] The term "basic pH" (e.g., as used in reference to improved stability to or increased tolerance of basic pH conditions) refers to a pH range of about 7 to 11, or in some embodiments, greater than pH 11.

[0070] The term "acidic pH" (e.g., as used in reference to improved stability to or increased tolerance to acidic pH conditions) refers to a pH range encompassing any pH value less than 7. In some embodiments, the acidic pH is less than 7, while in some other embodiments, the pH is less than about 6, 5, 4, 3, 2, or lower. In some alternative embodiments, the recombinant lipase of the invention is stable at pH levels between 2 and 4. However, it is not intended that the invention be limited to any particular pH value or range of values.

[0071] As used herein, the phrase "gastric challenge" refers to exposing a recombinant lipase of the present invention to a low pH environment and the presence of at least one enzyme (e.g., pepsin) such that the recombinant lipase is exposed to conditions that may be encountered in the stomach (e.g., the human stomach).

[0072] As used herein, the phrase "intestinal challenge" refers to exposing a recombinant lipase of the present invention to a neutral pH environment and the presence of intestinal proteases, such as trypsin and chymotrypsin, and at least one bile salt (e.g., sodium taurocholate), such that the recombinant lipase is exposed to conditions that may be encountered in the intestinal tract (e.g., the human intestine).

[0073] As used herein, the phrase "sequential multiple challenges" refers to exposing a recombinant lipase of the present invention to a series of challenge conditions. For example, in some embodiments, a 1-hour thermal challenge is followed by a 1-hour gastric challenge, and then a 1-hour intestinal challenge. It is not intended that the present invention be limited to any particular challenge and / or challenge condition or a particular order of challenges.

[0074] The terms "thermostable" and "thermostable" refer to the ability of a protein (e.g., a recombinant lipase of the present invention) to function at a particular temperature. In some embodiments, the terms refer to the ability of a protein to function after incubation at a particular temperature. In some embodiments, the recombinant lipases of the present invention are "thermostable" (i.e., the enzymes maintain their catalytic activity at high temperatures). In some embodiments, the recombinant lipases resist inactivation at high temperatures, and in some additional embodiments, maintain catalytic activity at high temperatures over extended exposure times. These terms are used interchangeably herein. It is not intended that the present invention be limited to any particular temperature and / or exposure time. Such stability can be measured by any method known in the art (e.g., methods described herein). It is not intended that the present invention be limited to any particular temperature stability level or temperature range. In some embodiments, thermal stability is measured after incubation of the protein (e.g., a recombinant lipase of the present invention) at a particular temperature.

[0075] The term "chemical stability" refers to the ability of a protein (e.g., a recombinant lipase of the present invention) to function in the presence of chemicals that adversely affect the function of another protein. It is not intended that the present invention be limited to any particular level or range of chemical stability.

[0076] "Conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product under specified conditions within a given period of time. Thus, the "enzyme activity" or "activity" of a lipase polypeptide can be expressed as the "percent conversion" of a substrate to a product over a specified period of time.

[0077] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are performed under lower stringency conditions, followed by washing at various, but higher stringencies. The term "moderately stringent hybridization" refers to conditions that allow a target DNA to bind a complementary nucleic acid with about 60% identity, preferably about 75% identity, or about 85% identity to the target DNA, with an identity greater than about 90% to the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by washing in 0.2x SSPE and 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization that achieves a thermal melting temperature T determined under solution conditions for a defined polynucleotide sequence. m To about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only 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, the hybrid is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w / v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions and moderately stringent conditions are described in the references cited above.

[0078] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence that can cause the expression of a polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, a plasmid is used as a vector. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also contains a transcription terminator sequence.

[0079] As used herein, "codon-optimized" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein is more efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons called "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for a given gene, for genes with a common function or ancestral origin, for highly expressed proteins versus low copy number proteins, and for the total protein-coding region of an organism's genome. In some embodiments, a polynucleotide encoding a lipase enzyme can be codon-optimized for optimal production from the host organism selected for expression.

[0080] As used herein, the term "control sequences" refers to all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present application. 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. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Linkers may be provided in the control sequences for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0081] "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned (i.e., in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0082] As used herein, "promoter sequence" refers to the nucleic acid sequence that is recognized by host cells for the expression of a polynucleotide of interest, such as a coding sequence.Promoter sequences contain transcriptional control sequences that mediate the expression of a polynucleotide of interest.Promoters can be any nucleic acid sequence that shows transcriptional activity in selected host cells, including mutant promoters, truncated promoters and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to host cells.

[0083] As used herein, the phrase "suitable reaction conditions" refers to conditions in an enzyme conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, co-solvent, etc.) under which a lipase polypeptide of the present application can convert a substrate to a desired product compound; exemplary "suitable reaction conditions" are provided herein and exemplified by the Examples.

[0084] As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0085] As used herein, the term "substrate" used in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by a lipase polypeptide.

[0086] As used herein, the term "product" used in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of a lipase polypeptide on a substrate.

[0087] As used herein, the term "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.

[0088] As used herein, the term "producing" refers to the production of a protein and / or other compound 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 the secretion of a polypeptide from the cell.

[0089] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature.

[0090] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide encoding a lipase variant) provided herein. 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 known in the art.

[0091] As used herein, the term "analog" refers to a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to a reference polypeptide. In some embodiments, analogs include polypeptides containing one or more non-naturally occurring amino acid residues as well as naturally occurring amino acids, including, but not limited to, homoarginine, ornithine, and norvaline. In some embodiments, analogs also include one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0092] As used herein, the term "culture" refers to the growth of a population of cells under any suitable conditions (e.g., using a liquid, gel, or solid medium). In some embodiments, the cells are microbial cells (e.g., bacteria), while in some other embodiments, the cells are mammalian cells, insect cells, or cells obtained from another animal. It is not intended that the present invention be limited to the culture of any particular cell or cell type or to any particular method of culture. Indeed, the present invention is intended to encompass any suitable cell type cultured under any suitable conditions.

[0093] The term "therapeutic agent" refers to a compound that provides a beneficial or desired effect, including a medical effect, administered to a subject that exhibits signs or symptoms of a pathology.

[0094] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject (e.g., a human) comprising a pharmaceutically effective amount of an engineered lipase polypeptide encompassed by the present invention and an acceptable carrier.

[0095] The term "effective amount" means an amount sufficient to bring about a desired result. One of ordinary skill in the art can determine an effective amount by using routine experimentation.

[0096] The term "subject" includes animals, including, but not limited to, mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). The term is intended to include females and males.

[0097] As used herein, the term "patient" means any subject being evaluated for, treated for, or experiencing a disease.

[0098] The term "infant" refers to a child between the ages of 1 month and about 1 year.

[0099] As used herein, the term "newborn" refers to an infant from birth through 28 days of age. The term "preterm infant" refers to an infant born after the completion of the 20th week of pregnancy but before full term, generally weighing from about 500 to about 2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 grams at birth.

[0100] As used herein, the term "child" refers to a person who has not reached the legal age for consent to a treatment or research procedure. In some embodiments, the term refers to a person between birth and adolescence.

[0101] As used herein, the term "adult" refers to a person who has reached the legal age of the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term "young adult" refers to a person who is under the age of 18 but has reached sexual maturity.

[0102] As used herein, "composition" and "formulation" encompass products (e.g., pharmaceutical compositions, dietary and / or nutritional supplements, feeds, etc.) comprising at least one engineered lipase of the present invention intended for any suitable use.

[0103] As used herein, the terms "administering" and "administering" a composition means providing a composition of the present invention to a subject (e.g., to one affected by pancreatic insufficiency).

[0104] The term "carrier" as used herein in reference to pharmaceutical compositions means any of the standard pharmaceutical carriers, buffers and excipients, such as stabilizers, preservatives and auxiliary agents.

[0105] The term "pharmaceutically acceptable" means a material that may be administered to a subject without causing undesired biological effects or interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0106] As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API; e.g., an engineered lipase polypeptide of the invention). Excipients are typically included for formulation and / or administration purposes.

[0107] The term "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 lipase polypeptide) that ameliorates, attenuates, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptoms.

[0108] The term "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 lipase 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 that is desired by a researcher, physician, veterinarian, or other clinician.

[0109] As used herein, the terms "treating," "treat," and "treatment" refer to medical care administered to a subject (e.g., a human patient), including the administration of a pharmaceutical composition, such as a pharmaceutical composition provided herein. The terms "treating," "treat," and "treatment" are intended to encompass preventive (e.g., prophylactic) and palliative treatment or care. In some embodiments, treatment is provided to prevent or ameliorate symptoms of disease. In some embodiments, the pharmaceutical compositions of the present invention find use in the treatment or prevention of pancreatic enzyme deficiency diseases or conditions.

[0110] Engineered lipases: The present invention provides engineered lipases suitable for a variety of uses, including the treatment of pancreatic enzyme insufficiency. In some embodiments, the engineered lipases exhibiting improved properties have at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868 or at least about 100% amino acid sequence identity to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868. and have an amino acid residue difference compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868 at one or more amino acid positions (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid positions) compared to a sequence having at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more amino acid sequence identity. In some embodiments, the residue differences compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868 at one or more positions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more conservative amino acid substitutions. However, it is not intended that the invention be limited to lipase variants having conservative amino acid substitutions, as other substitutions find use in the present invention. In some embodiments, the engineered lipase polypeptide is a polypeptide listed in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1.In some embodiments, the engineered lipase polypeptide comprises SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 868.

[0111] The present invention relates to at least one recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 2, 3, 4, 22, 27, 46, 97, 136, 1 49 / 385, 27 / 46 / 385, 27 / 70 / 136 / 231 / 385, 27 / 136 / 323 / 385, 27 / 149, 27 / 149 / 385, 27 / 385, 34, 38, 41, 44, 46 / 70, 46 / 149 / 183 / 231 / 385, 48, 70, 73, 73 / 305, 82, 82 / 94 / 101 / 194 / 199, 82 / 94 / 199, 83, 85, 87, 89 , 92, 94, 96, 97 / 149 / 385, 135, 136 / 385, 140, 141, 142, 144, 146, 149, 149 / 231 / 385, 151, 174, 175, 178, 181, 183 / 385, 189, 194, 194 / 199, 195, 195 / 231 / 385, 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 2 Also provided is at least one recombinant lipase comprising at least one substitution or set of substitutions at one or more positions selected from 19, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some further embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, wherein the recombinant lipase is selected from the group consisting of 2H, 2M, 2T, 3A, 3B, 3C, 3D, 3D, 3E, 3F, 3G, 3H, 3M, 3I, 3J, 3J, 3J, 3M, 3J ... , 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97A / 136V / 149E / 385P, 27V / 46T / 385P, 27V / 70N / 136V / 231E / 385P, 27V / 136V / 323I / 385P, 27V / 149E, 27V / 149E / 385P, 27V / 385P, 34D, 38A, 38H, 38L, 41V, 44E,46T / 70N, 46T / 149E / 183L / 231E / 385P, 48V, 70N, 73C / 305I, 73I, 73R, 82C, 82E, 82E / 94S / 101S / 194N / 199L, 82E / 94S / 1 99L, 82F, 82G, 82L, 83G, 83K, 85I, 85W, 87R, 89A, 89T, 89V, 89W, 92A, 94S, 96E, 96N, 96S, 97A / 149E / 385P, 135G, 135Q, 1 35S, 135T, 135V, 136V / 385P, 140T, 141A, 141F, 141L, 141S, 142A, 142F, 142I, 142L, 144M, 144R, 144W, 146A, 146F, 146 S, 146Y, 149E, 149E / 231E / 385P, 149K, 149R, 151A, 151I, 151L, 174H, 174R, 175G, 175L, 175R, 178R, 178T, 181H, 183L / 3 85P, 189A, 189E, 189W, 194N, 194N / 199L, 194T, 195D / 231E / 385P, 195V, 199L, 199L / 213A, 210A, 210V, 212C, 212G, 212 R, 212T, 213A / 330T, 213H, 216R, 216T, 216W, 218D, 218G, 218I, 218M, 218T, 219C, 219G, 219K, 219R, 226R, 231E / 385P, and at least one substitution or set of substitutions selected from 238S, 247R, 250T, 270V, 274D, 281K, 281R, 292A, 292C, 292L, 292V, 296M, 296R, 300A, 300D, 300T, 308A, 330Y, 338N, 379T, 385A, 385C, 385D, 385P, 385R, and 385T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of E2H, E2M, E2T, T3A, T3G, T3R, T3S, S4M, S4W, S4Y, G22N, G23N, G24N, G25N, G26N, G27N, G28N, G29N, G30N, G31N, G32N, G33N, G34N, G35N, G36N, G37N, G38N, G39 ...9N, G40N, G41N, G42N, G43N, G44N, G45N, G46N, G47N, G48N, G49N, G41N, G42N, G43N, G44N, G45N, G46N, G47N, G48N, G49N, G49N, G50N, G51N, G52N, G53N, G54N, G55N, G56N, G57N, G58N, G59N, G59N, G60N, G61N, G62N, G63N, G64N, G65N, G66N, G67N, G68N, G69N, GY27V / R46T / Y97A / Y136V / T149E / F385P、Y27V / R46T / F385P、Y27V / K70N / Y136V / Q231E / F385P、Y27V / Y136V / K323I / F385P、Y27V / T149E、Y27V / T149E / F385P、Y27V / F385P、K34D、F38A、F38H、F38L、N41V、G44E、R46T / K70N、R46T / T149E / F183L / Q231E / F385P、Y48V、K70N、T73C / V305I、T73I、T73R、K82C、K82E、K82E / P94S / D101S / K194N / I199L、K82E / P94S / I199L、K82F、K82G、K82L、E83G、E83K、G85I、G85W、A87R、F89A、F89T、F89V、F89W、T92A、P94S、I96E、I96N、I96S、Y97A / T149E / F385P、Y135G、Y135Q、Y135S、Y135T、Y135V、Y136V / F385P、P140T、E141A、E141F、E141L、E141S、E142A、E142F、E142I、E142L、I144M、I144R、I144W、P146A、P146F、P146S、P146Y、T149E、T149E / Q231E / F385P、T149K、T149R、G151A、G151I、G151L、E174H、E174R、Q175G、Q175L、Q175R、S178R、S178T、K181H、F183L / F385P、S189A、S189E、S189W、K194N、K194N / I199L、K194T、Q195D / Q231E / F385P、Q195V、I199L、I199L / P213A、K210A、K210V、Q212C、Q212G、Q212R、Q212T、P213A / N330T、P213H、S216R、S216T、S216W、H218D、H218G、H218I、H218M、H218T、A219C、A219G、A219K、A219R、T226R、Q231E / F385P、Y238S、E247R、Q250T、R270V、T274D、G281K、G281R、M292A、M292C、M292L、M292V、S296M、S296R、Q300A、Q300D、Q300T、R308A、N330Y、K338N、N379T、F385A、and at least one substitution or set of substitutions selected from F385C, F385D, F385P, F385R, and F385T, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.

[0112] In some embodiments, the recombinant lipase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 23 1 / 296 / 300, 4 / 96 / 149 / 292 / 296 / 300, 4 / 96 / 219 / 296, 4 / 96 / 231 / 296 / 300, 4 / 96 / 292 / 296 / 300, 4 / 149 / 174, 4 / 174 / 219 / 292 / 296, 4 / 231 / 296 / 300, 27, 27 / 34 / 82, 27 / 73 / 82 / 218, 27 / 89, 27 / 89 / 178, 27 / 89 / 218, 27 / 218, 34 / 73 / 218, 34 / 82, 34 / 218, 73 / 82 , 73 / 82 / 183, 94 / 146 / 175, 96 / 149 / 174 / 292 / 296, 96 / 149 / 231 / 292 / 296, 96 / 149 / 292 / 296, 96 / 174 / 219 / 231 / 292 / 296, 96 / 231 / 296, 146 / 175 / 189 / 281, 149 / 174 / 292 / 296, 149 / 174 / 300, 149 / 219 / 231 / 292 / 296 / 300, 149 / 231 / 292 / 296, 149 / 231 / 292 / 296 and 296 / 300, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 3S / 4W / 96E / 296R / 300D, 3S / 292L / 296R, 4W, 4W / 11A / 1 49E / 292L / 296R / 300D, 4W / 96E / 149E / 231E / 296R / 300D, 4W / 96E / 149E / 292L / 296R / 300D, 4W / 96E / 219K / 296R, 4W / 96E / 231E / 296R / 300D, 4W / 96E / 292L / 296R / 300D, 4W / 149E / 174R, 4W / 174H / 219K / 292L / 296R, 4W / 231E / 296R / 300D, 27V, 27 V / 34D / 82E, 27V / 73I / 82L / 218I, 27V / 73I / 82L / 218M, 27V / 89T, 27V / 89T / 178P, 27V / 89T / 218I, 27V / 218I, 34D / 73I / 21 8I, 34D / 82C, 34D / 218M, 73I / 82C, 73I / 82L / 183L, 94S / 146F / 175G, 96E / 149E / 174H / 292L / 296R, 96E / 149E / 231E / 292L / 296R, 96E / 149E / 292L / 296R, 96E / 174H / 219K / 231E / 292L / 296R, 96E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174H / 292L / 296R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 231E / 292L / 296R, 149E / 231E / 292L / 296R / . 300D, 149E / 296R, 149E / 296R / 300D, 174H / 296R, 174R / 231E, 218I, 231E, 231E / 292L / 296R, 231E / 292L / 296R / 300D, 231E / 296R, 231E / 300D, 292L / 296R, 292L / 296R / 300D, 296R, and 296R / 300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of T3S / S4W / I96E / S296R / Q300D, T3S / M292L / S296R , S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S29 6R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S296R / Q300D, S4W / T149E / E1 74R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73I / K82L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H218I, K34D / T73I / H218I, K34D / K 82C, K34D / H218M, T73I / K82C, T73I / K82L / F183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T14 9E / Q231E / M292L / S296R, I96E / T149E / M292L / S296R, I96E / E174H / A219K / Q231E / M292L / S296R, I96E / Q231E / S296R,P146F / Q175G / S189A / G281K, T149E / E174H / M292L / S296R, T149E / E174H / Q300D, T149E / A219K / Q231E / M292L / S296R / Q300 D, T149E / Q231E / M292L / S296R, T149E / Q231E / M292L / S296R / Q300D, T149E / S296R, T149E / S296R / Q300D, E174H / S296R, E17 and at least one substitution or set of substitutions selected from 4R / Q231E, H218I, Q231E, Q231E / M292L / S296R, Q231E / M292L / S296R / Q300D, Q231E / S296R, Q231E / Q300D, M292L / S296R, M292L / S296R / Q300D, S296R, and S296R / Q300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94.

[0113] In some embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350 or a functional fragment thereof, and the recombinant lipase is / 137, 4 / 175 / 189 / 218, 4 / 175 / 189 / 300, 4 / 175 / 218 / 224, 4 / 175 / 218 / 373, 4 / 185, 4 / 189 / 218 / 373, 4 / 193, 4 / 218 / 300 / 369 / 373, 4 / 228, 4 / 233, 4 / 243, 4 / 271, 4 / 303, 4 / 334, 4 / 336, 4 / 375, 25, 27 / 82 / 174 / 175 / 189 ... 18 / 300 / 369 / 373, 27 / 82 / 174 / 175 / 218 / 300 / 369, 27 / 82 / 174 / 175 / 218 / 300 / 373 / 382, 27 / 82 / 174 / 218 / 300 / 373, 27 / 189 / 218 / 373, 28, 33, 99, 102, 104, 134, 153, 174 / 175 / 189, 174 / 373, 175 / 189 / 218 / 300 / 373, 1 and at least one substitution or set of substitutions at one or more positions selected from 75 / 189 / 373, 175 / 218, 175 / 218 / 382, 189 / 218 / 300 / 373, 191, 193, 231, 233, 243, 293, 303, 331, 336, 339, 368, and 373, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 189A / 218M ... , 4W / 175G / 189A / 300D, 4W / 175G / 218M / 224A, 4W / 175G / 218M / 373F, 4W / 185L, 4W / 189A / 218M / 373F, 4W / 193T, 4W / 218M / 300D / 369 N / 373F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 175G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 382M, 27V / 82C / 174H / 2 18M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218M / 3 and 373F, 175G / 218M, 175G / 218M / 382M, 175L / 189A / 373F, 189A / 218M / 300D / 373F, 191L, 193L, 231Q, 233G, 233S, 233W, 243R, 293R, 303P, 331R, 336R, 339K, 368A, 368T, and 373F, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S189A / Q3 00D, S4W / Q175G / H218M / V224A, S4W / Q175G / H218M / Y373F, S4W / I185L, S4W / S189A / H218M / Y373F, S4W / Q193T, S4W / H218M / Q300D / S369N / Y373F, S4W / P228E, S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S4W / A 336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218 M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S369N, Y 27V / K82C / E174H / Q175G / H218M / Q300D / Y373F / H382M, Y27V / K82C / E174H / H218M / Q300D / Y373F, Y27V / S189A / H218M / Y373F, R28N, L33Y, Q99D, E102L , N104H, E134R, N153G, E174H / Q175L / S189A, E174H / Y373F, Q175G / S189A / H and at least one substitution or set of substitutions selected from 218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T, and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.

[0114] In some embodiments, the recombinant lipase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, and the recombinant lipase is 3, 33 / 174 / 193 / 243, 33 / 174 / 334, 33 / 175 / 218 / 303, 33 / 175 / 218 / 334 / 339, 33 / 193, 34, 38, 46, 48, 70 / 271 / 293 / 334, 144, 174, 174 / 175 / 193, 174 / 175 / 218 / 339, 174 / 193 / 303 / 375, 174 / 193 / 375, 174 / 218 / 233 / 271 / 293 / 303, 174 / 218 / 271 / 303, 175 / 193 / 218 / 233 / 243 / 375, 175 / 193 / 218 / 243, 175 / 218 / 375, 181, 189, 193, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 303, and at least one substitution or set of substitutions at one or more positions selected from 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281, and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / L243R, L33Y / E174H / D334T ... Y / L175G / H218M / A303P, L33Y / L175G / H218M / D334T / Q339K, L33Y / Q193L, K34L, F38A, F38G, R46P, Y48L, K70H / G271D / Q293R / D334T , I144L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E174H / H218 M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N375A, L175G / Q193L / H21 8M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303P / D334T, Q193L / Q293R / A303P, K1 and at least one substitution or set of substitutions selected from: 94T, Q195I, Q195L, Q195Y, I199H, K210V, H218C, H218D, H218M, H218M / L243R, H218M / L243R / A303P / D334T, H218M / A303P, H218P, M225L, Y238W, T274D, G281K, G281P, N330F, and N330H, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0115] In some embodiments, the recombinant lipase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345 ... 4 / 174, 34 / 174 / 193 / 195 / 243 / 281, 34 / 174 / 193 / 195 / 303 / 330, 34 / 193 / 195 / 225 / 243, 34 / 193 / 243 / 303 / 330, 34 / 281 / 330, 38, 38 / 174 / 193 / 195 / 243 / 330, 38 / 174 / 193 / 225 / 274 / 283 / 303 / 330, 38 / 174 / 193 / 303, 38 / 174 / 195 / 281 / 330, 38 / 174 / 225 / 243 / 281 / 330, 38 / 17 4 / 281, 38 / 174 / 281 / 303, 38 / 174 / 281 / 303 / 345, 38 / 193 / 195 / 225 / 303 / 345, 38 / 195 / 243 / 303, 38 / 195 / 281 / 303 / 330, 38 / 195 / 281 / 330, 38 / 195 / 303, 49, 49 / 51 / 98 / 252 / 31 1, 49 / 51 / 123 / 252 / 344, 49 / 98 / 120 / 252 / 344, 49 / 120 / 252 / 344, 49 / 123 / 252 / 344, 49 / 123 / 264 / 344, 49 / 123 / 311, 49 / 252 / 344, 49 / 311 / 344, 49 / 344, 51, 51 / 252 / 344, 51 / 34 and at least one substitution or set of substitutions at one or more positions selected from: 4, 98, 98 / 344, 123 / 252 / 344, 129, 160, 161, 174 / 193 / 195 / 225, 174 / 193 / 303 / 330, 174 / 195 / 225 / 281 / 303 / 330 / 345, 174 / 195 / 243 / 281 / 345, 174 / 195 / 281 / 303, 174 / 225 / 243 / 281 / 303 / 345, 174 / 281 / 330, 174 / 303, 195 / 225 / 303 / 330, 252, 268, and 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:540. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I , 34L / 38G / 174R / 225L / 303P, 34L / 174G, 34L / 174G / 193L / 195L / 243R / 281P, 34L / 174H / 193L / 195I / 303P / 330F, 34L / 193L / 195I / 225 L / 243R, 34L / 193L / 243R / 303P / 330F, 34L / 281P / 330H, 38A / 174H / 193L / 225L / 274D / 283I / 303P / 330F, 38A / 174R / 281K / 303P / 345I,38A / 193L / 195L / 225L / 303P / 345I, 38A / 195I / 303P, 38A / 195Y / 281K / 303P / 330F, 38G, 38G / 174G / 193L / 303P, 38G / 174G / 281K / 303P, 38G / 174H / 195L / 281P / 330F, 38G / 174H / 225L / 243R / 281K / 330H, 38G / 174H / 281K, 38G / 174R / 193L / 195L / 2 43R / 330H, 38G / 195I / 281P / 330F, 38G / 195Y / 243R / 303P, 49S, 49T, 49T / 51A / 98P / 252V / 311W, 49T / 51A / 123Q / 252V / 344 H, 49T / 98P / 120T / 252V / 344H, 49T / 120T / 252V / 344H, 49T / 123Q / 252V / 344H, 49T / 123Q / 264S / 344H, 49T / 123Q / 311W, 49 T / 252V / 344H, 49T / 311W / 344H, 49T / 344H, 51A / 252V / 344H, 51A / 344H, 51V, 98P / 344H, 98R, 123Q / 252V / 344H, 129F, 160 T, 161I, 174G / 195I / 225L / 281K / 303P / 330F / 345I, 174G / 225L / 243R / 281P / 303P / 345I, 174H / 195I / 243R / 281K / 345I, 1 and at least one substitution or set of substitutions selected from 74H / 281P / 330H, 174R / 193L / 195I / 225L, 174R / 193L / 303P / 330H, 174R / 195I / 281K / 303P, 174R / 303P, 195Y / 225L / 303P / 330H, 252V, 268T, 344I, 344V, and 344W, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:540. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F,<h2 style=";text-align:left;direction:ltr">K34L / F38A / E174R / A303P / F345I、K34L / F38G / E174R / M225L / A303P、K34L / E174G、K34L / E174G / Q193L / Q195L / L243R / G281P、K34L / E174H / Q193L / Q195I / A303P / N330F、K34L / Q193L / Q195I / M225L / L243R、K34L / Q193L / L243R / A303P / N330F、K34L / G281P / N330H、F38A / E174H / Q193L / M225L / T274D / M283I / A3 03P / N330F、F38A / E174R / G281K / A303P / F345I、F38A / Q193L / Q195L / M225L / A303P / F345I、F38A / Q195I / A303P、F38A / Q195Y / G281K / A303P / N330F、F38G、 F38G / E174G / Q193L / A303P、F38G / E174G / G281K / A303P、F38G / E174H / Q195L / G281P / N330F、F38G / E174H / M225L / L243R / G281K / N330H、F38G / E174H / G281 K、F38G / E174R / Q193L / Q195L / L243R / N330H、F38G / Q195I / G281P / N330F、F3 8G / Q195Y / L243R / A303P、V49S、V49T、V49T / T51A / G98P / M252V / L311W、V49T / T51A / E123Q / M252V / S344H、V49T / G98P / M120T / M252V / S344H、V49T / M120T / M252V / S344H、V49T / E123Q / M252V / S344H、V49T / E123Q / T264S / S344H、V49T / E123Q / L311W、V49T / M252V / S344H、V49T / L311W / S344H、V49T / S344H、T51A / M252V / S344H、T51A / S344H、T51V、G98P / S344H、G98R、E123Q / M252V / S344H、S 129F, S160T, L161I, E174G / Q195I / M225L / G281K / A303P / N330F / F345I, E174G / M225L / L243R / G281P / A303P / F345I, E174H / Q195I / L243R / G281K / F345Iand at least one substitution or set of substitutions selected from E174H / G281P / N330H, E174R / Q193L / Q195I / M225L, E174R / Q193L / A303P / N330H, E174R / Q195I / G281K / A303P, E174R / A303P, Q195Y / M225L / A303P / N330H, M252V, S268T, S344I, S344V, and S344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 540.

[0116] In some embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 237 / 287 / 291, 16 / 168 / 177, 24, 24 / 278, 34 / 49 / 161 / 193 / 243 / 344, 34 / 49 / 161 / 193 / 344, 34 / 49 / 161 / 243 / 344, 34 / 49 / 161 / 252 / 268 / 344, 34 / 49 / 193 / 243 / 252 / 344, 34 / 49 / 193 / 344, 34 / 49 / 243 / 252, 34 / 49 / 252 / 268 / 344, 34 / 161 / 193 / 243 / 252 / 268, 34 / 161 / 193 / 243 / 268 / 344, 34 / 161 / 193 / 243 / 344, 34 / 161 / 193 / 252 / 2 68 / 344, 34 / 161 / 193 / 252 / 344, 34 / 161 / 193 / 268 / 344, 34 / 161 / 243 / 281 / 344, 34 / 161 / 344, 34 / 193 / 243 / 252 / 344, 34 / 193 / 252 / 268 / 344, 34 / 193 / 268 / 281, 34 / 252 / 268 / 281 / 344, 34 / 252 / 344, 49 / 161 / 193 / 243 / 252 / 344, 49 / 161 / 193 / 252 / 281 / 344, 49 / 161 / 243 / 252 / 344, 49 / 193 / 197 / 243 / 252 / 281 / 3 44, 49 / 193 / 243 / 252 / 344, 49 / 193 / 243 / 344, 49 / 243 / 344, 67 / 154 / 237 / 287, 67 / 168 / 237, 67 / 168 / 237 / 287, 67 / 177 / 237, 154 / 237 / 286 / 287, 154 / 286 / 287, 161 / 193 / 252 / 344, 161 / 193 / 344, 161 / 344, 168 / 237, 186 / 187 / 278, 193 / 243 / 281 / 344, 193 / 252 / 268 / 344, 193 / 252 / 281 / 344, 237, 237 / 287 / 291,and 281 / 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646, wherein the recombinant lipase is selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 287L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 16F / 67A / 237Q / 287L / 291A, 16F / 168T / 177L, 24M, 24M / 278L, 34K / 49T / 161I / 193L / 243R / 344H, 34K / 49T / 161I / 193L / 243R / 344V, 34K / 49T / 161I / 193L / 344W, 34K / 49T / 161I / 243R / 344H, 34K / 49T / 161I / 252V / 268T / 344V, 34K / 49T / 193L / 2 43R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 2 52V / 268T, 34K / 161I / 193L / 243R / 268T / 344V, 34K / 161I / 193L / 243R / 344W, 34K / 161I / 193L / 252V / 268T / 344H, 34K / 161I / 193L / 252V / 26 8T / 344V, 34K / 161I / 193L / 252V / 344H, 34K / 161I / 193L / 252V / 344V, 34K / 161I / 193L / 252V / 344W, 34K / 161I / 193L / 268T / 344H, 34K / 161 I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,34K / 252V / 268T / 281P / 344V, 34K / 252V / 344V, 49T / 161I / 193L / 243R / 252V / 344W, 49T / 161I / 193L / 252V / 281P / 344H, 49T / 161I / 243R / 252V / 344V, 49T / 193L / 197G / 243R / 252V / 281P / 344H, 49T / 193L / 243R / 252V / 344V, 49T / 193L / 243R / 344W, 49T / 243R / 344V, 67A / 154Y / 237Q / 287L, 67A / 168T / 237Q, 67A / 168T / 237Q / 287L, 67A / 17 and at least one substitution or set of substitutions selected from 7L / 237Q, 154Y / 237Q / 286V / 287L, 154Y / 286V / 287L, 161I / 193L / 252V / 344V, 161I / 193L / 344W, 161I / 344W, 168T / 237Q, 186T / 187A / 278L, 193L / 243R / 281P / 344W, 193L / 252V / 268T / 344V, 193L / 252V / 281P / 344H, 237Q, 237Q / 287L / 291A, and 281P / 344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:646. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, V11I / Y67A / S168T / F 287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y67A / A237Q / F287L / S291A, L16F / S168T / V177L, F24M, F24M / Y278L, L34K / V49T / L161I / Q193L / L243R / S344H, L34K / V49T / L161I / Q193L / L243R / S344V, L34K / V49T / L161I / Q193L / S344W, L34K / V49T / L161I / L243R / S344H,<h2 style=";text-align:left;direction:ltr">L34K / V49T / L161I / M252V / S268T / S344V, L34K / V49T / Q193L / L243R / M252V / S344W, L34K / V49T / Q193L / S344H, L34K / V49T / Q193L / S344W, L34K / V49T / L2 43R / M252V, L34K / V49T / M252V / S268T / S344V, L34K / L161I / Q193L / L243R / M252V / S268T, L34K / L161I / Q193L / L243R / S268T / S344V, L34K / L161I / Q193L / L243R / S344W、L34K / L161I / Q193L / M252V / S268T / S344H、L34K / L161I / Q193L / M252V / S268T / S344V、L34K / L161I / Q193L / M252V / S344H、L34K / L161I / Q 193L / M252V / S344V,L34K / L161I / Q193L / M252V / S344W,L34K / L161I / Q193L / S268T / S344H,L34K / L161I / L243R / G281P / S344V,L34K / L161I / S344V,L34 K / L161I / S344W、L34K / Q193L / L243R / M252V / S344W、L34K / Q193L / M252V / S2 68T / S344H、L34K / Q193L / S268T / G281P、L34K / M252V / S268T / G281P / S344V、 L34K / M252V / S344V,V49T / L161I / Q193L / L243R / M252V / S344W,V49T / L161I / Q193L / M252V / G281P / S344H,V49T / L161I / L243R / M252V / S344V,V49T / Q19 3L / S197G / L243R / M252V / G281P / S344H,V49T / Q193L / L243R / M252V / S344V,V49T / Q193L / L243R / S344W,V49T / L243R / S344V,Y67A / W154Y / A237Q / F287L Y67A / S168T / A237Q, Y67A / S168T / A237Q / F287L, Y67A / V177L / A237Q, W154Y / A237Q / L286V / F287L, W154Y / L286V / F287L, L161I / Q193L / M252V / S344Vand at least one substitution or set of substitutions selected from L161I / Q193L / S344W, L161I / S344W, S168T / A237Q, H186T / L187A / Y278L, Q193L / L243R / G281P / S344W, Q193L / M252V / S268T / S344V, Q193L / M252V / G281P / S344H, A237Q, A237Q / F287L / S291A, and G281P / S344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646.

[0117] In some embodiments, the recombinant lipase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758 or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 758. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758 or a functional fragment thereof, wherein the recombinant lipase comprises 24M / 168T / 252V / 281P, ... and at least one substitution or set of substitutions selected from 4M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S and 281P, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758.In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of F24M / S168T / M252V / G281P, F24M / A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S and G281P, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758.

[0118] In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, and the recombinant lipase is selected from the group consisting of 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / 252 / 260 and 385, 38, 49, 70, 149, 161, 174, 175, 189, 193, 225, 231, 243, 252, 271, 287, 292, 293, 296, 303, 334, 344, 373, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70K / 149T / 161L / 174E / 175Q / 189S / 193Q / 225M / 231Q / 243L / 252M / 271G / 287F / 292M / 293Q / 296S / 303A / 334D / 344S / 373Y / 385F, 24G, 24G, 24F ... H, 24I, 24K, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24W, 24Y, 38A, 38C, 38D, 38E, 38F, 38H , 38I, 38K, 38L, 38M, 38N, 38P, 38Q, 38R, 38S, 38T, 38V, 38W, 38Y, 49A, 49C, 49D, 49E, 49F, 49G, 49H, 49I, 49K, 49L, 49M, 49N, 49P, 49Q, 49R, 49S, 49V, 49W, 49Y, 70A, 70C, 70D, 70E, 7 0F, 70G, 70I, 70K, 70L, 70M, 70N, 70P, 70Q, 70R, 70S, 70T, 70V, 70W, 70Y, 149A, 149C, 149D,149F、149G、149H、149I、149K、149L、149M、149N、149P、149Q、149R、149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M、271N、271P、271Q、271R, 271S, 271T, 271V, 271W, 271Y, 287A, 287C, 287D, 287E, 287F, 287G , 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 287S, 287T, 287V, 287W , 287Y, 292A, 292C, 292D, 292E, 292F, 292G, 292H, 292I, 292K, 292M, 292 N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293 E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293P, 293Q, 293S, 29 3T, 293V, 293W, 293Y, 296A, 296C, 296D, 296E, 296F, 296G, 296H, 296I, 29 6K, 296L, 296M, 296N, 296P, 296Q, 296S, 296T, 296V, 296W, 296Y, 303A, 3 03C, 303D, 303E, 303F, 303G, 303H, 303I, 303K, 303L, 303M, 303N, 303Q, 3 03R, 303S, 303T, 303V, 303W, 303Y, 334A, 334C, 334D, 334E, 334F, 334G, 334H, 334I, 334K, 334L, 334M, 334N, 334P, 334Q, 334R, 334S, 334V, 334W, 334Y, 344A, 344C, 344D, 344E, 344F, 344G, 344I, 344K, 344L, 344M, 344N , 344P, 344Q, 344R, 344S, 344T, 344V, 344W, 344Y, 373A, 373C, 373D, 373E , 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373P, 373Q, 373R, 373S, 373T, 373V, 373W, 373Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W, and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase has at least 85% amino acid sequence identity with SEQ ID NO: 868.and a polypeptide sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the recombinant lipase, or a functional fragment thereof, including any of M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / L287F / L292M / R293Q / R296S / P303A / T334. D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M2 4R, M24S, M24T, M24V, M24W, M24Y, G38A, G38C, G38D, G38E, G38F, G38H, G38I , G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T 49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P , T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H 70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70 Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I16 1C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I161N, I161P , I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C, R174D, R 174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R174P, R17 4Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L175E, L175F,<h2 style=";text-align:left;direction:ltr">L175G、L175H、L175I、L175K、L175M、L175N、L175P、L175Q、L175R、L175S、L1 75T、L175V、L175W、L175Y、A189C、A189D、A189E、A189F、A189G、A189H、A189 I、A189K、A189L、A189M、A189N、A189P、A189Q、A189R、A189S、A189T、A189V、 A189W、A189Y、L193A、L193C、L193D、L193E、L193F、L193G、L193H、L193I、L19 3K, L193M, L193N, L193P, L193Q, L193R, L193S, L193T, L193V, L193W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, L225H, L225I, L225K, L225M, L 225N、L225P、L225Q、L225R、L225S、L225T、L225V、L225W、L225Y、E231A、E23 1C、E231D、E231F、E231G、E231H、E231I、E231K、E231L、E231M、E231N、E231P、 E231Q, E231R, E231S, E231T, E231V, E231W, E231Y, R243A, R243C, R243D, R243E, R243F, R243G, R243H, R243I, R243K, R243L, R243M, R243N, R243P, R243 Q、R243S、R243T、R243V、R243W、R243Y、V252A、V252C、V252D、V252E、V252F、 V252G、V252H、V252I、V252K、V252L、V252M、V252N、V252P、V252Q、V252R、V25 2S、V252T、V252W、V252Y、D271A、D271C、D271E、D271F、D271G、D271H、D271I 、D271K、D271L、D271M、D271N、D271P、D271Q、D271R、D271S、D271T、D271V、D 271W, D271Y, L287A, L287C, L287D, L287E, L287F, L287G, L287H, L287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287YL292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292K, L292M, L292N , L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R29, 3D, R293E, R293F, R293G, R293H, R293I, R293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R 296C, R296D, R296E, R296F, R296G, R296H, R296I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R296T, R296V, R296W, R296Y , P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303S, P303T, P303V, P30 3W, P303Y, T334A, T334C, T334D, T334E, T334F, T334G, T334H, T334I, T334K, T334L, T334M, T334N, T334P, T334Q, T334R, T334S, T3 34V, T334W, T334Y, H344A, H344C, H344D, H344E, H344F, H344G, H344I, H344K, H344L, H344M, H344N, H344P, H344Q, H344R, H344S, H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F373Q, F373 and at least one substitution or set of substitutions selected from: R, F373S, F373T, F373V, F373W, F373Y, P385A, P385C, P385D, P385E, P385F, P385G, P385H, P385I, P385K, P385L, P385M, P385N, P385Q, P385R, P385S, P385T, P385V, P385W, and P385Y, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868.

[0119] In some embodiments, the recombinant lipase comprises a polypeptide sequence or functional fragment thereof having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, wherein the amino acid positions of the recombinant lipase polypeptide sequence are numbered with reference to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868.

[0120] In some embodiments, the recombinant lipase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the even-numbered sequences of SEQ ID NOs: 14-1796.

[0121] In some embodiments, engineered lipase polypeptides include functional fragments of engineered lipase polypeptides encompassed by the present invention. Functional fragments have 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 engineered lipase polypeptide (i.e., the parent engineered lipase) from which the functional fragment is derived. In some embodiments, functional fragments comprise at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% of the parent sequence of the engineered lipase. 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.

[0122] Polynucleotides encoding engineered lipase polypeptides, expression vectors and host cells: The present invention provides polynucleotides encoding the engineered lipase polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to generate recombinant polynucleotides capable of expressing the polypeptides. An expression construct containing a heterologous polynucleotide encoding the engineered lipase polypeptide can be introduced into a suitable host cell to express the corresponding lipase polypeptide.

[0123] 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 provides a description of all polynucleotides capable of encoding the present polypeptides. The degeneracy of the genetic code, in which the same amino acids are coded for by alternative or synonymous codons, allows for the creation of a vast number of nucleic acids, all of which encode engineered lipase polypeptides. Thus, with knowledge of a specific amino acid sequence, one skilled in the art could create any number of different nucleic acids by simply altering the sequence of one or more codons without changing the amino acid sequence of the protein. In this regard, the present invention specifically contemplates any and all possible variations of polynucleotides that can be made to encode the polypeptides 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 variants provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and 5-1.

[0124] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria, while preferred codons used in fungi are used for expression in fungi. As a result, a codon-optimized polynucleotide encoding an engineered lipase polypeptide contains preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions in the full-length coding region.

[0125] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867. In some embodiments, the recombinant polynucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the odd-numbered sequences of SEQ ID NOs: 13-1795.

[0126] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 2, 3, 4, 22, 27 / 46 / 97 / 136 / 149 / 385, 27 / 46 / 385, 27 / 70 / 136 / 231 / 385, 27 / 136 / 323 / 385, 27 / 149, 2 7 / 149 / 385, 27 / 385, 34, 38, 41, 44, 46 / 70, 46 / 149 / 183 / 231 / 385, 48, 70, 73, 73 / 305, 82, 82 / 94 / 101 / 194 / 199, 82 / 94 / 199, 83, 85, 87, 89, 92, 94, 96, 97 / 149 / 385, 135, 136 / 385, 140, 141, 142, 144, 146, 149, 149 / 231 / 385, 151, 174, 175, 178, 181, 183 / 385, 189, 194, 194 / 199, 195, 195 / 231 / 385 , 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 219, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 2H, 2M, 2T, 3A, 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97 A / 136V / 149E / 385P, 27V / 46T / 385P, 27V / 70N / 136V / 231E / 385P, 27V / 136V / 323I / 385P, 27V / 149E, 27V / 149E / 385P, 27V / 38 5P, 34D, 38A, 38H, 38L, 41V, 44E, 46T / 70N, 46T / 149E / 183L / 231E / 385P, 48V, 70N, 73C / 305I, 73I, 73R, 82C, 82E, 82E / 94S / 1 01S / 194N / 199L, 82E / 94S / 199L, 82F, 82G, 82L, 83G, 83K, 85I, 85W, 87R, 89A, 89T, 89V, 89W, 92A, 94S, 96E, 96N, 96S, 97A / 14 9E / 385P, 135G, 135Q, 135S, 135T, 135V, 136V / 385P, 140T, 141A, 141F, 141L, 141S, 142A, 142F, 142I, 142L, 144M, 144R, 144 W, 146A, 146F, 146S, 146Y, 149E, 149E / 231E / 385P, 149K, 149R, 151A, 151I, 151L, 174H, 174R, 175G, 175L, 175R, 178R, 178T , 181H, 183L / 385P, 189A, 189E, 189W, 194N, 194N / 199L, 194T, 195D / 231E / 385P, 195V, 199L, 199L / 213A, 210A, 210V, 212C,212G, 212R, 212T, 213A / 330T, 213H, 216R, 216T, 216W, 218D, 218G, 218I, 218M, 218T, 219C, 219G, 219K, 219R, 226R, 231E / 385P, 238S, 247R, 250T, 270V, 274D, 281K, 281R, 292A, 292C, and 385T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of E2H, E2M, E2T, T3A, T4A, T5A, T6A, T7A, T8A, T9A, T10A, T11A, T12A, T13A, T14A, T15A, T16A, T17A, T18A, T19A, T20A, T21A, T22A, T23A, T24A, T25A, T26A, T27A, T28A, T29A, T30A, T31A, T32A, T33A, T34A, T35A, T36A, T37A, T38A, T39A, T40A, T41A, T42A, T43A, T44A, T45A, T46A, T47A, T48A, T49A, T50A, T51A, T52A, T53A, T54A, T55A, T56A, T57A, T58A, T59A, T59B, T59C, T58D, T59D, T59P, T59R, T59R, T60A, T61A, T62A, T63A, T64A, T65A, T66A, T67A, T68A, 3G, T3R, T3S, S4M, S4W, S4Y, G22N, Y27V / R46T / Y97A / Y136V / T149E / F385P, Y27V / R46T / F385P, Y27V / K70N / Y136V / Q231E / F385P, Y27V / Y136V / K323I / F385P, Y27V / T149E, Y27V / T149E / F385P, Y27V / F385P, K34D, F38 A, F38H, F38L, N41V, G44E, R46T / K70N, R46T / T149E / F183L / Q231E / F385P, Y48V, K70N, T73C / V305I, T73I, T73R, K82C, K82E, K82E / P94S / D101S / K194N / I199L, K82E / P94S / I199L, K82F, K82G, K82L, E83G, E83K, G85I , G85W, A87R, F89A, F89T, F89V, F89W, T92A, P94S, I96E, I96N, I96S, Y97A / T149E / F385P, Y135G, Y135Q, Y1 35S, Y135T, Y135V, Y136V / F385P, P140T, E141A, E141F, E141L, E141S, E142A, E142F, E142I, E142L, I144M,I144R, I144W, P146A, P146F, P146S, P146Y, T149E, T149E / Q231E / F385P, T149K, T149R, G1 51A, G151I, G151L, E174H, E174R, Q175G, Q175L, Q175R, S178R, S178T, K181H, F183L / F385P , S189A, S189E, S189W, K194N, K194N / I199L, K194T, Q195D / Q231E / F385P, Q195V, I199L, I1 99L / P213A, K210A, K210V, Q212C, Q212G, Q212R, Q212T, P213A / N330T, P213H, S216R, S216T , S216W, H218D, H218G, H218I, H218M, H218T, A219C, A219G, A219K, A219R, T226R, Q231E / F3 85P, Y238S, E247R, Q250T, R270V, T274D, G281K, G281R, M292A, M292C, M292L, M292V, S296M , S296R, Q300A, Q300D, Q300T, R308A, N330Y, K338N, N379T, F385A, F385C, F385D, F385P, F385R, and F385T, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.

[0127] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 231 / 296 / 300, 4 / 96 / 149 / 292 / 296 / 300, 4 / 96 / 219 / 296, 4 / 96 / 231 / 296 / 300, 4 / 96 / 292 / 296 / 300, 4 / 149 / 174, 4 / 174 / 219 / 292 / 296, 4 / 231 / 296 / 300, 27, 27 / 34 / 82, 27 / 73 / 82 / 218, 27 / 89, 27 / 89 / 178, 27 / 89 / 218, 27 / 218, 34 / 73 / 218, 34 / 82, 34 / 218 , 73 / 82, 73 / 82 / 183, 94 / 146 / 175, 96 / 149 / 174 / 292 / 296, 96 / 149 / 231 / 292 / 296, 96 / 149 / 292 / 296, 96 / 174 / 219 / 231 / 292 / 296, 96 / 231 / 296, 146 / 175 / 189 / 281, 149 / 174 / 292 / 296, 149 / 174 / 300, 149 / 219 / 231 / 292 / 296 / 300, 149 / 231 / 292 / 296, 149 / 231 / 29 and 296 / 300, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94 or a functional fragment thereof, wherein the recombinant lipase3S / 4W / 96E / 296R / 300D, 3S / 292L / 296R, 4W, 4W / 11A / 149E / 292L / 296R / 300D, 4W / 96E / 149E / 231E / 296R / 300D, 4W / 96E / 149E / 292 L / 296R / 300D, 4W / 96E / 219K / 296R, 4W / 96E / 231E / 296R / 300D, 4W / 96E / 292L / 296R / 300D, 4W / 149E / 174R, 4W / 174H / 219K / 292L / 29 6R, 4W / 231E / 296R / 300D, 27V, 27V / 34D / 82E, 27V / 73I / 82L / 218I, 27V / 73I / 82L / 218M, 27V / 89T, 27V / 89T / 178P, 27V / 89T / 218I, 2 7V / 218I, 34D / 73I / 218I, 34D / 82C, 34D / 218M, 73I / 82C, 73I / 82L / 183L, 94S / 146F / 175G, 96E / 149E / 174H / 292L / 296R, 96E / 149E / 231E / 292L / 296R, 96E / 149E / 292L / 296R, 96E / 174H / 219K / 231E / 292L / 296R, 96E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174 H / 292L / 296R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 231E / 292L / 296R, 149E / 231E / 292L / 296R / 300D, 149E / 296R, 149E / 296R / 300D, 174H / 296R, 174R / 231E, 218I, 231E, 231E / 292L / 296R, 231E / 292L / 296R / 300D, 231E / 296R, 231E / 300D, 292L / 296R, 292L / 296R / 300D, 296R, and 296R / 300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94, or a functional fragment thereof;The recombinant lipases were T3S / S4W / I96E / S296R / Q300D, T3S / M292L / S296R, S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S296R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S296R / Q300D, S4W / T149E / E174R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73I / K82L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H218I, K34D / T73I / H218I, K34D / K8 2C, K34D / H218M, T73I / K82C, T73I / K82L / F183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / Q231E / M292L / S296R, I96E / T14 9E / M292L / S296R, I96E / E174H / A219K / Q231E / M292L / S296R, I96E / Q231E / S 296R, P146F / Q175G / S189A / G281K, T149E / E174H / M292L / S296R, T149E / E17 4H / Q300D, T149E / A219K / Q231E / M292L / S296R / Q300D, T149E / Q231E / M292L / S296R, T149E / Q231E / M292L / S296R / Q300D, T149E / S296R, T149E / S296R / Q300D, E174H / S296R, E174R / Q231E, H218I, Q231E, Q231E / M292L / S296R, Q231E / M292L / S296R / Q300D, Q231E / S296R, Q231E / Q300D, M292L / S296R, M292L / S296R / Q300D, S296R, and S296R / Q300D;The amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 94.

[0128] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 4, 4 / 27 / 189 / 30 ... 70, 4 / 102, 4 / 137, 4 / 175 / 189 / 218, 4 / 175 / 189 / 300, 4 / 175 / 218 / 224, 4 / 175 / 218 / 373, 4 / 185, 4 / 189 / 218 / 373, 4 / 193, 4 / 218 / 300 / 369 / 373, 4 / 228, 4 / 233, 4 / 243, 4 / 271, 4 / 303, 4 / 334, 4 / 336, 4 / 375, 25, 27 / 174 / 178 5 / 189 / 218 / 300 / 369 / 373, 27 / 82 / 174 / 175 / 218 / 300 / 369, 27 / 82 / 174 / 175 / 218 / 300 / 373 / 382, 27 / 82 / 174 / 218 / 300 / 373, 27 / 189 / 218 / 373, 28, 33, 99, 102, 104, 134, 153, 174 / 175 / 189, 174 / 373, 175 / 189 / 218 / 300 / 37 and 373, 175 / 189 / 373, 175 / 218, 175 / 218 / 382, 189 / 218 / 300 / 373, 191, 193, 231, 233, 243, 293, 303, 331, 336, 339, 368, and 373, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 18 9A / 218M, 4W / 175G / 189A / 300D, 4W / 175G / 218M / 224A, 4W / 175G / 218M / 373F, 4W / 185L, 4W / 189A / 218M / 373F, 4W / 193T, 4W / 218M / 30 0D / 369N / 373F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 175G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 382M, 27V / 82C / 17 4H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218 and 373F, 175G / 218M, 175G / 218M / 382M, 175L / 189A / 373F, 189A / 218M / 300D / 373F, 191L, 193L, 231Q, 233G, 233S, 233W, 243R, 293R, 303P, 331R, 336R, 339K, 368A, 368T, and 373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S 189A / Q300D, S4W / Q175G / H218M / V224A, S4W / Q175G / H218M / Y373F, S4W / I18 5L, S4W / S189A / H218M / Y373F, S4W / Q193T, S4W / H218M / Q300D / S369N / Y373F , S4W / P228E, S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S 4W / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S36 9N, Y27V / K82C / E174H / Q175G / H218M / Q300D / Y373F / H382M, Y27V / K82C / E17 4H / H218M / Q300D / Y373F, Y27V / S189A / H218M / Y373F, R28N, L33Y, Q99D, E10 2L, N104H, E134R, N153G, E174H / Q175L / S189A, E174H / Y373F, Q175G / S189A / H218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T, and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 350.

[0129] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, Parse is 26, 33, 33 / 174 / 193 / 243, 33 / 174 / 334, 33 / 175 / 218 / 303, 33 / 175 / 218 / 334 / 339, 33 / 193, 34, 38, 46, 48, 70 / 271 / 293 / 334, 144, 174, 174 / 175 / 193, 174 / 175 / 218 / 339, 174 / 193 / 303 / 37 5, 174 / 193 / 375, 174 / 218 / 233 / 271 / 293 / 303, 174 / 218 / 271 / 303, 175 / 193 / 218 / 233 / 243 / 375, 175 / 193 / 218 / 243, 175 / 218 / 375, 181, 189, 193, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 3 and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / L243R, L33Y / E174H / D3 34T, L33Y / L175G / H218M / A303P, L33Y / L175G / H218M / D334T / Q339K, L33Y / Q193L, K34L, F38A, F38G, R46P, Y48L, K70H / G271D / Q293R / D334T, I144L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E174H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N375A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303P / D334T, Q193L / Q293R / A303P, and at least one substitution or set of substitutions selected from K194T, Q195I, Q195L, Q195Y, I199H, K210V, H218C, H218D, H218M, H218M / L243R, H218M / L243R / A303P / D334T, H218M / A303P, H218P, M225L, Y238W, T274D, G281K, G281P, N330F, and N330H, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442 or a functional fragment thereof, wherein the recombinant lipase is G26A, G26R, G26S, L33Y, L33Y / E174H / Q1. 93L / L243R, L33Y / E174H / D334T, L33Y / L175G / H218M / A303P, L33Y / L175G / H218M / D334T / Q339K, L33Y / Q193L, K34L, F38A, F38G, R46P, Y48L, K70H / G271D / Q293R / D334T, I144L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E174H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N375A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303P / D334T, Q193L / Q293R / A303P, K194T, Q195I, Q195L, Q195Y, I199H, K210V, H218C, H218D, and at least one substitution or set of substitutions selected from H218M, H218M / L243R, H218M / L243R / A303P / D334T, H218M / A303P, H218P, M225L, Y238W, T274D, G281K, G281P, N330F, and N330H, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0130] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174, 34 / 174 / 193 / 1 95 / 243 / 281, 34 / 174 / 193 / 195 / 303 / 330, 34 / 193 / 195 / 225 / 243, 34 / 193 / 243 / 303 / 330, 34 / 281 / 330, 38, 38 / 174 / 193 / 195 / 243 / 330, 38 / 174 / 193 / 225 / 274 / 283 / 303 / 330, 38 / 174 / 193 / 303, 38 / 174 / 195 / 281 / 330, 38 / 174 / 225 / 243 / 281 / 330, 38 / 174 / 281, 38 / 174 / 281 / 303, 38 / 174 / 281 / 303 / 345, 38 / 193 / 195 / 225 / 303 / 345, 38 / 195 / 243 / 303, 38 / 195 / 281 / 303 / 330, 38 / 195 / 281 / 330, 38 / 195 / 303, 49, 49 / 51 / 98 / 252 / 311, 49 / 51 / 123 / 252 / 344, 49 / 98 / 120 / 252 / 344, 49 / 120 / 252 / 344, 49 / 123 / 252 / 344, 49 / 123 / 264 / 344, 49 / 123 / 311, 49 / 252 / 344, 49 / 311 / 344, 49 / 344, 51, 51 / 252 / 344, 51 / 344, 98, 98 / 344, 1 and at least one substitution or set of substitutions at one or more positions selected from: 23 / 252 / 344, 129, 160, 161, 174 / 193 / 195 / 225, 174 / 193 / 303 / 330, 174 / 195 / 225 / 281 / 303 / 330 / 345, 174 / 195 / 243 / 281 / 345, 174 / 195 / 281 / 303, 174 / 225 / 243 / 281 / 303 / 345, 174 / 281 / 330, 174 / 303, 195 / 225 / 303 / 330, 252, 268, and 344, wherein the amino acid positions of the polypeptide sequence areand are numbered with reference to SEQ ID NO: 540. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 174G, 34L / 174G / 193L / 195L / 243R / 281P, 34L / 174H / 193L / 19 5I / 303P / 330F, 34L / 193L / 195I / 225L / 243R, 34L / 193L / 243R / 303P / 330F, 34L / 281P / 330H, 38A / 174H / 193L / 225L / 274D / 283I / 303P / 330F, 38A / 174R / 281K / 303P / 345I, 38A / 193L / 195L / 225L / 303P / 345I, 38A / 195I / 303P, 38 A / 195Y / 281K / 303P / 330F, 38G, 38G / 174G / 193L / 303P, 38G / 174G / 281K / 3 03P, 38G / 174H / 195L / 281P / 330F, 38G / 174H / 225L / 243R / 281K / 330H, 38G / 174H / 281K, 38G / 174R / 193L / 195L / 243R / 330H, 38G / 195I / 281P / 330F, 38G / 195Y / 243R / 303P, 49S, 49T, 49T / 51A / 98P / 252V / 311W, 49T / 51A / 123Q / 25 2V / 344H, 49T / 98P / 120T / 252V / 344H, 49T / 120T / 252V / 344H, 49T / 123Q / 25 2V / 344H, 49T / 123Q / 264S / 344H, 49T / 123Q / 311W, 49T / 252V / 344H, 49T / 31 1W / 344H, 49T / 344H, 51A / 252V / 344H, 51A / 344H, 51V, 98P / 344H, 98R, 123Q / 252V / 344H, 129F, 160T, 161I, 174G / 195I / 225L / 281K / 303P / 330F / 345I,and at least one substitution or set of substitutions selected from 174G / 225L / 243R / 281P / 303P / 345I, 174H / 195I / 243R / 281K / 345I, 174H / 281P / 330H, 174R / 193L / 195I / 225L, 174R / 193L / 303P / 330H, 174R / 195I / 281K / 303P, 174R / 303P, 195Y / 225L / 303P / 330H, 252V, 268T, 344I, 344V, and 344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:540. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, wherein the recombinant lipase is / L243R / G281P / A303P / N330F, K34L / F38A / E174R / A303P / F345I, K34L / F38G / E174R / M225L / A303P, K34L / E174G, K 34L / E174G / Q193L / Q195L / L243R / G281P, K34L / E174H / Q193L / Q195I / A303P / N330F, K34L / Q193L / Q195I / M225L / L2 43R, K34L / Q193L / L243R / A303P / N330F, K34L / G281P / N330H, F38A / E174H / Q193L / M225L / T274D / M283I / A303P / N3 30F, F38A / E174R / G281K / A303P / F345I, F38A / Q193L / Q195L / M225L / A303P / F345I, F38A / Q195I / A303P, F38A / Q195 Y / G281K / A303P / N330F, F38G, F38G / E174G / Q193L / A303P, F38G / E174G / G281K / A303P, F38G / E174H / Q195L / G281P / N330F, F38G / E174H / M225L / L243R / G281K / N330H, F38G / E174H / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H,F38G / Q195I / G281P / N330F, F38G / Q195Y / L243R / A303P, V49S, V49T, V49T / T51A / G98P / M252V / L311W, V4 9T / T51A / E123Q / M252V / S344H, V49T / G98P / M120T / M252V / S344H, V49T / M120T / M252V / S344H, V49T / E123 Q / M252V / S344H, V49T / E123Q / T264S / S344H, V49T / E123Q / L311W, V49T / M252V / S344H, V49T / L311W / S344 H, V49T / S344H, T51A / M252V / S344H, T51A / S344H, T51V, G98P / S344H, G98R, E123Q / M252V / S344H, S129F, S160T, L161I, E174G / Q195I / M225L / G281K / A303P / N330F / F345I, E174G / M225L / L243R / G281P / A303P / F 345I, E174H / Q195I / L243R / G281K / F345I, E174H / G281P / N330H, E174R / Q193L / Q195I / M225L, E174R / Q19 3L / A303P / N330H, E174R / Q195I / G281K / A303P, E174R / A303P, Q195Y / M225L / A303P / N330H, M252V, S268T, S344I, S344V, and S344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 540.

[0131] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 23 7 / 287 / 291, 16 / 168 / 177, 24, 24 / 278, 34 / 49 / 161 / 193 / 243 / 344, 34 / 49 / 161 / 193 / 344, 34 / 49 / 161 / 243 / 344, 34 / 49 / 161 / 252 / 268 / 344, 34 / 49 / 193 / 243 / 252 / 344, 34 / 49 / 193 / 344, 34 / 49 / 243 / 252, 34 / 49 / 252 / 268 / 344, 34 / 161 / 193 / 243 / 252 / 268, 34 / 161 / 193 / 243 / 268 / 344, 34 / 161 / 193 / 243 / 344, 34 / 1 61 / 193 / 252 / 268 / 344, 34 / 161 / 193 / 252 / 344, 34 / 161 / 193 / 268 / 344, 34 / 161 / 243 / 281 / 344, 34 / 161 / 344, 34 / 193 / 243 / 252 / 344, 34 / 193 / 252 / 268 / 34 4, 34 / 193 / 268 / 281, 34 / 252 / 268 / 281 / 344, 34 / 252 / 344, 49 / 161 / 193 / 243 / 252 / 344, 49 / 161 / 193 / 252 / 281 / 344, 49 / 161 / 243 / 252 / 344, 49 / 193 / 197 / 2 43 / 252 / 281 / 344, 49 / 193 / 243 / 252 / 344, 49 / 193 / 243 / 344, 49 / 243 / 344, 67 / 154 / 237 / 287, 67 / 168 / 237, 67 / 168 / 237 / 287, 67 / 177 / 237, 154 / 237 / 286 / 287, 154 / 286 / 287, 161 / 193 / 252 / 344, 161 / 193 / 344, 161 / 344, 168 / 237, 186 / 187 / 278, 193 / 243 / 281 / 344, 193 / 252 / 268 / 344, 193 / 252 / 281 / 344, 237,and 281 / 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 2 ... 7L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 16F / 67A / 237Q / 287L / 291A, 16F / 168T / 177L, 24M, 24M / 278L, 34K / 49T / 161I / 193L / 243R / 344 H, 34K / 49T / 161I / 193L / 243R / 344V, 34K / 49T / 161I / 193L / 344W, 34K / 49T / 161I / 243R / 344H, 34K / 49T / 161I / 252V / 268T / 344V, 34K / 49T / 1 93L / 243R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 24 3R / 252V / 268T, 34K / 161I / 193L / 243R / 268T / 344V, 34K / 161I / 193L / 243R / 344W, 34K / 161I / 193L / 252V / 268T / 344H, 34K / 161I / 193L / 252V / 268T / 344V, 34K / 161I / 193L / 252V / 344H, 34K / 161I / 193L / 252V / 344V, 34K / 161I / 193L / 252V / 344W, 34K / 161I / 193L / 268T / 344H, 34K / 16 1I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,34K / 252V / 268T / 281P / 344V, 34K / 252V / 344V, 49T / 161I / 193L / 243R / 252V / 344W, 49T / 161I / 193L / 252V / 281P / 344H, 49T / 161I / 243R / 252V / 344V, 49T / 193L / 197G / 243R / 252V / 281P / 344H, 49T / 193L / 243R / 252V / 344V, 49T / 193L / 243R / 344W, 49T / 243R / 344V, 67A / 154Y / 237Q / 287L, 67A / 168T / 237Q, 67A / 168T / 237Q / 287L, 67A / 17 and at least one substitution or set of substitutions selected from 7L / 237Q, 154Y / 237Q / 286V / 287L, 154Y / 286V / 287L, 161I / 193L / 252V / 344V, 161I / 193L / 344W, 161I / 344W, 168T / 237Q, 186T / 187A / 278L, 193L / 243R / 281P / 344W, 193L / 252V / 268T / 344V, 193L / 252V / 281P / 344H, 237Q, 237Q / 287L / 291A, and 281P / 344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:646. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, ... , V11I / Y67A / S168T / F287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y67A / A237Q / F287L / S291A, L16F / S168T / V177L, F2 4M, F24M / Y278L, L34K / V49T / L161I / Q193L / L243R / S344H, L34K / V49T / L161I / Q193L / L243R / S344V, L34K / V49T / L161I / Q193L / S344W,<h2 style=";text-align:left;direction:ltr">L34K / V49T / L161I / L243R / S344H、L34K / V49T / L161I / M252V / S268T / S344V、 L34K / V49T / Q193L / L243R / M252V / S344W、L34K / V49T / Q193L / S344H、L34K / V 49T / Q193L / S344W、L34K / V49T / L243R / M252V、L34K / V49T / M252V / S268T / S3 44V、L34K / L161I / Q193L / L243R / M252V / S268T、L34K / L161I / Q193L / L243R / S 268T / S344V, L34K / L161I / Q193L / L243R / S344W, L34K / L161I / Q193L / M252V / S268T / S344H, L34K / L161I / Q193L / M252V / S268T / S344V, L34K / L161I / Q19 3L / M252V / S344H、L34K / L161I / Q193L / M252V / S344V、L34K / L161I / Q193L / M252V / S344W、L34K / L161I / Q193L / S268T / S344H、L34K / L161I / L243R / G281P / S344V, L34K / L161I / S344V, L34K / L161I / S344W, L34K / Q193L / L243R / M252V / S344W, L34K / Q193L / M252V / S268T / S344H, L34K / Q193L / S268T / G281P, L34 K / M252V / S268T / G281P / S344V、L34K / M252V / S344V、V49T / L161I / Q193L / L243R / M252V / S344W、V49T / L161I / Q193L / M252V / G281P / S344H、V49T / L161I / L 243R / M252V / S344V, V49T / Q193L / S197G / L243R / M252V / G281P / S344H, V49T / Q193L / L243R / M252V / S344V, V49T / Q193L / L243R / S344W, V49T / L243R / S34 4V、Y67A / W154Y / A237Q / F287L、Y67A / S168T / A237Q、Y67A / S168T / A237Q / F2 87L、Y67A / V177L / A237Q、W154Y / A237Q / L286V / F287L、W154Y / L286V / F287L、and at least one substitution or set of substitutions selected from L161I / Q193L / M252V / S344V, L161I / Q193L / S344W, L161I / S344W, S168T / A237Q, H186T / L187A / Y278L, Q193L / L243R / G281P / S344W, Q193L / M252V / S268T / S344V, Q193L / M252V / G281P / S344H, A237Q, A237Q / F287L / S291A, and G281P / S344W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646.

[0132] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:758 or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24M / 168T / 252V / 2 and at least one substitution or set of substitutions selected from 81P, 24M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S and 281P, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of F24M / S168T / M252V / G281P, ... and at least one substitution or set of substitutions selected from A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S and G281P, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758.

[0133] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / and 385, 38, 49, 70, 149, 161, 174, 175, 189, 193, 225, 231, 243, 252, 271, 287, 292, 293, 296, 303, 334, 344, 373, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868 or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70K / 149T / 161L / 174E / 175Q / 189S / 193Q / 225M / 231Q / 243L / 252M / 271G / 287F / 292M / 293Q / 296S / 303A / 334D / 344S / 37 3Y / 385F, 24G, 24H, 24I, 24K, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24W, 24Y, 38A, 38C, 38D, 38E, 38F, 38H, 38I, 38K, 38L, 38M, 38N, 38P, 38Q, 38R, 38S, 38T, 38V, 38W, 38Y, 49A, 49C, 49D, 49E, 49F, 49G, 49H, 49I, 49K, 49L, 49M, 49N, 49P, 49Q, 49R, 49S, 49V, 49W, 49Y, 70A, 70C, 70D, 70E, 70F, 70G, 70I, 70K, 70L, 70M, 70N, 70P, 70Q, 70R, 70S, 70T, 70V, 70W,70Y、149A、149C、149D、149F、149G、149H、149I、149K、149L、149M、149N、149P、149Q、149R、149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M, 271N, 271P, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 287A, 287C, 287D, 287E, 287F, 287G, 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 2 87S, 287T, 287V, 287W, 287Y, 292A, 292C, 292D, 292E, 292F, 292G, 292H, 29 2I, 292K, 292M, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293 A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293 P, 293Q, 293S, 293T, 293V, 293W, 293Y, 296A, 296C, 296D, 296E, 296F, 296G , 296H, 296I, 296K, 296L, 296M, 296N, 296P, 296Q, 296S, 296T, 296V, 296W, 296Y, 303A, 303C, 303D, 303E, 303F, 303G, 303H, 303I, 303K, 303L, 303M, 3 03N, 303Q, 303R, 303S, 303T, 303V, 303W, 303Y, 334A, 334C, 334D, 334E, 3 34F, 334G, 334H, 334I, 334K, 334L, 334M, 334N, 334P, 334Q, 334R, 334S, 33 4V, 334W, 334Y, 344A, 344C, 344D, 344E, 344F, 344G, 344I, 344K, 344L, 344 M, 344N, 344P, 344Q, 344R, 344S, 344T, 344V, 344W, 344Y, 373A, 373C, 373D , 373E, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373P, 373Q, 373R, 373S, 373T, 373V, 373W, 373Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W, and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868.and a recombinant lipase encoding the recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868, or a functional fragment thereof, wherein the recombinant lipase is selected from the group consisting of M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / L2 87F / L292M / R293Q / R296S / P303A / T334D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M24R, M24S, M24T, M24V, M24W, M24Y, G38 A, G38C, G38D, G38E, G38F, G38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49 H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H7 0Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G , E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E1 49T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161 H, I161K, I161L, I161M, I161N, I161P, I161Q, I161R, I161S, I161T, I161V, I 161W, I161Y, R174A, R174C, R174D, R174E, R174F, R174G, R174H, R174I, R17 4K, R174L, R174M, R174N, R174P, R174Q, R174S, R174T, R174V, R174W, R174Y,<h2 style=";text-align:left;direction:ltr">L175A、L175C、L175D、L175E、L175F、L175G、L175H、L175I、L175K、L175M、L1 75N、L175P、L175Q、L175R、L175S、L175T、L175V、L175W、L175Y、A189C、A189 D, A189E, A189F, A189G, A189H, A189I, A189K, A189L, A189M, A189N, A189P, A189Q, A189R, A189S, A189T, A189V, A189W, A189Y, L193A, L193C, L193D, L19 3E、L193F、L193G、L193H、L193I、L193K、L193M、L193N、L193P、L193Q、L193R 、L193S、L193T、L193V、L193W、L193Y、L225A、L225C、L225D、L225E、L225F、L 225G, L225H, L225I, L225K, L225M, L225N, L225P, L225Q, L225R, L225S, L225T, L225V, L225W, L225Y, E231A, E231C, E231D, E231F, E231G, E231H, E231I E231K、E231L、E231M、E231N、E231P、E231Q、E231R、E231S、E231T、E231V、E2 31W、E231Y、R243A、R243C、R243D、R243E、R243F、R243G、R243H、R243I、R243 K、R243L、R243M、R243N、R243P、R243Q、R243S、R243T、R243V、R243W、R243Y、 V252A、V252C、V252D、V252E、V252F、V252G、V252H、V252I、V252K、V252L、V25 2M, V252N, V252P, V252Q, V252R, V252S, V252T, V252W, V252Y, D271A, D271C, D271E, D271F, D271G, D271H, D271I, D271K, D271L, D271M, D271N, D271P, D 271Q, D271R, D271S, D271T, D271V, D271W, D271Y, L287A, L287C, L287D, L287E, L287F, L287G, L287H, L287I, L287K, L287M, L287N, L287P, L287Q, L287R<h2 style=";text-align:left;direction:ltr">L287S, L287T, L287V, L287W, L287Y, L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292K, L292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V<h2 style=";text-align:left;direction:ltr"> , L292W, L292Y, R293A, R293C, R293D, R293E, R293F, R293G, R293H, R293I, R293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R296C, R296D, R296E, R296F, R296G, R296H, R296I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R2 96T, R296V, R296W, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303 S, P303T, P303V, P303W, P303Y, T334A, T334C, T334D, T334E, T334F, T334G, T334H, T334I, T334K, T334L, T334M, T334N, T334P, T334Q, T334R, T334S, T334V, T334W, T334Y, H344A, H344C, H344D, H344E, H344F, H344G, H344I, H344K, H344L, H344M, H344N, H344P, H344Q, H 344R, H344S, H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F37 and at least one substitution or set of substitutions selected from P385A, P385C, P385D, P385E, P385F, P385G, P385H, P385I, P385K, P385L, P385M, P385N, P385Q, P385R, P385S, P385T, P385V, P385W, and P385Y, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868.

[0134] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having lipase activity with the properties disclosed herein, wherein the polypeptide has a sequence identity at least 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, and / or 868, or any of the variants disclosed in the tables above. In some embodiments, the polynucleotide encodes an engineered polypeptide having lipase activity with the properties disclosed herein, wherein the polypeptide has a sequence identity that is at least 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%, 1111%, 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, 158, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and one or more residue differences compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758 and / or 868 at residue positions selected from the residue positions provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1.

[0135] In some additional embodiments, the polynucleotide comprises a sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to reference sequence 1, 93, 349, 441, 539, 645, 757 and / or 867. In some additional embodiments, the polynucleotide comprises a sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to at least one polynucleotide sequence provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1. In some additional embodiments, the polynucleotide comprises a 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 reference sequence 1, 93, 349, 441, 539, 645, 757 and / or 867. In some additional embodiments, the polynucleotide comprises a 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 at least one polynucleotide sequence provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1. In some embodiments, the polynucleotide encoding the engineered lipase polypeptide comprises the polynucleotide sequence of SEQ ID NO: 93, 349, 441, 539, 645, 757, and / or 867.

[0136] In some embodiments, a polynucleotide is capable of hybridizing to a reference polynucleotide sequence under high stringency conditions. In some embodiments, the reference sequence is selected from a polynucleotide sequence encoding SEQ ID NO: 1, 93, 349, 441, 539, 645, 757, and / or 867, or their complements, or any of the variant lipase polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes a lipase polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NO: 1, 93, 349, 441, 539, 645, 757, and / or 867 at a residue position selected from any of the positions set forth in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1. In some further embodiments, the engineered polynucleotide comprises a polynucleotide 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 selected from those provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1, or selected from SEQ ID NOs: 1, 93, 349, 441, 539, 645, 757 and / or 867. In some additional embodiments, the polynucleotide comprises a 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 at least one polynucleotide sequence provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1 and / or SEQ ID NOs: 1, 93, 349, 441, 539, 645, 757 and / or 867.In some further embodiments, the engineered polynucleotide sequence comprises 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 base changes compared to the reference polynucleotide sequence. In yet some other embodiments, the engineered polynucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 40, 45, or 50 base changes compared to the reference polynucleotide sequence. In some embodiments, the engineered polynucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 base changes compared to the reference polynucleotide sequence.

[0137] In some embodiments, an isolated polynucleotide encoding any of the engineered lipase polypeptides provided herein is manipulated in various ways to provide for gene expression and polypeptide production. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to regulate expression of the polynucleotide and / or polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0138] In some embodiments, control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As is known in the art, appropriate control sequences can be selected based on the host cell used. It is not intended that the present invention be limited to any particular control sequence.

[0139] Exemplary promoters for bacterial host cells include, but are not limited to, promoters obtained from the genes of the Escherichia coli lactose operon, tryptophan operon, arabinose operon, the T7 promoter from T7 bacteriophage, and the Saccharopolyspora erythraea erythromycin resistance gene.

[0140] Exemplary promoters for filamentous fungal host cells include the NA2-tpi promoter (Aspergillus niger neutral α-amylase and Aspergillus niger), as well as promoters from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787). Examples of useful promoters for yeast host cells include promoters from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase genes. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0141] Exemplary promoters for use in mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), Homo Sapiens phosphoglycerate kinase, beta-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or Gallus gallus beta-actin.

[0142] In some embodiments, the control sequence is a suitable transcription terminator sequence, i.e., a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for bacterial host cells can be obtained from the T7 bacteriophage for the T7 terminator, or from Escherichia coli ribosomal RNA, such as the rrnB terminator. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), or Homo sapiens growth hormone.

[0143] In some embodiments, the control sequence is a suitable leader sequence, a nontranslated region of an mRNA important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) genes.

[0144] In some embodiments, the regulatory sequence may also be a polyadenylation sequence, i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, when transcribed, is recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the selected host cell may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, eg, Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0145] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of a nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence.

[0146] Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell can be used to express the engineered lipase polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the immunoglobulin gamma (IgG) gene.

[0147] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is sometimes referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0148] In another aspect, the present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered lipase polypeptide and one or more expression control regions, such as a promoter, terminator, or origin of replication, depending on the type of host into which the vector will be introduced. In some embodiments, the various nucleic acids and control sequences described above are ligated together to create a recombinant expression vector containing one or more convenient restriction sites to allow for the insertion or substitution of a nucleic acid sequence encoding a variant lipase polypeptide at one or more convenient restriction sites. Alternatively, the polynucleotide sequences of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct containing the polynucleotide sequence into an appropriate vector for expression. In creating an expression vector, the coding sequence is placed in the vector so that the coding sequence is operably linked to appropriate control sequences for expression.

[0149] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can result in expression of the variant lipase polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0150] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0151] In some embodiments, the expression vector preferably contains one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Suitable markers for bacterial host cells include, but are not limited to, carbenicillin, ampicillin, chloramphenicol, tetracycline, kanamycin, and zeocin. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one engineered lipase polypeptide of the present application, the polynucleotide operably linked to one or more control sequences for expression of the engineered lipase enzyme in the host cell.

[0152] Host cells for use in expressing the polypeptides encoded by the expression vectors of the invention are well known in the art and include bacterial cells (e.g., E. coli); fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC Accession No. 201178]); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat, and Bowes melanoma cell lines).

[0153] Thus, in another aspect, the present invention provides methods for producing an engineered lipase polypeptide, comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered lipase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the lipase polypeptide as described herein.

[0154] Appropriate culture media and growth conditions for the above host cells are well known in the art. Polynucleotides for expressing lipases can be introduced into cells by various methods known in the art. Techniques include, inter alia, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0155] Engineered lipases having the properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered lipase polypeptides to mutagenesis and / or directed evolution methods known in the art and described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0156] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host, such as E. coli. Variants of recombinant polypeptides can be produced by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques known to those of skill in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (region-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Numerous suitable methods for generating enzyme variants are known to those of skill in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be readily applied to enzyme-encoding polynucleotides to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method can be used in the present invention and is well known in the art (e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,262,676, 6,270,677, 6,280,678, 6,290,681, 6,300,682, 6,310,683, 6,320,684, 6,330,685, 6,340,686, 6,350,687, 6,360,689, 6,372,689, 6,380,689, 6,390,689, 6,391,689, 6,392,689, 6,393,689, 6,394,689, 6,395,696, 6,396,689, 6,397,697, 6,398,699, 6,400,699, 6,400,699, 6,400,699, 6,410,699, 6,410,699, 6,420,699, 6,420,699, 6,430,699, 6,440,699 65,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,31 No. 9,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,No. 484, same as No. 6,358,740, same as No. 6,358,742, same as No. 6,365,377, same as No. 6,365,408, same as No. 6,368,861, same as No. 6,372,497, same as No. 6,337,186, same as No. 6,376,246, same as No. 6,379,964, same as No. 6 ,387,702, same as No. 6,391,552, same as No. 6,391,640, same as No. 6,395,547, same as No. 6,406,855, same as No. 6,406,910, same as No. 6,413,745, same as No. 6,413,774, same as No. 6,420,175, same as No. 6,423,542 No., same as No. 6,426,224, same as No. 6,436,675, same as No. 6,444,468, same as No. 6,455,253, same as No. 6,479,652, same as No. 6,482,647, same as No. 6,483,011, same as No. 6,484,105, same as No. 6,489,146, same as No. 6,50 No. 0,617, same as No. 6,500,639, same as No. 6,506,602, same as No. 6,506,603, same as No. 6,518,065, same as No. 6,519,065, same as No. 6,521,453, same as No. 6,528,311, same as No. 6,537,746, same as No. 6,573,098, same as No. 6,576,467, same as No. 6,579,678, same as No. 6,586,182, same as No. 6,602,986, same as No. 6,605,430, same as No. 6,613,514, same as No. 6,653,072, same as No. 6,686,515, same as No. 6,703,240, same as No. 6,716,6 No. 31, same as No. 6,825,001, same as No. 6,902,922, same as No. 6,917,882, same as No. 6,946,296, same as No. 6,961,664, same as No. 6,995,017, same as No. 7,024,312, same as No. 7,058,515, same as No. 7,105,297, same as No. 7, No. 148,054, same as No. 7,220,566, same as No. 7,288,375, same as No. 7,384,387, same as No. 7,421,347, same as No. 7,430,477, same as No. 7,462,469, same as No. 7,534,564, same as No. 7,620,500, same as No. 7,620,502 , same as No. 7,629,170, same as No. 7,702,464, same as No. 7,747,391, same as No. 7,747,393, same as No. 7,751,986, same as No. 7,776,598, same as No. 7,783,428, same as No. 7,795,030, same as No. 7,853,410, same as No. 7,868,No. 138, No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7, 957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806, No. 8,224,58 No. 0, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, No. 8,589 ,085, 8,762,066, 8,768,871, 8,849,575, 9,593,326, 9,665,694, 9,684,771, 9,803,224, 9,864,833, 9,821,613, 9,996,661, 10,738,286, and all related U.S. and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Op. Chem. Biol. 3:284-290

[1999] ; Christians et al., Nat. Biotechnol. 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; Stemmer, Nature, 370:389-391

[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA,91:10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and WO 2009 / 152336, all of which are incorporated herein by reference.

[0157] In some embodiments, the resulting enzyme variants after mutagenesis are screened by subjecting the enzyme variants to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after the heat treatment or other assay conditions. DNA containing the polynucleotide encoding the lipase polypeptide is then isolated from the host cell, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a different or the same host cell. Measuring enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0158] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase synthesis methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then linked (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method, since the classical phosphoramidite method is typically performed using automated synthesis methods (see, e.g., Beaucage et al., Tetra. Lett., 22:1859-69

[1981] ; and Matthes et al., EMBO J., 3:801-05

[1984] ). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector. However, it is not intended that the present invention be limited to any particular method for the production of polynucleotides and oligonucleotides, as any suitable method finds use in the present invention.

[0159] Thus, in some embodiments, a method for preparing an engineered lipase polypeptide can include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1 and SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868, and (b) expressing the lipase polypeptide encoded by the polynucleotide. In some embodiments of this method, the amino acid sequence encoded by the polynucleotide comprises one or several (e.g., up to 3, 4, 5, or up to 10) deletions, insertions, and / or substitutions of amino acid residues. In some embodiments, the amino acid sequence comprises deletions, insertions and / or substitutions of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues. In some embodiments, the amino acid sequence comprises deletions, insertions and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues. In some embodiments, the amino acid sequence comprises deletions, insertions and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residues. In some embodiments, substitutions include conservative and / or non-conservative substitutions.

[0160] The expressed engineered lipase polypeptides can be evaluated for any desired improved properties (e.g., activity, selectivity, stability, acid tolerance, protease susceptibility, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0161] In some embodiments, any engineered lipase polypeptide expressed in the host cells is recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, and chromatography, among others.

[0162] Chromatographic techniques for isolating lipase polypeptides include, among others, reverse-phase chromatography, high-performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate improved variant lipase enzymes. In some embodiments utilizing affinity chromatography purification, any antibody that specifically binds the variant lipase polypeptide is used.

[0163] In some embodiments utilizing affinity chromatography purification, a protein that binds to the glycan covalently attached to the lipase is used. In yet other embodiments utilizing affinity chromatography purification, any small molecule that binds to the lipase active site is used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with a polypeptide (e.g., a lipase variant) or a fragment thereof. In some embodiments, the lipase polypeptide or fragment is bound to a suitable carrier, such as BSA, by a side chain functional group or a linker attached to the side chain functional group.

[0164] In some embodiments, the engineered lipase polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., E. coli, S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotide sequence encoding an engineered lipase polypeptide described herein under conditions promoting production of the engineered lipase polypeptide, and recovering the engineered lipase polypeptide from the cells and / or culture medium.

[0165] In some embodiments, the present invention provides a method for identifying a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence (e.g., SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868) when optimally aligned with the amino acid sequence of SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868, and an amino acid sequence as provided in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, and / or 5-1. and / or combinations thereof, under suitable culture conditions that allow for the production of the engineered lipase polypeptide; and optionally recovering the engineered lipase polypeptide from the culture and / or the cultured bacterial cells.

[0166] In some embodiments, once the engineered polypeptide is recovered from the recombinant host cell or cell culture medium, the engineered polypeptide is further purified by any suitable method known in the art. In some additional embodiments, the purified lipase polypeptide is combined with other ingredients and compounds to provide compositions and formulations (e.g., pharmaceutical compositions) comprising the engineered lipase polypeptide as appropriate for different applications and uses. In some additional embodiments, the purified or formulated lipase polypeptide is lyophilized.

[0167] Composition: The present invention provides a variety of compositions and formats, including but not limited to those described below. In some embodiments, the present invention provides engineered lipase polypeptides suitable for use in pharmaceutical and other compositions, such as dietary supplements and / or nutraceuticals.

[0168] Depending on the mode of administration, these compositions comprising a therapeutically effective amount of an engineered lipase according to the present invention are in solid, semi-solid, or liquid form. In some embodiments, the compositions contain other pharmaceutically acceptable ingredients, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details regarding techniques for formulation and administration are well known in the art and described in the literature.

[0169] In some embodiments, the engineered lipase polypeptide is formulated for use in a pharmaceutical composition. Any suitable format for use in delivering the engineered lipase polypeptide finds use in the present invention, including, but not limited to, pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, liniments, aerosols, chewing gums, demulcents, sticks, solutions, suspensions (including, but not limited to, oil suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled-release formulations, suppositories, and the like. In some embodiments, the engineered lipase polypeptide is provided in a format suitable for injection or infusion (i.e., in an injectable formulation). In some embodiments, the engineered lipase polypeptide is provided in a biocompatible matrix, such as a sol-gel, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered lipase polypeptides are encapsulated and / or enteric coated. In some alternative embodiments, the engineered lipase polypeptides are entrapped in nanostructures (e.g., nanotubules, nanocapsules or microcapsules, microspheres, liposomes, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery formulation and / or means of delivery. It is intended that the engineered lipase polypeptides be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc.

[0170] In some embodiments, the engineered lipase polypeptides are chemically modified by glycosylation, chemical cross-linking reagents, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.), or other compounds (see, e.g., Ikeda, Amino Acids 29:283-287

[2005] ; U.S. Patent Nos. 7,531,341, 7,534,595, 7,560,263, and 7,53,653; U.S. Patent Application Publication Nos. 2013 / 0039898, 2012 / 0177722, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery method and / or mechanism.

[0171] In some additional embodiments, the engineered lipase polypeptide is provided in a formulation comprising matrix-stabilized enzyme crystals. In some embodiments, the formulation comprises a crosslinked crystalline engineered lipase enzyme and a polymer having a reactive moiety attached to the enzyme crystal. The present invention also provides engineered lipase polypeptides in a polymer.

[0172] In some embodiments, compositions comprising engineered lipase polypeptides of the invention comprise one or more commonly used carrier compounds, including, but not limited to, sugars (e.g., lactose, sucrose, mannitol and / or sorbitol), starches (e.g., corn, wheat, rice, potato, or other vegetable starches), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., acacia, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.).

[0173] In some embodiments, the engineered lipase polypeptides are suitable for use in improving dietary fat absorption and reducing dietary lipids in feces. In some embodiments, the present invention provides engineered lipase polypeptides suitable for use in reducing glycolipid concentrations in fluids such as blood and cerebrospinal fluid. The dosage of the engineered lipase polypeptide administered depends on the condition or disease, the general condition of the subject, and other factors known to those of skill in the art. In some embodiments, the composition is intended for single or multiple administration. In some embodiments, it is contemplated that the concentration of the engineered lipase polypeptide in a composition administered to a human with pancreatic insufficiency disease is sufficient to effectively treat and / or ameliorate the disease (e.g., pancreatic insufficiency disease). In some embodiments, the engineered lipase polypeptides are used in combination with other enzymes, such as amylases and / or proteases, for the treatment of diseases such as pancreatic enzyme insufficiency.

[0174] In some embodiments, the engineered lipase polypeptides are administered in combination with other pharmaceutical and / or dietary compositions, including, but not limited to, dietary supplements, nutraceuticals, etc. It is not intended that the present invention be limited to any particular method or mode of administration, as any suitable method and / or mode may be used. [Example]

[0175] experiment The following examples (including experiments and results achieved) are provided for illustrative purposes only and are not to be construed as limiting the invention. In the experimental disclosure that follows, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM, and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees centigrade); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (Col. Genetic Stock Center [CGSC], New Commonly used laboratory E. coli strains available from Haven, CT); EPI (exocrine pancreatic insufficiency); LIP and lip (lipases); btLIP (B. thermoamylovorans) lipase); HPLC (high-pressure liquid chromatography); ms (mass spectrometry or mass spectrometry); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PES (polyethersulfone); ACN (acetonitrile); IPA (isopropyl alcohol); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); LB (Luria Broth); PBS (phosphate-buffered saline); MeOH (methanol); TAG (triolein); DAG (diolein); MAG (monoolein); OA (oleic acid); FIOPC and FIOP (fold improvement over positive control) improvement); HTP (high throughput); CAV (cell accelerator voltage; collision cell accelerator voltage); CE (collision energy); RF (radio frequency); Sinclair (Sinclair Research, and Sinclair Bio Resources, Auxvasse, MO); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Molecular Devices (Molecular Devices, LLC, Sunnyvale, CA); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, CA); RAPIDFIRE® MS (RAPIDFIRE® Mass Spectrometer, Agilent); Thermo Scientific (part of ThermoFisher Scientific, Waltham, MA); Gibco (ThermoFisher Scientific); Pierce (Pierce Biotechnology (now part of Thermo Fisher Scientific), Rockford, IL); ThermoFisher. Scientific (Thermo Fisher Scientific, Waltham, MA); Corning (Corning, Inc., Palo Alto, CA); AbbVie (AbbVie, Inc., North Chicago, IL); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA).

[0176] Example 1 Isolation of bacterial lipase genes and construction of expression vectors A DNA sequence encoding Bacillus thermoamylovorans lipase (SEQ ID NO: 2) was codon-optimized for expression in E. coli and cloned into the E. coli expression vector pCK110900 vector system (see, e.g., U.S. Patent No. 7,629,157, U.S. Patent No. 9,714,437, and U.S. Patent Application Publication No. 2006 / 0195947, all of which are incorporated herein by reference) or pJV110900 vector system (see, e.g., U.S. Patent Application Publication No. 2017 / 213758, also incorporated herein by reference). However, it is not intended that the present invention be limited to any particular vector. Furthermore, in some embodiments, expression vectors lacking antimicrobial resistance markers are used. Plasmid constructs were transformed into E. coli strains derived from W3110. Directed evolution techniques well known to those skilled in the art were used to generate libraries of genetic variants from this plasmid construct (see, e.g., U.S. Pat. No. 8,383,346, WO 2010 / 144103) and derivatives thereof.

[0177] Example 2 Obtaining mammalian lipase genes, constructing expression vectors, and HTP growth and activity screening The genes encoding the mammalian lipases in Table 1 (SEQ ID NOs: 1, 3, 5, 7, 9, and 11) were codon-optimized for expression in Saccharomyces cerevisiae. For secretory expression of the lipases, the S. cerevisiae mating factor alpha (MFα) signal peptide was genetically fused to the mature form of the lipase. These yeast strains were grown in HTP as described in WO 2016 / 105889 (incorporated herein by reference). Supernatants from these yeast cultures were assayed for activity as described in Example 3. The activities obtained from these lipases are shown in Table 2-1. [Table 2-1]

[0178] Example 3 High-throughput (HTP) growth and screening conditions for lipase variants The experiments carried out for propagation and screening of lipase variants are described below.

[0179] High-throughput (HTP) propagation of B. thermoamylovorans lipase (btLIP) and lipase (LIP) variants: Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and supplemented with selection. After overnight incubation at 37°C, colonies were placed into wells of 96-well shallow flat-bottom plates (NUNC™, Thermo-Scientific) filled with 180 μl / well of LB supplemented with 1% glucose and selection. Cultures were grown overnight for 18-20 hours in a shaking incubator (200 rpm, 30°C, 85% relative humidity; Kuhner). An overnight growth sample (20 μL) was transferred to a COSTAR® 96-well deep plate filled with 380 μL of Terrific Broth supplemented with selection compound (e.g., chloramphenicol). Plates were incubated for 135 minutes in a shaking incubator (250 rpm, 30°C, 85% relative humidity; Kuhner). Expression of the lipase variants was then induced with 40 μL of 10 mM IPTG in sterile water, and the cultures were incubated overnight in a shaking incubator (250 rpm, 30°C, 85% relative humidity; Kuhner) for 20–24 h. Cells were pelleted by centrifugation (4000 rpm × 20 min), the supernatant was discarded, and the cells were frozen at −80°C prior to analysis.

[0180] Dissolving HTP pellets: First, 200–400 μL of lysis buffer (1x PBS, 1 mg / ml lysozyme, and 0.5 mg / ml polymyxin B sulfate) was added to the cell pellet. The cell pellet and buffer were gently shaken at room temperature for 1.5–2 hours and then centrifuged (4000 rpm for 5 minutes) before using the clarified lysate in the various HTP assays described herein. Analysis of these lysates by SDS-PAGE revealed the presence of an overexpressed protein with an apparent MW of approximately 45 kDa, consistent with the predicted MW of btLIP.

[0181] Analysis of clarified lysates for lipase activity: btLIP variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein through a time course of changes in the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL triolein, pH 7.0, were mixed with 10 μL of lysate, added to wells of a polyacrylate 96-well microtiter plate (COSTAR® Plate #3635; Corning), and incubated at 37°C for 30 minutes to 1 hour. Reactions were quenched with 600 μL of ACN:IPA (1:3), clarified by centrifugation, and separated on a C18 POROSHELL® 5 μm 150 mm column run with an isocratic flow of 55% IPA in acetonitrile to detect oleic acid, diolein, monoolein, and triolein at 214 nm. Alternatively, the quenched reaction was clarified and 10 μL was diluted in 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then diluted again by adding 10 μL to 190 μL of 50:25:25 HO:IPA:MeOH. Samples were then analyzed by RAPIDFIRE® MS. In the following examples, "activity without challenge" refers to lipase activity determinations performed without external pretreatment / challenge, as described in the assay method description below. [Table 3-1] JPEG2025121900000003.jpg53149

[0182] HTP analysis of clarified lysates pretreated with heat pretreatment: The activity of btLIP variants was determined after incubation at 37°C to 65°C. First, 100 μL of clarified lysate was added to the wells of a 96-well BioRad Hard-Shell PCR thin-walled microtiter plate (#hsp9601; BioRad). The plate was sealed and incubated in a thermocycler at 37°C to 65°C for 1 hour before analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by measuring the time course of oleic acid, diolein, and monoolein abundance. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL triolein pH 7.0 were mixed with 10 μL of lysate and added to the wells of a polyacrylate 96-well microtiter plate (COSTAR® plate #3635; Corning) and incubated at 37°C for 30 minutes to 1 hour. The reaction was quenched with 600 μL of ACN:IPA (1:3), clarified, and 10 μL was diluted in 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then diluted again by adding 10 μL to 190 μL of 50:25:25 HO:IPA:MeOH. The sample was then analyzed by RAPIDFIRE® MS. The results are shown in the table in Example 4.

[0183] HTP analysis of clarified lysates at low pH and with or without pepsin pretreatment: The activity of btLIP variants was determined after incubation with pepsin to simulate the gastric environment. First, 90 μL of 100 mM sodium citrate, 2.5 mg TAG pH 2.0-5.0 with or without 1.5 mg / mL pepsin (P7000 Sigma), and 10 μL of clarified lysate were added to wells of a 96-well round-bottom microtiter plate (COSTAR® Plate #3798; Corning). The plate was sealed and incubated at 37°C with shaking (THERMOTRON® Shaker HT Infors AJ185, 400 rpm, 1-inch throw) for 1 hour before analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by measuring the time course of the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL triolein pH 7.0 were mixed with 10 μL of pepsin reaction mixture and added to wells of a polyacrylate 96-well microtiter plate (COSTAR® Plate #3635; Corning) and incubated at 37°C for 30 minutes to 1 hour. The reaction was quenched with 600 μL of ACN:IPA (1:3), clarified, and 10 μL was diluted in 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then diluted again by adding 10 μL to 190 μL of 50:25:25 HO:IPA:MeOH. Samples were then analyzed by RAPIDFIRE® MS. Results are shown in the table in Example 4.

[0184] HTP analysis of clarified lysates with protease pretreatment: To simulate the lower intestinal environment, the activity of btLIP variants was determined after incubation with chymotrypsin and trypsin. First, 50 μL of protease mix (0.01–100 mg / ml chymotrypsin (C4129, Sigma), 0.01–100 mg / ml trypsin (T7409, Sigma), 0–30 μL of 20 mM sodium taurocholate in 100 mM sodium phosphate pH 6.5–7.0, and 50 μL of clarified lysate were added to wells of a 96-well round-bottom microtiter plate (COSTAR® Plate #3798; Corning). The plates were sealed and shaken (THERMOTRON® Shaker HT Infors) prior to analysis. The reaction mixture was incubated at 37°C for 1 hour with a 1-inch throw (AJ185, 400 rpm). Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein via a time course of the amount of oleic acid, diolein, and monoolein present. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL triolein pH 7.0 were mixed with 10 μL of the protease reaction mixture and added to a well of a polyacrylate 96-well microtiter plate (COSTAR® Plate #3635; Corning) and incubated at 37°C for 30 minutes to 1 hour. The reaction was quenched with 600 μL of ACN:IPA (1:3), clarified, and 10 μL was diluted in 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then 10 μL was added to 190 μL of 50:25:25 IPA:MeOH. The sample was diluted again by addition to HO:IPA:MeOH. The sample was then analyzed by RAPIDFIRE® MS. The results are shown in the table in Example 4.

[0185] HTP analysis of clarified lysates from a multi-stage gastrointestinal (GI) challenge: The activity of the btLIP variants was determined after incubation at 37°C to 65°C. First, 100 μL of the clarified lysate was placed in a 96-well BioRad Hard-Shell plate. The btLIP variants were then added to wells of a thin-walled PCR microtiter plate (#hsp9601; BioRad). The plate was sealed and incubated in a thermocycler at 37°C to 65°C for 1 hour prior to gastric challenge. Next, to simulate the gastric environment, the btLIP variants were incubated with pepsin. First, 50 μL of 100 mM sodium citrate pH 2.0 to 5.0 containing 3.0 mg / mL pepsin (P7000 Sigma) and 50 μL of the heat-treated lysate were added to wells of a 96-well round-bottom microtiter plate (COSTAR® plate #3798; Corning). The plate was sealed and incubated with shaking (THERMOTRON® shaker HT Infors AJ185, 400 rpm, 1-inch throw) at 37°C for 1 hour prior to analysis. Next, the btLIP variants were incubated with chymotrypsin and trypsin to simulate the lower intestinal environment. In this step, 100 μL of protease mix (0.01–100 mg / ml chymotrypsin [C4129; Sigma] and 0.01–100 mg / ml trypsin [T7409; Sigma]), 0–30 μL of 20 mM sodium taurocholate in 100 mM sodium phosphate pH 6.5–7.0, and 100 μL of heat-treated, gastric challenge lysate were added to wells of a 96-well round-bottom microtiter plate (COSTAR® Plate #3798; Corning). The plates were sealed and incubated at 37°C with shaking (THERMOTRON® Shaker HT Infors AJ185, 400 rpm, 1-inch throw) for 1 h before analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by measuring the time course of the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL triolein pH 7.0 were mixed with 10 μL of gastrointestinal response and added to wells of a polyacrylate 96-well microtiter plate (COSTAR® Plate #3635; Corning) and incubated at 37°C. The reaction was quenched with 600 μL of ACN:IPA (1:3), clarified, and diluted 10 μL into 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then diluted again by adding 10 μL into 190 μL of 50:25:25 HO:IPA:MeOH. The sample was then analyzed by RAPIDFIRE® MS. The results are shown in the table in Example 4.

[0186] Example 4 Screening results for lipase variants The variants generated from homologous diversity and saturation mutagenesis were screened under several different conditions as described in Example 3. The results (compared to SEQ ID NO:2) are shown in Table 4-1, where amino acid differences are shown relative to SEQ ID NO:2. [Table 4-1] JPEG2025121900000005.jpg226149 JPEG2025121900000006.jpg226149 JPEG2025121900000007.jpg112149

[0187] Based on the results shown in Table 4-1, SEQ ID NO:94 was selected as the parent sequence for the next iteration of protein optimization. Beneficial mutations identified from the results shown in Table 4-1 were engineered into the backbone. Variants were screened under the same conditions as described in Example 3. The only difference was that the acid challenge screening was performed at pH 3.5 instead of pH 3. Data compared to SEQ ID NO:94 are listed in Table 4-2. In this table, amino acid differences are shown relative to SEQ ID NO:94. [Table 4-2] JPEG2025121900000009.jpg205149

[0188] Based on the results shown in Table 4-2, SEQ ID NO:350 was selected as the next parent sequence for the next iteration of protein optimization. Beneficial mutations identified from Table 4-2 were recombined into the backbone. In addition, variants were also constructed using SEQ ID NO:350 through saturation mutagenesis at different positions. The variants were assayed as described in Example 3, and the results are provided in Table 4-3. In this table, amino acid differences are shown relative to SEQ ID NO:350. [Table 4-3] JPEG2025121900000011.jpg146149

[0189] Based on the results from Table 4-3, SEQ ID NO:442 was selected as the next parent sequence for the next iteration of protein optimization. Beneficial mutations identified based on the results shown in Table 4-3 were engineered into the backbone. Additionally, variants were also constructed for SEQ ID NO:442 through saturation mutagenesis at different positions. The variants were assayed as described in Example 3, and the results are provided in Table 4-4. In this table, amino acid differences are shown relative to SEQ ID NO:442. [Table 4-4] JPEG2025121900000013.jpg131149

[0190] Based on the results shown in Table 4-4, SEQ ID NO:540 was selected as the next parent sequence for the next iteration of protein optimization. Beneficial mutations identified based on the results shown in Table 4-4 were engineered into the backbone. Additionally, variants were also constructed for SEQ ID NO:540 through saturation mutagenesis at different positions. The variants were assayed as described in Example 3, and the results are provided in Table 4-5. In this table, amino acid differences are shown relative to SEQ ID NO:540. [Table 4-5] JPEG2025121900000015.jpg147149

[0191] Based on the results shown in Tables 4-5, SEQ ID NO:646 was selected as the next parent sequence for the next iteration of protein optimization. Beneficial mutations identified based on the results shown in Tables 4-5 were engineered into the backbone. The variants were assayed as described in Example 3, and the results are provided in Tables 4-6, where amino acid differences are shown relative to SEQ ID NO:646. [Table 4-6] JPEG2025121900000017.jpg155149

[0192] Based on the results shown in Tables 4-6, SEQ ID NO:758 was selected as the next parent sequence for the next iteration of protein optimization. Beneficial mutations identified based on the results shown in Tables 4-6 were engineered into the backbone. The variants were assayed as described in Example 3, and the results are provided in Tables 4-7, where amino acid differences are shown relative to SEQ ID NO:758. [Table 4-7]

[0193] Example 5 Screening results of individual mutation variants against SEQ ID NO: 868 Single point mutations were constructed on SEQ ID NO:868 through site-saturation mutagenesis at 24 positions different from SEQ ID NO:2. These variants were assayed for lipase activity after being subjected to various pretreatments as described in Example 3. Each variant was tested in triplicate, and an analysis of the activity data for both SEQ ID NO:868 and SEQ ID NO:2 is listed in Table 5-1. [Table 5-1] JPEG2025121900000020.jpg212149 JPEG2025121900000021.jpg212149 JPEG2025121900000022.jpg212149 JPEG2025121900000023.jpg211149 JPEG2025121900000024.jpg211149 JPEG2025121900000025.jpg211149 JPEG2025121900000026.jpg211149 JPEG2025121900000027.jpg212149 JPEG2025121900000028.jpg212149 JPEG2025121900000029.jpg211149 JPEG2025121900000030.jpg211149 JPEG2025121900000031.jpg212149 JPEG2025121900000032.jpg49149

[0194] Example 6 Validation of a surgical model of exocrine pancreatic insufficiency in minipigs A surgical model of exocrine pancreatic insufficiency (EPI) was created in minipigs by pancreatic duct ligation. Starting 7 days before surgery and continuing throughout the experiment, 3-4 month-old female SINCLAIR™ minipigs were fed a high-fat diet (HFD; Sinclair standard diet S-9 mixed with BERTOLLI® olive oil at a 10:1 ratio w / w) once daily. Total fecal output over a 24-hour period was collected on three consecutive days before surgery and on days 15, 16, and 17 after surgery. Daily total fecal collections from each animal were prepared for subsequent analysis by adding distilled water in a volume approximately 1.5 times the net weight of the feces, homogenizing, dividing into 3 x 50 mL aliquots, and freezing at -20°C until analysis. The model was validated by measuring the coefficient of fat absorption (CFA; modified Van de Kamer method; see Van de Kamer, in Seligson (ed.), Standard Methods of Clinical Chemistry, volume 2, Academic Press, New York, NY, 1958, pp. 34–39) and the coefficient of nitrogen absorption (CNA; Kjeldahl total nitrogen using a Vario Max CN instrument with a combustion method; see Watson et al., in Peters et al. (eds.), Recommended Methods of Manure Analysis, University of Wisconsin Cooperative Extension Publishing, Publication No. A3769, Madison, WI, 2003, pp. 18–24) in fecal samples and assessing the percent changes before and after surgery. Before surgery, the CFA and CNA of healthy minipigs were 91.3% ± 1.1 SEM and 84.2% ± 1.2 SEM, respectively. After surgery, CFA decreased by 53.2% (p<0.0001, n=11) and CNA decreased by 28.7% (p<0.0001, n=11), thus confirming the validity of this surgical model for EPI studies.

[0195] Example 7 In vivo characterization of SEQ ID NO: 868 compared to CREON® Pancrelipase To evaluate the engineered lipase variant (SEQ ID NO: 868) compared to the current standard of care (CREON® pancrelipase; AbbVie, Inc.), a validated SINCLAIR™ minipig surgical model of EPI (female, 4-5 months old) was developed for in vivo studies. Starting at least 8 days prior to dosing, animals were fed a HFD (Sinclair standard S-9 diet mixed with BERTOLLI® plain olive oil, 10:1 w / w) combined with one 3.9 oz cup of unsweetened applesauce once daily. During dosing days, diets were prepared sequentially for each animal to minimize ex vivo lipase interactions with dietary fat prior to ingestion. Briefly, the HFD was first prepared in a clean feeding bowl. The acidic pH protected against premature enzyme activation, so the enzymes (0.5 g of lyophilized SEQ ID NO: 868 powder, or 5.6 g of microspheres pooled from 7 x 36,000 U CREON® pancrelipase capsules, equivalent to 258,000 U and 252,000 U lipase, respectively) were then mixed into one 3.9 oz cup of unsweetened applesauce. The units of enzyme administered per meal were based on the specific activity of lipase as determined by the USP lipase assay (United States Pharmacopeia and National Formulary (USP42-NF37) Rockville, MD: United States Pharmacopeial Convention; 2016; available online at uspnf.com / uspnf / document / GUID-AC788D41-90A2-4F36-A6E7-769954A9ED09_1_en-US) and the total calculated fat provided in the meal. The lipase-applesauce mixture was then transferred to the HFD and quickly mixed. Finally, the feeding bowl was immediately presented to the animals before proceeding with the next preparation. After 2 hours, the feeding bowl was removed. The animals consumed the entire meal in 30 minutes or less. A crossover design was used to maximize the number of minipigs per group (n = 3). The study included an 8-day "washout" (HFD + enzyme-free applesauce once daily) between each 10-day treatment period.Total feces were collected for three consecutive days, immediately before the start of treatment and 24 hours after treatment on days 9, 10, and 11 of each period. Stool samples were prepared by adding distilled water in a volume approximately 1.5 times the net weight of the feces to the total fecal collection each day, homogenizing, dividing into 3 x 50 mL portions, freezing at -20°C until analysis, and discarding the remainder. Analysis was completed as described for model validation above. Both CREON® pancrelipase and SEQ ID NO: 868 similarly improved CFA to values that were not significantly different from the pre-operative baseline.

[0196] Example 8 In vivo dose response characterization of SEQ ID NO: 868 For further in vivo SEQ ID NO:868 dose-response studies, we used the validated SINCLAIR™ minipig surgical model (female, 4-6 months of age) for EPI. The HFD and feeding regimen, lipase mixing protocol, fecal sample collection and preparation, and analysis were the same as described above. Postoperative CFA measured immediately before administration was reduced to 39.9% + / - 13.2 SD (p<0.0001, n=8) compared to preoperative values (92.8% + / - 3.2 SD, n=8). SEQ ID NO:868 was administered at 258,000 U (0.5 g of lyophilized powder) to bridge previous studies, and at 86,000 U (167 mg) and 29,000 U (55.5 mg) to evaluate lower-dose efficacy. All doses evaluated had a dose-dependent positive effect on CFA. From high to low doses, CFA values increased to 81.3% + / - 3.9 SD (p<0.0001, n=3), 74% + / - 3.6 SD (p<0.0001, n=3), and 65.5% + / - 3.7 SD (p<0.0001, n=2), respectively, compared to baseline.

[0197] All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0198] While various particular embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention.

Claims

1. 1. A recombinant lipase comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 868, The recombinant lipase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to acid pH; iii) increased tolerance to pH 3.5; iv) increased tolerance to pH 3; v) increased tolerance to at least one protease; vi) increased tolerance to at least one bile salt; vii) increased thermostability; or any combination of i), ii), iii), iv), v), vi) and vii), compared to the lipase of SEQ ID NO:

2.

2. 2. The recombinant lipase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions at one or more positions selected from 24, 38, 49, 70, 149, 161, 174, 175, 189, 193, 225, 231, 243, 252, 271, 287, 292, 293, 296, 303, 334, 344, 373, and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

868.

3. The recombinant lipase of any one of claims 1 to 2, wherein the recombinant lipase is more thermostable than the lipase of SEQ ID NO:

2.

4. 4. The recombinant lipase of any one of claims 1 to 3, wherein the recombinant lipase is more stable at acidic pH levels than the lipase of SEQ ID NO:

2.

5. The recombinant lipase of any one of claims 1 to 4, wherein the recombinant lipase is more resistant to proteolysis than the lipase of SEQ ID NO:

2.

6. 6. The recombinant lipase of any one of claims 1 to 5, wherein the recombinant lipase is more active in the presence of at least one bile salt than the lipase of SEQ ID NO:

2.

7. The recombinant lipase of any one of claims 1 to 6, wherein the recombinant lipase is purified.

8. A composition comprising at least one recombinant lipase according to any one of claims 1 to 7.

9. A recombinant polynucleotide encoding at least one recombinant lipase according to any one of claims 1 to 7.

10. 10. The recombinant polynucleotide of claim 9, wherein the sequence of the polynucleotide is codon-optimized.

11. An expression vector comprising the sequence of at least one recombinant polynucleotide according to claim 9 or 10.

12. The expression vector of claim 11 , wherein the sequence of the recombinant polynucleotide is operably linked to a control sequence.

13. 13. The expression vector of claim 11 or 12, wherein the control sequence is a promoter.

14. The expression vector of claim 13 , wherein the promoter is a heterologous promoter.

15. A host cell comprising the expression vector according to any one of claims 11 to 14.

16. The host cell of claim 15 , wherein the host cell is a eukaryote or a prokaryote.

17. 17. A method for producing a recombinant lipase, comprising culturing a host cell according to claim 15 or 16 under conditions in which the recombinant lipase encoded by the recombinant polynucleotide is produced.

18. 18. The method of claim 17, further comprising recovering the lipase.

19. 20. The method of claim 18, further comprising purifying the lipase.

20. A pharmaceutical composition for the treatment of pancreatic insufficiency, comprising the composition of claim 8.

21. 21. The pharmaceutical composition of claim 20, further comprising a pharmaceutically acceptable carrier and / or excipient.

22. 22. The pharmaceutical composition of claim 20 or 21, further comprising at least one additional enzyme selected from at least one protease and at least one amylase.

23. 22. The pharmaceutical composition of claim 20 or 21, further comprising a pancreatic extract containing lipase, protease and amylase activity.

24. 24. The pharmaceutical composition according to any one of claims 20 to 23, wherein the composition is suitable for parenteral injection or infusion, subcutaneous injection, inhalation or dermal application to humans.

25. The pharmaceutical composition of any one of claims 20 to 23, wherein the composition is suitable for oral administration to humans.

26. 26. The pharmaceutical composition of claim 25, wherein the composition further comprises an enteric coating.

27. 27. A pharmaceutical composition according to any one of claims 20 to 26 for use in a method for treating and / or preventing symptoms of pancreatic insufficiency in a subject.

28. 28. The pharmaceutical composition of claim 27, wherein the symptoms of pancreatic insufficiency are ameliorated.

29. 29. The pharmaceutical composition of claim 27 or 28, wherein the subject is able to eat a diet whose lipid content is less restricted than the diet required by subjects exhibiting the symptoms of pancreatic insufficiency.

30. The pharmaceutical composition of any one of claims 27 to 29, wherein the subject is an infant or a child.

31. 30. The pharmaceutical composition of any one of claims 27 to 29, wherein the subject is an adult or a young adult.

32. A recombinant lipase according to any one of claims 1 to 7 or a composition according to any one of claims 8 and 20 to 26 for use as a medicament.

33. A recombinant lipase according to any one of claims 1 to 7 or a composition according to any one of claims 8 and 20 to 26 for use as a nutritional supplement.

34. A recombinant lipase according to any one of claims 1 to 7 or a composition according to any one of claims 8 and 20 to 26 for use in the treatment or prevention of symptoms of pancreatic insufficiency.