Operated lipase variant

Abstract

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) and basic (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.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 62 / 894,019, filed Aug. 30, 2019, which is hereby incorporated by reference in its entirety 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 thermal stability, protease stability, and stability under a broad range of pH conditions, including acidic (pH < 7) conditions. The present invention also relates to the use of compositions comprising 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 A copy of the Sequence Listing in the form of a formal copy is submitted herewith as an ASCII text file named "CX7-187WO2_ST25.txt", created on Aug. 27, 2020, and having a size of 4.30 megabytes, via EFS-Web simultaneously with this specification. The Sequence Listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0004] Background of the Invention Pancreatic enzyme replacement therapy (PERT) has been found to be useful in the treatment of pancreatic exocrine insufficiency (PEI). A variety of disorders, including pancreatitis, cystic fibrosis, celiac disease, inflammatory bowel disease, and pancreatic cancer, can result in PEI as a consequence of reduced secretion of pancreatic enzymes into the duodenum. This leads to poor digestion of food and inadequate absorption of fat, protein, carbohydrates, and vitamins by the intestine, which can lead to malnutrition. Although orally administered PERT treatments are currently available, in some people the condition may not be alleviated due to insufficient activity of PERT in the gastrointestinal tract and / or poor treatment compliance by patients due to the significant pill burden associated with current treatment protocols. In some cases, the coefficient of fat absorption (CFA) and / or the coefficient of nitrogen absorption (CNA) are inferior to those of healthy patients, leading to weight loss and other health concerns. Thus, a need remains in the art for improved PERT treatments. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] SUMMARY OF THE INVENTION The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides are optimized to provide improved thermal stability, protease stability, and stability under a broad range of pH conditions, including acidic (pH < 7) conditions. The present invention also relates to the use of compositions comprising 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 a recombinant lipase and / or a biologically active recombinant lipase fragment comprising an amino acid sequence having 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 the 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 NO: 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 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,comprising at least one substitution or set of substitutions at one or more positions selected from 385, and 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 2H, 2M, 2T, 3A, 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 / 199L, 82F, 82G, 82L, 83G, 83K, 85I, 85W, 87R, 89A, 89T, 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,Comprising at least one substitution or combination 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, and 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 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, 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,Comprising at least one substitution or combination of substitutions selected from 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, 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 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 / 296 / 300, 149 / 296, 149 / 296 / 300, 174 / 231, 174 / 296, 218, 231, 231 / 292 / 296, 231 / 292 / 296 / 300, 231 / 296, 231 / 300, 292 / 296, 292 / 296 / 300, 296, and 296 / 300, and 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 comprises 3S / 4W / 96E / 296R / 300D,3S / 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 / 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 at least one substitution or combination of substitutions selected from 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 or a 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: 94, and the recombinant lipase is 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 / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / 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 / 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 at least one substitution or combination of substitutions selected from 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 4, 4 / 27 / 189 / 300, 4 / 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 / 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, 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, 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 comprises at least one substitution or combination of substitutions selected from 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 / 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 / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218M / 300D / 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 comprises at least one substitution or combination of substitutions selected from S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S189A / Q300D, 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 / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S369N, Y27V / 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 / 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, and 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 at least one substitution or combination of substitutions at one or more positions selected from 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 / 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, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281, and 330, and 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 comprises at least one substitution or combination of substitutions selected from 26A, 26R, 26S, 33Y, 33Y / 174H / 193L / 243R, 33Y / 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, 174H / 218M / 233R / 271D / 293R / 303P, 174H / 218M / 271D / 303P, 174R, 175G / 193L / 218M / 233R / 243R / 375A, 175G / 193L / 218M / 243R, 175G / 218M / 375A, 181Q, 189H, 193L, 193L / 218M / 243R, 193L / 271D / 303P / 334T, 193L / 293R / 303P, 194T, 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, and 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 comprises at least one substitution or combination of substitutions selected from G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / 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, 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, and the recombinant lipase comprises at least one substitution or combination of substitutions at one or more positions selected from 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, 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, 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, and the amino acid positions of the polypeptide sequence areIt 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 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 / 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, 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 / 243R / 330H, 38G / 195I / 281P / 330F, 38G / 195Y / 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 / 311W / 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,Comprising at least one substitution or combination 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, and the recombinant lipase is K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, 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 / A303P / 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 / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H,At least one substitution or combination of substitutions selected from F38G / Q195I / G281P / N330F, F38G / 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, S129F, S160T, L161I, E174G / Q195I / M225L / G281K / A303P / N330F / F345I, E174G / M225L / L243R / G281P / A303P / F345I, E174H / Q195I / L243R / G281K / F345I, 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.,

[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 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 / 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 / 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, 237 / 287 / 291,comprising at least one substitution or set of substitutions at one or more positions selected from 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 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 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 / 243R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 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 / 161I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,Comprising at least one substitution or combination of substitutions selected from 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 / 177L / 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 or a 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, and the recombinant lipase is 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, 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 / Q193L / S344H, L34K / V49T / Q193L / S344W, L34K / V49T / L243R / 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 / 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, 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 / Q193L / 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 / S344VComprising at least one substitution or combination 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., 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, and 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 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, and the recombinant lipase comprises at least one substitution or set of substitutions selected from 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, 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, the recombinant lipase comprises at least one substitution or combination 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 comprises at least one substitution or set of substitutions at one or more positions selected from 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / 252 / 271 / 287 / 292 / 293 / 296 / 303 / 334 / 344 / 373 / 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, and the recombinant lipase comprises 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, 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, 271QAt least one substitution or combination of substitutions selected from 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, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293P, 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, 303N, 303Q, 303R, 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, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase is at least 85% relative to SEQ ID NO: 868,A polypeptide sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, or a functional fragment thereof, and the recombinant lipase is 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 / T334D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M24R, M24S, M24T, M24V, M24W, M24Y, G38A, G38C, G38D, G38E, G38F, G38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I161N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C, R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R174P, R174Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L175E, L175F,L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R, L175S, L175T, L175V, L175W, L175Y, A189C, A189D, A189E, A189F, A189G, A189H, A189I, A189K, A189L, A189M, A189N, A189P, A189Q, A189R, A189S, A189T, A189V, A189W, A189Y, L193A, L193C, L193D, L193E, L193F, L193G, L193H, L193I, L193K, L193M, L193N, L193P, L193Q, L193R, L193S, L193T, L193V, L193W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, 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, E231W, E231Y, R243A, R243C, R243D, R243E, R243F, R243G, R243H, R243I, R243K, R243L, R243M, R243N, R243P, R243Q, R243S, R243T, R243V, R243W, R243Y, V252A, V252C, V252D, V252E, V252F, V252G, V252H, V252I, V252K, V252L, V252M, V252N, V252P, V252Q, V252R, V252S, V252T, V252W, V252Y, D271A, D271C, D271E, D271F, D271G, D271H, D271I, D271K, D271L, D271M, D271N, D271P, D271Q, D271R, D271S, D271T, D271V, D271W, D271Y, L287A, L287C, L287D, L287E, L287F, L287G, L287H, L287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287Y,L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292K, L292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R2 at least one substitution or combination of substitutions selected from 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, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303S, 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, H344R, H344S, H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F373Q, F373R, 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, wherein 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 shown in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1, said positions being 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 its enzyme activity after exposure to high and / or low temperatures. In some additional embodiments, the recombinant lipase retains a higher enzyme activity after exposure to high and / or low temperatures as compared to a 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 both low pH and high pH environments, and in some additional embodiments, the lipase is stable and / or active in low pH, neutral pH, and high pH environments. In some embodiments, the recombinant lipase retains enzyme activity after exposure to low pH, high pH, and / or neutral pH environments. In some additional embodiments, the recombinant lipase is more stable and / or active in high pH, neutral pH, and / or low pH environments compared to a 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 less than 7 (i.e., below acidic pH conditions or levels) compared to the lipase of SEQ ID NO: 2. In some additional embodiments, the recombinant lipase is more stable and / or active at a pH less than 7 (i.e., below acidic pH conditions or levels) compared to the lipases of SEQ ID NOs: 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 less than 5 compared to the lipase of SEQ ID NO: 2. In some additional embodiments, the recombinant lipase is more stable and / or active at pH 5 compared to the lipase of SEQ ID NO: 2. In some additional embodiments, the recombinant lipase is more stable and / or active at pH 5 compared to the lipases of SEQ ID NOs: 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 compared to 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 lipases of SEQ ID NOs: 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 lipases of SEQ ID NOs: 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 lipases of SEQ ID NOs: 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 embodiments, the recombinant lipase retains its enzyme activity after exposure to bile salts. In some additional embodiments, the recombinant lipase retains a higher enzyme activity after exposure to bile salts as compared to a reference sequence. In some embodiments, the reference sequence is the lipase of SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868. In some additional embodiments, the bile salt is taurocholate.

[0018] In some additional embodiments, the recombinant lipase is more stable and / or active at acidic pH, more heat resistant, 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 some further embodiments, the lipase is stable in food and / or beverage. In some additional embodiments, the recombinant lipase is more stable and / or active at acidic pH, more heat resistant, more resistant to proteolysis, and / or more active in the presence of at least one bile salt than the lipases 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt as 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt as compared to the lipases 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt as 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as compared to the lipases of SEQ ID NOs: 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as compared to the lipases of SEQ ID NOs: 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 thermal stability, improved resistance to proteolysis, and improved activity in the presence of at least one bile salt, as 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 thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as compared to the lipases of SEQ ID NOs: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least one additional improved property in addition to at least one improved property selected from improved stability and / or activity at acidic pH, improved thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as compared to the lipase of SEQ ID NO: 2.In some embodiments, the recombinant lipase exhibits at least one additional improved property in addition to at least one improved property selected from improved stability and / or activity at acidic pH, improved thermal stability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, as compared to the lipases 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 heat resistance; or any combination of i), ii), iii), iv), v), vi), and vii), as compared to a reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 2, although 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 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 heat resistance; or any combination of i), ii), iii), iv), v), vi), and vii), as compared to at least one reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 2, although 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 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), when 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 a recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. In some embodiments, the recombinant polynucleotide sequence is 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 to 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 the odd-numbered sequences of SEQ ID NOs: 13 to 1795. In some additional embodiments, the present invention provides at least one sequence set forth in the odd-numbered sequences of SEQ ID NOs: 13 to 1795. In some additional embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences of SEQ ID NOs: 13 to 1795. In some embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences of SEQ ID NOs: 13 to 1795, wherein said 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 to 1795, wherein said sequence encodes a recombinant polypeptide provided in the even-numbered sequences of SEQ ID NOs: 14 to 1796. In some further embodiments, the recombinant polynucleotide encoding the recombinant lipase provided herein 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 to 1795.In some further embodiments, the recombinant polynucleotide encoding the 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 to 1795. In some further embodiments, the recombinant polynucleotide encoding the 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 to 1795, and the recombinant lipase comprises a polypeptide sequence comprising the even-numbered sequences provided in SEQ ID NOs: 14 to 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 control sequence. In some embodiments, the control 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 a recombinant lipase provided herein. In some embodiments, the host cell is eukaryotic, but 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 a method for producing at least one recombinant lipase, comprising culturing at least one host cell provided herein under conditions in which a recombinant lipase encoded by a recombinant polynucleotide is produced. In some embodiments, these methods further comprise the step of recovering the lipase. In some further additional embodiments, these methods further comprise the step of purifying the lipase.

[0023] The present invention also provides a composition 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 the treatment of pancreatic insufficiency. In some additional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion into a human. In some additional embodiments, the pharmaceutical composition is suitable for oral administration to a human. In some further embodiments, the pharmaceutical composition is suitable for other modes of administration to a human. The present invention also provides a composition comprising at least one recombinant lipase provided herein, which is suitable for other uses.

[0024] The present invention also provides a method for treating and / or preventing symptoms of pancreatic insufficiency in a subject, the method comprising providing a subject having pancreatic insufficiency and providing the pharmaceutical composition provided herein to the subject. In some embodiments, when a composition comprising at least one recombinant lipase is administered to a subject, the symptoms of pancreatic insufficiency are improved 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 some further 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 that is less restricted in its lipid content than the diet 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 pediatric patient. In still 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 medicament comprising at least one recombinant lipase provided herein.

[0026] In some embodiments, the present invention provides a method for degrading fat and / or lipid, 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 where the fat and / or lipid is degraded. In some additional embodiments, the present invention provides a method for degrading fat and / or lipid, 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 where the fat and / or lipid is degraded. The present invention also provides the use of a composition 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 such that the fat and / or lipid is hydrolyzed by the engineered amylase. In some further embodiments, the present invention provides a method for fat and / or lipid hydrolysis, comprising providing a composition comprising a 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 thermal stability, protease stability, and stability under a broad range of pH conditions, including acidic (pH < 7) conditions. The present invention also relates to the use of compositions comprising engineered lipase polypeptides for therapeutic purposes. In some embodiments, the lipase variants of the present invention have been found to be useful in PERT treatment in the PEI state. In some additional embodiments, the lipase is administered in a form that does not require enteric coating and / or proton pump inhibitors (PPIs). 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 have the same meaning as commonly understood by one of ordinary skill in the art. In general, the nomenclature used herein, as well as the laboratory procedures in cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry, and nucleic acid chemistry described below, are well known and commonly employed in the art. Such techniques are well known and described in numerous textbooks and references that are well known to those of skill in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned in both the above and below specification are hereby expressly incorporated herein by reference.

[0029] Any suitable methods and materials similar or equivalent to those described herein may be used in the practice of the invention, although several methods and materials are described herein. Specific methodologies, protocols, and reagents may vary depending on the circumstances in which they are used by those skilled in the art, so it should be understood that the invention is not limited to the specific methodologies, protocols, and reagents described. Accordingly, the terms defined immediately below are more fully explained by reference to the entire present application. All patents, patent applications, papers, and publications referred to in both the above and below of this specification are hereby 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] Numeric ranges are inclusive of the numbers defining the range. Accordingly, all numeric ranges disclosed herein are intended to include all narrower numeric ranges that fall within such broader numeric ranges as if all such narrower numeric ranges were expressly written herein. All maximum (or minimum) numeric limitations disclosed herein are also intended to include all lower (or higher) numeric limitations as if all such lower (or higher) numeric limitations were expressly written herein.

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

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

[0034] Unless otherwise indicated, each nucleic acid is written left to right in the 5' to 3' direction, and each amino acid sequence is written left to right in the amino to carboxy direction.

[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, the "EC" number refers 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 of enzymes based on the chemical reactions catalyzed by the enzymes.

[0037] As used herein, "ATCC" refers to the American Type Culture Collection whose biological repository collection includes 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 polymers that includes fatty acids and their esters, sterols, prenols, certain fat-soluble vitamins, and other related compounds. "Fat" is a subpopulation of lipids composed of fatty acid esters (e.g., triglycerides made from glycerol and three fatty acids). The present invention is not intended to be limited to any particular lipid and / or fat. Considering the context, the terms "fat" and "lipid" 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 a number of 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 proteins, protein catabolism, and cell signaling.

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

[0043] "Amino acid" is represented herein by either the generally known three-letter symbol or one-letter symbol recommended by the Biochemical Nomenclature Commission of IUPAC-IUB. Similarly, nucleotides can be represented by the generally accepted one-letter code.

[0044] The terms "engineered", "recombinant", "non-naturally occurring" and "variant", when used with respect to a cell, polynucleotide or polypeptide, refer to a material that has been modified in a manner that does not otherwise occur in nature or that is identical to a material in its native or natural form, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques, or a material corresponding to the native or natural form of such material.

[0045] Recombinant polypeptides can be produced using any suitable method known in the art. The gene encoding the 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, S. cerevisiae. Variants of the recombinant polypeptide can be made by a variety of methods known in the art. In fact, there are a wide variety of different mutagenesis techniques well known to those skilled in the art. Furthermore, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for performing specific substitutions (site-directed) at defined amino acids, specific or random mutations (region-directed) in local regions of the gene, or random mutagenesis across the entire gene (e.g., saturation mutagenesis). A number of suitable methods for generating enzyme variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single-stranded 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 the manipulation of DNA and proteins are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After making the variant, the variant can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability and / or acid pH stability, etc.). In some embodiments, a "recombinant lipase polypeptide" (also referred to herein as an "engineered lipase polypeptide", a "variant lipase enzyme", and a "lipase variant") is 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 that exists in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

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

[0048] The term "percent(%) sequence identity" is used herein to refer to the comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues are present to obtain the number of matching positions, dividing the number of matching 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 where identical nucleic acid bases or amino acid residues are present in both sequences, or where nucleic acid bases or amino acid residues are aligned with gaps, to obtain the number of matching positions, dividing the number of matching 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. The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

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

[1970] ), by the similarity search method of Pearson and Lipman (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 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), but are not limited thereto. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al. supra). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs that contain them. These word hits are then extended in both directions along each sequence as far as possible while increasing the cumulative alignment score. The cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for non-matching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative alignment score drops by an amount X from its maximum achieved value; when the cumulative score goes to zero or below due to the accumulation of one 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 an 11-word length (W), an expectation value (E) of 10, M = 5, N = -4, and a comparison of both strands as the default settings.In the case of amino acid sequences, the BLASTP program uses an initial setting of a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determination of sequence alignment and % sequence identity can be made using the BESTFIT or GAP programs of the GCG Wisconsin Software package (Accelrys, Madison WI) using the provided initial setting parameters.

[0049] As used herein, "reference sequence" refers to a defined sequence used as a basis for array comparison. The reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, the 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 full-length, of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences that are similar between the two sequences (i.e., part of the complete sequence) and (2) can further contain sequences that differ between the two polynucleotides, sequence comparison between two (or more) polynucleotides or polypeptides typically involves comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions where the sequences are similar. In some embodiments, the "reference sequence" can be based on a primary amino acid sequence, and the reference sequence is a sequence that can have one or more changes in the primary sequence. A "comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, within which a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and the portion of the sequence within the comparison window can contain additions or deletions (i.e., gaps) of 20% or less compared to the reference sequence (without including additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 consecutive residues and can include windows of 30, 40, 50, 100, or longer as needed.

[0050] As used in the context of numbering of a given amino acid or polynucleotide sequence, "corresponding to", "reference to", or "relative to" refers to the numbering of the residues of the designated reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is specified with respect to the reference sequence, rather than by the actual numerical position of the residues 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 matches between the two sequences. In these cases, gaps are present, but the numbering of the residues in the 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., 1 or more) of substitutions, insertions, deletions, and / or rearrangements that are present in the sequence (i.e., as compared to the starting or reference sequence). Thus, a mutation in a sequence results in the production of a variant polypeptide (e.g., a variant or recombinant lipase) as provided herein.

[0052] As used herein, "amino acid difference" or "residue difference" refers to a difference in the 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 the 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 a difference in the amino acid residue at the polypeptide position corresponding to position 92 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has threonine at position 92, "a residue difference at position X92 compared to SEQ ID NO:2" means that an amino acid residue other than threonine is present at the position of the polypeptide corresponding to position 92 of SEQ ID NO:2 (e.g., T92A). In most examples herein, a particular amino acid residue difference at a position is shown as "XnY", where "Xn" designates the corresponding position as described above and "Y" is the one-letter identifier of the amino acid found in the engineered polypeptide (i.e., a residue different from the residue in the reference polypeptide). In some examples (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 a particular amino acid difference denoted by the conventional notation "AnB", where A is the one-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 one-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some examples, the polypeptides of the disclosure can include one or more amino acid residue differences relative to a reference sequence, as indicated by a list of specified positions at which residue differences exist relative to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position of a 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 variants include more than one substitution. These substitutions are separated by slashes for readability (e.g., Y27V / F385P).This application includes engineered polypeptide sequences that contain one or more amino acid differences, including one or both of conservative amino acid substitutions 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 having a similar side chain, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid within the same or a similar defined class of amino acids. By way of example, and not limitation, an amino acid having an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain may be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain may be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain may be substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain may be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid may be replaced 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 characteristics. Non-conservative substitutions may use amino acids from outside of the defined groups, rather than within the defined groups, and affect (a) the structure of the peptide backbone in the region of the 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 the removal of 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 a maximum of 10% of the total number of amino acids, or up to a maximum of 20% of the total number of amino acids, from the reference enzyme while retaining the enzyme activity of the engineered enzyme and / or while retaining improved properties. Deletions can target internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous.

[0056] As used herein, "insertion" refers to a modification to a polypeptide by the addition of one or more amino acids to a reference polypeptide. Insertions can be to an internal portion of the polypeptide or to the carboxy or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be a contiguous segment of amino acids or can be separated by one or more amino acids present 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 compared to the starting or reference sequence due to the presence of a stop codon.

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

[0059] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with the polypeptide. The term encompasses polypeptides that have been removed from or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). A recombinant lipase polypeptide can be present intracellularly, can be present in the cell culture medium, or can be prepared in various forms such as lysates or isolated preparations. Thus, in some embodiments, a recombinant lipase polypeptide can 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 is naturally associated with the molecule. The term "purified" is not intended to 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., is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis), and generally, when the species of interest constitutes at least about 50% of the macromolecular species present, on a molar or weight percentage basis, is a composition that is substantially purified. Generally, a substantially pure lipase composition constitutes 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 basis. In some embodiments, the species of interest is purified to essential homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods) from the starting preparation, and the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the 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, a substantially pure recombinant lipase polypeptide preparation (e.g., polysaccharides, surfactants, etc.) added to a formulation suitable for use in the present invention.

[0062] As used herein, the terms "improved enzyme characteristics" and "improved characteristics" refer to the characteristics of an engineered lipase polypeptide that include an improvement in any enzyme characteristic as compared to a reference lipase polypeptide and / or a wild-type lipase polypeptide or another engineered lipase polypeptide. Improved characteristics include, but are not limited to, increased protein expression, increased thermoactivity, increased thermal stability, increased pH activity, increased stability, increased enzyme 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., decreased susceptibility to proteolysis), decreased aggregation, increased solubility, decreased immunogenicity, improved post-translational modification (e.g., glycosylation), and altered temperature profile.

[0063] As used herein, "increased enzyme activity" or "enhanced catalytic activity" refers to an improved characteristic of an engineered lipase polypeptide that can be represented by an increase in specific activity (e.g., product generated / time / weight of protein) or an increase in the percent conversion of substrate to product (e.g., percent conversion of the starting amount of substrate to product over a specified period using a specified amount of lipase) as compared to a reference lipase enzyme. Exemplary methods for determining enzyme activity are provided in the Examples. m , V max or k cat Any characteristic associated with enzyme activity, including classical enzyme characteristics of K

[0064] Lipase activity can be measured by any suitable method known in the art, such as standard assays that monitor changes in the spectrophotometric properties of reactants or products. In some embodiments, the amount of product generated can be measured by high performance liquid chromatography (HPLC) separation combined with UV absorbance. In some embodiments, the amount of product generated can be measured using a RAPIDFIRE® mass spectrometer, although in some other embodiments, the product can be measured using alternative methods known in the art. Comparison of enzyme activities is done using a defined preparation of the enzyme, a defined assay under defined conditions, and one or more defined substrates, as described in more detail herein.

[0065] As used herein, the terms “protease stable” and “stability to proteolysis” refer to the ability of a protein (e.g., a recombinant lipase of the invention) to function and withstand proteolysis mediated by any protease or other proteolytic compound or factor and retain its function after exposure to the protease. The term is not intended to be limited to the use of any particular protease for assessing the stability of a protein. Indeed, engineered lipases of the invention are stable in the presence of or after exposure to various proteases and retain their enzyme activity. In some embodiments, the engineered lipase is stable in the presence of trypsin, chymotrypsin, and / or pepsin. However, the invention is not intended to be limited to any particular protease or any particular method for 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 present invention) to function after incubation at a specific pH. In some embodiments, the present invention provides a recombinant lipase that is stable in a pH range including but not limited to the range of pH 2 to pH 7. In some embodiments, the recombinant lipase is stable in different pH ranges as shown in the examples provided herein. It is not intended that the present invention be limited to any particular pH stability level or pH range.

[0067] As used herein, the term "improved tolerance to acidic pH" means that the 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 specific period [e.g., 1 hour, up to 24 hours, etc.]) compared to a reference lipase or another enzyme.

[0068] As used herein, "physiological pH" means the pH range generally found in the blood of a subject (e.g., human) (e.g., pH 7.2 - 7.4).

[0069] The term "basic pH" (used, for example, with respect to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH range of about 7 to 11, or in some embodiments, a pH range greater than pH 11.

[0070] The term "acidic pH" (used, for example, with respect to improved stability to acidic pH conditions or increased tolerance to acidic pH) means a pH range encompassing any pH value less than 7. In some embodiments, the pH of the acid is less than 7, but 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 present invention is stable at pH levels of 2 - 4. However, it is not intended that the present invention be limited to any particular pH value or range of values.

[0071] As used herein, the phrase "gastric challenge" refers to exposing the 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 it might encounter in the stomach (e.g., the human stomach).

[0072] As used herein, the phrase "intestinal challenge" refers to exposing the 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 it might encounter in the intestinal tract (e.g., the human intestine).

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

[0074] The terms "thermal stability" and "heat stability" 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 term refers 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 are resistant to inactivation at high temperatures and, in some additional embodiments, maintain their catalytic activity at high temperatures over extended exposure times. These terms are used interchangeably herein. The present invention is not intended to be limited to any particular temperature and / or exposure time. Such stability can be measured by any method known in the art (e.g., the methods described herein). The present invention is not intended to be limited to any particular level of temperature stability or temperature range. In some embodiments, thermal stability is measured after incubation of a 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 chemical substances that adversely affect the function of another protein. The present invention is not intended to be limited to any particular level of chemical stability or range of chemical stability.

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

[0077] As used herein, "hybridization stringency" relates to hybridization conditions such as washing conditions in nucleic acid hybridization. Generally, a hybridization reaction is performed under lower stringency conditions followed by washing at various, but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow a target DNA to bind to a complementary nucleic acid having greater than about 90% identity to the target polynucleotide and having about 60% identity, preferably about 75% identity, about 85% identity to the target DNA. Exemplary moderately stringent conditions are equivalent to hybridization in 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS at 42°C, followed by washing in 0.2× SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to conditions that are about 10°C or less below the thermal melting temperature T m determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if the hybrid is not stable in 0.018 M 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 at 42°C in conditions equivalent to 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS followed by washing at 65°C in 0.1× SSPE and 0.1% SDS. Another high stringency condition is hybridization in 5× SSC containing 0.1% (w / v) SDS at 65°C and washing in 0.1× SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions and moderately stringent conditions are described in the references cited above.

[0078] As used herein, "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 appropriate control sequences for effecting expression of a polypeptide encoded by the DNA sequence in an appropriate host. In some embodiments, a plasmid is used as the vector. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) for driving expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0079] As used herein, "codon-optimized" refers to the change of codons of a polynucleotide encoding a protein to codons that are preferentially used in a particular organism such that the encoded protein is expressed more efficiently 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, codon usage by a particular organism is non-random and is known to be biased towards certain codon triplets. This codon usage bias can be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the total protein coding regions of the genome of an organism. 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, "control sequence" refers to all components that are 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, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences and transcription terminators. At a minimum, the control sequences include a promoter, as well as transcription and translation stop signals. Linkers may be provided in the control sequences for the purpose of introducing specific restriction sites that facilitate the ligation of the control sequences to 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 appropriately positioned relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest (i.e., in a functional relationship).

[0082] As used herein, "promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for the expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, 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 the host cell.

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

[0084] As used herein, "loading" 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" when used in the context of an enzyme conversion reaction process refers to a compound or molecule acted upon by a lipase polypeptide.

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

[0087] As used herein, the term "expression" includes any process involved in the production of 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 a cell.

[0088] As used herein, the term "produce" refers to the production of proteins and / or other compounds by a cell. This term is intended to encompass any process involved in the production of 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 a 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 naturally associated.

[0090] As used herein, the terms "host cell" and "host strain" refer to a host suitable for an expression vector containing the DNA provided herein (e.g., a polynucleotide encoding a lipase variant). In some embodiments, the 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) with a reference polypeptide. In some embodiments, the analogs include polypeptides containing one or more unnatural amino acid residues (including but not limited to homoarginine, ornithine, and norvaline) as well as naturally occurring amino acids. In some embodiments, the 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 liquid, gel, or solid media). 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. The present invention is not intended to be limited to the culture of any particular cell or cell type or 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, when administered to a subject exhibiting signs or symptoms of a pathology, provides a beneficial or desirable effect, including a medical effect.

[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 produce a desired result. One of ordinary skill in the art can determine the effective amount by using routine experimentation.

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

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

[0098] The term "infant" refers to a child in the period from 1 month to about 1 year after birth.

[0099] As used herein, the term "newborn" refers to a child in the period from birth to 28 days after birth. The term "premature infant" refers to an infant born after the 20th week of pregnancy but before the due date, and generally has a birth weight of about 500 to about 2499 grams. A "very low birth weight infant" is an infant with a weight of less than 1500 g at birth.

[0100] As used herein, the term "pediatric" refers to a person who has not reached the legal age for consent to treatment or research procedures. 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 old in the United States). In some embodiments, the term refers to any fully grown and mature organism. In some embodiments, the term "young adult" refers to a person who is under 18 years old but has reached sexual maturity.

[0102] As used herein, the terms "composition" and "formulation" encompass a product (e.g., a pharmaceutical composition, a dietary supplement and / or a nutritional supplement, a feed, etc.) containing at least one engineered lipase of the present invention, which is intended for any suitable use.

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

[0104] The term "carrier", as used herein with respect to a pharmaceutical composition, means any standard pharmaceutical carrier, buffer, and excipient, such as a stabilizer, preservative, and adjuvant.

[0105] The term "pharmaceutically acceptable" means a material that can be administered to a subject without causing undesirable biological effects or interacting in a harmful manner with any of the components contained therein, and has the desired biological activity.

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

[0107] The term "therapeutically effective amount", when used with respect to the symptoms of a disease / condition, refers to the amount and / or concentration of a compound (e.g., an engineered lipase polypeptide) that improves, 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 with respect to a disease / condition, refers to the amount and / or concentration of a composition (e.g., an engineered lipase polypeptide) that improves, attenuates, or eliminates the disease / condition. In some embodiments, the term is used with respect to the amount of a composition that elicits a biological (e.g., medical) response by a tissue, system, or animal subject as determined 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) that includes administration of a pharmaceutical composition such as the pharmaceutical compositions 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 improve the symptoms of a disease. In some embodiments, the pharmaceutical compositions of the invention are found to be useful in the treatment or prevention of a pancreatic enzyme insufficiency disease or condition.

[0110] Engineered lipase: The present invention provides engineered lipases suitable for a variety of uses including the treatment of pancreatic enzyme insufficiency. In some embodiments, an engineered lipase that exhibits improved properties has 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 with SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868, and has an amino acid residue difference compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868 or 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 than that amino acid sequence identity at 1 or more amino acid positions (e.g., at amino acid positions 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 than that). In some embodiments, the residue difference compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758, and / or 868 at 1 or more positions includes 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 than that conservative amino acid substitutions. However, since other substitutions are found to be used in the present invention, the present invention is not intended to be limited to lipase variants having conservative amino acid substitutions. 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 NOs: 94, 350, 442, 540, 646, 758, and / or 868.

[0111] The present invention provides 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 comprises at least one substitution or set of substitutions at one or more positions selected from 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, and 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 comprises 2H, 2M, 2T, 3A, 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,At least one substitution or combination of substitutions selected from 46T / 70N, 46T / 149E / 183L / 231E / 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, 89T, 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, 270V, 274D, 281K, 281R, 292A, 292C, 292L, 292V, 296M, 296R, 300A, 300D, 300T, 308A, 330Y, 338N, 379T, 385A, 385C, 385D, 385P, 385R, and 385T, and 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 a 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, and the recombinant lipase is 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, 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, F385AComprising at least one substitution or combination 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 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 / 296 / 300, 149 / 296, 149 / 296 / 300, 174 / 231, 174 / 296, 218, 231, 231 / 292 / 296, 231 / 292 / 296 / 300, 231 / 296, 231 / 300, 292 / 296, 292 / 296 / 300, 296, and 296 / 300, and 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 comprises 3S / 4W / 96E / 296R / 300D,3S / 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 / 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 at least one substitution or combination of substitutions selected from 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 or a 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: 94, and the recombinant lipase is 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 / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / 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 / 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 at least one substitution or combination of substitutions selected from 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 4, 4 / 27 / 189 / 300, 4 / 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 / 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, 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, 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 comprises at least one substitution or combination of substitutions selected from 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 / 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 / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218M / 300D / 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 comprises at least one substitution or combination of substitutions selected from S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S189A / Q300D, 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 / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S369N, Y27V / 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 / 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, and 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 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 / 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, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281, and 330, and 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 comprises at least one substitution or combination of substitutions selected from G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / 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, 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 comprises at least one substitution or combination of substitutions at one or more positions selected from 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, 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, 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, and the amino acid positions of the polypeptide sequence areIt 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 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 / 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, 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 / 243R / 330H, 38G / 195I / 281P / 330F, 38G / 195Y / 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 / 311W / 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,Comprising at least one substitution or combination 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, and the recombinant lipase is K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, 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 / A303P / 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 / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H,Comprising at least one substitution or combination of substitutions selected from F38G / Q195I / G281P / N330F, F38G / 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, S129F, S160T, L161I, E174G / Q195I / M225L / G281K / A303P / N330F / F345I, E174G / M225L / L243R / G281P / A303P / F345I, E174H / Q195I / L243R / G281K / F345I, 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 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 / 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 / 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, 237 / 287 / 291,comprising at least one substitution or set of substitutions at one or more positions selected from 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 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 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 / 243R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 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 / 161I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,Comprising at least one substitution or combination of substitutions selected from 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 / 177L / 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, and the recombinant lipase is 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, 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 / Q193L / S344H, L34K / V49T / Q193L / S344W, L34K / V49T / L243R / 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 / 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, 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 / Q193L / 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 / S344VComprising at least one substitution or combination 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, and the recombinant lipase comprises at least one substitution or a combination 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 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, and the recombinant lipase comprises at least one substitution or a combination of substitutions selected from 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, 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, the recombinant lipase comprises at least one substitution or combination 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.

[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 comprises at least one substitution or combination of substitutions at one or more positions selected from 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / 252 / 271 / 287 / 292 / 293 / 296 / 303 / 334 / 344 / 373 / 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, and 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, and the recombinant lipase comprises 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, 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,At least one substitution or combination of substitutions selected from 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, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293P, 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, 303N, 303Q, 303R, 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, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some further embodiments, the recombinant lipase is at least 85% relative to SEQ ID NO: 868,A polypeptide sequence having a sequence identity of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or a functional fragment thereof, and the recombinant lipase is 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 / T334D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M24R, M24S, M24T, M24V, M24W, M24Y, G38A, G38C, G38D, G38E, G38F, G38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I161N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C, R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R174P, R174Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L175E, L175F,L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R, L175S, L175T, L175V, L175W, L175Y, A189C, A189D, A189E, A189F, A189G, A189H, A189I, A189K, A189L, A189M, A189N, A189P, A189Q, A189R, A189S, A189T, A189V, A189W, A189Y, L193A, L193C, L193D, L193E, L193F, L193G, L193H, L193I, L193K, L193M, L193N, L193P, L193Q, L193R, L193S, L193T, L193V, L193W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, 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, E231W, E231Y, R243A, R243C, R243D, R243E, R243F, R243G, R243H, R243I, R243K, R243L, R243M, R243N, R243P, R243Q, R243S, R243T, R243V, R243W, R243Y, V252A, V252C, V252D, V252E, V252F, V252G, V252H, V252I, V252K, V252L, V252M, V252N, V252P, V252Q, V252R, V252S, V252T, V252W, V252Y, D271A, D271C, D271E, D271F, D271G, D271H, D271I, D271K, D271L, D271M, D271N, D271P, D271Q, D271R, D271S, D271T, D271V, D271W, 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 including at least one substitution or combination of substitutions selected from 3D, 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, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303S, 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, H344R, H344S, H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F373Q, F373R, 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 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, or a functional fragment thereof, and the amino acid positions of the recombinant lipase and the 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, the engineered lipase polypeptide comprises a functional fragment of the engineered lipase polypeptide encompassed by the present invention. The functional fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity of the engineered lipase polypeptide from which the functional fragment is derived (i.e., the parent engineered lipase). In some embodiments, the functional fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even 99% of the parent sequence of the engineered lipase. In some embodiments, the functional fragment has less than 5, less than 10, less than 15, less than 20, less than 25, less than 30, less than 35, less than 40, less than 45 and less than 50 amino acids cleaved.

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

[0123] As will be apparent to those skilled in the art, the availability of the protein sequence and knowledge of the codons corresponding to the various amino acids provide a description of all polynucleotides capable of encoding this polypeptide. The degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, allows for the creation of a very large number of nucleic acids, all of which encode the engineered lipase polypeptide. Thus, with knowledge of a specific amino acid sequence, one skilled in the art will be able to create any number of different nucleic acids by simply modifying the sequence of one or more codons so as not to change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates every possible variation of a polynucleotide that can be made to encode the polypeptides described herein by selecting combinations based on possible codon choices, and all such variations are to 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, the codons are preferably selected to be compatible with the host cell in which the protein is being produced. For example, the preferred codons used in bacteria are those used for expression in bacteria, while the preferred codons used in fungi are those used for expression in fungi. As a result, the codon-optimized polynucleotide encoding the engineered lipase polypeptide contains preferred codons in more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions of the full-length coding region.

[0125] In some embodiments, the invention provides a recombinant polynucleotide 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 SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867. In some embodiments, the invention provides a recombinant polynucleotide 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: 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, and the recombinant lipase comprises at least one substitution or combination of substitutions at one or more positions selected from 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, and 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, and the recombinant lipase comprises 2H, 2M, 2T, 3A, 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,Comprising at least one substitution or combination of substitutions selected from 46T / 70N, 46T / 149E / 183L / 231E / 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, 89T, 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, 270V, 274D, 281K, 281R, 292A, 292C, 292L, 292V, 296M, 296R, 300A, 300D, 300T, 308A, 330Y, 338N, 379T, 385A, 385C, 385D, 385P, 385R, and 385T, and 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 or a 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, and the recombinant lipase is 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, 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, F385AComprising at least one substitution or combination of substitutions selected from F385C, F385D, F385P, F385R, and F385T, wherein 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, and the recombinant lipase comprises at least one substitution or a combination of substitutions at one or more positions selected from 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 / 296 / 300, 149 / 296, 149 / 296 / 300, 174 / 231, 174 / 296, 218, 231, 231 / 292 / 296, 231 / 292 / 296 / 300, 231 / 296, 231 / 300, 292 / 296, 292 / 296 / 300, 296, and 296 / 300, and 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, and the recombinant lipase comprisesAt least one substitution or combination of substitutions selected from 3S / 4W / 96E / 296R / 300D, 3S / 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 / 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, and 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 lipase contains at least one substitution or combination of substitutions selected from 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 / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / 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 / 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, and the recombinant lipase comprises at least one substitution or combination of substitutions at one or more positions selected from 4, 4 / 27 / 189 / 300, 4 / 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 / 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, 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, and 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, and the recombinant lipase comprises at least one substitution or combination of substitutions selected from 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 / 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 / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 189A, 174H / 373F, 175G / 189A / 218M / 300D / 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 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, and the recombinant lipase comprises at least one substitution or combination of substitutions selected from S4W, S4W / Y27V / S189A / Q300D, S4W / K70H, S4W / E102T, S4W / S137R, S4W / Q175G / S189A / H218M, S4W / Q175G / S189A / Q300D, 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 / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S189A / H218M / Q300D / S369N / Y373F, Y27V / K82C / E174H / Q175G / H218M / Q300D / S369N, Y27V / 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 / 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, and 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, and the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from 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 / 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, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281, and 330, and 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, and the recombinant lipase comprises at least one substitution or combination of substitutions selected from G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / 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, 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.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, and the recombinant lipase comprises at least one substitution or combination of substitutions selected from G26A, G26R, G26S, L33Y, L33Y / E174H / Q193L / 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, 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.

[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, and the recombinant lipase comprises at least one substitution or combination of substitutions at one or more positions selected from 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, 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, 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, and the amino acid positions of the polypeptide sequence areIt is 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, and the recombinant lipase is 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 / 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, 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 / 243R / 330H, 38G / 195I / 281P / 330F, 38G / 195Y / 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 / 311W / 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,Comprising at least one substitution or combination 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, and the recombinant lipase is K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, 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 / A303P / 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 / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H,Comprising at least one substitution or combination of substitutions selected from F38G / Q195I / G281P / N330F, F38G / 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, S129F, S160T, L161I, E174G / Q195I / M225L / G281K / A303P / N330F / F345I, E174G / M225L / L243R / G281P / A303P / F345I, E174H / Q195I / L243R / G281K / F345I, 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.,

[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, and the recombinant lipase is 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 / 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 / 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,Comprising at least one substitution or set of substitutions at one or more positions selected from 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 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, and the recombinant lipase is 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 / 243R / 252V / 344W, 34K / 49T / 193L / 344H, 34K / 49T / 193L / 344W, 34K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 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 / 161I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P,At least one substitution or combination of substitutions selected from 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 / 177L / 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, and the recombinant lipase is 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, 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 / Q193L / S344H, L34K / V49T / Q193L / S344W, L34K / V49T / L243R / 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 / 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, 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 / Q193L / 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 / F287LComprising at least one substitution or combination 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, and the recombinant lipase comprises at least one substitution or combination 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 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, and the recombinant lipase comprises at least one substitution or combination of substitutions selected from 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, 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, and the recombinant lipase comprises at least one substitution or combination 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.

[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, and the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 193 / 225 / 231 / 243 / 252 / 271 / 287 / 292 / 293 / 296 / 303 / 334 / 344 / 373 / 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, and 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, and the recombinant lipase comprises 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, 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,At least one substitution or combination of substitutions selected from 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, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 293L, 293M, 293N, 293P, 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, 303N, 303Q, 303R, 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, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 868. In some embodiments, the polynucleotide isEncoding 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, the recombinant lipase being 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 / T334D / H344S / F373Y / P385F, M24G, M24H, M24I, M24K, M24L, M24N, M24P, M24Q, M24R, M24S, M24T, M24V, M24W, M24Y, G38A, G38C, G38D, G38E, G38F, G38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I161N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C, R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R174P, R174Q, R174S, R174T, R174V, R174W, R174Y,L175A, L175C, L175D, L175E, L175F, L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R, L175S, L175T, L175V, L175W, L175Y, A189C, A189D, A189E, A189F, A189G, A189H, A189I, A189K, A189L, A189M, A189N, A189P, A189Q, A189R, A189S, A189T, A189V, A189W, A189Y, L193A, L193C, L193D, L193E, L193F, L193G, L193H, L193I, L193K, L193M, L193N, L193P, L193Q, L193R, L193S, L193T, L193V, L193W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, 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, E231W, E231Y, R243A, R243C, R243D, R243E, R243F, R243G, R243H, R243I, R243K, R243L, R243M, R243N, R243P, R243Q, R243S, R243T, R243V, R243W, R243Y, V252A, V252C, V252D, V252E, V252F, V252G, V252H, V252I, V252K, V252L, V252M, V252N, V252P, V252Q, V252R, V252S, V252T, V252W, V252Y, D271A, D271C, D271E, D271F, D271G, D271H, D271I, D271K, D271L, D271M, D271N, D271P, D271Q, D271R, D271S, D271T, D271V, D271W, D271Y, L287A, L287C, L287D, L287E, L287F, L287G, L287H, L287I, L287K, L287M, L287N, L287P, L287Q, L287R,L287S, L287T, L287V, L287W, L287Y, L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292K, L292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V, , including at least one substitution or combination of substitutions selected from 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, R296T, R296V, R296W, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P303H, P303I, P303K, P303L, P303M, P303N, P303Q, P303R, P303S, 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, H344R, H344S, H344T, H344V, H344W, H344Y, F373A, F373C, F373D, F373E, F373G, F373H, F373I, F373K, F373L, F373M, F373N, F373P, F373Q, F373R, 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.

[0134] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having lipase activity with the characteristics disclosed herein, and the polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence (e.g., SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868) or an amino acid sequence of any variant disclosed in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7 and / or 5-1, and has 1 or more residue differences (e.g., 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, or more amino acid residue positions) compared to the amino acid sequence of the reference polypeptide of SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868 or any variant disclosed in said tables. In some embodiments, the polynucleotide encodes an engineered polypeptide having lipase activity with the characteristics disclosed herein, and the polypeptide has at least 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 SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868 when optimally aligned with the polypeptide of SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, and has 1 or more residue differences compared to SEQ ID NO: 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 sequences 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 sequences 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 sequences of SEQ ID NOs: 93, 349, 441, 539, 645, 757 and / or 867.

[0136] In some embodiments, the polynucleotide can hybridize to a reference polynucleotide sequence under high stringency conditions. In some embodiments, the reference sequence is selected from the polynucleotide sequences encoding any of SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867, or their complements, or the modified lipase polypeptide provided herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes a lipase polypeptide having an amino acid sequence with 1 or more residue differences compared to SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867 at residue positions selected from any positions described 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 is 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 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence selected from SEQ ID NO: 1, 93, 349, 441, 539, 645, 757 and / or 867. In some additional embodiments, the polynucleotide comprises 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 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 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 as compared to the reference polynucleotide sequence. In some further 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 as 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 as compared to the reference polynucleotide sequence.

[0137] In some embodiments, an isolated polynucleotide encoding any of the engineered lipase polypeptides provided herein is engineered in various ways to provide for gene expression and polypeptide production. In some embodiments, a polynucleotide encoding a polypeptide is provided as an expression vector having in it one or more control sequences to regulate the 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 polynucleotide and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0138] In some embodiments, control sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. As is known in the art, appropriate control sequences can be selected based on the host cell being used. The present invention is not intended to be limited to any particular control sequence.

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

[0140] Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes of 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), in addition to the NA2-tpi promoter (a hybrid of the promoters from the genes of Aspergillus niger neutral α-amylase and Aspergillus oryzae triosephosphate isomerase), and variants, truncated forms, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes of 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. 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, cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), Homo Sapiens phosphoglycerate kinase, β-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or those derived from Gallus gallus β-actin.

[0142] In some embodiments, the control sequence is a suitable transcription terminator sequence, i.e., a sequence recognized by the 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 is used in the present invention. For example, exemplary transcription terminators for bacterial host cells can be obtained from T7 bacteriophage for the T7 terminator, or 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 of 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 of 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, e.g., 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, an untranslated region of the mRNA that is 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 selected host cell can be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the genes of 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).

[0144] In some embodiments, the control sequence is a polyadenylation sequence, i.e., a sequence operably linked to the 3' end of the nucleic acid sequence that can also be recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA when transcribed. Any polyadenylation sequence that is functional in the selected host cell can 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 of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Polyadenylation sequences useful for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0145] In some embodiments, the control sequence encodes an amino acid sequence linked to the amino terminus of the polypeptide and is a signal peptide coding region that directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region that is naturally linked in the translation reading frame to a segment of the coding region encoding 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 the selected host cell is used for the expression of the engineered lipase polypeptide provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Signal peptides useful for yeast host cells include, but are not limited to, those derived from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Signal peptides useful for mammalian host cells include, but are not limited to, those derived from the gene of immunoglobulin gamma (IgG).

[0147] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is, in some cases, referred to as a "proenzyme", "propolypeptide" or "zymogen". The 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 a recombinant expression vector comprising a polynucleotide encoding an engineered lipase polypeptide and one or more expression regulatory regions such as a promoter, a terminator, an origin of replication, etc., depending on the type of host to be introduced. In some embodiments, the various nucleic acids and control sequences described above are ligated together to create a recombinant expression vector that includes one or more convenient restriction sites for insertion or substitution of a nucleic acid sequence encoding a variant lipase polypeptide at one or more convenient restriction sites. Alternatively, the polynucleotide sequence of the present invention is expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into a suitable vector for expression. In the construction of an expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to a suitable control sequence for expression.

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

[0150] In some embodiments, the expression vector is a vector that replicates autonomously (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, episome, minichromosome or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector may be one that integrates into the genome when introduced into the host cell and is replicated along with the chromosome into which the vector has integrated. Further, a single vector or plasmid, or two or more vectors or plasmids that together contain all the DNA 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 enable easy selection of the transformed cells. A "selectable marker" is a gene whose product provides biocide resistance or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, and the like. Markers suitable for bacterial host cells include, but are not limited to, carbenicillin, ampicillin, chloramphenicol, tetracycline, kanamycin, and zeocin. Markers suitable 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 adenylyltransferase), and trpC (anthranilate synthase) and their equivalents. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered lipase polypeptide of the present application, the polynucleotide being operably linked to one or more control sequences for the expression of the engineered lipase enzyme in the host cell.

[0152] Host cells for use in expressing the polypeptide encoded by the expression vector of the present 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 number 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 fibroblasts, THP-1, Jurkat, and Bowes melanoma cell lines), but are not limited thereto.

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

[0154] Suitable culture media and growth conditions for the above host cells are well known in the art. The polynucleotide for the expression of lipase 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] An engineered lipase having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered lipase polypeptide 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, inter alia, the staggered extension process (StEP), in vitro recombination (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. The gene encoding the 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 the recombinant polypeptide can be produced by various methods known in the art. In fact, there are a wide variety of different mutagenesis techniques well known to those skilled in the art. Furthermore, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for performing specific substitutions (site-directed) at defined amino acids, specific or random mutations in local regions of the gene (region-directed), or random mutagenesis across the entire gene (e.g., saturation mutagenesis). A number of suitable methods for generating enzyme variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single-stranded 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 the polynucleotide encoding the enzyme to generate a library of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method is used in the present invention and is well known in the art (e.g., U.S. Patent 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, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,No. 484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,483,011, No. 6,484,105, No. 6,489,146, No. 6,500,617, No. 6,500,639, No. 6,506,602, No. 6,506,603, No. 6,518,065, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, No. 6,613,514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6,825,001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,220,566, No. 7,288,375, No. 7,384,387, No. 7,421,347, No. 7,430,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,U.S. Patent Nos. 7,783,428; 7,873,477; 7,873,499; 7,904,249; 7,957,912; 7,981,614; 8,014,961; 8,029,988; 8,048,674; 8,058,001; 8,076,138; 8,108,150; 8,170,806; 8,224,580; 8,377,681; 8,383,346; 8,457,903; 8,504,498; 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. U.S.A., 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,See 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, enzyme variants obtained after mutagenesis treatment are screened by subjecting the enzyme variants to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other assay conditions. Next, DNA containing a polynucleotide encoding the lipase polypeptide is isolated from the host cell, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in different or the same host cells. Measuring enzyme activity from an expression library can be done using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0158] In the case of an engineered polypeptide of a known sequence, the polynucleotide encoding the enzyme can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical ligation methods, or methods mediated by polymerase) to form any desired contiguous sequence. The classical phosphoramidite method is typically performed by an automated synthetic method, so that, for example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (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., on an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector. However, since any suitable method can be used in the present invention, it is not intended that the present invention be limited to any particular method for the manufacture of polynucleotides and oligonucleotides.

[0159] Accordingly, 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 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 an amino acid sequence selected from SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868; and (b) expressing the lipase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide includes deletions, insertions and / or substitutions of one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residues. In some embodiments, the amino acid sequence includes deletions, insertions and / or substitutions of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the amino acid sequence includes 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 includes 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, the substitutions include conservative substitutions and / or non-conservative substitutions.

[0160] The expressed and engineered lipase polypeptide can be evaluated for any desired improved properties (e.g., activity, selectivity, stability, acid tolerance, protease sensitivity, 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 one or more of the well-known techniques for protein purification, including but not limited to lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, and chromatography, are used to recover the engineered lipase polypeptide expressed in the host cell from the cells and / or the culture medium.

[0162] Chromatography 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. The conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate the improved variant lipase enzyme. 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 a glycan covalently attached to the lipase is used. In still 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 conjugated 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, an 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 the engineered lipase polypeptide under conditions that promote the production of the engineered lipase polypeptide, and recovering the engineered lipase polypeptide from the cells and / or the culture medium.

[0165] In some embodiments, the present invention, when optimally aligned with the amino acid sequences of SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758 and / or 868, has 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 NOs: 2, 94, 350, 442, 540, 646, 758 and / or 868), and amino acid residue differences 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, and has 1 or more amino acid residue differences compared to SEQ ID NOs: 2, 94, 350, 442, 540, 646, 758 and / or 868, and culturing a recombinant eukaryotic cell comprising a polynucleotide sequence encoding an engineered lipase polypeptide 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, when the engineered polypeptide is recovered from a 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 components and compounds to provide compositions and formulations (e.g., pharmaceutical compositions) containing the engineered lipase polypeptide suitable for different applications and uses. In some additional embodiments, the purified v polypeptide or formulated lipase polypeptide is lyophilized.

[0167] Composition: The present invention provides various 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 nutraceuticals and / or dietary supplements.

[0168] Depending on the mode of administration, these compositions containing a therapeutically effective amount of the engineered lipase according to the present invention are in solid, semi-solid or liquid form. In some embodiments, the composition includes other pharmaceutically acceptable components 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 are 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 is used 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, coatings, aerosols, chewing gums, lubricants, sticks, solutions, suspensions (including, but not limited to, oily suspensions, water-in-oil 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., an injectable formulation). In some embodiments, the engineered lipase polypeptide is provided in a biocompatible matrix such as a sol-gel comprising a silica-based (e.g., oxysilane) sol-gel. In some embodiments, the engineered lipase polypeptide is encapsulated and / or enteric coated. In some alternative embodiments, the engineered lipase polypeptide is encapsulated within nanostructures (e.g., nanotubes, 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. The engineered lipase polypeptide is intended to be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implant, and the like.

[0170] In some embodiments, the engineered lipase polypeptide is chemically modified by glycosylation, chemical crosslinking reagents, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (e.g., see 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.). In fact, the present invention is not intended to 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 enzyme crystals stabilized in a matrix. 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 an engineered lipase polypeptide in a polymer.

[0172] In some embodiments, the composition comprising the engineered lipase polypeptide of the present invention comprises 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 plant starches), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.).

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

[0174] In some embodiments, the engineered lipase polypeptide is administered in combination with other pharmaceutical compositions and / or dietary compositions including, but not limited to, dietary supplements, nutraceuticals, etc. Since any suitable method and / or form is used, it is not intended that the present invention be limited to any particular method or form of administration.

Examples

[0175] Experiment The following examples (including experiments and achieved results) are provided for illustrative purposes only and should not be construed as limiting the present invention. In the disclosure of the following experiments, the following abbreviations apply: ppm (parts per million); M (molarity); mM (millimolarity), uM and μM (micromolarity); nM (nanomolarity); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used experimental E. coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); EPI (exocrine pancreatic insufficiency); LIP and lip (lipase); btLIP (B. thermoamylovorans lipase); HPLC (high performance liquid chromatography); ms (mass spectrometry or mass spectroscopy); 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); 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, Life Technologies, Corp., part of 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) Acquisition of Bacterial Lipase Gene and Construction of Expression Vector The 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, for example, 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 hereby incorporated by reference) or the pJV110900 vector system (see, for example, U.S. Patent Application Publication No. 2017 / 213758, which is also hereby incorporated by reference). However, it is not intended that the present invention be limited to any particular vector. Further, in some embodiments, expression vectors lacking an antimicrobial agent resistance marker are used. The plasmid construct was transformed into an E. coli strain derived from W3110. Using directed evolution techniques well known to those skilled in the art, a library of gene variants (see, for example, U.S. Patent No. 8,383,346, International Publication No. 2010 / 144103) and derivatives thereof was generated from this plasmid construct.

[0177] (Example 2) Acquisition of Mammalian Lipase Genes, Construction of Expression Vectors, and HTP Propagation 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 the secretory expression of lipase, the S. cerevisiae mating factor alpha (MFα) signal peptide was genetically fused to the mature form of the lipase. These yeast strains were propagated by HTP as described in International Publication No. 2016 / 105889 (incorporated herein by reference). The 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 Experiments conducted on the growth and screening of lipase variants are described below.

[0179] High-throughput (HTP) growth of B. thermoamylovorans lipase (btLIP) and lipase (LIP) variants: Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose with selection added. After incubation overnight 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. The cultures were grown overnight for 18 - 20 h in a shaking incubator (200 rpm, 30 °C, 85% relative humidity; Kuhner). Overnight growth samples (20 μl) were transferred into COSTAR® 96-well deep plates filled with 380 μL of Terrific Broth supplemented with the selection compound (e.g., chloramphenicol). The plates were incubated for 135 min 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 for 20 - 24 h in a shaking incubator (250 rpm, 30 °C, 85% relative humidity; Kuhner). 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] Lysis of HTP pellets: First, 200 - 400 μL of lysis buffer (1×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 centrifuged (4000 rpm × 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 a protein overexpressed with an apparent MW of approximately 45 kDa, which is 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 by the time - dependent changes in the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein pH 7.0 were mixed with 10 μL of the lysate, 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 by centrifugation, and separated on a C18 POROSHELL® 5 μm 150 mm column running with a homogeneous concentration 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 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 H2O:IPA:MeOH. The samples were then analyzed by RAPIDFIRE® MS. In the following examples, "activity without challenge" refers to the lipase activity determination performed without external pretreatment / challenge as described in the explanation of the following assay method.

Table 3 - 1 - 1

Table 3-1-2

[0182] HTP analysis of the clarified lysate pretreated by heat pretreatment: After incubation at 37 °C to 65 °C, the activity of the btLIP variant was determined. First, 100 μL of the clarified lysate was added to the wells of a 96-well BioRad Hard-Shell PCR thin-wall microtiter plate (#hsp9601; BioRad). The plate was sealed and incubated in a thermocycler at 37 °C to 65 °C for 1 hour before analysis. The variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by the time-dependent changes in the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein pH 7.0 were mixed with 10 μL of the 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, 10 μL was diluted into 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then 10 μL was added to 190 μL of 50:25:25 H2O:IPA:MeOH for further dilution. The samples were then analyzed by RAPIDFIRE® MS. The results are shown in the table of Example 4.

[0183] HTP analysis of the clarified lysate at low pH and with or without pepsin pretreatment: The activity of the 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 the wells of a 96-well round-bottom microtiter plate (COSTAR® plate #3798; Corning). The plate was sealed and incubated at 37 °C for 1 h with shaking (THERMOTRON® shaker HT Infors AJ185, 400 rpm, stroke 1 inch) prior to analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by the time-dependent 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 the pepsin reaction mixture, added to the wells of a polyacrylate 96-well microtiter plate (COSTAR® plate #3635; Corning), and incubated at 37 °C for 30 min - 1 h. The reaction was quenched with 600 μL of ACN:IPA (1:3), clarified, 10 μL was diluted into 190 μL of 50:50 IPA:MeOH, shaken for 1 min, and then 10 μL was added and diluted again into 190 μL of 50:25:25 H2O:IPA:MeOH. The samples were then analyzed by RAPIDFIRE® MS. The results are shown in the table of Example 4.

[0184] HTP analysis of clarified lysates by protease pretreatment: To simulate the environment of the lower intestine, the activity of the 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) was added to the wells of a 96-well round-bottom microtiter plate (COSTAR® plate #3798; Corning). The plate was sealed and incubated at 37 °C for 1 hour with shaking (THERMOTRON® shaker HT Infors AJ185, 400 rpm, 1-inch stroke) before analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by the time-dependent change in the abundance of oleic acid, diolein, and monoolein. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein pH 7.0 were mixed with 10 μL of the protease reaction mixture, 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, 10 μL was diluted into 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then 10 μL was added and diluted again into 190 μL of 50:25:25 H2O:IPA:MeOH. The samples were then analyzed by RAPIDFIRE® MS. The results are shown in the table of Example 4.

[0185] HTP analysis of clarified lysate by multi-stage gastrointestinal (GI) challenge: After incubation at 37°C to 65°C, the activity of the btLIP variant was determined. First, 100 μL of the clarified lysate was added to the wells of a 96-well BioRad Hard-Shell PCR thin-wall microtiter plate (#hsp9601; BioRad). The plate was sealed and incubated at 37°C to 65°C for 1 hour in a thermocycler prior to the gastric challenge. Next, to simulate the gastric environment, the btLIP variant was 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 the wells of a 96-well round-bottom microtiter plate (COSTAR® plate #3798; Corning). The plate was sealed and incubated at 37°C for 1 hour with shaking (THERMOTRON® shaker HT Infors AJ185, 400 rpm, stroke 1 inch) prior to analysis. Next, to simulate the lower intestinal environment, the btLIP variant was incubated with chymotrypsin and trypsin. In this step, 100 μL of protease mix (0.01 to 100 mg / ml chymotrypsin [C4129; Sigma], and 0.01 to 100 mg / ml trypsin [T7409; Sigma]), 0 to 30 μL of 20 mM sodium taurocholate in 100 mM sodium phosphate pH 6.5 to 7.0, and 100 μL of the heat-treated, gastric-challenged lysate were added to the wells of a 96-well round-bottom microtiter plate (COSTAR® plate #3798; Corning). The plate was sealed and incubated at 37°C for 1 hour with shaking (THERMOTRON® shaker HT Infors AJ185, 400 rpm, stroke 1 inch) prior to analysis. The variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by the time-dependent change in the abundance of oleic acid, diolein, and monoolein.For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein pH 7.0 were mixed with 10 μL of gastrointestinal reaction 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, 10 μL was diluted into 190 μL of 50:50 IPA:MeOH, shaken for 1 minute, and then 10 μL was added to 190 μL of 50:25:25 H2O:IPA:MeOH for further dilution. The samples were then analyzed by RAPIDFIRE® MS. The results are shown in the table of Example 4.

[0186] (Example 4) Screening results for lipase variants Variants generated from homology 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. In this table, the amino acid differences are shown compared to SEQ ID NO: 2. [Table 4-1-1] [Table 4-1-2] [Table 4-1-3] [Table 4-1-4]

[0187] Based on the results shown in Table 4-1, SEQ ID NO: 94 was selected as the parental sequence for the next iteration of protein optimization. The beneficial mutations identified from the results shown in Table 4-1 were recombined into the backbone. The 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. The data compared to SEQ ID NO: 94 are listed in Table 4-2. In this table, the amino acid differences are shown compared to SEQ ID NO: 94.

Table 4-2-1

Table 4-2-2

[0188] Based on the results shown in Table 4-2, SEQ ID NO: 350 was selected as the next parental sequence for the next iteration of protein optimization. The beneficial mutations identified from Table 4-2 were recombined into the backbone. Furthermore, 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, the amino acid differences are shown compared to SEQ ID NO: 350.

Table 4-3-1

Table 4-3-2

[0189] Based on the results from Table 4-3, SEQ ID NO: 442 was selected as the next parental sequence for the next iteration of protein optimization. The beneficial mutations identified based on the results shown in Table 4-3 were recombined into the backbone. Furthermore, 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, the amino acid differences are shown compared to SEQ ID NO: 442.

Table 4-4-1

Table 4-4-2

[0190] Based on the results shown in Table 4-4, SEQ ID NO: 540 was selected as the next parental sequence for the next iteration of protein optimization. The beneficial mutations identified based on the results shown in Table 4-4 were recombined into the backbone. Additionally, variants were 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, the amino acid differences are shown relative to SEQ ID NO: 540.

Table 4-5-1

Table 4-5-2

[0191] Based on the results shown in Table 4-5, SEQ ID NO: 646 was selected as the next parental sequence for the next iteration of protein optimization. The beneficial mutations identified based on the results shown in Table 4-5 were recombined into the backbone. The variants were assayed as described in Example 3, and the results are provided in Table 4-6. In this table, the amino acid differences are shown relative to SEQ ID NO: 646.

Table 4-6-1

Table 4-6-2

[0192] Based on the results shown in Table 4-6, SEQ ID NO: 758 was selected as the next parental sequence for the next iteration of protein optimization. The beneficial mutations identified based on the results shown in Table 4-6 were recombined into the backbone. The variants were assayed as described in Example 3, and the results are provided in Table 4-7. In this table, the amino acid differences are shown compared to SEQ ID NO: 758. [Table 4-7]

[0193] (Example 5) Screening results of individual mutant 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. After being subjected to various pre-treatments as described in Example 3, these variants were assayed for lipase activity. Each variant was tested in triplicate, and the analysis of the activity data regarding both SEQ ID NO: 868 and SEQ ID NO: 2 is listed in Table 5-1. [Table 5-1-1] [Table 5-1-2] [Table 5-1-3] [Table 5-1-4] [Table 5-1-5] [Table 5-1-6] [Table 5-1-7] [Table 5-1-8]

Table 5-1-9

Table 5-1-10

Table 5-1-11

Table 5-1-12

Table 5-1-13

Table 5-1-14

[0194] (Example 6) Verification of a Surgical Model of Exocrine Pancreatic Insufficiency in Miniature Pigs 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, female Sinclair (trademark) minipigs, 3 - 4 months of age, were given a high - fat diet (HFD; Sinclair standard diet S - 9 mixed with BERTOLLI (registered trademark) olive oil at 10:1 w / w) once daily. Total fecal output over 24 hours was collected on 3 consecutive days before surgery and on days 15, 16, and 17 after surgery. Distilled water in an amount approximately 1.5 times the net weight of the feces was added, homogenized, divided into 3×50 mL aliquots, and frozen at - 20°C until analysis to prepare daily total fecal collections from each animal for subsequent analysis. In fecal samples, the coefficient of fat absorption (CFA; by the 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 by Vario Max CN instrument by combustion method; see Watson et al., in Peters et al. (eds.) Recommended Methods of Manure Analysis, Univ. of Wisconsin Cooperative Extension Publishing, Publication No. A3769. Madison WI.

[2003] , p. 18 - 24) were measured, and the model was validated by evaluating the percent change 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 that this surgical model is effective for EPI research.

[0195] (Example 7) In vivo characterization of SEQ ID NO: 868 compared to CREON (registered trademark) Pancrelipase To evaluate the engineered lipase variant (SEQ ID NO: 868) compared to the current standard of care (CREON® pancrelipase; AbbVie, Inc.), the validated Sinclair™ minipig surgical model (female, 4 - 5 months of age) of EPI was advanced to an in vivo study. Starting at least 8 days prior to dosing, animals were given once daily a HFD (Sinclair standard S-9 diet mixed with 10:1 w / w BERTOLLI® unstimulated olive oil) combined with 1 3.9 ounce cup of unsweetened applesauce. During the dosing days, the diet was prepared sequentially for each animal to minimize ex vivo lipase interaction with dietary fat prior to ingestion. Briefly, first the HFD was prepared in a clean feeding bowl. Since acidic pH protected against early enzyme activation, next the enzyme (0.5 g of lyophilized SEQ ID NO: 868 powder, corresponding to 258,000 U and 252,000 U of lipase respectively, or 5.6 g of microspheres pooled from 7 × 36,000 U CREON® pancrelipase capsules) was mixed into 1 3.9 ounce cup of unsweetened applesauce. The units of enzyme administered per meal were based on the specific activity of lipase determined by the USP lipase assay (available online from United States Pharmacopeial Convention, Rockville, MD: United States Pharmacopeial Convention; 2016; uspnf.com / uspnf / document / GUID-AC788D41-90A2-4F36-A6E7-769954A9ED09_1_en-US) and the total calculated fat provided in the diet. The lipase - applesauce mixture was then transferred to the HFD and mixed rapidly. Finally, the feeding bowl was immediately given to the animals before proceeding to the next preparation. After 2 hours, the feeding bowl was removed. 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). This study incorporated an 8-day "washout" (once daily HFD + applesauce without enzyme) during each 10-day dosing period.Immediately before the start of administration and 24 hours after administration on days 9, 10, and 11 of each period, total feces were collected for 3 consecutive days. Distilled water with a volume approximately 1.5 times the net weight of the feces was added to each day's total feces collection, homogenized, divided into 3 × 50 mL, frozen at -20°C until analysis, and the remainder was discarded to prepare fecal samples. Analysis was completed as described for the above model validation. Both CREON® pancrelipase and SEQ ID NO: 868 similarly improved CFA to values not significantly different from the pre-surgical baseline.

[0196] (Example 8) Characterization of the In Vivo Dose Response of SEQ ID NO: 868 For further in vivo SEQ ID NO: 868 dose response studies, the validated SINCLAIR™ mini-pig surgical model for EPI (female, 4 - 6 months old) was used. The HFD and feeding regimen, lipase mixing protocol, fecal sample collection and preparation, and analysis were the same as above. Post-operative CFA measured immediately before dosing decreased to 39.9% ± 13.2 SD (p < 0.0001, n = 8) compared to pre-operative 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 with previous studies and at 86,000 U (167 mg) and 29,000 U (55.5 mg) to evaluate low-dose efficacy. All doses evaluated had a positive effect on CFA in a dose-dependent manner. From high dose to low dose, 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) compared to before dosing, respectively.

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

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

Claims

1. A recombinant lipase comprising a polypeptide sequence having 100% sequence identity to SEQ ID NO:

868.

2. The recombinant lipase according to claim 1, wherein the recombinant lipase is purified.

3. A composition comprising the recombinant lipase according to claim 1 or 2.

4. A recombinant polynucleotide encoding the recombinant lipase according to claim 1.

5. The recombinant polynucleotide according to claim 4, wherein the sequence of the polynucleotide is codon-optimized.

6. An expression vector comprising the sequence of the recombinant polynucleotide according to claim 4 or 5.

7. The expression vector according to claim 6, wherein the sequence of the recombinant polynucleotide is operably linked to a control sequence.

8. The expression vector according to claim 7, wherein the control sequence is a promoter.

9. The expression vector according to claim 8, wherein the promoter is a heterologous promoter.

10. A host cell comprising the expression vector according to any one of claims 6 to 9.

11. The host cell according to claim 10, wherein the host cell is a eukaryote or a prokaryote.

12. A method for producing a recombinant lipase, comprising culturing the host cell according to claim 10 or 11 under conditions such that a recombinant lipase encoded by the recombinant polynucleotide is produced.

13. The method according to claim 12, further comprising the step of recovering the lipase.

14. The method according to claim 13, further comprising the step of purifying the lipase.

15. A pharmaceutical composition for the treatment of pancreatic insufficiency, comprising the composition according to claim 3.

16. The pharmaceutical composition according to claim 15, further comprising a pharmaceutically acceptable carrier and / or excipient.

17. The pharmaceutical composition according to claim 15 or 16, further comprising at least one additional enzyme selected from at least one protease and at least one amylase.

18. The pharmaceutical composition according to claim 15 or 16, further comprising a pancreatic extract containing lipase, protease and amylase activities.

19. The pharmaceutical composition according to any one of claims 15 to 18, wherein the composition is suitable for parenteral injection or infusion, subcutaneous injection, inhalation or topical application to humans.

20. The pharmaceutical composition according to any one of claims 15 to 18, wherein the composition is suitable for oral administration to humans.

21. The pharmaceutical composition according to claim 20, wherein the composition further comprises an enteric coating.

22. The pharmaceutical composition according to any one of claims 15 to 21, which is for treating and / or preventing symptoms of pancreatic insufficiency in a subject.

23. The pharmaceutical composition according to claim 22, wherein the symptoms of pancreatic insufficiency are improved.

24. The pharmaceutical composition according to claim 22 or 23, wherein the subject can eat a diet that is less restricted in its lipid content than the diet required by a subject exhibiting the symptoms of pancreatic insufficiency.

25. The pharmaceutical composition according to any one of claims 22 to 24, wherein the subject is an infant or a child.

26. The pharmaceutical composition according to any one of claims 22 to 24, wherein the subject is an adult or a young adult.

27. The recombinant lipase according to claim 1 or 2, or the composition according to claim 3 and any one of claims 15 to 21, for use as a medicament.

28. The recombinant lipase according to claim 1 or 2, or the composition according to claim 3 and any one of claims 15 to 21, for use as a nutritional supplement.

29. The recombinant lipase according to claim 1 or 2, or the composition according to claim 3 and any one of claims 15 to 21, for use in treating or preventing symptoms of pancreatic insufficiency.

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