Engineered lipase variants

Abstract

The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides have been optimized to provide improved thermostability, protease stability, and stability under a range of pH conditions including acidic (pH <7) and alkaline (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, and methods for preparing the engineered polynucleotides and lipase polypeptides.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 62 / 894,019, filed on August 30, 2019, which is hereby incorporated by reference in its entirety for all purposes. Field of the Invention

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

[0003] Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification as an ASCII formatted text file via EFS-Web, with the file name "CX7-187WO2_ST25.txt", the creation date of August 27, 2020, and the size of 4.30 megabytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0006] Pancreatic enzyme replacement therapy (PERT) can be used to treat pancreatic enzyme insufficiency (PEI). Various diseases, including pancreatitis, cystic fibrosis, celiac disease, inflammatory bowel disease and pancreatic cancer, can lead to PEI as a result of reduced secretion of pancreatic enzymes into the duodenum. This leads to poor digestion of food, insufficient intestinal absorption of fats, proteins, carbohydrates and vitamins, which can lead to malnutrition. Although orally administered PERT treatment is currently available, the condition may not be alleviated in some people due to insufficient activity of PERT in the gastrointestinal tract and / or insufficient patient compliance with treatment due to the significant drug burden associated with current treatment regimens. In some cases, the coefficient of fat absorption (CFA) and / or the coefficient of nitrogen absorption (CNA) are not as good as those of healthy patients, leading to weight loss and other health problems. Therefore, there is still a need for improved PERT treatment in the art. SUMMARY OF THE INVENTION

[0008] The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides have been optimized to provide improved thermostability, protease stability, and stability under a range of pH conditions including acidic (pH <7) conditions. The present invention also relates to the use of compositions comprising the engineered lipase polypeptides for therapeutic and / or nutritional purposes. The present invention also provides polynucleotides encoding the engineered lipase polypeptides, and methods for preparing the engineered polynucleotides and lipase polypeptides.

[0009] The present invention provides recombinant lipases and / or biologically active recombinant lipase fragments 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 lipases and / or biologically active recombinant lipase fragments comprise 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 lipases comprise 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. NO:2 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, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 2, 3, 4, 22, 27 / 46 / 97 / 1 36 / 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, 1 95, 195 / 231 / 385, 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 219, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379 and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the recombinant lipase comprises a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 2H, 2M, 2T, 3A, 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97A / 136V / 149E / 385P, 27V / 46T / 385P, 27V / 70N / 136V / 231E / 385P, 27V / 136V / 323I / 385P, 27V / 149E, 27V / 149E / 385P, 27V / 385P, 34D, 38A, 38H, 38L, 41V, 44E, 46T / 70N, 46T / 70N, 46T / 70 6T / 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, 14 1S, 142A, 142F, 142I, 142L, 144M, 144R, 144W, 146A, 146F, 146S, 146Y, 149E, 149E / 231E / 385P, 149K, 149R, 151A, 151I, 151L, 174H, 174R, 17 5G, 175L, 175R, 178R, 178T, 181H, 183L / 385P, 189A, 189E, 189W, 194N, 194N / 199L, 194T, 195D / 231E / 385P, 195V, 199L, 199L / 213A, 210A, 210 V, 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, 29 6R, 300A, 300D, 300T, 308A, 330Y, 338N, 379T, 385A, 385C, 385D, 385P,385R and 385T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of E2H, E2M, E2T, T3A, T3G, T3R, T3S, S4M, S4W, S4Y, G22N, Y27V / R46T / Y97A / Y136V / T149E / F385P, Y27V / R46T / F385P, Y27V / K70N / Y136V / Q231E / F 385P, Y27V / Y136V / K323I / F385P, Y27V / T149E, Y27V / T149E / F385P, Y2 7V / F385P, K34D, F38A, F38H, F38L, N41V, G44E, R46T / K70N, R46T / T149 E / F183L / Q231E / F385P, Y48V, K70N, T73C / V305I, T73I, T73R, K82C, K82E, K82E / P94S / D101S / K194N / I199L, K82E / P94S / I199L, K82F, K82G, K 82L, 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, T1 49E, T149E / Q231E / F385P, T149K, T149R, G151A, G151I, G151L, E174H, E174R, Q175G, Q175L, Q175R, S178R, S178T, K181H, F183L / F385P, S189 A. 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, A219 C. A219G, A219K, A219R, T226R, Q231E / F385P, Y238S, E247R, Q250T, R270V, T274D, G2 81K, G281R, M292A, M292C, M292L, M292V, S296M, S296R, Q300A, Q300D, Q300T, R308A, N330Y, K338N, N379T, F385A, F385C, F385D, F385P, F385R and F385T, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 2. 、

[0010] 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: 94, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 231 / 296 / 300, 4 / 96 / 149 / 292 / 296 / 300, 4 / 96 / 219 / 296, 4 / 96 / 231 / 296 / 300, 4 / 96 / 292 / 296 / 300, 4 / 149 / 174, 4 / 174 / 219 / 292 / 296, 4 / 231 / 296 / 300, 27, 27 / 34 / 82, 27 / 73 / 82 / 218, 27 / 89, 27 / 89 / 178, 27 / 89 / 218, 27 / 218, 34 / 73 / 21 8, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. 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: 94, or a functional fragment thereof,and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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. W / 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、96 E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174H / 292L / 296R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 2 0D, 292L / 296R, 292L / 296R / 300D, 296R and 296R / 300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some 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: 94, or a functional fragment thereof,and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: T3S / S4W / I96E / S296R / Q300D, T3S / M292L / S296R, S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S296R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S296R / Q300 D. S4W / T149E / E174R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73I / K82 L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H218I, K34D / T73I / H218I, K34D / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183 L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / Q2 31E / M292L / S296R, I96E / T149E / M292L / S296R, I96E / E174H / A219K / Q231E / M292L / S296R, I96E / Q231E / S296R, P146F / Q175G / S189A / G281K, T149E / E 174H / M292L / S296R, T149E / E174H / Q300D, T149E / A219K / Q231E / M292L / S2 96R / 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.

[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: 350, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 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、1 74 / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 189A / 218M, 4W / 175G / 189A / 300D、4W / 175G / 218M / 224A、4W / 175G / 218M / 373F、4W / 185L、4W / 189A / 218M / 373F、4W / 193T、4W / 218M / 300D / 369N / 3 73F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 17 5G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 3 82M, 27V / 82C / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 18 9A, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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 / P228 E. S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S4W / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S18 9A / 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 / S18 9A, E174H / Y373F, Q175G / S189A / H218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.

[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: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of 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、19 3, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 303, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281 and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some 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: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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 / 29 3R / 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 / 243 R / 375A, 175G / 193L / 218M / 243R, 175G / 218M / 375A, 181Q, 189H, 193L, 193L / 218M / 243R, 193L / 271D / 3 03P / 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, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:442.

[0013] 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: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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, I1 44L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E17 4H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N37 5A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303 P / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0014] 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174 / 193 / 195 / 243 / 281 / 303 / 330. 1. 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 / 252 / 344, 49 / 123 / 264 / 344, 49 / 123 / 311, 49 / 252 / 344, 49 / 311 / 344 4, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 540. 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 174G, 34L / 174G / 193L / 195L / 243R / 281P、34L / 174H / 193L / 195I / 303P / 330F、34L / 193L / 195I / 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 / 28 1P / 330F, 38G / 174H / 225L / 243R / 281K / 330H, 38G / 174H / 281K, 38G / 174 R / 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 / 34 4H, 49T / 344H, 51A / 252V / 344H, 51A / 344H, 51V, 98P / 344H, 98R, 123Q / 2 52V / 344H, 129F, 160T, 161I, 174G / 195I / 225L / 281K / 303P / 330F / 345I,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 additional embodiments, the recombinant lipase comprises the amino acid residue of SEQ ID NO: 541. NO:540 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38A / E174R / A303 P / F345I, K34L / F38G / E174R / M225L / A303P, K34L / E174G, K34L / E174G / Q193L / Q195L / L243R / G281P, K34L / E174H / Q 193L / Q195I / A303P / N330F, K34L / Q193L / Q195I / M225L / L243R, K34L / Q193L / L243R / A303P / N330F, K34L / G281P / N33 0H, F38A / E174H / Q193L / M225L / T274D / M283I / A303P / N330F, F38A / E174R / G281K / A303P / F345I, F38A / Q193L / Q195 L / M225L / A303P / F345I, F38A / Q195I / A303P, F38A / Q195Y / G281K / A303P / N330F, F38G, F38G / E174G / Q193L / A303P, F 38G / E174G / G281K / A303P, F38G / E174H / Q195L / G281P / N330F, F38G / E174H / M225L / L243R / G281K / N330H, F38G / E17 4H / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H, F38G / Q195I / G281P / N330F, F38G / Q195Y / L243R / A303P, V49S,V49T, V49T / T51A / G98P / M252V / L311W, V49T / T51A / E123Q / M252V / S344H, V49T / G98P / M12 0T / M252V / S344H, V49T / M120T / M252V / S344H, V49T / E123Q / M252V / S344H, V49T / E123Q / T 264S / 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 / G 281P / A303P / F345I, E174H / Q195I / L243R / G281K / F345I, E174H / G281P / N330H, E174R / Q1 93L / 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 sequences are numbered with reference to SEQ ID NO: 540.

[0015] 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 237 / 287 / 291, 16 / 168 / 177 / 247 / 251 / 257. / 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 / 34 4. 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 / 28 1 / 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 and 281 / 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some additional embodiments, the recombinant lipase comprises NO:646 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 287L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 11I / 16F / 67A / 154Y / 168T / 237Q / 241S / 287L. 87L / 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, 3 4K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 252V / 268T, 34K / 161I / 193L / 243R / 268T / 344V, 34K / 161I / 193L / 243 R / 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、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 1 In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646, wherein the amino acid sequence of SEQ ID NO: 646 is numbered with reference to SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646, wherein the amino acid sequence of SEQ ID NO: 646 is numbered with reference to SEQ ID NO: 646. NO:646 has 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, V11I / Y67A / S168T / F287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y67A / A237Q / F287L / S291A, L 16F / S168T / V177L, F24M, F24M / Y278L, L34K / V49T / L161I / Q193L / L243R / S344H, L 34K / V49T / L161I / Q193L / L243R / S344V, L34K / V49T / L161I / Q193L / S344W, L34K / V 49T / L161I / L243R / S344H, L34K / V49T / L161I / M252V / S268T / S344V, L34K / V49T / Q 193L / L243R / M252V / S344W, L34K / V49T / Q193L / S344H, L34K / V49T / Q193L / S344W,<h2 style=";text-align:left;direction:ltr">L34K / V49T / L243R / M252V、L34K / V49T / M252V / S268T / S344V、L34K / L161I / Q193L / L243R / M252V / S268T、L34K / L161I / Q193L / L243R / S268T / S344V、L34 K / L161I / Q193L / L243R / S344W、L34K / L161I / Q193L / M252V / S268T / S344H、L 34K / 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 / L 161I / S344V, L34K / L161I / S344W, L34K / Q193L / L243R / M252V / S344W, L34K / Q193L / M252V / S268T / S344H, L34K / Q193L / S268T / G281P, L34K / M252V / S268 T / G281P / S344V、L34K / M252V / S344V、V49T / L161I / Q193L / L243R / M252V / S 344W、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 / W1 54Y / A237Q / F287L、Y67A / S168T / A237Q、Y67A / S168T / A237Q / F287L、Y67A / V177L / A237Q、W154Y / A237Q / L286V / F287L、W154Y / L286V / F287L、L161I / Q1 93L / 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. 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:758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some 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:758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24M / 168T / 252V / 281P, 24M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S, and 281P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some 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: 758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: F24M / S168T / M252V / G281P, F24 M / A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S and G281P, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 758.

[0016] 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: 868, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 19 3 / 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 additional embodiments, the recombinant lipase comprises IDNO: 868 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70 K / 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, 24 W, 24Y, 38A, 38C, 38D, 38E, 38F, 38H, 38I, 38K, 38L, 38M, 38N, 38P, 38Q, 38R, 38S, 38T, 38V, 38 W, 38Y, 49A, 49C, 49D, 49E, 49F, 49G, 49H, 49I, 49K, 49L, 49M, 49N, 49P, 49Q, 49R, 49S, 49V, 49 W, 49Y, 70A, 70C, 70D, 70E, 70F, 70G, 70I, 70K, 70L, 70M, 70N, 70P, 70Q, 70R, 70S, 70T, 70V, 70 W, 70Y, 149A, 149C, 149D, 149F, 149G, 149H, 149I, 149K, 149L, 149M, 149N, 149P, 149Q, 149R,149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M、271N、271P、271Q、271R、271S、271T、271V、271W、271Y、287A、287C、287D、287E、287F、287G, 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 287S, 287T, 287V, 287W, 287Y, 292A, 292C, 292D, 292E, 292F, 292G, 292H, 292I, 292K, 292M, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 2 93L, 293M, 293N, 293P, 293Q, 293S, 293T, 293V, 293W, 293Y, 296A, 296C, 296D, 296E, 296F, 296G, 296H, 296I, 296K, 296L, 296M, 29 6N, 296P, 296Q, 296S, 296T, 296V, 296W, 296Y, 303A, 303C, 303D, 303E, 303F, 303G, 303H, 303I, 303K, 303L, 303M, 303N, 303Q, 303 R, 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, 543A, 343C, 343D, 343E, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343P, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 343A, 343C, 343D, 343E, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343P, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:868. In some additional embodiments, the recombinant lipase comprises a lipase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%,99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / 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, G 38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38 W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70 F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149 L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I16 1N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C , R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R 174P, R174Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L17 5E, L175F, L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R,<h2 style=";text-align:left;direction:ltr">L175S、L175T、L175V、L175W、L175Y、A189C、A189D、A189E、A189F、A189G、A1 89H、A189I、A189K、A189L、A189M、A189N、A189P、A189Q、A189R、A189S、A189 T、A189V、A189W、A189Y、L193A、L193C、L193D、L193E、L193F、L193G、L193H、 L193I、L193K、L193M、L193N、L193P、L193Q、L193R、L193S、L193T、L193V、L19 3W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, L225H, L225I, L225K, L225M, L225N, L225P, L225Q, L225R, L225S, L225T, L225V, L225W, L225Y, E 231A、E231C、E231D、E231F、E231G、E231H、E231I、E231K、E231L、E231M、E23 1N、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, V252 E、V252F、V252G、V252H、V252I、V252K、V252L、V252M、V252N、V252P、V252Q、 V252R、V252S、V252T、V252W、V252Y、D271A、D271C、D271E、D271F、D271G、D27 1H、D271I、D271K、D271L、D271M、D271N、D271P、D271Q、D271R、D271S、D271T 、D271V、D271W、D271Y、L287A、L287C、L287D、L287E、L287F、L287G、L287H、L 287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287Y, L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292KL292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R293D, R293E, R293F, R293G, R293H, R293I, R 293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R296C, R296D, R296E, R296F, R296G, R296H, R2 96I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R296T, R296V, R296W, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P30 3H, 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, F3 73G, 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.

[0017] In some additional embodiments, the recombinant lipase comprises at least one mutation in at least one position provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7, and / or Table 5-1, wherein the positions are numbered with reference to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868. In some additional 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 listed in the even-numbered sequences in SEQ ID NO: 14-1796. 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 at least one sequence listed in the even-numbered sequences in SEQ ID NOs: 14-1796. In some additional embodiments, the recombinant lipase comprises at least one sequence listed in the even-numbered sequences in SEQ ID NOs: 14-1796.

[0018] In some other embodiments, the recombinant lipase is more thermostable than the lipase of SEQ ID NO:2. In some other embodiments, the recombinant lipase is more thermostable than the lipase of SEQ ID NO:94,350,442,540,646,758 and / or 868. In some embodiments, the recombinant lipase retains enzymatic activity after being exposed to high temperature and / or low temperature. In some other embodiments, the recombinant lipase retains greater enzymatic activity after being exposed to high temperature and / or low temperature compared to the reference sequence. In some embodiments, the reference sequence is a wild-type lipase, and in some other embodiments, the reference sequence is another recombinant lipase.

[0019] In some embodiments, recombinant lipase is stable and / or active in low pH environment, and in other embodiments, recombinant lipase is stable and / or active in high pH environment, and in other embodiments, recombinant lipase is stable and / or active in neutral pH environment. In some embodiments, recombinant lipase is stable and / or active in low pH environment and high pH environment, and in some other embodiments, lipase is stable and / or active in low pH environment, neutral pH environment and high pH environment. In some embodiments, recombinant lipase retains enzymatic activity after being exposed to low, high and / or neutral pH environment. In some other embodiments, compared with reference sequence, recombinant lipase is more stable and / or more active in high pH environment, neutral pH environment and / or low pH environment. In some embodiments, reference sequence is wild-type lipase, and in other embodiments, reference sequence is another engineered lipase. In some other embodiments, recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO:2 at pH less than 7 (that is, under acidic pH conditions or levels). In some additional embodiments, the recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 868 at a pH less than pH 7 (i.e., under acidic pH conditions or levels). In some additional embodiments, the recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO: 2 at a pH less than 5. In some additional embodiments, the recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO: 2 at pH 5. In some additional embodiments, the recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 688 at pH 5. In some additional embodiments, the recombinant lipase is more stable and / or more active than the lipase of SEQ ID NO: 2 at pH 3.5. In some additional embodiments, the recombinant lipase is more stable and / or more active at pH 3.5 than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 868. In some additional embodiments, the recombinant lipase is more stable and / or more active at pH 3 than the lipase of SEQ ID NO: 2. In some additional embodiments, the recombinant lipase is more stable and / or more active at pH 3 than the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 868. In some embodiments, the recombinant lipase is more resistant to proteolysis than the lipase of SEQ ID NO: 2.In some embodiments, the recombinant lipase is more tolerant to proteolysis than the lipase of SEQ ID NO:94,350,442,540,646,758 and / or 868. In some other embodiments, the recombinant lipase is more tolerant to proteolysis by trypsin. In some other embodiments, the recombinant lipase is more tolerant to proteolysis by trypsin than the lipase of SEQ ID NO:2. In some embodiments, the recombinant lipase is more tolerant to proteolysis by trypsin than the lipase of SEQ ID NO:94,350,442,540,646,758 and / or 868. In some embodiments, the recombinant lipase is more tolerant to proteolysis by chymotrypsin. In some other embodiments, the recombinant lipase is more tolerant to proteolysis by chymotrypsin than the lipase of SEQ ID NO:2. In some embodiments, the recombinant lipase is more tolerant to proteolysis by chymotrypsin than the lipase of SEQ ID NO:94,350,442,540,646,758 and / or 868. In some additional embodiments, the recombinant lipase is resistant to proteolysis by 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 lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758 and / or 868.

[0020] In some further embodiments, the recombinant lipase is more active than the lipase of SEQ ID NO:2 in the presence of at least one bile salt. In some further embodiments, the recombinant lipase is more active than the lipase of SEQ ID NO:94, 350, 442, 540, 646, 758 and / or 868 in the presence of at least one bile salt. In some embodiments, the recombinant lipase retains enzymatic activity after exposure to bile salts. In some further embodiments, the recombinant lipase retains greater enzymatic activity after exposure to bile salts compared to the reference sequence. In some embodiments, the reference sequence is the lipase of SEQ ID NO:2, 94, 350, 442, 540, 646, 758 and / or 868. In some further embodiments, the bile salt is taurocholate.

[0021] In some other embodiments, the recombinant lipase is more stable and / or more active, more thermostable, more tolerant to proteolysis and / or more active in the presence of at least one bile salt than the lipase of SEQ ID NO:2 at acidic pH. In some other embodiments, the lipase is stable in food and / or beverage. In some other embodiments, the recombinant lipase is more stable and / or more active, more thermostable, more tolerant to proteolysis and / or more active in the presence of at least one bile salt than the lipase of SEQ ID NO:94,350,442,540,646,758 and / or 868 at acidic pH. In some embodiments, compared with the lipase of SEQ ID NO:2, the recombinant lipase shows at least one improved characteristic selected from the following: improved stability and / or activity at acidic pH, improved thermostable, improved tolerance to proteolysis and / or improved activity in the presence of at least one bile salt. In some embodiments, the recombinant lipase exhibits at least one improved property selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least two improved properties selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits at least two improved properties selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least three improved properties selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits at least three improved properties selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868.In some embodiments, the recombinant lipase exhibits the following improved properties compared to the lipase of SEQ ID NO: 2: improved stability and / or activity at acidic pH, improved thermostability, improved tolerance to proteolysis, and / or improved activity in the presence of at least one bile salt. In some embodiments, the recombinant lipase exhibits the following improved properties compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868: improved stability and / or activity at acidic pH, improved thermostability, improved tolerance to proteolysis, and / or improved activity in the presence of at least one bile salt. In some embodiments, the recombinant lipase exhibits at least one improved property selected from the group consisting of improved stability and / or activity at acidic pH, improved thermostability, improved tolerance to proteolysis, and / or improved activity in the presence of at least one bile salt, and at least one additional improved property compared to the lipase of SEQ ID NO: 2. In some embodiments, the recombinant lipase exhibits at least one improved property selected from the group consisting of: improved stability and / or activity at acidic pH, improved thermostability, improved resistance to proteolysis, and / or improved activity in the presence of at least one bile salt, and at least one additional improved property, compared to the lipase of SEQ ID NO: 94, 350, 442, 540, 646, 758, and / or 868. In some embodiments, the recombinant lipase exhibits at least one improved property selected from the group consisting of: 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 thermal tolerance; or any combination of i), ii), iii), iv), v), vi) and vii). In some embodiments, the reference sequence is SEQ ID NO: 2, while in some alternative embodiments, the reference sequence is selected from SEQ ID NO: 94, 350, 442, 540, 646, 758 and 868. In some embodiments, the recombinant lipase exhibits at least one improved property selected from the group consisting of: 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 thermal tolerance; or any combination of i), ii), iii), iv), v), vi) and vii).In some embodiments, the reference sequence is SEQ ID NO: 2, and in some optional embodiments, the reference sequence is selected from SEQ ID NO: 94, 350, 442, 540, 646, 758 and 868. In some embodiments, compared with at least two or more reference sequences, the recombinant lipase exhibits at least one improved property selected from the following: 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 thermal tolerance; or any combination of i), ii), iii), iv), v), vi) and vii). In some embodiments, the protease is selected from pepsin, trypsin and / or chymotrypsin. In some embodiments, the reference sequence is selected from SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and 868. In some additional embodiments, the recombinant lipase is purified.

[0022] The present invention also provides the polynucleotide sequence of encoding at least one recombinant lipase provided herein.In some embodiments, the recombinant polynucleotide sequence is codon optimized.In some other embodiments, the recombinant polynucleotide comprises at least one sequence listed in the odd numbered sequence in SEQ ID NO:13-1795 and has 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.In some other embodiments, the polynucleotide comprises at least one sequence listed in the odd numbered sequence in SEQ ID NO:13-1795 and has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some additional embodiments, the present invention provides at least one sequence listed in the odd-numbered sequences in SEQ ID NOs: 13-1795. In some additional embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences in SEQ ID NOs: 13-1795. In some embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences in SEQ ID NOs: 13-1795, wherein the sequence encodes a recombinant polypeptide provided herein. In some embodiments, the recombinant polynucleotide sequence comprises a sequence selected from the odd-numbered sequences in SEQ ID NOs: 13-1795, wherein the sequence encodes a recombinant polypeptide provided in the even-numbered sequences provided in SEQ ID NOs: 14-1796. In some additional 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 with at least one sequence listed in the odd-numbered sequences in SEQ ID NO: 13-1795. In some additional 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 with at least one sequence listed in the odd-numbered sequences in SEQ ID NO: 13-1795.In some additional 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 listed in the odd-numbered sequences in SEQ ID NOs: 13-1795, wherein the recombinant lipase comprises a polypeptide sequence comprising the even-numbered sequences provided in SEQ ID NOs: 14-1796.

[0023] 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 other embodiments, the recombinant polynucleotide sequence is operably connected to a control sequence. In some embodiments, the control sequence is a promoter. In some other embodiments, the promoter is a heterologous promoter.

[0024] 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, wherein the at least one expression vector comprises 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, wherein the expression vector comprises 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, wherein the expression vector comprises at least one recombinant polynucleotide sequence encoding at least one recombinant lipase provided herein. In some embodiments, the host cell is eukaryotic, and in some optional embodiments, the host cell is prokaryotic. In some embodiments, the host cell is Escherichia coli. In some optional embodiments, the host cell is Saccharomyces cerevisiae.

[0025] The present invention also provides a method for producing at least one recombinant lipase, the method being included in cultivating at least one host cell provided herein under the conditions of producing the recombinant lipase encoded by the recombinant polynucleotide. In some embodiments, the method also includes the step of reclaiming the lipase. In some other embodiments, the method also includes the step of purifying the lipase.

[0026] 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 includes a pharmaceutical composition. In some other embodiments, the pharmaceutical composition is applicable to the treatment of pancreatic insufficiency (pancreatic insufficiency). In some other embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition is applicable to parenteral injection and / or infusion to the mankind. In some other embodiments, the pharmaceutical composition is applicable to oral administration to the mankind. In some other embodiments, the pharmaceutical composition is applicable to administration to the mankind in other ways. The present invention also provides a composition comprising at least one recombinant lipase provided herein, wherein the composition is applicable to other purposes.

[0027] The present invention also provides a method for treating and / or preventing the symptoms of pancreatic insufficiency in a subject, the method comprising providing a subject with pancreatic insufficiency and providing the subject with a pharmaceutical composition provided herein. In some embodiments, after administering a composition comprising at least one recombinant lipase to the subject, the subject's pancreatic insufficiency symptoms are improved. In some other embodiments, the pharmaceutical composition also comprises at least one protease. In some other embodiments, the pharmaceutical composition also comprises at least one amylase. In some other embodiments, the pharmaceutical composition also comprises at least one protease and at least one amylase.

[0028] In some other embodiments, the subject can eat a diet that is less restricted in lipid content than the diet required by the subject showing the symptoms of pancreatic insufficiency. In some other embodiments, the subject is an infant, and in some other embodiments, the subject is a child. In some other embodiments, the subject is an adult, and in some optional embodiments, the subject is a young person. The present invention also provides a medicine comprising at least one recombinant lipase provided herein.

[0029] In some embodiments, the present invention provides a method for decomposing fat and / or lipid, the method comprising providing at least one fat and / or lipid and at least one engineered lipase of the present invention, and exposing fat and / or lipid to at least one engineered lipase under conditions where fat and / or lipid are decomposed. In some other embodiments, the present invention provides a method for decomposing fat and / or lipid, the method comprising providing at least one fat and / or lipid and a composition comprising at least one engineered lipase of the present invention, and exposing fat and / or lipid to a composition comprising at least one engineered lipase under conditions where fat and / or lipid are decomposed. 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 hydrolyzing fat and / or lipid, the method comprising providing 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 where the fat and / or lipid are hydrolyzed by the engineered lipase. In some additional embodiments, the present invention provides a method for hydrolysis of fat and / or lipids, comprising providing a composition of fat and / or lipids and comprising 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.

[0030] Description of the invention

[0031] The present invention provides engineered lipase polypeptides and compositions thereof. The engineered lipase polypeptides have been optimized to provide improved thermal stability, protease stability, and stability under a 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 can be used for PERT treatment of PEI conditions. In some other 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 engineered lipase polypeptides, and methods for preparing engineered polynucleotides and lipase polypeptides.

[0032] Abbreviations and definitions:

[0033] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis. All patents, patent applications, articles and publications mentioned herein (both above and below) are clearly incorporated herein by reference.

[0034] Although any suitable method and material similar to or equal to those described herein can be used in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to the described ad hoc method, scheme and reagent, because these can change according to the situation that those skilled in the art use them.Therefore, the following terms of definition are more fully described by reference to the entire application.All patents, patent applications, articles and publications mentioned herein (both above and below) are expressly incorporated herein by reference.

[0035] As used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] Numerical ranges include the numbers that define the ranges. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as if such a narrower numerical range is fully and clearly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as if such a lower (or higher) numerical limit is clearly written herein.

[0037] The term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0038] In addition, the titles provided herein are not limitations of the various aspects or embodiments of the present invention that may be possessed by reference to the present application as a whole. Therefore, the terms to be defined below are more fully described by reference to the present application as a whole. Nevertheless, for ease of understanding the present invention, many terms are defined as follows.

[0039] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0040] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0041] As used herein, "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that they catalyze.

[0042] As used herein, "ATCC" refers to the American Type Culture Collection, whose biological deposit collections include genes and strains.

[0043] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0044] 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 breaking down fats into fatty acids, glycerol, and other alcohols. Lipases are essential in the digestion, transport, and processing of dietary lipids in most organisms.

[0045] As used herein, the term "lipid" refers to a class of water-insoluble macromolecules, including fatty acids and esters thereof, sterols, terpenoids, some poorly soluble vitamins and other related compounds." fat " is a subset of the lipid composed of fatty acid esters (e.g., triglycerides, which are composed of glycerol and three fatty acids). It is not intended that the present invention is limited to any specific lipid and / or fat. Considering the context, the terms "fat" and "lipid" are used interchangeably herein.

[0046] As used herein, "protease" (as well as "proteinase" and "peptidase") refers to a number of enzymes that hydrolyze proteins. There are many proteases involved in the breakdown of proteins into smaller polypeptide units or individual amino acids. Proteases are important for many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling.

[0047] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0048] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0049] The terms "engineered," "recombinant," "non-naturally occurring," and "variant" when used with respect to cells, polynucleotides, or polypeptides refer to materials that are or correspond to the native or natural form of that material: have been modified or are identical to a manner not originally found in nature but are produced or derived from synthetic materials and / or through manipulation using recombinant techniques.

[0050] Recombinant polypeptide can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned in a vector such as a plasmid and expressed in a desired host, such as Escherichia coli (E.coli), Saccharomyces cerevisiae (S.cerevisiae), etc. The variant of the recombinant polypeptide can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method of generating a specific replacement at a determined amino acid (site-directed), the specificity (region-specificity) or random mutation in the local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene is available. Many suitable methods for producing enzyme variants known to those skilled in the art include, but are not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering 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 the variants are generated, they can be screened for any desired properties (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability and / or acidic pH stability, etc.). In some embodiments, "recombinant lipase polypeptides" (also referred to herein as "engineered lipase polypeptides", "variant lipases" and "lipase variants") can be used.

[0051] As used herein, "wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0052] As used herein, "coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0053] The term "percentage (%) of sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align two sequences. 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 percentages of sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215:403-410

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

[1977] ). Software for performing BLAST analysis is available to the public through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word 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 act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score reaches 0 or less than 0 due to the accumulation of one or more negative scoring residue alignments; or 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 the following as defaults: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

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

[0054] As used herein, "reference sequence" refers to a determined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the total length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) comprise a similar sequence (i.e., a part of a complete sequence) between the two sequences, and (2) can also comprise a different (divergent) sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in a primary sequence. "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein a portion of the sequence in the comparison window may include 20% or less additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0055] In the case of numbering for a specified amino acid or polynucleotide sequence, "corresponding to," "referenced to," or "relative to" refers to the numbering of the residues of the specified reference sequence when the specified amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered lipase, the residue matching between the two sequences can be optimized by introducing gaps to align with the reference sequence. In these cases, the numbering of the residues in a given amino acid or polynucleotide sequence is performed with respect to the reference sequence to which it is aligned, despite the presence of gaps.

[0056] As used herein, "mutation" refers to any change in a polypeptide or polynucleotide sequence. It is intended to include any number (i.e., one or more) of substitutions, insertions, deletions, and / or rearrangements (i.e., compared to a starting or reference sequence) present in a 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.

[0057] As used herein, "amino acid difference" or "residue difference" refers to the difference of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, wherein n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X92 compared to SEQ ID NO:2" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 92 of SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has threonine at position 92, then "the residue difference at position X92 compared to SEQ ID NO:2" means the amino acid residue (e.g., T92A) at the polypeptide position corresponding to position 92 of SEQ ID NO:2. In most cases herein, the specific amino acid residue difference at one position is indicated as "XnY", wherein "Xn" specifies the corresponding position as described above, and "Y" is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some cases (e.g., as in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1), the disclosure also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some cases, a polypeptide of the disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, when more than one amino acid can be used in a specific 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, an enzyme variant includes more than one substitution. For ease of reading, these substitutions are separated by slashes (e.g., Y27V / F385P). The present application includes engineered polypeptide sequences comprising one or more amino acid differences, including either or both of conservative amino acid substitutions and non-conservative amino acid substitutions.

[0058] As used herein, "conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, an amino acid with an aliphatic side chain can be replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain can be replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain can be replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid can be replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0059] As used herein, "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of basic or aliphatic amino acids for acidic amino acids; substitutions of small amino acids for aromatic amino acids; and substitutions of hydrophobic amino acids for hydrophilic amino acids.

[0060] As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered enzyme. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous.

[0061] As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of the polypeptide or to the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0062] As used herein, an asterisk (*) used in the context of a polynucleotide sequence indicates the presence of a stop codon in 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.

[0063] "Functional fragment" and "biologically active fragment", which are used interchangeably herein, refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion and / or an internal deletion (e.g., the sequence is truncated), but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., the full-length engineered lipase of the invention) and retains substantially all of the activity of the full-length polypeptide.

[0064] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term includes polypeptides that have been taken out or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The recombinant lipase polypeptide can be present in a cell, in a cell culture medium, or prepared in various forms (such as lysates or isolated products). Therefore, in some embodiments, the recombinant lipase polypeptide can be an isolated polypeptide.

[0065] The terms "isolated" and "purified" are used to refer to a molecule (e.g., isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0066] As used herein, "substantially pure polypeptide" refers to following composition, in which polypeptide substance is the main substance present (that is, on a molar or weight basis, it is more abundant than any other independent macromolecular substance in said composition), and when the target substance constitutes the macromolecular substance present by mole or % weight at least about 50%, it is generally a composition that is substantially purified.Usually, substantially pure lipase composition constitutes all macromolecular substances present in said composition by mole or % weight 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.In some embodiments, the target substance is purified to basic homogeneity (that is, pollutant substances can not be detected in the composition by conventional detection methods) from the starting product, wherein said composition is substantially composed of single macromolecular substance.Solvent substances, small molecules (<500 dalton) and elemental ion substances are not considered to be macromolecular substances.In some embodiments, the recombinant lipase polypeptide of separation is a substantially pure polypeptide composition.In some embodiments, the recombinant lipase polypeptide of separation is a substantially pure polypeptide composition. In some embodiments, substantially pure recombinant lipase polypeptide preparations are added to formulations suitable for use in the present invention (eg, polysaccharides, surfactants, etc.).

[0067] As used herein, the terms "improved enzyme properties" and "improved properties" refer to the properties of engineered lipase polypeptides including improvements in any enzyme properties compared to a reference lipase polypeptide and / or a wild-type lipase polypeptide or another engineered lipase polypeptide. Improved properties include, but are not limited to, properties such as: increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased tolerance to substrate or end product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic or alkaline pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation) and altered temperature profiles.

[0068] As used herein, "increased enzymatic activity" or "enhanced catalytic activity" refers to an improved property of an engineered lipase polypeptide, which can be expressed as an increase in specific activity (e.g., product / time / weight protein produced) or an increase in the percentage conversion of substrate to product (e.g., the percentage conversion of a starting amount of substrate to product over a specified time period using a specified amount of lipase) compared to a reference lipase. Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity can be affected, including classical enzyme properties such as lipase activity, lipase activity, and lipase activity. m 、V max or k cat , which changes can result in an increase in enzymatic activity. The improvement in enzymatic activity can be from about 1.1 times the enzymatic activity of the corresponding wild-type enzyme to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more of the enzymatic activity of a naturally occurring lipase or another engineered lipase from which a lipase polypeptide is derived.

[0069] Lipase activity can be measured by any suitable method known in the art (e.g., standard assays such as monitoring changes in spectrophotometric properties of reactants or products). In some embodiments, the amount of product produced can be measured by high performance liquid chromatography (HPLC) separation combined with UV absorbance. In some embodiments, the amount of product produced can be measured using The amount of product produced can be measured by mass spectrometry, while in some other embodiments, alternative methods known in the art can be used to measure the product. Comparison of enzyme activity is performed using defined enzyme preparations, defined assays under set conditions, and one or more defined substrates, as described in further detail herein.

[0070] As used herein, the terms "protease-stable" and "proteolytic stability" refer to the ability of a protein (e.g., a recombinant lipase of the present invention) to function and withstand proteolysis mediated by any proteolytic enzyme or other proteolytic compound or factor, and to retain its function after exposure to a protease. It is not intended that the term be limited to the use of any particular protease to assess the stability of a protein. In fact, the engineered lipase of the present invention is stable and retains enzymatic activity in the presence of or after exposure to various proteases. In some embodiments, the engineered lipase is stable in the presence of trypsin, chymotrypsin and / or pepsin. However, it is not intended that the present invention be limited to any particular protease or any particular method for assessing proteolytic stability.

[0071] 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 at a certain range of pH (including but not limited to a range of pH 2 to pH 7). In some embodiments, the recombinant lipase is stable at different pH ranges, as shown in the examples provided herein. It is not intended that the present invention be limited to any pH stability level or pH range.

[0072] As used herein, the term "improved tolerance to acidic pH" means that the recombinant lipase according to the present invention will have increased stability (retain higher activity at about pH 7, 6, 5, 4, 3, 2 or even lower pH after exposure to acidic pH for a specified period of time [e.g., 1 hour, up to 24 hours, etc.]) compared to a reference lipase or another enzyme.

[0073] As used herein, "physiological pH" means the pH range (eg, pH 7.2-7.4) normally found in the blood of a subject (eg, a human).

[0074] The term "alkaline pH" (eg, used in reference to improved stability to alkaline pH conditions or increased tolerance to alkaline pH) means a pH range of about 7 to 11, or in some embodiments, greater than pH 11.

[0075] The term "acidic pH" (e.g., used in reference to improved stability to acidic pH conditions or increased tolerance to acidic pH) is meant to include a pH range of any pH value less than 7. In some embodiments, the acidic pH is less than 7, while in some other embodiments, the pH is less than about 6, 5, 4, 3, 2 or less. In some optional embodiments, the recombinant lipase of the present invention is stable at a pH level of 2 to 4. However, it is not intended that the present invention be limited to any particular pH value or range of values.

[0076] As used herein, the expression "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 that may be encountered in the stomach (e.g., the human stomach).

[0077] 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), as well as at least one bile salt (e.g., sodium taurocholate), such that the recombinant lipase is exposed to conditions that may be encountered in the intestine (e.g., the intestine of a human).

[0078] As used herein, the phrase "multiple sequential challenges" refers to exposing the recombinant lipase of the present invention to a series of challenge conditions. For example, in some embodiments, a one-hour heat challenge is followed by a one-hour gastric challenge, and then a one-hour intestinal challenge. It is not intended that the present invention be limited to any particular challenge and / or challenge conditions or a particular challenge sequence.

[0079] The terms "thermal stability" and "thermostability" refer to the ability of a protein (e.g., a recombinant lipase of the present invention) to function at a specific temperature. In some embodiments, the term refers to the ability of a protein to function after incubation at a specific temperature. In some embodiments, the recombinant lipase of the present invention is "thermostable" (i.e., the enzyme maintains its catalytic activity at elevated temperatures). In some embodiments, the recombinant lipase tolerates inactivation at elevated temperatures, and in some other embodiments, maintains catalytic activity at elevated temperatures for extended exposure times. These terms are used interchangeably herein. It is not intended that the present invention be limited to any specific temperature and / or exposure time. Such stability can be measured by any method known in the art (e.g., methods described herein). It is not intended that the present invention be limited to any specific temperature stability level 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 specific temperature.

[0080] The term "chemical stability" refers to the ability of a protein (eg, a recombinant lipase of the invention) to function in the presence of a chemical that adversely affects the function of another protein. It is not intended that the present invention be limited to any particular level of chemical stability or range of chemical stability.

[0081] "Conversion" refers to the enzymatic conversion (or bioconversion) of a substrate to a corresponding product. "Conversion percentage" refers to the percentage of substrate converted to product under specified conditions within a certain period of time. Therefore, the "enzymatic activity" or "activity" of a lipase polypeptide can be expressed as the "conversion percentage" of substrate to product within a specific period of time.

[0082] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washings of different but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity with the target DNA, and greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions with a thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids at 65°C in 0.018M NaCl (i.e., if the hybrid is unstable at 65°C in 0.018M NaCl, it will be unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS at 42°C, followed by washing in 0.1×SSPE and 0.1%SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization at 65°C in 5X SSC containing 0.1% (w / v) SDS and washing at 65°C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited above.

[0083] As used herein, "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operably connected to a suitable control sequence capable of achieving expression of a polypeptide encoded in a DNA sequence in a suitable host. In some embodiments, a plasmid can be used as a vector. In some embodiments, an "expression vector" has a promoter sequence operably connected to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0084] As used herein, "codon optimized" refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a particular organism, so that the encoded protein is more effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a particular organism is non-random and biased for specific codon triplets. With respect to the aggregated protein coding region of a given gene, a gene with a common function or ancestral origin, a high-expression protein contrast low copy number protein, and an organism's genome, this codon usage bias may be higher. In some embodiments, the polynucleotides encoding lipase may be codon optimized for optimized production from a selected host organism for expression.

[0085] As used herein, "control sequences" refer in this article to all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present application. Each control sequence can be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. For the purpose of introducing specific restriction sites, control sequences can be provided together with joints that promote the connection of control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0086] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0087] As used herein, "promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutants, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0088] As used herein, the phrase "suitable reaction conditions" refers to those conditions (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, etc.) in an enzymatic conversion reaction solution under which the lipase polypeptide of the present application is able to convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present application and are illustrated by the Examples.

[0089] As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0090] As used herein, in the context of an enzymatic conversion reaction process, "substrate" refers to the compound or molecule upon which the lipase polypeptide acts.

[0091] As used herein, in the context of an enzymatic conversion process, "product" refers to the compound or molecule resulting from the action performed by the enzyme polypeptide on the substrate.

[0092] As used herein, the term "expression" includes any step involved in the production of the polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0093] As used herein, the term "production" refers to the production of proteins and / or other compounds from a cell. It is intended that the term encompass any step 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 secretion of a polypeptide from a cell.

[0094] As used herein, an amino acid or nucleotide sequence (eg, a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature.

[0095] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector comprising a DNA as 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 as known in the art.

[0096] As used herein, the term "analog" refers to a polypeptide having more than 70% sequence identity, but less than 100% sequence identity (e.g., more 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, "analogs" include polypeptides containing one or more non-naturally occurring amino acid residues (including but not limited to homoarginine, ornithine and norvaline) and naturally occurring amino acids. In some embodiments, analogs also include one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0097] As used herein, the term "culture" refers to the growth of a cell colony under any suitable conditions (e.g., using a liquid, gel, or solid culture medium). In some embodiments, the cell is a microbial cell (e.g., a bacterium), and in some other embodiments, the cell is a mammalian cell, an insect cell, or a cell obtained from another animal. It is not intended that the present invention be limited to the cultivation of any specific cell or cell type or any specific culture method. In fact, it is intended that the present invention include any suitable cell type cultivated under any suitable conditions.

[0098] The term "therapeutic agent" refers to a compound that is administered to a subject displaying signs or symptoms of a pathology to provide a beneficial or desired effect, including a medical effect.

[0099] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject (eg, a human) comprising a pharmaceutically effective amount of an engineered lipase polypeptide encompassed by the present invention and an acceptable carrier.

[0100] The term "effective amount" means an amount sufficient to produce the desired result. One of ordinary skill in the art can determine what the effective amount is by using routine experimentation.

[0101] The term "subject" includes animals, including but not limited to mammals such as humans, non-human primates, livestock, pets, and laboratory animals (eg, rodents and lagomorphs). It is intended that the term includes females as well as males.

[0102] As used herein, the term "patient" means any subject being evaluated, treated, or experiencing a disease.

[0103] The term "infant" refers to children between the first month of life and about one (1) year of age.

[0104] As used herein, the term "neonatal" refers to a child in the period from birth to the 28th day of life. The term "preterm infant" refers to an infant born after the twentieth completed week of gestation but before the expiration of gestation, typically weighing ˜500 to ˜2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 g at birth.

[0105] As used herein, the term "child" refers to a person who has not reached the legal age to consent to treatment or research procedures. In some embodiments, the term refers to a person between the ages of birth and puberty.

[0106] As used herein, the term "adult" refers to a person who has reached the legal age for the relevant jurisdiction (e.g., 18 years old in the United States). In some embodiments, the term refers to any fully grown organism. In some embodiments, the term "young person" refers to a person who is less than 18 years old but has reached sexual maturity.

[0107] As used herein, "composition" and "formulation" include products comprising at least one engineered lipase of the invention intended for any suitable use (eg, pharmaceutical composition, dietary / nutritional supplement, feed, etc.).

[0108] As used herein, the terms "administration" and "administering" a composition means providing a composition of the invention to a subject (eg, a person suffering from the effects of pancreatic insufficiency).

[0109] The term "carrier" when used herein with reference to pharmaceutical compositions means any of the standard pharmaceutical carriers, buffers, and excipients such as stabilizers, preservatives and adjuvants.

[0110] The term "pharmaceutically acceptable" means a material that can be administered to a subject without causing any adverse biological effect or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.

[0111] As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient other than an active pharmaceutical ingredient (API; e.g., an engineered lipase polypeptide of the invention). Excipients are typically included for formulation and / or administration purposes.

[0112] When used in reference to symptoms of a disease / condition, the term "therapeutically effective amount" refers to the amount and / or concentration of a compound (e.g., an engineered lipase polypeptide) that improves, reduces or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of symptoms.

[0113] When used in reference to a disease / condition, the term "therapeutically effective amount" 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 to refer to the amount of a composition that elicits a biological (e.g., medical) response in a tissue, system, or animal subject that is sought by a researcher, physician, veterinarian, or other clinician.

[0114] As used herein, the terms "treating," "treat," and "treatment" refer to medical care given to a subject (e.g., a human patient), including the administration of a pharmaceutical composition (such as those provided herein). It is intended that the terms "treating," "treat," and "treatment" include preventive treatments (e.g., prophylactics) as well as palliative care or care. In some embodiments, treatment is provided to prevent or ameliorate symptoms of a disease. In some embodiments, the pharmaceutical compositions of the present invention can be used to treat or prevent a pancreatic enzyme deficiency disease or symptom.

[0115] Engineered lipase:

[0116] The present invention provides engineered lipases suitable for various uses, including treatment of pancreatic enzyme deficiency. In some embodiments, the engineered lipases exhibiting improved properties have at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868, and at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758, and / or 868, and at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity to SEQ ID NO: NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, an amino acid residue difference at one or more amino acid positions (such as, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid positions) compared to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, or a difference from SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868. NO: 2, 94, 350, 442, 540, 646, 758 and / or 868 have 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 greater amino acid sequence identity. In some embodiments, the residue difference at one or more positions compared to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868 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 conservative amino acid substitutions. However, it is not intended that the present invention is limited to the lipase variants with conservative amino acid substitutions, because other substitutions can be used for the present invention. In some embodiments, the engineered lipase polypeptide is a polypeptide listed in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1. In some embodiments, the engineered lipase polypeptide comprises SEQ ID NO:94, 350, 442, 540, 646, 758 and / or 868.

[0117] The present invention also 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, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 2, 3, 4, 22, 27 / 46 / 97 / 1 36 / 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, 1 95, 195 / 231 / 385, 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 219, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379 and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 2. NO:2 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 2H, 2M, 2T, 3A, 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97A / 136V / 149E / 385P, 27V / 46T / 385P, 27V / 70N / 136V / 231E / 385P, 27V / 136V / 323I / 385P, 27V / 149E, 27V / 149E / 385P, 27V / 385P, 3 4D, 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, 141 F, 141L, 141S, 142A, 142F, 142I, 142L, 144M, 144R, 144W, 146A, 146F, 146S, 146Y, 149E, 149E / 231E / 3 85P, 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, 21 2G, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the recombinant lipase comprises NO:2 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, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of E2H, E2M, E2T, T3A, T3G, T3R, T3S, S4M, S4W , S4Y, G22N, Y27V / R46T / Y97A / Y136V / T149E / F385P, Y27V / R46T / F385P, Y27V / K70N / Y136V / Q231E / F385P, Y27V / Y136V / K323I / F385P, Y27V / T149E, 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, K82 E / P94S / D101S / K194N / I199L, K82E / P94S / I199L, K82F, K82G, K82L, E83G, E83K, G85I, G85W, A87R, F89A, F89T, F89V, F89W, T92A, P94S, I96 E, I96N, I96S, Y97A / T149E / F385P, Y135G, Y135Q, Y135S, Y135T, Y135V, Y136V / F385P, P140T, E141A, E141F, E141L, E141S, E142A, E142F, E142I, E142L, I144M, I144R, I144W, P146A, P146F, P146S, P146Y, T149E, T149E / Q231E / F385P, T149K, T149R, G151A, G151I, G151L, E174H, E174R, Q175G, Q175L, Q175R, S178R, S178T, K181H, F183L / F385P, S189A, S189E, S189W, K194N, K194N / I199L, K194T, Q195D / Q231E / F385P , Q195V, I199L, I199L / P213A, K210A, K210V, Q212C, Q212G, Q212R, Q212T, P213A / N330T, P213H, S216R, S216T, S216W, H218D, H218G, H218I , H218M, H218T, A219C, A219G, A219K, A219R, T226R, Q231E / F385P, Y238S, E247R, Q250T, R270V, T274D, G281K, G281R, M292A, M292C, M292L, M292V, S296M, S296R, Q300A, Q300D, Q300T, R308A, N330Y, K338N, N379T, F385A, F385C, F385D, F385P, F385R and F385T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. ,

[0118] 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: 94, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 231 / 296 / 300, 4 / 96 / 149 / 292 / 296 / 300, 4 / 96 / 219 / 296, 4 / 96 / 231 / 296 / 300, 4 / 96 / 292 / 296 / 300, 4 / 149 / 174, 4 / 174 / 219 / 292 / 296, 4 / 231 / 296 / 300, 27, 27 / 34 / 82, 27 / 73 / 82 / 218, 27 / 89, 27 / 89 / 178, 27 / 89 / 218, 27 / 218, 34 / 73 / 21 8, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. 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: 94, or a functional fragment thereof,and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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. W / 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、96 E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174H / 292L / 296R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 2 0D, 292L / 296R, 292L / 296R / 300D, 296R and 296R / 300D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some 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: 94, or a functional fragment thereof,and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: T3S / S4W / I96E / S296R / Q300D, T3S / M292L / S296R, S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S296R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S296R / Q300 D. S4W / T149E / E174R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73I / K82 L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H218I, K34D / T73I / H218I, K34D / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183 L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / Q2 31E / M292L / S296R, I96E / T149E / M292L / S296R, I96E / E174H / A219K / Q231E / M292L / S296R, I96E / Q231E / S296R, P146F / Q175G / S189A / G281K, T149E / E 174H / M292L / S296R, T149E / E174H / Q300D, T149E / A219K / Q231E / M292L / S2 96R / 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.

[0119] 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 4, 4 / 27 / 189 / 300, 4 / 70, 4 / 102, 4 / 479 / 189 / 200, 4 / 27 / 189 / 300, 4 / 27 ... / 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, 2 5. 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 189A / 218M, 4W / 175G / 189A / 300D、4W / 175G / 218M / 224A、4W / 175G / 218M / 373F、4W / 185L、4W / 189A / 218M / 373F、4W / 193T、4W / 218M / 300D / 369N / 3 73F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 17 5G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 3 82M, 27V / 82C / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 18 9A, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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 / P228 E. S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S4W / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S18 9A / 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 / S18 9A, E174H / Y373F, Q175G / S189A / H218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.

[0120] 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: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 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、19 3, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 303, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281 and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some 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: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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, I1 44L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E17 4H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N37 5A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303 P / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0121] 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: 540, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174 / 193 / 195 / 243 / 281 / 303 / 330. 74 / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:540. In some additional embodiments, the recombinant lipase comprises a lipase having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 174G, 34L / 174G / 193L / 195L / 243R / 281P, 34L / 174H / 193L / 195 I / 303P / 330F, 34L / 193L / 195I / 225L / 243R, 34L / 193L / 243R / 303P / 330F, 3 4L / 281P / 330H, 38A / 174H / 193L / 225L / 274D / 283I / 303P / 330F, 38A / 174R / 2 81K / 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 / 34 4H, 49T / 98P / 120T / 252V / 344H, 49T / 120T / 252V / 344H, 49T / 123Q / 252V / 344 H, 49T / 123Q / 264S / 344H, 49T / 123Q / 311W, 49T / 252V / 344H, 49T / 311W / 344 H, 49T / 344H, 51A / 252V / 344H, 51A / 344H, 51V, 98P / 344H, 98R, 123Q / 252V / 3 44H, 129F, 160T, 161I, 174G / 195I / 225L / 281K / 303P / 330F / 345I, 174G / 225 L / 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 sequences are numbered with reference to SEQ ID NO: 540. In some additional embodiments, the recombinant lipase comprises NO:540 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38A / E174R / A303P / F345I, K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F 8G / 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 / T274 D / M283I / A303P / N330F, F38A / E174R / G281K / A303P / F345I, F38A / Q193L / Q195L / M225L / A303P / F345I, F38A / Q195I / A303P, F 38A / Q195Y / G281K / A303P / N330F, F38G, F38G / E174G / Q193L / A303P, F38G / E174G / G281K / A303P, F38G / E174H / Q195L / G281P / N330F, F38G / E174H / M225L / L243R / G281K / N330H, F38G / E174H / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H, F38G / Q195 I / 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 / M25 2V / 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 sequences are numbered with reference to SEQ ID NO: 540. ,

[0122] 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 237 / 287 / 291, 16 / 168 / 177 / 247 / 251 / 257. / 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 / 34 4. 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 / 28 1 / 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 and 281 / 344, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 646. In some additional embodiments, the recombinant lipase comprises NO:646 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 287L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 11I / 16F / 67A / 154Y / 168T / 237Q / 241S / 287L. 87L / 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, 3 4K / 49T / 243R / 252V, 34K / 49T / 252V / 268T / 344V, 34K / 161I / 193L / 243R / 252V / 268T, 34K / 161I / 193L / 243R / 268T / 344V, 34K / 161I / 193L / 243 R / 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、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 1 In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646, wherein the amino acid sequence of SEQ ID NO: 646 is numbered with reference to SEQ ID NO: 646. In some further embodiments, the recombinant lipase comprises the amino acid sequence of SEQ ID NO: 646, wherein the amino acid sequence of SEQ ID NO: 646 is numbered with reference to SEQ ID NO: 646. NO:646 has 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, V11I / Y67A / S168T / F287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y67A / A237Q / F287L / S291A, L 16F / S168T / V177L, F24M, F24M / Y278L, L34K / V49T / L161I / Q193L / L243R / S344H, L 34K / V49T / L161I / Q193L / L243R / S344V, L34K / V49T / L161I / Q193L / S344W, L34K / V 49T / L161I / L243R / S344H, L34K / V49T / L161I / M252V / S268T / S344V, L34K / V49T / Q 193L / L243R / M252V / S344W, L34K / V49T / Q193L / S344H, L34K / V49T / Q193L / S344W,<h2 style=";text-align:left;direction:ltr">L34K / V49T / L243R / M252V、L34K / V49T / M252V / S268T / S344V、L34K / L161I / Q193L / L243R / M252V / S268T、L34K / L161I / Q193L / L243R / S268T / S344V、L34 K / L161I / Q193L / L243R / S344W、L34K / L161I / Q193L / M252V / S268T / S344H、L 34K / 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 / L 161I / S344V, L34K / L161I / S344W, L34K / Q193L / L243R / M252V / S344W, L34K / Q193L / M252V / S268T / S344H, L34K / Q193L / S268T / G281P, L34K / M252V / S268 T / G281P / S344V、L34K / M252V / S344V、V49T / L161I / Q193L / L243R / M252V / S 344W、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 / W1 54Y / A237Q / F287L、Y67A / S168T / A237Q、Y67A / S168T / A237Q / F287L、Y67A / V177L / A237Q、W154Y / A237Q / L286V / F287L、W154Y / L286V / F287L、L161I / Q1 93L / 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 sequences are numbered with reference to SEQ ID NO:646.

[0123] 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:758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some 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:758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24M / 168T / 252V / 281P, 24M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S, and 281P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some 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: 758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: F24M / S168T / M252V / G281P, F24 M / A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S and G281P, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:758.

[0124] 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: 868, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 24, 24 / 38 / 49 / 70 / 149 / 161 / 174 / 175 / 189 / 19 3 / 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 additional embodiments, the recombinant lipase comprises IDNO: 868 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70 K / 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, 24 W, 24Y, 38A, 38C, 38D, 38E, 38F, 38H, 38I, 38K, 38L, 38M, 38N, 38P, 38Q, 38R, 38S, 38T, 38V, 38 W, 38Y, 49A, 49C, 49D, 49E, 49F, 49G, 49H, 49I, 49K, 49L, 49M, 49N, 49P, 49Q, 49R, 49S, 49V, 49 W, 49Y, 70A, 70C, 70D, 70E, 70F, 70G, 70I, 70K, 70L, 70M, 70N, 70P, 70Q, 70R, 70S, 70T, 70V, 70 W, 70Y, 149A, 149C, 149D, 149F, 149G, 149H, 149I, 149K, 149L, 149M, 149N, 149P, 149Q, 149R,149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M、271N、271P、271Q、271R、271S、271T、271V、271W、271Y、287A、287C、287D、287E、287F、287G, 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 287S, 287T, 287V, 287W, 287Y, 292A, 292C, 292D, 292E, 292F, 292G, 292H, 292I, 292K, 292M, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 293I, 293K, 2 93L, 293M, 293N, 293P, 293Q, 293S, 293T, 293V, 293W, 293Y, 296A, 296C, 296D, 296E, 296F, 296G, 296H, 296I, 296K, 296L, 296M, 29 6N, 296P, 296Q, 296S, 296T, 296V, 296W, 296Y, 303A, 303C, 303D, 303E, 303F, 303G, 303H, 303I, 303K, 303L, 303M, 303N, 303Q, 303 R, 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, 543A, 343C, 343D, 343E, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343P, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 343A, 343C, 343D, 343E, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343P, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:868. In some additional embodiments, the recombinant lipase comprises a lipase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%,99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / 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, G 38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38 W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70 F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149 L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I16 1N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C , R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R 174P, R174Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L17 5E, L175F, L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R,<h2 style=";text-align:left;direction:ltr">L175S、L175T、L175V、L175W、L175Y、A189C、A189D、A189E、A189F、A189G、A1 89H、A189I、A189K、A189L、A189M、A189N、A189P、A189Q、A189R、A189S、A189 T、A189V、A189W、A189Y、L193A、L193C、L193D、L193E、L193F、L193G、L193H、 L193I、L193K、L193M、L193N、L193P、L193Q、L193R、L193S、L193T、L193V、L19 3W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, L225H, L225I, L225K, L225M, L225N, L225P, L225Q, L225R, L225S, L225T, L225V, L225W, L225Y, E 231A、E231C、E231D、E231F、E231G、E231H、E231I、E231K、E231L、E231M、E23 1N、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, V252 E、V252F、V252G、V252H、V252I、V252K、V252L、V252M、V252N、V252P、V252Q、 V252R、V252S、V252T、V252W、V252Y、D271A、D271C、D271E、D271F、D271G、D27 1H、D271I、D271K、D271L、D271M、D271N、D271P、D271Q、D271R、D271S、D271T 、D271V、D271W、D271Y、L287A、L287C、L287D、L287E、L287F、L287G、L287H、L 287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287Y, L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292KL292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R293D, R293E, R293F, R293G, R293H, R293I, R 293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R296C, R296D, R296E, R296F, R296G, R296H, R2 96I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R296T, R296V, R296W, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P30 3H, 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, F3 73G, 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.

[0125] 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 wherein the amino acid positions of the polypeptide sequence are numbered with reference to 2, 94, 350, 442, 540, 646, 758 and / or 868.

[0126] 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 in SEQ ID NOs: 14-1796.

[0127] In some embodiments, the engineered lipase polypeptide comprises the functional fragment of the engineered lipase polypeptide comprised by the present invention.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 (that is, parental engineered lipase) derived from it.In some embodiments, functional fragment constitutes 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 parental sequence of engineered lipase. In some embodiments, the functional fragment is truncated by 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.

[0128] Polynucleotides, expression vectors and host cells encoding engineered lipase polypeptides:

[0129] The invention provides polynucleotides encoding engineered lipase polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous or homologous regulatory sequences controlling gene expression to create a recombinant polynucleotide capable of expressing a polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered lipase polypeptide can be introduced into a suitable host cell to express the corresponding lipase polypeptide.

[0130] As will be apparent to the technician, the availability of protein sequences and the understanding of the codons corresponding to various amino acids provide descriptions of all polynucleotides that can encode the subject polypeptide. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative codons or synonymous codons, allows the preparation of a very large number of nucleic acids, all of which encode engineered lipase polypeptides. Therefore, after knowing the specific amino acid sequence, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention particularly contemplates that each and every possible change of the polynucleotides that can be selected to combine to prepare the polypeptide described herein encoding based on possible codon selections, and for any polypeptide described herein, all such changes, including the variants provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and Table 5-1, should be considered to be particularly disclosed.

[0131] In various embodiments, codons are preferably selected to adapt to the host cell producing protein therein. For example, the preferred codons used in the bacterium are used for expression in bacteria, and the preferred codons used in the fungi are used for expression in the fungi. Therefore, the codon-optimized polynucleotides encoding the engineered lipase polypeptides comprise preferred codons at about 40%, 50%, 60%, 70%, 80% or greater than 90% of the codon positions in the full-length coding region.

[0132] In some embodiments, the present 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 present 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 in SEQ ID NOs: 13-1795.

[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: 2, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 2, 3, 4, 22, 27 / 46 / 97 / 1 36 / 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, 1 95, 195 / 231 / 385, 199, 199 / 213, 210, 212, 213, 213 / 330, 216, 218, 219, 226, 231 / 385, 238, 247, 250, 270, 274, 281, 292, 296, 300, 308, 330, 338, 379 and 385, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a recombinant lipase comprising the amino acid sequence of SEQ ID NO: 2. NO:2 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, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 2H, 2M, 2T, 3A, 3G, 3R, 3S, 4M, 4W, 4Y, 22N, 27V / 46T / 97A / 136V / 149E / 3 85P, 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, 14 1A, 141F, 141L, 141S, 142A, 142F, 142I, 142L, 144M, 144R, 144W, 146A, 146F, 146S, 146Y, 149E, 149E / 2 31E / 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, 219 , 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a recombinant lipase comprising the amino acid sequence of SEQ ID NO: 2. NO:2 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or its functional fragment, and wherein the recombinant lipase comprises at least one substitution or substitution set selected from the group consisting of E2H, E2M, E2T, T3A, T3G, T3R, T3S, S4M, S4W, S4Y, G22N, Y27V / R46T / Y97A / Y136V / T149E / F385P, Y27V / R46T / F385P, Y27V / K70N / Y136V / Q231E / F385P, Y27V / Y136V / K323I / F385P, Y27V / T149E,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, F8 9V, F89W, T92A, P94S, I96E, I96N, I96S, Y97A / T149E / F385P, Y135G, Y1 35Q, Y135S, Y135T, Y135V, Y136V / F385P, P140T, E141A, E141F, E141L, E 141S, E142A, E142F, E142I, E142L, I144M, I144R, I144W, P146A, P146F , P146S, P146Y, T149E, T149E / Q231E / F385P, T149K, T149R, G151A, G151 I, G151L, E174H, E174R, Q175G, Q175L, Q175R, S178R, S178T, K181H, F1 83L / F385P, S189A, S189E, S189W, K194N, K194N / I199L, K194T, Q195D / Q 231E / F385P, Q195V, I199L, I199L / P213A, K210A, K210V, Q212C, Q212G , Q212R, Q212T, P213A / N330T, P213H, S216R, S216T, S216W, H218D, H218 G, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. 、

[0134] 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 3 / 4 / 96 / 296 / 300, 3 / 292 / 296, 4 / 11 / 149 / 292 / 296 / 300, 4 / 96 / 149 / 231 / 149 / 296 / 301. / 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 / 21 8, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:94. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 94, or a functional fragment thereof,and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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. W / 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、96 E / 231E / 296R, 146F / 175G / 189A / 281K, 149E / 174H / 292L / 296R, 149E / 174H / 300D, 149E / 219K / 231E / 292L / 296R / 300D, 149E / 2 31E / 292L / 296R, 149E / 231E / 292L / 296R / 300D, 149E / 296R, 149E / 296R / 300D, 174H / 296R, 174R / 231E, 218I, 231E, 231E / 292L / 296R, 231E / 292L / 296R / 300D, 231E / 296R, 231E / 300D, 292L / 296R, 292L / 296R / 300D, 296R and 296R / 300D, wherein the amino acid positions of the polypeptide sequences 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: T3S / S4W / I96E / S296R / Q300D, T3S / M292L / S296R, S4W, S4W / V11A / T149E / M292L / S296R / Q300D, S4W / I96E / T149E / Q231E / S296R / Q300D, S4W / I96E / T149E / M292L / S296R / Q300D, S4W / I96E / A219K / S296R, S4W / I96E / Q231E / S296R / Q300D, S4W / I96E / M292L / S296R / Q300 D. S4W / T149E / E174R, S4W / E174H / A219K / M292L / S296R, S4W / Q231E / S296R / Q300D, Y27V, Y27V / K34D / K82E, Y27V / T73I / K82L / H218I, Y27V / T73I / K82 L / H218M, Y27V / F89T, Y27V / F89T / S178P, Y27V / F89T / H218I, Y27V / H218I, K34D / T73I / H218I, K34D / K82C, K34D / H218M, T73I / K82C, T73I / K82L / F183 L, P94S / P146F / Q175G, I96E / T149E / E174H / M292L / S296R, I96E / T149E / Q2 31E / M292L / S296R, I96E / T149E / M292L / S296R, I96E / E174H / A219K / Q231E / M292L / S296R, I96E / Q231E / S296R, P146F / Q175G / S189A / G281K, T149E / E 174H / M292L / S296R, T149E / E174H / Q300D, T149E / A219K / Q231E / M292L / S2 96R / 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.

[0135] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 4, 4 / 27 / 189 / 300, 4 / 70, 4 / 102, 4 / 123, 4 / 179, 4 / 280, 4 / 301, 4 / 402, 4 / 503, 4 / 604, 4 / 705, 4 / 806, 4 / 907, 4 / 108, 4 / 112, 4 / 129, 4 / 130, 4 / 141, 4 / 152, 4 / 163, 4 / 27 / 189. 37, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 4W, 4W / 27V / 189A / 300D, 4W / 70H, 4W / 102T, 4W / 137R, 4W / 175G / 189A / 218M, 4W / 175G / 189A / 312M. / 300D、4W / 175G / 218M / 224A、4W / 175G / 218M / 373F、4W / 185L、4W / 189A / 218M / 373F、4W / 193T、4W / 218M / 300D / 369N / 3 73F, 4W / 228E, 4W / 233R, 4W / 243R, 4W / 271D, 4W / 303P, 4W / 334T, 4W / 336S, 4W / 336T, 4W / 375A, 25V, 27V / 82C / 174H / 17 5G / 189A / 218M / 300D / 369N / 373F, 27V / 82C / 174H / 175G / 218M / 300D / 369N, 27V / 82C / 174H / 175G / 218M / 300D / 373F / 3 82M, 27V / 82C / 174H / 218M / 300D / 373F, 27V / 189A / 218M / 373F, 28N, 33Y, 99D, 102L, 104H, 134R, 153G, 174H / 175L / 18 9A, 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 350, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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 / P228 E. S4W / N233R, S4W / L243R, S4W / G271D, S4W / A303P, S4W / D334T, S4W / A336S, S4W / A336T, S4W / N375A, L25V, Y27V / K82C / E174H / Q175G / S18 9A / 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 / S18 9A, E174H / Y373F, Q175G / S189A / H218M / Q300D / Y373F, Q175G / H218M, Q175G / H218M / H382M, Q175L / S189A / Y373F, S189A / H218M / Q300D / Y373F, A191L, Q193L, E231Q, N233G, N233S, N233W, L243R, Q293R, A303P, S331R, A336R, Q339K, F368A, F368T and Y373F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:350.

[0136] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a nucleotide sequence of SEQ ID NO:442 has 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 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 the group consisting of: 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, 19 3, 193 / 218 / 243, 193 / 271 / 303 / 334, 193 / 293 / 303, 194, 195, 199, 210, 218, 218 / 243, 218 / 243 / 303 / 334, 218 / 303, 225, 238, 274, 281 and 330, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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, I1 44L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E17 4H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N37 5A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303 P / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 442, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 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, I1 44L, E174H, E174H / L175G / Q193L, E174H / L175G / H218M / Q339K, E174H / Q193L / A303P / N375A, E174H / Q193L / N375A, E17 4H / H218M / N233R / G271D / Q293R / A303P, E174H / H218M / G271D / A303P, E174R, L175G / Q193L / H218M / N233R / L243R / N37 5A, L175G / Q193L / H218M / L243R, L175G / H218M / N375A, K181Q, A189H, Q193L, Q193L / H218M / L243R, Q193L / G271D / A303 P / 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, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:442.

[0137] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 540, or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 34 / 38 / 174 / 193 / 195 / 243 / 281 / 303 / 330, 34 / 38 / 174 / 225 / 303, 34 / 38 / 174 / 303 / 345, 34 / 174 / 193 / 195 / 243 / 281 / 303 / 330. 、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 / 252 / 344、49 / 123 / 264 / 344、49 / 123 / 311、49 / 252 / 344、49 / 311 / 34 4, 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, 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 34L / 38A / 174G / 193L / 195L / 243R / 281P / 303P / 330F, 34L / 38A / 174R / 303P / 345I, 34L / 38G / 174R / 225L / 303P, 34L / 174G, 34L / 174G / 193L / 195L / 243R / 281P, 34L / 174H / 193L / 195I / 303P / 330F, 34L / 193L / 195I / 225L / 243R, 34L / 193L / 243R / 303P / 330F, 34L / 281P / 330H, 3 8A / 174H / 193L / 225L / 274D / 283I / 303P / 330F, 38A / 174R / 281K / 303P / 345I, 38A / 193L / 195L / 225L / 303P / 345I, 38A / 195I / 303P, 38A / 195 Y / 281K / 303P / 330F, 38G, 38G / 174G / 193L / 303P, 38G / 174G / 281K / 303P, 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, 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of K34L / F38A / E174G / Q193L / Q195L / L243R / G281P / A303P / N330F, K34L / F38 A / 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 / A 303P / N330F, K34L / G281P / N330H, F38A / E174H / Q193L / M225L / T274D / M283I / A303P / N330F, F38A / E174R / G2 81K / A303P / F345I, F38A / Q193L / Q195L / M225L / A303P / F345I, F38A / Q195I / A303P, F38A / Q195Y / G281K / A30 3P / N330F, F38G, F38G / E174G / Q193L / A303P, F38G / E174G / G281K / A303P, F38G / E174H / Q195L / G281P / N330F , F38G / E174H / M225L / L243R / G281K / N330H, F38G / E174H / G281K, F38G / E174R / Q193L / Q195L / L243R / N330H,F38G / Q195I / G281P / N330F, F38G / Q195Y / L243R / A303P, V49S, V49T, V49T / T51A / G98P / M252V / L3 11W, V49T / T51A / E123Q / M252V / S344H, V49T / G98P / M120T / M252V / S344H, V49T / M120T / M252V / S34 4H, V49T / E123Q / M252V / S344H, V49T / E123Q / T264S / S344H, V49T / E123Q / L311W, V49T / M252V / S3 44H, 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 / N3 30H, 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 sequences are numbered with reference to SEQ ID NO: 540. ,

[0138] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 646, or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 11 / 16 / 67 / 168 / 180 / 287, 11 / 16 / 237 / 241 / 287, 11 / 67 / 168 / 287, 16 / 67 / 154 / 168 / 237 / 241 / 287, 16 / 67 / 237 / 287 / 291, 16 / 168 / 177 / 247 / 251 / 257. 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 / 28 1 / 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 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 the amino acid sequence of SEQ ID NO: 646. NO:646 has 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 11I / 16F / 67A / 168T / 180V / 287L, 11I / 16F / 237Q / 241S / 287L, 11I / 67A / 168T / 287L, 16F / 67A / 154Y / 168T / 237Q / 241S / 287L, 16F / 67 A / 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 / 1 61I / 193L / 252V / 344V, 34K / 161I / 193L / 252V / 344W, 34K / 161I / 193L / 268T / 344H, 34K / 161I / 243R / 281P / 344V, 34K / 161I / 344V, 34K / 161 I / 344W, 34K / 193L / 243R / 252V / 344W, 34K / 193L / 252V / 268T / 344H, 34K / 193L / 268T / 281P, 34K / 252V / 268T / 281P / 344V, 34K / 252V / 344V,49T / 161I / 193L / 243R / 252V / 344W, 49T / 161I / 193L / 252V / 281P / 344H, 49T / 161I / 243R / 252V / 344V, 49T / 193L / 197G / 243R / 252V / 281P / 34 4H, 49T / 193L / 243R / 252V / 344V, 49T / 193L / 243R / 344W, 49T / 243R / 344V, 67A / 154Y / 237Q / 287L, 67A / 168T / 237Q, 67A / 168T / 237Q / 287L, 6 7A / 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 IDNO: 646 has 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 or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of V11I / L16F / Y67A / S168T / I180V / F287L, V11I / L16F / A237Q / T241S / F287L, V11I / L16F / Y67A / S168T / F287L, L16F / Y67A / W154Y / S168T / A237Q / T241S / F287L, L16F / Y 67A / A237Q / F287L / S291A, L16F / S168T / V177L, F24M, F24M / Y278L, L34K / V 49T / L161I / Q193L / L243R / S344H, L34K / V49T / L161I / Q193L / L243R / S344V, L34K / V49T / L161I / Q193L / S344W, L34K / V49T / L161I / L243R / S344H, L34K / V 49T / L161I / M252V / S268T / S344V, L34K / V49T / Q193L / L243R / M252V / S344W,<h2 style=";text-align:left;direction:ltr">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 / L24 3R / S344W、L34K / L161I / Q193L / M252V / S268T / S344H、L34K / L161I / Q193L / M 252V / S268T / S344V,L34K / L161I / Q193L / M252V / S344H,L34K / L161I / Q193L / M252V / S344V,L34K / L161I / Q193L / M252V / S344W,L34K / L161I / Q193L / S2 68T / S344H, L34K / L161I / L243R / G281P / S344V, L34K / L161I / S344V, L34K / L161I / S344W, L34K / Q193L / L243R / M252V / S344W, L34K / Q193L / M252V / S268 T / S344H、L34K / Q193L / S268T / G281P、L34K / M252V / S268T / G281P / S344V、L 34K / 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, W15 4Y / A237Q / L286V / F287L、W154Y / L286V / F287L、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 sequences are numbered with reference to SEQ ID NO:646.

[0139] In some embodiments, the polynucleotide encodes a recombinant lipase, which comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:758, or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 24 / 168 / 252 / 281, 24 / 237 / 287 / 344, 24 / 252, 24 / 252 / 287, 24 / 344, 213 / 252 / 278 / 344, 252 / 278 / 344, 252 / 287 / 344 and 281, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:758. In some embodiments, the polynucleotide encodes a recombinant lipase, the recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 758, or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24M / 168T / 252V / 281P, 24M / 237Q / 287L / 344S, 24M / 252V, 24M / 252V / 287L, 24M / 344W, 213S / 252V / 278L / 344S, 252V / 278L / 344W, 252V / 287L / 344S, and 281P, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO: 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 wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: F24M / S168T / M252V / G281P, F24 M / A237Q / F287L / H344S, F24M / M252V, F24M / M252V / F287L, F24M / H344W, P213S / M252V / Y278L / H344S, M252V / Y278L / H344W, M252V / F287L / H344S and G281P, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:758.

[0140] In some embodiments, the polynucleotide encodes a recombinant lipase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 868, or a functional fragment thereof, wherein the recombinant lipase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 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 embodiments, the polynucleotide encodes a recombinant lipase comprising the amino acid sequence of SEQ ID NO: 868. NO:868 has 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, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of 24A, 24C, 24D, 24E, 24F, 24F / 38F / 49V / 70K / 149T / 161L / 174E / 175Q / 189S / 193Q / 225M / 231Q / 243L / 252M / 271G / 287F / 292M / 293Q / 296S / 303A / 334D / 344S / 373Y / 385F, 24G, 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, 3 8T, 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, 7 0S, 70T, 70V, 70W, 70Y, 149A, 149C, 149D, 149F, 149G, 149H, 149I, 149K, 149L, 149M, 149N,149P、149Q、149R、149S、149T、149V、149W、149Y、161A、161C、161D、161E、161F、161G、161H、161K、161L、161M、161N、161P、161Q、161R、161S、161T、161V、161W、161Y、174A、174C、174D、174E、174F、174G、174H、174I、174K、174L、174M、174N、174P、174Q、174S、174T、174V、174W、174Y、175A、175C、175D、175E、175F、175G、175H、175I、175K、175M、175N、175P、175Q、175R、175S、175T、175V、175W、175Y、189C、189D、189E、189F、189G、189H、189I、189K、189L、189M、189N、189P、189Q、189R、189S、189T、189V、189W、189Y、193A、193C、193D、193E、193F、193G、193H、193I、193K、193M、193N、193P、193Q、193R、193S、193T、193V、193W、193Y、225A、225C、225D、225E、225F、225G、225H、225I、225K、225M、225N、225P、225Q、225R、225S、225T、225V、225W、225Y、231A、231C、231D、231F、231G、231H、231I、231K、231L、231M、231N、231P、231Q、231R、231S、231T、231V、231W、231Y、243A、243C、243D、243E、243F、243G、243H、243I、243K、243L、243M、243N、243P、243Q、243S、243T、243V、243W、243Y、252A、252C、252D、252E、252F、252G、252H、252I、252K、252L、252M、252N、252P、252Q、252R、252S、252T、252W、252Y、271A、271C、271E、271F、271G、271H、271I、271K、271L、271M、271N、271P、271Q、271R、271S、271T、271V、271W、271Y、287A、287C、287D, 287E, 287F, 287G, 287H, 287I, 287K, 287M, 287N, 287P, 287Q, 287R, 287S, 287T, 287V, 287W, 287Y, 292A, 292C, 292D, 292E, 292 F, 292G, 292H, 292I, 292K, 292M, 292N, 292P, 292Q, 292R, 292S, 292T, 292V, 292W, 292Y, 293A, 293C, 293D, 293E, 293F, 293G, 293H, 2 93I, 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, 30 3Q, 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, 344 573A, 373C, 373D, 373E, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373P, 373Q, 373R, 373S, 373T, 373V, 373W, 373Y, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N, 385Q, 385R, 385S, 385T, 385V, 385W and 385Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:868. In some embodiments, the polynucleotide encodes a recombinant lipase comprising a recombinant lipase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%,99% or more sequence identity or a functional fragment thereof, and wherein the recombinant lipase comprises at least one substitution or set of substitutions selected from the group consisting of: M24A, M24C, M24D, M24E, M24F, M24F / G38F / T49V / H70K / E149T / I161L / R174E / L175Q / A189S / L193Q / L225M / E231Q / R243L / V252M / D271G / 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, G 38H, G38I, G38K, G38L, G38M, G38N, G38P, G38Q, G38R, G38S, G38T, G38V, G38 W, G38Y, T49A, T49C, T49D, T49E, T49F, T49G, T49H, T49I, T49K, T49L, T49M, T49N, T49P, T49Q, T49R, T49S, T49V, T49W, T49Y, H70A, H70C, H70D, H70E, H70 F, H70G, H70I, H70K, H70L, H70M, H70N, H70P, H70Q, H70R, H70S, H70T, H70V, H70W, H70Y, E149A, E149C, E149D, E149F, E149G, E149H, E149I, E149K, E149 L, E149M, E149N, E149P, E149Q, E149R, E149S, E149T, E149V, E149W, E149Y, I161A, I161C, I161D, I161E, I161F, I161G, I161H, I161K, I161L, I161M, I16 1N, I161P, I161Q, I161R, I161S, I161T, I161V, I161W, I161Y, R174A, R174C , R174D, R174E, R174F, R174G, R174H, R174I, R174K, R174L, R174M, R174N, R 174P, R174Q, R174S, R174T, R174V, R174W, R174Y, L175A, L175C, L175D, L17 5E, L175F, L175G, L175H, L175I, L175K, L175M, L175N, L175P, L175Q, L175R,<h2 style=";text-align:left;direction:ltr">L175S、L175T、L175V、L175W、L175Y、A189C、A189D、A189E、A189F、A189G、A1 89H、A189I、A189K、A189L、A189M、A189N、A189P、A189Q、A189R、A189S、A189 T、A189V、A189W、A189Y、L193A、L193C、L193D、L193E、L193F、L193G、L193H、 L193I、L193K、L193M、L193N、L193P、L193Q、L193R、L193S、L193T、L193V、L19 3W, L193Y, L225A, L225C, L225D, L225E, L225F, L225G, L225H, L225I, L225K, L225M, L225N, L225P, L225Q, L225R, L225S, L225T, L225V, L225W, L225Y, E 231A、E231C、E231D、E231F、E231G、E231H、E231I、E231K、E231L、E231M、E23 1N、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, V252 E、V252F、V252G、V252H、V252I、V252K、V252L、V252M、V252N、V252P、V252Q、 V252R、V252S、V252T、V252W、V252Y、D271A、D271C、D271E、D271F、D271G、D27 1H、D271I、D271K、D271L、D271M、D271N、D271P、D271Q、D271R、D271S、D271T 、D271V、D271W、D271Y、L287A、L287C、L287D、L287E、L287F、L287G、L287H、L 287I, L287K, L287M, L287N, L287P, L287Q, L287R, L287S, L287T, L287V, L287W, L287Y, L292A, L292C, L292D, L292E, L292F, L292G, L292H, L292I, L292KL292M, L292N, L292P, L292Q, L292R, L292S, L292T, L292V, L292W, L292Y, R293A, R293C, R293D, R293E, R293F, R293G, R293H, R293I, R 293K, R293L, R293M, R293N, R293P, R293Q, R293S, R293T, R293V, R293W, R293Y, R296A, R296C, R296D, R296E, R296F, R296G, R296H, R2 96I, R296K, R296L, R296M, R296N, R296P, R296Q, R296S, R296T, R296V, R296W, R296Y, P303A, P303C, P303D, P303E, P303F, P303G, P30 3H, 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, F3 73G, 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.

[0141] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having a lipase activity with properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having 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 Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1, and ... NO:2, 94, 350, 442, 540, 646, 758 and / or 868, or one 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 any variant disclosed in the table. In some embodiments, the polynucleotide encodes an engineered polypeptide having lipase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having 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 the reference sequence SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, and one or more residue differences compared to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, wherein the one or more residue differences are located at a position selected from the group consisting of: The polypeptides of NO: 2, 94, 350, 442, 540, 646, 758 and / or 868 are optimally aligned at residue positions as those provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1.

[0142] 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 the reference sequence SEQ ID NO: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 Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 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 the reference sequence SEQ ID NO:1, 93, 349, 441, 539, 645, 757 and / or 867. In some other 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 Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1. In some embodiments, the polynucleotide encoding the engineered lipase polypeptide comprises a polynucleotide sequence of SEQ ID NO:93, 349, 441, 539, 645, 757 and / or 867.

[0143] In some embodiments, polynucleotide can hybridize with reference polynucleotide sequence under high stringency conditions.In some embodiments, reference sequence is selected from SEQ ID NO:1,93,349,441,539,645,757 and / or 867 or its complementary sequence or the polynucleotide sequence of any variant lipase polypeptide provided herein encoding.In some embodiments, the polynucleotide encoding lipase polypeptide that can hybridize under high stringency conditions, described lipase polypeptide comprises and SEQ ID NO:1,93,349,441,539,645,757 and / or 867 compared to the residue position of any position selected from as listed in table 4-1, table 4-2, table 4-3, table 4-4, table 4-5, table 4-6, table 4-7 and / or table 5-1 has one or more residue difference amino acid sequence. In some additional embodiments, the engineered polynucleotide is selected from those provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1, or comprises a polynucleotide having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence selected from SEQ ID NO: 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 Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1 and / or SEQ ID NO:1, 93, 349, 441, 539, 645, 757 and / or 867. In some additional embodiments, the engineered polynucleotide sequence comprises 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 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 base changes compared to a reference polynucleotide sequence.In yet other embodiments, the engineered polynucleotide sequence comprises 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 40, 45, or 50 base changes compared to a reference polynucleotide sequence. In some embodiments, the engineered polynucleotide sequence comprises 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 base changes compared to a reference polynucleotide sequence.

[0144] In some embodiments, the polynucleotide of the separation of any engineered lipase polypeptide provided herein is operated in various ways, to provide the expression of polypeptide gene and the generation of polypeptide.In some embodiments, the polynucleotide of encoding polypeptide provides with expression vector, wherein there is one or more control sequences to regulate the expression of polynucleotide and / or polypeptide.Depend on expression vector, before the polynucleotide of separation is inserted in the carrier, it is operated and can be desirable or necessary.It is well known in the art to be used for utilizing the technology of recombinant DNA method to modify polynucleotide and nucleotide sequence.

[0145] In some embodiments, control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As known in the art, suitable control sequences can be selected based on the host cell used. It is not intended that the present invention be limited to any specific control sequence.

[0146] Exemplary promoters for bacterial host cells include, but are not limited to, promoters obtained from the E. coli lactose operon, the tryptophan operon, the arabinose operon, the T7 promoter from T7 bacteriophage, and the Saccharopolyspora erythraea erythromycin resistance gene.

[0147] Exemplary promoters for filamentous fungal host cells include those obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral alpha-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for 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] ).

[0148] Exemplary promoters for use in mammalian cells include, but are not limited to, a promoter from cytomegalovirus (CMV), a promoter from simian vacuolating virus 40 (SV40), a promoter from phosphoglycerate kinase, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase of Homo sapiens, or a promoter from β-actin of Gallus gallus.

[0149] In some embodiments, the control sequence is a suitable transcription terminator sequence, and the transcription terminator sequence is a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that has a function in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for bacterial host cells can be obtained from T7 bacteriophage (e.g., T7 terminator) or Escherichia coli ribosomal RNA (e.g., rrnB terminator). For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: 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 following genes: 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., above). Exemplary terminators for mammalian cells include, but are not limited to, terminators from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), or from Homo sapiens growth hormone.

[0150] In some embodiments, the control sequence is a suitable leader sequence, and the leader sequence is the non-translational region of the mRNA important for the translation of the host cell. The leader sequence is operably connected to the 5' end of the nucleotide sequence of the encoded polypeptide. Any leader sequence that works in the selected host cell can be used. The exemplary leader sequence for filamentous fungal host cells is obtained from the following gene: 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 following genes: 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).

[0151] In some embodiments, the control sequence can also be a polyadenylation sequence, which is a sequence that is operably connected to the 3' end of the nucleic acid sequence and is recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA when transcribed. Any polyadenylation sequence that works 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 obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 15: 5983-5990

[1995] ).

[0152] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region that is naturally linked in 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.

[0153] Any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for the expression of the engineered lipase polypeptide provided herein. The effective signal peptide coding region for filamentous fungal host cells includes but is not limited to the signal peptide coding region obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include but are not limited to those from the following genes: Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to signal peptides from immunoglobulin gamma (IgG) genes.

[0154] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or in some cases a "zymogen." The propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0155] On the other hand, the present invention also provides recombinant expression vector, described recombinant expression vector comprises the polynucleotide of encoding engineering lipase polypeptide and depends on one or more expression control regions of the type of the host cell that the polynucleotide is introduced into, such as promoter and terminator, replication origin etc.In some embodiments, various nucleic acids described above and control sequence are linked together to produce recombinant expression vector, described recombinant expression vector comprises one or more convenient restriction sites to allow the nucleotide sequence of encoding variant lipase polypeptide to insert or replace at such site.Alternatively, polynucleotide sequence of the present invention is expressed by inserting the polynucleotide sequence or nucleic acid construct comprising this polynucleotide sequence into the suitable vector for expression.When producing expression vector, encoding sequence is located in carrier, makes encoding sequence operably connected with the suitable control sequence for expression.

[0156] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can result in the expression of the variant lipase polynucleotide sequence. The selection of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0157] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some optional embodiments, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates with the chromosome into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids or transposons that together contain the total DNA to be introduced into the host cell genome may be used.

[0158] In some embodiments, the expression vector preferably comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral tolerance, tolerance to heavy metals, prototrophy to auxotrophs, etc. Suitable markers for bacterial host cells include, but are not limited to, those that provide resistance to: carbenicillin, ampicillin, chloramphenicol, tetracycline, kanamycin, zeocin. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the 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 expressing the engineered lipase in the host cell.

[0159] Host cells for expressing polypeptides encoded by the expression vectors of the invention are well known in the art, and include, but are not limited to, bacterial cells (e.g., E. coli); fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC Accession No. 201178]); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, CHO-K1, COS and BHK) and human cells (e.g., HEK293T, human fibroblasts, THP-1, Jurkat and Bowes melanoma cell lines).

[0160] Therefore, on the other hand, the invention provides a method for producing an engineered lipase polypeptide, wherein the method comprises cultivating a host cell capable of expressing a polynucleotide encoding an engineered lipase polypeptide under conditions suitable for expressing the polypeptide. In some embodiments, the method further comprises the step of separating and / or purifying the lipase polypeptide as described herein.

[0161] Suitable culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expressing lipase can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0162] The engineered lipase with characteristics disclosed herein can be obtained by subjecting the polynucleotides encoding the naturally occurring or engineered lipase polypeptides to mutagenesis and / or directed evolution methods known in the art and / or as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, 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 United States Patent (USP) 6,537,746). Other directed evolution procedures that can be used include the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

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

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

[1996] ), among others.

[0163] 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 in a vector such as a plasmid and expressed in a desired host such as Escherichia coli. Variants of recombinant polypeptides can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specificity (region-specificity) or random mutations in a local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene. Many suitable methods for producing enzyme variants are known to those skilled in the art, including but not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be easily applied to polynucleotides encoding enzymes to produce variant libraries that can be expressed, screened and measured. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165 ,793, No. 6,180,406, No. 6,251,674, No. 6,265,201, No. 6,277,638, No. 6,287,861, No. 6,287,862, No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030 No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, 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,No. 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. 6,6 02,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,2 No. 96, 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. 7,77 No. 6,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 7,981,614 No. 8,014,961, No. 8,029,988, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806, No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 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,No. 833, No. 9,821,613, No. 9,996,661, No. 10,738,286 and all related U.S. and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0164] In some embodiments, the enzyme variants obtained after the mutagenesis treatment are screened by subjecting the enzyme variants to a determined temperature (or other assay conditions) and measuring the amount of the remaining enzymatic activity after thermal treatment or other assay conditions. The DNA of the polynucleotides comprising the coding lipase polypeptide is then separated from the host cell, sequenced to identify nucleotide sequence changes (if any), and used to express enzymes in different or identical host cells. Measuring the enzymatic activity from the expression library can be carried out using any suitable method known in the art (for example, standard biochemical techniques, such as HPLC analysis).

[0165] For engineered polypeptides of known sequences, polynucleotides encoding enzymes 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 separately and then connected (for example, by enzymatic or chemical connection methods or polymerase-mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can use classical phosphoramidite methods (see, for example, Beaucage et al., Tetra. Lett., 22: 1859-69

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

[1984] ), as usually practiced in automated synthesis methods, prepared by chemical synthesis. According to the phosphoramidite method, oligonucleotides are synthesized (for example, in an automatic DNA synthesizer), purified, annealed, connected and cloned into appropriate vectors. However, it is not intended that the present invention is limited to any specific method for producing polynucleotides or oligonucleotides, because any suitable method can be used in the present invention.

[0166] Therefore, in some embodiments, the method for preparing an engineered lipase polypeptide can include: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any variant provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1 and SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, and (b) expressing a lipase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide comprises one or several (e.g., up to 3, 4, 5 or up to 10) amino acid residues missing, inserted and / or substituted. In some embodiments, the amino acid sequence comprises 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 residue deletions, insertions and / or substitutions. In some embodiments, the amino acid 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, 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 residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid 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 amino acid residue deletions, insertions and / or substitutions. In some embodiments, substitutions include conservative substitutions and / or non-conservative substitutions.

[0167] The expressed engineered lipase polypeptides can be evaluated for any desired improved property (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.

[0168] In some embodiments, any of the engineered lipase polypeptides expressed in host cells is recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, and chromatography.

[0169] The chromatographic techniques used to separate the 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 used to purify the specific enzymes 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 separate improved variant lipases. In some embodiments utilizing affinity chromatography purification, any antibody of the specific binding variant lipase polypeptide can be used.

[0170] In some embodiments utilizing affinity chromatography purification, a protein that is covalently attached to a polysaccharide bound to a lipase can be used. In other embodiments utilizing affinity chromatography purification, any small molecule that binds to the lipase active site can be used. In order to produce antibodies, various host animals are immunized by injection of polypeptides (e.g., lipase variants) or fragments thereof, including but not limited to rabbits, mice, rats, etc. In some embodiments, the lipase polypeptide or fragment is attached to a suitable carrier, such as BSA, by means of a side chain functional group or a linker attached to a side chain functional group.

[0171] In some embodiments, the engineered lipase polypeptide is produced in a host cell by a method comprising: culturing a host cell (e.g., E. coli, Saccharomyces cerevisiae, Daucus carota, Nicotiana tabacum, Homo sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotide sequence encoding an engineered lipase polypeptide as described herein under conditions favorable for production of the engineered lipase polypeptide, and recovering the engineered lipase polypeptide from the cells and / or culture medium.

[0172] In some embodiments, the invention includes a method for producing an engineered lipase polypeptide, the method comprising culturing a recombinant eukaryotic cell comprising a polynucleotide sequence encoding an engineered lipase polypeptide under suitable culture conditions to allow production of the engineered lipase polypeptide, and optionally recovering the engineered lipase polypeptide from the culture medium and / or the cultured eukaryotic cells, wherein the engineered lipase polypeptide 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 NO: 2, 94, 350, 442, 540, 646, 758 and / or 868), and one or more amino acid residue differences compared to SEQ ID NO: 2, 94, 350, 442, 540, 646, 758 and / or 868, wherein the one or more amino acid residue differences are selected from the group consisting of: When the amino acid sequences of NO:2, 94, 350, 442, 540, 646, 758 and / or 868 are optimally aligned, the amino acid residue differences and / or combinations thereof provided in Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7 and / or Table 5-1.

[0173] In some embodiments, after the engineered polypeptide is recovered from recombinant host cells or cell culture medium, they are further purified by any suitable method known in the art. In some other embodiments, the purified lipase polypeptide is combined with other compositions and compounds to provide compositions and preparations (e.g., pharmaceutical compositions) comprising the engineered lipase polypeptide applicable to different applications and purposes. In some other embodiments, the purified lipase polypeptide or the prepared lipase polypeptide are lyophilized.

[0174] Composition:

[0175] The invention provides various compositions and forms, including but not limited to those described below.In some embodiments, the invention provides engineered lipase polypeptides suitable for use in pharmaceutical compositions and other compositions such as dietary and / or nutritional supplements.

[0176] Depending on the mode of administration, these compositions comprising a therapeutically effective amount of engineered lipase according to the present invention are in the form of solid, semisolid or liquid. In some embodiments, the compositions include other pharmaceutically acceptable components, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers and other ingredients. The details of the technology for preparation and administration are well known in the art and are described in the literature.

[0177] In some embodiments, the engineered lipase polypeptide is prepared for use in a pharmaceutical composition. Any suitable form used in delivering the engineered lipase polypeptide can be used for the present invention, and the form includes but is not limited to pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, gels, coatings, aerosols, chewing gums, demulcents, strips, solutions, suspensions (including but not limited to oil-based suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, etc. In some embodiments, the engineered lipase polypeptide is provided in a form suitable for injection or infusion (that is, with injectable preparations). In some embodiments, the engineered lipase polypeptide is provided with a biocompatible matrix such as sol-gel, including a sol-gel based on silicon dioxide (for example, oxysilane (oxysilane)). In some embodiments, the engineered lipase polypeptide is encapsulated and / or enteric coated. In some optional embodiments, the engineered lipase polypeptide is encapsulated in a nanostructure (e.g., nanotubes, nanotubes, nanocapsules or microcapsules, microspheres, liposomes, etc.). In fact, it is not intended that the present invention be limited to any particular delivery formulation and / or mode of delivery. It is intended that the engineered lipase polypeptide be administered by any suitable means known in the art, including but not limited to: parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc.

[0178] In some embodiments, the engineered lipase polypeptide is chemically modified by glycosylation, chemical cross-linking agents, pegylation (i.e., modification with polyethylene glycol [PEG] or activated PEG, etc.), or other compounds (see, e.g., Ikeda, Amino Acids 29:283-287

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

[0179] In some other embodiments, the engineered lipase polypeptide is provided in a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises a cross-linked crystalline engineered lipase and a polymer with a reactive portion attached to the enzyme crystal. The present invention also provides an engineered lipase polypeptide with a polymer.

[0180] In some embodiments, compositions comprising an engineered lipase polypeptide of the invention comprise one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol and / or sorbitol), starches (e.g., corn starch, wheat starch, rice starch, potato starch or other plant starches), celluloses (e.g., methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, tragacanth, guar gum, etc.), and / or proteins (e.g., gelatin, collagen, etc.).

[0181] In some embodiments, engineered lipase polypeptides are suitable for improving dietary fat absorption and reducing dietary lipids in feces. In some embodiments, the invention provides engineered lipase polypeptides suitable for reducing glycolipid concentration in fluids (such as blood, cerebrospinal fluid, etc.). The dosage of the engineered lipase polypeptides used depends on the general condition of the condition or disease, the experimenter, and other factors known to those skilled in the art. In some embodiments, it is intended that the composition is used once or more than once. In some embodiments, it is envisioned that the concentration of one or more engineered lipase polypeptides in one or more compositions used by the human beings suffering from pancreatic insufficiency is enough to effectively treat and / or improve disease (for example, pancreatic insufficiency disease). In some embodiments, engineered lipase polypeptides can be used for treating diseases (such as pancreatic enzyme deficiency) in combination with other enzymes (such as amylase and / or protease).

[0182] In some embodiments, the engineered lipase polypeptides are administered in combination with other drugs and / or dietary compositions (including but not limited to dietary supplements, nutraceuticals, etc.). It is not intended that the present invention be limited to any particular method or form of administration, as any suitable method and / or form may be used.

[0183] experiment

[0184] The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0185] In the following experimental disclosure, the following abbreviations are used: ppm (parts per million); M (mole / liter); mM (millimol / liter), uM and μM (micromol / liter); nM (nanomoles / liter); 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(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (a commonly used laboratory strain of E. coli available from the Coli Genetic Stock Center [CGSC], New Haven, CT); EPI (exocrine pancreatic insufficiency); LIP and lip (lipase); btLIP ( Thermophage B. thermoamylovorans lipase); HPLC (high pressure liquid chromatography); ms (mass spectrometry or mass spectrometry); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PES (polyether sulfone); ACN (acetonitrile); IPA (isopropanol); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphonic acid); 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 accelerating voltage; collision cell accelerating voltage); CE (collision energy); RF (radio frequency); Sinclair (Sinclair Research and Sinclair Bio Resources, Auxvasse, MO); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Molecular Devices (Molecular Devices, LLC, Sunnyvale, CA); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, CA); MS( Mass spectrometers, 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).

[0186] Example 1

[0187] Obtaining bacterial lipase gene and constructing its expression vector

[0188] The DNA sequence encoding Bacillus thermoamylovorans lipase (SEQ ID NO: 2) is codon optimized for expression in Escherichia coli and cloned into the Escherichia coli expression vector pCK110900 vector system (see, e.g., U.S. Patent Nos. 7,629,157 and 9,714,437, and U.S. Patent Application Publication No. 2006 / 0195947, all of which are incorporated herein by reference) or pJV110900 vector system (see, e.g., U.S. Patent Application Publication No. 2017 / 213758, which is also incorporated herein by reference). However, it is not intended that the present invention be limited to any specific vector. In addition, in some embodiments, an expression vector lacking an antimicrobial resistance marker may be used. The plasmid construct is transformed into an Escherichia coli strain derived from W3110. A library of gene variants was generated from this plasmid construct using directed evolution techniques generally known to those skilled in the art (see, eg, US Pat. No. 8,383,346 and WO2010 / 144103) and its derivatives.

[0189] Example 2

[0190] Acquisition of mammalian lipase gene, construction of expression vector and screening of HTP growth and activity

[0191] The genes encoding the mammalian lipases in Table 2-1 (SEQ ID NO: 1, 3, 5, 7, 9, 11) were codon optimized for expression in Saccharomyces cerevisiae. The Saccharomyces cerevisiae mating factor alpha (MFα) signal peptide was genetically fused to the mature form of the lipase for secretory expression of the lipase. These yeast strains were grown with HTP as described in WO 2016 / 105889 (incorporated herein by reference). The activity of the supernatants of these yeast cultures was determined as described in Example 3. The activities obtained from these lipases are shown in Table 2-1.

[0192]

[0193] Example 3

[0194] High-throughput (HTP) growth and screening conditions for lipase variants

[0195] The experiments performed for the growth and screening of lipase variants are described below.

[0196] High Throughput (HTP) Growth of Bacillus thermoamylovorus lipase (btLIP) and lipase (LIP) variants:

[0197] The transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and selection. After incubation at 37°C overnight, colonies were plated into 96-well shallow bottom plates (NUNC TM , Thermo-Scientific) wells filled with 180 μl / well of LB supplemented with 1% glucose and selection. The cultures were allowed to grow overnight in a shaking incubator (200 rpm, 30°C and 85% relative humidity; Kuhner) for 18-20 hours. Samples (20 μL) of the overnight growth were transferred to a 380 μL Terrific Broth supplemented with selection compound (e.g., chloramphenicol). 96-well deep plates. The plates were incubated in a shaking incubator (250 rpm, 30°C and 85% relative humidity; Kuhner) for 135 minutes. The expression of the lipase variants was then induced with 40 μL of 10 mM IPTG in sterile water, and the cultures were incubated overnight in a shaking incubator (250 rpm, 30°C and 85% relative humidity; Kuhner) for 20-24 hours. The cells were precipitated by centrifugation (4000 rpm x 20 min), the supernatant was discarded, and the cells were frozen at -80°C and then analyzed.

[0198] Lysis of HTP precipitate:

[0199] First, 200-400 μL of lysis buffer (1X PBS, 1 mg / ml lysozyme, and 0.5 mg / ml polymyxin B sulfate) was added to the cell pellet. The cell pellet and buffer were gently shaken at room temperature for 1.5-2 h and centrifuged (4000 rpm x 5 min), and the clarified lysate was then used in each HTP assay described herein. Analysis of these lysates by SDS-PAGE revealed that the overexpressed protein was present with an apparent MW of ~45 kDa, consistent with the expected MW of btLIP.

[0200] Analysis of Lipase Activity of Clarified Lysates:

[0201] btLIP variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by their changes in abundance over time. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein at pH 7.0 were mixed with 10 μL of lysate and added to a polyacrylate 96-well microtiter plate ( The reaction was quenched with 600 μL ACN:IPA (1:3), clarified by centrifugation, and plated on C18 Separation was performed on a 5 μm 150 mm column running an isocratic flow of 55% IPA in acetonitrile to detect oleic acid, diolein, monoolein, and triolein at 214 nm. Alternatively, the quenched reaction was clarified and 10 μL was diluted into 190 μL of 50:50 IPA:MeOH, shaken for 1 min, and then diluted again by adding 10 μL to 190 μL of 50:25:25 H2O:IPA:MeOH. The reaction was then stirred by The samples were analyzed by MS. In the following examples, "unchallenged activity" refers to the lipase activity determination as described in the assay description below, performed without any external pretreatment / challenge.

[0202]

[0203]

[0204] HTP analysis of clarified lysates pretreated with heat pretreatment:

[0205] The activity of btLIP variants was determined after incubation at 37°C to 65°C. First, 100 μL of clarified lysate was added to the wells of a 96-well BioRad Hard-Shell PCR thin-wall microtiter plate (#hsp9601; BioRad). The plate was sealed and incubated in a thermal cycler at 37°C to 65°C for 1 h before analysis. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein (via their changes in abundance over time). 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 lysate and added to a polyacrylate 96-well microtiter plate ( The reaction was quenched with 600 μL ACN:IPA (1:3), clarified and 10 μL diluted into 190 μL 50:50 IPA:MeOH, shaken for 1 min, and then diluted again by adding 10 μL to 190 μL 50:25:25 H2O:IPA:MeOH. Then, the reaction was continued by The samples were analyzed by MS. The results are provided in the table in Example 4.

[0206] HTP analysis of clarified lysates with low pH and with or without pepsin pretreatment:

[0207] The activity of the btLIP variants was determined after incubation with pepsin to simulate the environment of the stomach. First, 90 μL of 100 mM sodium citrate, 2.5 mg TAG at pH 2.0 to 5.0, with or without 1.5 mg / mL pepsin (P7000 Sigma), and 10 μL of clarified lysate were added to a 96-well round-bottom microtiter plate ( The plate was sealed and incubated at 37°C with shaking ( The samples were incubated for 1 h on a shaker (HT Infors AJ185, 400 rpm, 1" swing) and then analyzed. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by their abundance changes over time. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein at pH 7.0 were mixed with 10 μL of pepsin reaction mixture and added to a polyacrylate 96-well microtiter plate ( The reaction was quenched with 600 μL ACN:IPA (1:3), clarified and 10 μL diluted into 190 μL 50:50 IPA:MeOH, shaken for 1 min, and then diluted again by adding 10 μL to 190 μL 50:25:25 H2O:IPA:MeOH. Then, the reaction was continued by The samples were analyzed by MS. The results are provided in the table in Example 4.

[0208] HTP analysis of clarified lysates pretreated with protease:

[0209] The activity of the btLIP variants was determined after incubation with chymotrypsin and trypsin to simulate the lower intestine environment. First, 50 μL of a protease mixture (0.01-100 mg / ml chymotrypsin (C4129, Sigma), 0.01-100 mg / ml trypsin (C4129, Sigma), 0-30 μL of 20 mM sodium taurocholate in 100 mM sodium phosphate at pH 6.5 to 7.0, and 50 μL of clarified lysate were added to a 96-well round-bottom microtiter plate ( The plate was sealed and incubated at 37°C with shaking ( The cells were incubated for 1 h on a shaker (HTInfors AJ185, 400 rpm, 1" swing) and then analyzed. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by their abundance changes over time. 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 and added to a polyacrylate 96-well microtiter plate ( The reaction was quenched with 600 μL ACN:IPA (1:3), clarified and 10 μL diluted into 190 μL 50:50 IPA:MeOH, shaken for 1 min, and then diluted again by adding 10 μL to 190 μL 50:25:25 H2O:IPA:MeOH. Then, the reaction was continued by The samples were analyzed by MS. The results are provided in the table in Example 4.

[0210] HTP analysis of clarified lysates with multi-step gastrointestinal (GI) challenge:

[0211] The activity of the btLIP variants was determined after incubation at 37°C to 65°C. 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 thermal cycler at 37°C to 65°C for 1 h before gastric challenge. Next, the btLIP variants were incubated with pepsin to simulate the environment of the stomach. First, 50 μL of 100 mM sodium citrate at 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 a 96-well round-bottom microtiter plate ( The plate was sealed and incubated at 37°C with shaking ( The cells were incubated for 1 h on a shaker (HTInfors AJ185, 400 rpm, 1" swing) and then analyzed. Next, the btLIP variants were incubated with chymotrypsin and trypsin to simulate the lower intestinal environment. In this step, 100 μL of a protease mixture (0.01-100 mg / ml chymotrypsin [C4129; Sigma] and 0.01-100 mg / ml trypsin [T7409; Sigma]), 0-30 μL of 20 mM sodium taurocholate in 100 mM sodium phosphate at pH 6.5 to 7.0, and 100 μL of heat-treated gastric challenge lysate were added to a 96-well round-bottom microtiter plate ( The plate was sealed and incubated at 37°C with shaking ( The samples were incubated for 1 h on a shaker (HT Infors AJ185, 400 rpm, 1" swing) and then analyzed. Variant activity was determined by measuring the formation of oleic acid, diolein, and monoolein by their abundance changes over time. For this assay, 90 μL of 100 mM sodium phosphate and 2.5 μL of triolein at pH 7.0 were mixed with 10 μL of the gastrointestinal reaction and added to a polyacrylate 96-well microtiter plate ( The reaction was quenched with 600 μL ACN:IPA (1:3), clarified and 10 μL diluted into 190 μL 50:50 IPA:MeOH, shaken for 1 min, and then diluted again by adding 10 μL to 190 μL 50:25:25 H2O:IPA:MeOH. Then, the reaction was continued by The samples were analyzed by MS. The results are provided in the table in Example 4.

[0212] Example 4

[0213] Screening results of lipase variants

[0214] Variants generated by homologs diversity and saturation mutagenesis were screened under several different conditions as described in Example 3. The results (relative to SEQ ID NO: 2) are provided in Table 4-1. In this table, amino acid differences are indicated relative to SEQ ID NO: 2.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] Based on the results shown in Table 4-1, SEQ ID NO: 94 was selected as the parent sequence for the next iteration of protein optimization. The beneficial mutations identified from the results shown in Table 4-1 were recombined into the backbone. Variants were screened under the same conditions as described in Example 3. The only difference was that the acid challenge screen was performed at pH 3.5 instead of pH 3. The data relative to SEQ ID NO: 94 were

[0221] Listed in Table 4-2. In this table, the amino acid differences are indicated relative to SEQ ID NO:94.

[0222]

[0223]

[0224]

[0225] Based on the results shown in Table 4-2, SEQ ID NO:350 was selected as the parent sequence for the next protein optimization iteration. The beneficial mutations identified from Table 4-2 were recombined into the backbone. In addition, variants were constructed by saturation mutagenesis at different positions using SEQ ID NO:350. Variants were measured as described in Example 3, and the results are provided in Table 4-3. In this table, the amino acid differences are indicated relative to SEQ ID NO:350.

[0226]

[0227]

[0228]

[0229] Based on the results from Table 4-3, SEQ ID NO:442 was selected as the parental sequence for the next protein optimization iteration. The beneficial mutations identified based on the results shown in Table 4-3 were recombined into the backbone. In addition, variants were constructed by saturation mutagenesis at different positions on SEQ ID NO:442. Variants were measured as described in Example 3, and the results are provided in Table 4-4. In this table, the amino acid differences are indicated relative to SEQ ID NO:442.

[0230]

[0231]

[0232]

[0233] Based on the results shown in Table 4-4, SEQ ID NO:540 was selected as the parental sequence for the next protein optimization iteration. The beneficial mutations identified based on the results shown in Table 4-4 were recombined into the backbone. In addition, variants were constructed by saturation mutagenesis at different positions on SEQ ID NO:540. Variants were measured as described in Example 3, and the results are provided in Table 4-5. In this table, the amino acid differences are indicated relative to SEQ ID NO:540.

[0234]

[0235]

[0236]

[0237] Based on the results shown in Tables 4-5, SEQ ID NO: 646 was selected as the parent sequence for the next protein optimization iteration. Beneficial mutations identified based on the results shown in Tables 4-5 were recombined into the backbone. Variants were assayed as described in Example 3, and the results are provided in Tables 4-6. In this table, the amino acid differences are indicated relative to SEQ ID NO: 646.

[0238]

[0239]

[0240]

[0241] Based on the results shown in Tables 4-6, SEQ ID NO: 758 was selected as the parent sequence for the next protein optimization iteration. Beneficial mutations identified based on the results shown in Tables 4-6 were recombined into the backbone. Variants were assayed as described in Example 3, and the results are provided in Tables 4-7. In this table, the amino acid differences are indicated relative to SEQ ID NO: 758.

[0242]

[0243] Example 5

[0244] Screening results of individual mutant variants on SEQ ID NO: 868

[0245] Single point mutations were constructed on SEQ ID NO: 868 by site saturation mutations at 24 positions different from SEQ ID NO: 2. Lipase activity was determined for these variants after they were subjected to various pretreatments as described in Example 3. Each variant was tested in triplicate, and the activity data were analyzed with reference to both SEQ ID NO: 868 and SEQ ID NO: 2, as listed in Table 5-1.

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] Example 6

[0266] Validation of a surgical model of exocrine pancreatic insufficiency in miniature pigs

[0267] A surgical model of exocrine pancreatic insufficiency (EPI) was produced in miniature pigs by pancreatic duct ligation. Female pigs aged 3-4 months were given SINCLAIR® starting 7 days before surgery and continuing throughout the experiment. TM Miniature pigs were fed a high-fat diet (HFD; Sinclair standard diet S-9 and Olive oil 10:1 w / w mixture) once a day. Total fecal output within 24 h was collected for 3 consecutive days before surgery and on days 15, 16, and 17 after surgery. The daily total fecal collection from each animal was prepared for subsequent analysis by adding a volume of distilled water approximately 1.5x the net weight of feces, homogenizing, dividing into 3x50 mL aliquots, and freezing at -20°C until analysis. The model was validated by measuring the coefficient of fat absorption (CFA; by a modified Van de Kamer method; see 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 combustion method from Vario Max CN machine; see Peters et al. (eds.) Recommended Methods of Manure Analysis , Watson et al., Univ. of Wisconsin Cooperative Extension Publishing, Publication No. A3769. Madison, WI.

[2003] , p. 18-24, and the percentage changes before and after surgery were evaluated. Before surgery, CFA and CNA in healthy miniature pigs 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 demonstrating that this surgical model can be used for EPI research.

[0268] Example 7

[0269] and In vivo characterization of SEQ ID NO: 868 compared to pancreatic lipase

[0270] Proven SINCLAIR TM Minipig EPI surgical model (female, 4-5 months old) was further used for in vivo studies to evaluate the efficacy of EPI in comparison with the current standard of care ( Starting at least 8 days prior to dosing, animals were provided a HFD (Sinclair standard S-9 diet with Olive oil 10:1, w / w mixture). During dosing days, meals were prepared sequentially for each animal to ensure minimal interaction of the ex vivo lipase with dietary fat prior to ingestion. Briefly, HFD was first prepared in a clean feeding bowl. Next, the enzyme (0.5 g lyophilized SEQ ID NO: 868 powder or 7 x 36,000 U 5.6 g of microspheres collected from pancrelipase capsules, which are equivalent to 258,000 U and 252,000 U of lipase, respectively, were mixed into a 3.9 oz. cup of unsweetened applesauce because the acidic pH prevents early enzyme activation. The enzyme units administered per meal are based on the specific activity of the lipase, determined by the USP lipase assay (United States Pharmacopeia and National Formulary (USP 42-NF 37). Rockville, MD: United States Pharmacopeial Convention; 2016; available at online.uspnf.com / uspnf / document / GUID-AC788D41-90A2-4F36-A6E7-769954A9ED09_1_en-us) and the total fat provided in the calculated diet. The lipase-applesauce mixture was then transferred to the HFD and mixed quickly. Finally, the feeding bowl was immediately provided to the animal before proceeding to the next preparation. After 2 hours, the feeding bowl was removed; the animals ate the entire meal in 30 minutes or less. A crossover design was used to maximize the number of miniature pigs in each group (n=3). The study introduced an 8-day "washout" (HFD + applesauce without enzymes once a day) between each 10-day dosing phase. Total fecal material was collected 24 hours after dosing for 3 consecutive days before the start of dosing and on the 9th, 10th and 11th days of each phase. Fecal samples were prepared by adding a volume of distilled water 1.5x the net weight of feces to each daily total fecal collection, homogenized, divided into 3x50mL, and frozen at -20°C until analysis, discarding the remainder. The analysis was completed as described above for model validation. Both pancrelipase and SEQ ID NO:868 resulted in similar improvements in CFA, with values ​​not significantly different from pre-surgery baseline.

[0271] Example 8

[0272] In vivo dose response characterization of SEQ ID NO:868

[0273] Proven SINCLAIR TMMinipig EPI surgery model (female, 4-6 months old) was used for additional in vivo SEQ ID NO: 868 dose response studies. HFD and feeding regimens, lipase mixing regimens, fecal sample collection and preparation, and analysis were the same as described above. Compared with preoperative values ​​(92.8% + / - 3.2SD, n = 8), the postoperative CFA measured just before administration dropped to 39.9% + / - 13.2SD (p < 0.0001, n = 8). SEQ ID NO: 868 was administered at 258,000U (0.5g lyophilized powder) to connect with previous studies, and at 86,000U (167mg) and 29,000U (55.5mg) to evaluate low-dose efficacy. All doses evaluated resulted in a positive effect of CFA in a dose-dependent manner. Compared with before administration, CFA values ​​increased from high dose to low dose to 81.3% + / - 3.9 SD (p<0.0001, n=3), 74% + / - 3.6 SD (p<0.0001, n=3) and 65.5% + / - 3.7 SD (p<0.0001, n=2), respectively.

[0274] 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 was individually indicated to be incorporated by reference for all purposes.

[0275] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. A recombinant lipase, wherein the recombinant lipase consists of the same amino acid sequence as SEQ ID NO:

868.

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

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

4. The recombinant polynucleotide of claim 3, wherein the polynucleotide is codon optimized. An expression vector comprising the recombinant polynucleotide according to claim 3 . The expression vector of claim 5 , wherein the recombinant polynucleotide is operably linked to a control sequence.

7. The expression vector according to claim 5 or 6, wherein the control sequence is a promoter. The expression vector according to claim 7 , wherein the promoter is a heterologous promoter.

9. A host cell for producing recombinant lipase, the host cell comprising the expression vector according to claim 5.

10. The host cell of claim 9, wherein the host cell is eukaryotic or prokaryotic.

11. A method for producing a recombinant lipase, the method comprising culturing the host cell according to claim 9 under conditions where the recombinant lipase encoded by the recombinant polynucleotide is produced.

12. The method according to claim 11, further comprising the step of recovering the lipase.

13. The method according to claim 11 or 12, further comprising the step of purifying the lipase.

14. A composition comprising the recombinant lipase according to claim 1.

15. The composition of claim 14, further comprising a pharmaceutically acceptable carrier and / or excipient.

16. The composition of claim 15, further comprising at least one additional enzyme selected from the group consisting of proteases and amylases.

17. Use of the recombinant lipase according to claim 1 in the preparation of a medicament for treating pancreatic insufficiency.

18. The use according to claim 17, wherein the medicament further comprises a pancreatic extract containing lipase, protease and amylase activities.

19. The use according to claim 17, wherein the medicament is suitable for parenteral injection or infusion, subcutaneous injection, inhalation or skin application to humans.

20. The use according to claim 17, wherein the medicament is suitable for oral administration to humans.

21. The use according to claim 20, wherein the medicament further comprises an enteric coating.

22. Use of the recombinant lipase of claim 1 in the preparation of a medicament for treating and / or preventing symptoms of pancreatic insufficiency in a subject.

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

24. The use according to claim 22, wherein the subject is able to consume a diet that is less restricted in lipid content than that required for a subject displaying symptoms of said pancreatic insufficiency.

25. The use according to claim 22, wherein the subject is a child.

26. The use according to claim 22, wherein the subject is an infant.

27. The use according to claim 22, wherein the subject is an adult.

28. The use according to claim 22, wherein the subject is a young person.

29. Use of the recombinant lipase according to claim 1 in the preparation of a medicament for treating exocrine pancreatic insufficiency.

30. Use of the recombinant lipase according to claim 1 in preparing a nutritional supplement.

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