Carboxylesterase Polypeptides for Amide Coupling

Abstract

The present invention provides engineered carboxylesterase enzymes having improved properties compared to naturally occurring wild-type carboxylesterase enzymes, as well as polynucleotides encoding the engineered carboxylesterase enzymes, host cells capable of expressing the engineered carboxylesterase enzymes, and methods of using the engineered carboxylesterase enzymes in amidation reactions. [Solution] The present invention provides engineered carboxylesterases (EC 3.1.1) with improved non-native properties compared to naturally occurring wild-type (WT) carboxylesterase enzymes, as well as polynucleotides encoding the engineered carboxylesterase enzymes, host cells capable of expressing the engineered carboxylesterase enzymes, and methods for applying the engineered carboxylesterase enzymes in amidation reactions.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 598,189, filed December 13, 2017, which is incorporated by reference in its entirety for all purposes.

[0002] Reference to sequence listing, tables or computer programs Pursuant to 37 CFR § 1.821, the Sequence Listing in computer readable form (CRF) contemporaneously submitted herewith via EFS-Web under filename CX2-165US1_ST25.txt is hereby incorporated by reference. The electronic copy of the Sequence Listing was created on December 7, 2018 and has a file size of 420 Kbytes.

[0003] FIELD OF THEINVENTION The present invention provides engineered carboxylesterases (EC 3.1.1) having improved non-native properties compared to naturally occurring wild-type (WT) carboxylesterase enzymes, as well as polynucleotides encoding the engineered carboxylesterase enzymes, host cells capable of expressing the engineered carboxylesterase enzymes, and methods of applying the engineered carboxylesterase enzymes to amidation reactions. [Background technology]

[0004] Amide bonds are important functional moieties in a variety of synthetic molecules, including polymers (e.g., proteins, nylons), pesticides (e.g., propanil, chlorpropham), and pharmaceuticals (e.g., valsartan, lisdexamfetamine). A recent survey of the prevalence of reaction types used in the search for novel drug candidates listed N-acylamidations at approximately 16% of all of these reactions (Roughley and Jordan, J. Med. Chem., 54:3451-3479

[2011] ). When produced using traditional chemical methods, amide bond formation is a resource-intensive transformation. Amide bonds are typically synthesized from carboxylic acids and amines. However, at ambient temperatures, the reaction between these two functional groups does not occur spontaneously, and the necessary water elimination occurs only at elevated temperatures (e.g., 200°C). These conditions tend to be detrimental to the substrate and product.

[0005] For amidation to occur under more suitable conditions, activation of the carboxylic acid is generally required for coupling to the amine. Carboxylic acid activation usually occurs with a coupling reagent to form an activated ester or anhydride, or by converting the carboxylic acid to the corresponding acid chloride (i.e., via the Schotten-Baumann reaction; see El-Faham and Albericio, Chem. Rev., 111:6557-6602

[2011] ). These reactions are carried out with excess or stoichiometric concentrations of expensive coupling reagents that utilize atom-inefficient synthetic routes (see Pattabiraman and Bode, Nature, 480:471-479

[2011] ). In addition, the reagents and the resulting waste products can be highly toxic and environmentally unfriendly. At least one equivalent of waste is generated per product molecule formed in these reactions, resulting in very poor atom economy. Removal of the waste products from the reaction mixture is a laborious and expensive process. Thus, there is a need in the art for more efficient and environmentally friendly means for the generation of amide bonds in a variety of situations.

[0006] Lipases have been applied on a commercial scale for the hydrolysis of fatty acid esters and have been used in the amidation of esters (Seem Faber, Biotransformations in Organic Chemistry, In Special Techniques, Springer-Verlag, New York, NY,

[2011] ; and Kalkote et al., Asian J. Biochem., 2:279-283

[2007] ). The formation of amide bonds using enzymes is a very atom-economical process since there is no need to activate the carboxylic acid as in typical coupling approaches. The use of enzymes in these reactions offers great industrial value since they are environmentally friendly, occur under mild conditions, and are less expensive than currently available chemical routes. Although eukaryotic proteases and lipases are capable of forming amide bonds (see Adolfsson et al., Chem. Soc. Rev., 43:2714-2742

[2014] ; Guzman et al., Elec. J. Biotech., 10(2)

[2007] ; and Asano et al., J. Biosci. Bioeng., 100(6):662-666

[2005] ), typically these enzymes are poorly expressed in prokaryotic expression systems and may perform poorly in organic solvents. In contrast, through directed evolution methods, the carboxylesterases of the invention are readily produced in prokaryotic (E. coli) expression systems, increasing their tolerance and utility in organic solvent regimes more appropriate for this reaction type. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Roughley and Jordan, J.Med.Chem.,54:3451-3479

[2011] [Non-Patent Document 2] El-Faham and Albericio,Chem.Rev.,111:6557-6602

[2011] [Non-Patent Document 3] Pattabiraman and Bode,Nature,480:471-479

[2011] [Non-Patent Document 4] Seem Faber, Biotransformations in Organic Chemistry, In Special Techniques, Springer-Verlag, New York, NY,

[2011] [Non-Patent Document 5] Kalkote et al., Asian J. Biochem., 2:279-283

[2007] [Non-Patent Document 6] Adolfsson et al., Chem.Soc.Rev.,43:2714-2742

[2014] [Non-Patent Document 7] Guzman et al., Elec.J.Biotech., 10(2)

[2007] [Non-Patent Document 8] Asano et al., J. Biosci. Bioeng., 100(6):662-666

[2005] Summary of the Invention [Means for solving the problem]

[0008] The present invention provides engineered carboxylesterases (EC 3.1.1) having improved non-native properties compared to naturally occurring wild-type (WT) carboxylesterase enzymes, as well as polynucleotides encoding the engineered carboxylesterase enzymes, host cells capable of expressing the engineered carboxylesterase enzymes, and methods of applying the engineered carboxylesterase enzymes to amidation reactions.

[0009] The present invention relates to engineered carboxylesterases or functional fragments thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:2, wherein said polypeptide sequence has at least In some embodiments, the at least one substitution or set of substitutions in the polypeptide sequence is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429 4, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372, 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 32 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 39 / 323, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109 / 117, 110, 11 1, 113, 114, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 153, 153 / 215, 164 / 271, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 21 5 / 271, 216, 217, 217 / 231, 224 / 268 / 372, 249 / 284, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 31 7, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, 320 / 376 / 377, 321, 323, 324, 372, 372 / 376, 373, 376, 377, 405, 420, 427, 428 and 429, where the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 39M / 323I, 62H / 117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H / 263R, 71P, 71R, 71V, 71Y, 77S / 184G, 103P, 103R, 103T / 147S, 104P, 104Q / 429V, 105L, 107D / 185W, 107L, 107P, 107S, 108G, 108K, 108Q, 108R, 108S, 108W, 109G / 117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111V , 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F, 118G / 349V, 118I, 118N, 118N / 269T, 119G, 119P, 119S, 126C, 153H / 215P, 153L, 164R / 271T, 174D / 282V, 183P, 184F, 184 G, 184P, 184S / 249T, 184Y, 185A, 185T, 186C, 186G, 186P, 186R, 186T, 187P, 188E, 188G, 19. 0H, 190K, 190L, 190M, 190Q, 190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T, 210W, 211I, 211L, 211R, 211V, 212A, 212P, 21 2R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q, 213R / 345G, 213S, 213T / 271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M, 215P, 21 5R, 215R / 271R, 215W, 216P, 217G, 217L, 217P, 217R, 217R / 231V, 217S, 217V, 217W, 224I / 268S / 372F, 249V / 284P, 269N, 269V, 27 0I, 270I / 470M, 270R, 271A, 271K, 271L, 271P, 271Q / 416V, 271S, 271T, 276F, 277M, 278H, 278S, 279C, 279E, 279G, 279L / 280G / 282 M, 279V, 280E, 280G, 280S, 281P, 281V, 281Y / 374N, 282A, 282C, 282Q, 282R, 282S, 282T, 282W, 283C, 283D, 283K, 283R / 429V, 283 T, 283V, 283Y, 284C, 284T, 284T / 438T, 284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F, 320G, 320G / 323S, 320S, 320S / 323S / 372A, 320S / 372A / 376G, 320S / 376G / 377V, 320W, 321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372A / 376A, 372L, 373G, 376A, 376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V and 429L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of T39M / F323I, R62H / P117G, P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G, Y65S, Y65T, Y65W, P66N, A68L, A68P,I69F、I69G、I69H、I69L、I69V、I69W、I69Y、G70L、G70R、G70T、G70W、A71F、A71G、A71H / Q263R、A71P、A71R、A71V、A71Y、F77S / E184G、W103P、W103R、W103T / P147S、I104P、I104Q / A429V、H105L、G107D / S185W、G107L、G107P、G107S、A108G、A108K、A108Q、A108R、A108S、A108W、F109G / P117M、T110A、T110H、T110P、T110S、N111L、N111M、N111R、N111S、N111V、N111W、S113P、G114A、G114H、G114Q、S115H、S115T、S115V、P117A、P117F、V118G / A349V、V118I、V118N、V118N / A269T、Y119G、Y119P、Y119S、R126C、R153H / N215P、R153L、W164R / W271T、G174D / L282V、G183P、E184F、E184G、E184P、E184S / A249T、E184Y、S185A、S185T、A186C、A186G、A186P、A186R、A186T、G187P、A188E、A188G、S190H、S190K、S190L、S190M、S190Q、S190R、S190W、L209E、L209G、L209P、L209S、L209V、Q210P、Q210T、Q210W、S211I、S211L、S211R、S211V、G212A、G212P、G212R、G212S、A213C、A213E、A213L、A213N、A213P、A213Q、A213R / S345G、A213S、A213T / W271K、A213V、G214K、G214L、G214T、G214V、N215K、N215M、N215P、N215R、N215R / W271R、N215W、M216P、A217G、A217L、A217P、A217R、A217R / A231V、A217S、A217V、A217W、T224I / P268S / I372F、A249V / F284P、A269N、A269V、V270I、V270I / V470M、V270R、W271A、W271K、W271L、W271P、W271Q / A416V、W271S、W271、 T, A276F, G277M, G278H, G278S, S279C, S279E, S279G, S279L / V280G / L282M, S279V, V 280E, V280G, V280S, L281P, L281V, L281Y / D374N, L282A, L282C, L282Q, L282R, L282S , L282T, L282W, P283C, P283D, P283K, P283R / A429V, P283T, P283V, P283Y, F284C, F2 84T, F284T / P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P, Y320A, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered carboxylesterase comprises a substitution at position 282, where positions are numbered with reference to SEQ ID NO: 2. In some further embodiments, the substitution at position 282 is aliphatic, non-polar, basic, polar, or aromatic. In still some further embodiments, the substitution is selected from X282T, X282G, X282A, X282V, X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

[0010] The present invention provides an engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, wherein the engineered carboxylesterase or functional fragment thereof comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, at least one substitution or set of substitutions in a polypeptide sequence is 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372, 65 / 320, 65 / 320 / 323, 68, 68 / 69 / 189 / 214, 68 / 69 / 189 / 214 / 215, 68 / 69 / 189 / 214 / 215 / 271, 68 / 69 / 189 / 214 / 215 / 271 / 281 / 282 / 343 / 381, 68 / 69 / 189 / 214 / 271 / 280, 68 / 69 / 189 / 214 / 372, 68 / 69 / 189 / 214 / 377 / 381, 68 / 69 / 189 / 271, 68 / 69 / 189 / 271 / 280 / 372 / 381, 68 / 69 / 189 / 280 / 281 / 282 / 372 / 377, 68 / 69 / 189 / 281 / 282 / 372 / 377 / 381, 68 / 69 / 189 / 343 / 381, 68 / 69 / 189 / 372, 68 / 69 / 189 / 381, 68 / 69 / 214 / 215 / 271, 68 / 69 / 214 / 343 , 68 / 69 / 215, 68 / 69 / 271, 68 / 69 / 282 / 287, 68 / 69 / 343 / 372, 68 / 108 / 377, 68 / 184, 68 / 184 / 189, 68 / 189 / 271 / 372, 68 / 189 / 343, 68 / 214 / 215 / 271 / 281 / 282 / 372, 68 / 215 / 271 / 343 / 372 / 381, 68 / 215 / 377, 68 / 271 / 372, 68 / 377, 69, 69 / 70, 69 / 70 / 331 / 372, 69 / 70 / 372, 69 / 70 / 459, 69 / . 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 282、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320、108 / 189 / 377、108 / 215、108 / 215 / 377、108 / 269 / 270、108 / 270、108 / 282 / 285 / 377、108 / 285、108 / 320 / 323、108 / 377、126、126 / 184 / 213 / 280 / 281 / 285 / 320、126 / 184 / 213 / 372、126 / 189 / 285 / 372、126 / 215、126 / 372、181 / 215、189、189 / 214、189 / 214 / 215 / 271 / 282、189 / 215、189 / 215 / 249 / 277、189 / 215 / 271 / 281 / 282 / 377、189 / 215 / 343 / 372、189 / 270 / 285、189 / 270 / 372、189 / 280 / 282、189 / 320 / 377、189 / 343、189 / 343 / 377、189 / 372 / 377、189 / 377、189 / 381、213 / 215 / 320, 214 / 215 / 271, 214 / 215 / 271 / 377, 214 / 271, 214 / 280 / 282 / 343 / 377 / 381, 215, 215 / 249 / 280 / 281 / 285 / 372, 215 / 271 / 372, 215 / 280 / 281 / 285 / 372, 215 / 281 / 285 / 372, 215 / 281 / 285 / 373, 215 / 281 / 373, 215 / 285, 215 / 285 / 317, 215 / 285 / 346, 215 / 285 / 445, 215 / 320, 215 / 320 / 372, 215 / 323, 215 / 37 and 377, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 63A, 63A / 189A, 63A / 215R / 343V, 63R, 63R / 65G / 108G, 63R / 65G / 108G / 189L, 63R / 65G / 108G / 377I, 63R / 65G / 282A / 285L / 320W / 323I, 63R / 65G / 320W / 323I, 63R / 108G, 63R / 108G / 282A / 285L / 377L, 63R / 108G / 285L / 377I, 63R / 108G / 320W / 323C, 63R / 377I, 63T / 215R, 63Y, 63Y / 189L, 63Y / 212P / 215R, 63Y / 212P / 215R / 268A / 269N / 343V, 63Y / 215P / 269N, 63Y / 215R, 63Y / 215R / 270I / 27 1S, 63Y / 268A / 269N / 270I / 429V, 65G / 320W, 65G / 320W / 323I, 65W / 69L / 3 72L, 65W / 69M / 70A / 281P / 372L, 65W / 69W / 70L / 372L, 65W / 70L / 372M, 68P,68P / 69L / 189E / 214R / 271Y / 2、 <h2 style=";text-align:left;direction:ltr">80G、68P / 69L / 189E / 214R / 372L、68P / 69L / 189I / 214R / 215P / 271Y、68P / 69L / 189I / 281P / 282C / 372L / 377Y / 381L、68P / 69L / 189Q / 214R、68P / 69L / 189Q / 271Y / 280G / 372L / 381L、68P / 69L / 215P、68P / 69L / 271Y、68P / 69L / 282C / 287I、68P / 69L / 343V / 372L、68P / 69W / 189E / 214R / 215P / 271Y / 281P / 282G / 343V / 381L、68P / 69W / 189E / 280G / 281P / 282A / 372L / 377Y、68P / 69W / 189E / 343V / 3 81L、68P / 69W / 189I / 214R / 215P、68P / 69W / 189I / 214R / 377Y / 381L、68P / 69W / 189I / 271Y、68P / 69W / 189I / 372L、68P / 69W / 189I / 381L、68P / 69W / 214R / 215 P / 271Y、68P / 69W / 214R / 343V、68P / 69W / 215P、68P / 108G / 377L、68P / 184S、68 P / 184S / 189E、68P / 189I / 271Y / 372L、68P / 189I / 343V、68P / 214R / 215P / 271 Y / 281P / 282A / 372L、68P / 215P / 271Y / 343V / 372L / 381L、68P / 215P / 377L、68P / 271Y / 372L、68P / 377L、69F / 108G / 270E / 372L / 377L、69F / 189L、69F / 215K、69F / 215K / 269L / 270I / 377L、69F / 215R、69F / 285L / 373G、69L、69L / 70L / 331Q / 372M、69L / 189E / 271Y / 281P / 282A、69L / 189I、69L / 189I / 214R / 271Y / 281P / 282A / 343V、69L / 189I / 271Y / 343V / 381L、69L / 189I / 280G / 282G / 343V / 372L / 381L、69L / 189I / 282A、69L / 189Q / 377Y、69L / 215P / 271Y / 280G / 281P / 282C、69L / 215P / 271Y / 282A、69L / 215P / 271Y / 372L、69L / 215P / 343V / 372L / 381L、<h2 style=";text-align:left;direction:ltr">69L / 215R / 285P / 317P, 69L / 271Y, 69L / 271Y / 372L, 69L / 282C / 343V / 372L, 69L / 372L, 69M / 70A / 372M, 69W, 69W / 70L, 69W / 70L / 372M, 69W / 70L / 459R 69W / 108S, 69W / 189E / 214R / 343V / 372L, 69W / 189E / 271Y / 343V, 69W / 189E / 372L, 69W / 189I, 69W / 189I / 215P / 343V, 69W / 189I / 271Y, 69W / 189I / 34 3V / 381L、69W / 189Q / 372L / 377Y、69W / 212A / 213L / 215R / 280G / 281P、69W / 214R / 215P / 271Y / 372L / 377Y / 381L、69W / 214R / 271Y / 282A、69W / 214R / 27 1Y / 343V, 69W / 215K / 343V, 69W / 215P, 69W / 215R, 69W / 215R / 323Y, 69W / 282A, 69W / 372M, 69Y / 108G / 281P / 285P, 69Y / 110A / 215R / 281P, 69Y / 189L / 28 1P / 373G、70L、70L / 212P、108G、108G / 189I / 282A / 285L / 320W、108G / 189L 、108G / 189L / 320W、108G / 189L / 377I、108G / 215K、108G / 215P / 377L、108G / 269L / 270E、108G / 270E、108G / 282A / 285L / 377L、108G / 285L、108G / 320W / 323I、108G / 377I、108G / 377L、126C、126C / 184S / 213S / 280G / 281P / 285L / 320G、126C / 184S / 213S / 372L、126C / 189I / 285L / 372L、126C / 215P、126C / 372L、181L / 215P、189E / 372L / 377Y、189I、189I / 214R / 215P / 271Y / 282G 189I / 215K, 189I / 215P / 343V / 372L, 189I / 215R / 249T / 277M, 189I / 270E / 285L, 189I / 270E / 372L, 189I / 280G / 282A, 189I / 320W / 377I, 189I / 343V,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 189I / 377I, 189L, 189Q, 189Q / 214R, 189Q / 215P / 271Y / 281P / 282C / 377Y, 189Q / 343V, 189Q / 343V / 377Y, 189Q / 381L, 213S / 215P / 320G, 214R / 215P / 271Y, 214R / 215P / 271Y / 377Y, 214R / 271Y, 214R / 280G / 282A / 343V / 377Y / 381L, 215K, 215K / 281P / 285L / 372L, 215K / 281P / 373G, 215K / 285L / 317P, 215K / 285L / 445L, 215K / 323Y, 215K / 372L, 215K / 372L / 377L, 215K / 373G, 215P, 215P / 271Y / 372L, 215P / 320G, 215P / 320G / 372L, 215P / 372L, 215P / 372L / 377L, 215P / 377L, 215P / 381L, 215R, 215R / 2 49T / 280G / 281P / 285L / 372L, 215R / 280G / 281P / 285L / 372L, 215R / 281P / 285L / 373G, 215R / 285P, 215R / 320G, 215R / 372L, 2 15W, 215W / 285L / 346S, 215W / 285P, 215W / 373G, 249T / 377L, 269L / 270E / 281P / 372L / 377L, 270E / 377L, 271Y, 271Y / 343V, 27 and at least one substitution selected from 1Y / 343V / 372L, 271Y / 343V / 372L / 381L, 280G / 285L / 372L, 281P / 372L, 282A / 285L / 320W / 323I, 285L / 323I, 320W, 343V / 372L, 372L, 372L / 377L, 372L / 381L, 372M, 373G and 377L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of P63A, P63A / M189A, P63A / N215R / A343V, P63R, P63R / Y65G / A108G, P63R / Y65G / A108G / M189L, P63R / Y65G / A108G / F377I, P63R / Y65G / T282A / A285L / Y320W / F323I,P63R / Y65G / Y320W / F323I、P63R / A108G、P63R / A108G / T282A / A285L / F377L、P63R / A108G / A285L / F377I、P63R / A108G / Y320W / F323C、P63R / F377I、P63T / N215R、P63Y、P63Y / M189L、P63Y / G212P / N215R、P63Y / G212P / N215R / P268A / A269N / A343V、P63Y / N215P / A269N、P63Y / N215R、P63Y / N215R / V270I / W271S、P63Y / P268A / A269N / V270I / A429V、Y65G / Y320W、Y65G / Y320W / F323I、Y65W / I69L / I372L、Y65W / I69M / G70A / L281P / I372L、Y65W / I69W / G70L / I372L、Y65W / G70L / I372M、A68P、A68P / I69L / M189E / G214R / W271Y / V280G、A68P / I69L / M189E / G214R / I372L、A68P / I69L / M189I / G214R / N215P / W271Y、A68P / I69L / M189I / L281P / T282C / I372L / F377Y / A381L、A68P / I69L / M189Q / G214R、A68P / I69L / M189Q / W271Y / V280G / I372L / A381L、A68P / I69L / N215P、A68P / I69L / W271Y、A68P / I69L / T282C / V287I、A68P / I69L / A343V / I372L、A68P / I69W / M189E / G214R / N215P / W271Y / L281P / T282G / A343V / A381L、A68P / I69W / M189E / V280G / L281P / T282A / I372L / F377Y、A68P / I69W / M189E / A343V / A381L、A68P / I69W / M189I / G214R / N215P、A68P / I69W / M189I / G214R / F377Y / A381L、A68P / I69W / M189I / W271Y、A68P / I69W / M1、 <h2 style=";text-align:left;direction:ltr">89I / I372L、A68P / I69W / M189I / A381L、A68P / I69W / G214R / N215P / W271Y、A6 8P / I69W / G214R / A343V、A68P / I69W / N215P、A68P / A108G / F377L、A68P / E184 S、A68P / E184S / M189E、A68P / M189I / W271Y / I372L、A68P / M189I / A343V、A68 P / G214R / N215P / W271Y / L281P / T282A / I372L、A68P / N215P / W271Y / A343V / I3 72L / A381L、A68P / N215P / F377L、A68P / W271Y / I372L、A68P / F377L、I69F / A1 08G / V270E / I372L / F377L、I69F / M189L、I69F / N215K、I69F / N215K / A269L / V 270I / F377L, I69F / N215R, I69F / A285L / T373G, I69L, I69L / G70L / P331Q / I372M, I69L / M189E / W271Y / L281P / T282A, I69L / M189I, I69L / M189I / G214R / W2 71Y / L281P / T282A / A343V、I69L / M189I / W271Y / A343V / A381L、I69L / M189I / V280G / T282G / A343V / I372L / A381L、I69L / M189I / T282A、I69L / M189Q / F377 Y, I69L / N215P / W271Y / V280G / L281P / T282C, I69L / N215P / W271Y / T282A, I69L / N215P / W271Y / I372L, I69L / N215P / A343V / I372L / A381L, I69L / N215R / A2 85P / T317P、I69L / W271Y、I69L / W271Y / I372L、I69L / T282C / A343V / I372L、I69L / I372L、I69M / G70A / I372M、I69W、I69W / G70L、I69W / G70L / I372M、I69W / G70L / G459R、I69W / A108S、I69W / M189E / G214R / A343V / I372L、I69W / M189E / W271Y / A343V、I69W / M189E / I372L、I69W / M189I、I69W / M189I / N215P / A343V、<h2 style=";text-align:left;direction:ltr">I69W / M189I / W271Y、I69W / M189I / A343V / A381L、I69W / M189Q / I372L / F377 Y、I69W / G212A / A213L / N215R / V280G / L281P、I69W / G214R / N215P / W271Y / I3 72L / F377Y / A381L、I69W / G214R / W271Y / T282A、I69W / G214R / W271Y / A343V、I69W / N215K / A343V、I69W / N215P、I69W / N215R、I69W / N215R / F323Y、I69W / T282A、I69W / I372M、I69Y / A108G / L281P / A285P、I69Y / T110A / N215R / L281 P、I69Y / M189L / L281P / T373G、G70L、G70L / G212P、A108G、A108G / M189I / T28 2A / A285L / Y320W, A108G / M189L, A108G / M189L / Y320W, A108G / M189L / F377I, A108G / N215K, A108G / N215P / F377L, A108G / A269L / V270E, A108G / V270E A108G / T282A / A285L / F377L, A108G / A285L, A108G / Y320W / F323I, A108G / F377I, A108G / F377L, R126C, R126C / E184S / A213S / V280G / L281P / A285L / Y32 0G、R126C / E184S / A213S / I372L、R126C / M189I / A285L / I372L、R126C / N215 P、R126C / I372L、V181L / N215P、M189E / I372L / F377Y、M189I、M189I / G214R / N215P / W271Y / T282G、M189I / N215K、M189I / N215P / A343V / I372L、M189I / N215R / A249T / G277M、M189I / V270E / A285L、M189I / V270E / I372L、M189I / V28 0G / T282A、M189I / Y320W / F377I、M189I / A343V、M189I / F377I、M189L、M189Q、M189Q / G214R、M189Q / N215P / W271Y / L281P / T282C / F377Y、M189Q / A343V、M189Q / A343V / F377Y, M1, 89Q / A381L, A213S / N215P / Y320G, G214R / N215P / W271Y, G214R / N215P / W271Y / F377Y, G214R / W271Y, G214R / V280G / T282A / A343V / F377Y / A381L, N215K, N215K / L281P / A285L / I372L, N215K / L281P / T373G, N215K / A285L / T317P, N215K / A285L / V445L, N215K / F323 Y, N215K / I372L, N215K / I372L / F377L, N215K / T373G, N215P, N215P / W271Y / I372L, N215P / Y320G, N215P / Y320G / I372L, N215P / I3 72L, N215P / I372L / F377L, N215P / F377L, N215P / A381L, N215R, N215R / A249T / V280G / L281P / A285L / I372L, N215R / V280G / L281P / A285L / I372L, N215R / L281P / A285L / T373G, N215R / A285P, N215R / Y320G, N215R / I372L, N215W, N215W / A285L / G346S, N215W / A285 P, N215W / T373G, A249T / F377L, A269L / V270E / L281P / I372L / F377L, V270E / F377L, W271Y, W271Y / A343V, W271Y / A343V / I372L, W2 and at least one substitution selected from 71Y / A343V / I372L / A381L, V280G / A285L / I372L, L281P / I372L, T282A / A285L / Y320W / F323I, A285L / F323I, Y320W, A343V / I372L, I372L, I372L / F377L, I372L / A381L, I372M, T373G and F377L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0011] The present invention also provides engineered carboxylesterases or functional fragments thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:138, and comprising at least one substitution or set of substitutions in said polypeptide sequence, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:138.

[0012] The present invention also provides an engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:140, wherein the engineered carboxylesterase or functional fragment thereof comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:140.

[0013] The present invention also provides engineered carboxylesterases comprising a polypeptide sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136.

[0014] The present invention further provides engineered carboxylesterases that exhibit at least one improved property compared to the wild-type T. fusca carboxylesterase of SEQ ID NO: 2. In some embodiments, the improved property is improved amidation activity, solvent tolerance, thermal The engineered carboxylesterase exhibits improved solvent tolerance to at least one solvent selected from acetone, acetonitrile, toluene, tetrahydrofuran, isopropanol, isopropyl acetate, dimethyl sulfoxide, and / or methyl ethyl ketone. In some further embodiments, the engineered carboxylesterase exhibits greater activity towards at least one substrate selected from aniline, isobutylamine, n-butylamine, t-butylamine, N'-t-butoxycarbonyl-benzhydrazide, 4-methylpiperidine, Ot-butylhydroxylamine, benzylamine, 2,6-dimethylaniline, (S)-(-)-α-methylbenzylamine, (R)-(+)-α-methylbenzylamine, methyl phenylacetate, ethyl acetate, ethyl benzoate, 2-pyrazinylethyl ester, 4-ethyl-1H-indole ester, N,N-diethylglycylmethyl ester than the wild-type T. fusca carboxylesterase. In some further embodiments, the engineered carboxylesterase exhibits greater activity than the wild-type T. fusca carboxylesterase for at least one substrate or set of substrates selected from aniline, isobutylamine, n-butylamine, t-butylamine, N'-t-butoxycarbonyl-benzhydrazide, 4-methylpiperidine, Ot-butylhydroxylamine, benzylamine, 2,6-dimethylaniline, (S)-(-)-α-methylbenzylamine, (R)-(+)-α-methylbenzylamine, methyl phenylacetate, ethyl acetate, ethyl benzoate, 2-pyrazinylethyl ester, 4-ethyl-1H-indole ester, N,N-diethylglycylmethyl ester.In yet some further embodiments, the engineered carboxylesterase is selected from the group consisting of acetanilide, Nn-butyl-benzylacetamide, N-[(S)-1-phenylethyl]-pyrazinylamide, N-[(S)-1-phenylethyl]-benzamide, N-[(R)-1-phenylethyl]-benzamide, N'-t-butoxycarbonyl-benzhydrazide, 1-benzoyl-4-methylpiperidine, 2-pyrazinyl-4-methylpiperidine, N-isobutyl-benzamide, Nt and N',N'-(diethylamino)-N-phenylacetamide, N',N'-(diethylamino)-N-benzylacetamide, N',N'-(diethylamino)-N-2,6-dimethylphenylacetamide (i.e., lidocaine) (Table 1). In some embodiments, the engineered carboxylesterases of the invention comprise at least one substitution selected from X343V, X372L, X320W / G, X214R, X282C, X271Y, X65G, where the substitutions are numbered with reference to SEQ ID NO:2, and as shown in the schematic diagram below, the engineered carboxylesterases exhibit greater activity towards hindered amines for the formation of N',N'-(diethylamino)-N-2,6-dimethylphenylacetamide from ethyl benzoate and 2,6-dimethylaniline than wild-type T. fusca carboxylesterase. [ka]

[0015] In yet some further embodiments, the engineered carboxylesterase is The engineered carboxylesterase comprises at least one substitution selected from 68A, X63A / R, X189Q / I / E, X214R, X282G / C, X381L and X69W, where the substitutions are numbered with reference to SEQ ID NO:2, and as shown in the schematic diagram below, the engineered carboxylesterase exhibits greater activity towards secondary amines for the formation of 1-benzoyl-4-methyl-piperidine from ethyl benzoate and 4-methylpiperidine than the wild-type T. fusca carboxylesterase. [ka]

[0016] In yet some further embodiments, the engineered carboxylesterases provided herein are purified. In yet some further embodiments, the engineered carboxylesterases are immobilized. The present invention also provides compositions comprising at least one engineered carboxylesterase provided herein.

[0017] The present invention also provides polynucleotide sequences encoding at least one engineered carboxylesterase provided herein. In some embodiments, the polynucleotide sequence encodes at least one engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 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, wherein the engineered carboxylesterase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some embodiments, the polynucleotide sequence is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 118 / 269 , 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372, 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, the polynucleotide sequence is 39 / 323, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 153, 153 / 215, 164 / 271, 174 / 282, 183. , 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224 / 268 / 372, 249 / 284, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 4 and 429, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, the polynucleotide sequence is 39M / 323I, 62H / 117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W , 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H / 263R, 71P, 71R, 71V, 71Y, 77S / 184G, 103P, 10 3R, 103T / 147S, 104P, 104Q / 429V, 105L, 107D / 185W, 107L, 107P, 107S, 108G, 108K, 108 Q, 108R, 108S, 108W, 109G / 117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111 V, 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F, 118G / 349V, 118I, 118N , 118N / 269T, 119G, 119P, 119S, 126C, 153H / 215P, 153L, 164R / 271T, 174D / 282V, 183P , 184F, 184G, 184P, 184S / 249T, 184Y, 185A, 185T, 186C, 186G, 186P, 186R, 186T, 187P,188E, 188G, 190H, 190K, 190L, 190M, 190Q, 190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T, 210W, 211I, 211L, 211R, 211V, 212A, 2 12P, 212R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q, 213R / 345G, 213S, 213T / 271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M, 215P, 21 5R, 215R / 271R, 215W, 216P, 217G, 217L, 217P, 217R, 217R / 231V, 217S, 217V, 217W, 224I / 268S / 372F, 249V / 284P, 269N, 269V, 270I, 270 I / 470M, 270R, 271A, 271K, 271L, 271P, 271Q / 416V, 271S, 271T, 276F, 277M, 278H, 278S, 279C, 279E, 279G, 279L / 280G / 282M, 279V, 280E, 280G, 280S, 281P, 281V, 281Y / 374N, 282A, 282C, 282Q, 282R, 282S, 282T, 282W, 283C, 283D, 283K, 283R / 429V, 283T, 283V, 283Y, 284C, 2 84T, 284T / 438T, 284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F, 320G, 320G / 323S, 320S, 320S / 323S / 372A, 320S / 372A / 376 376A, 372L, 373G, 376A, 376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V and 429L, wherein the amino acid positions are numbered with reference to SEQ ID NO:2. In some further embodiments, the polynucleotide sequence is T39M / F323I, R62H / P117G, P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G,、Y65S、Y65T、Y65W、P66N、A68L、A68P、I69F、I69G、I69H、I69L、I69V、I69W、I69Y、G70L、G70R、G70T、G70W、A71F、A71G、A71H / Q263R、A71P、A71R、A71V、A71Y、F77S / E184G、W103P、W103R、W103T / P147S、I104P、I104Q / A429V、H105L、G107D / S185W、G107L、G107P、G107S、A108G、A108K、A108Q、A108R、A108S、A108W、F109G / P117M、T110A、T110H、T110P、T110S、N111L、N111M、N111R、N111S、N111V、N111W、S113P、G114A、G114H、G114Q、S115H、S115T、S115V、P117A、P117F、V118G / A349V、V118I、V118N、V118N / A269T、Y119G、Y119P、Y119S、R126C、R153H / N215P、R153L、W164R / W271T、G174D / L282V、G183P、E184F、E184G、E184P、E184S / A249T、E184Y、S185A、S185T、A186C、A186G、A186P、A186R、A186T、G187P、A188E、A188G、S190H、S190K、S190L、S190M、S190Q、S190R、S190W、L209E、L209G、L209P、L209S、L209V、Q210P、Q210T、Q210W、S211I、S211L、S211R、S211V、G212A、G212P、G212R、G212S、A213C、A213E、A213L、A213N、A213P、A213Q、A213R / S345G、A213S、A213T / W271K、A213V、G214K、G214L、G214T、G214V、N215K、N215M、N215P、N215R、N215R / W271R、N215W、M216P、A217G、A217L、A217P、A217R、A217R / A231V、A217S、A217V、A217W、T224I / P268S / I372F、A249V / F284P、A269N、A269V、V270I、V270I / V470M、V270R、W271A、W271K、W271L, W271P, W271Q / A416V, W271S, W271T, A276F, G277M, G278H, G278S, S279C, S279E, S279G, S279L / V280G / L282M, S279V, V280E, V280G, V280S, L281P, L281V, L281Y / D374N, L282A, L282C, L282Q, L282R, L282S, L282T, L282W, P283C, P283D, P283K, P283R / A429V, P283T, P283V, P283Y, F284C, F284T, F284T / P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P, Y320A, Y320F, Y320G, Y320G / F323S, Y320S, Y320S / F323S / I372A, Y320S / I372A / V376G, Y320S / V376G / F377V, Y320W, R321L, R321S, F323C, F323I, F323R, F323Y, L324A, I372A / V376A, I372L, T373G, The polynucleotide sequence encodes at least one engineered carboxylesterase comprising at least one substitution or set of substitutions selected from V376A, V376G, V376L, V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V and A429L, where the amino acids are numbered with reference to SEQ ID NO:2. In some embodiments, the polynucleotide sequence encodes at least one engineered carboxylesterase comprising a substitution at position 282, where the positions are numbered with reference to SEQ ID NO:2. In some further embodiments, the substitution at position 282 is aliphatic, non-polar, basic, polar or aromatic. In yet some further embodiments, the substitution is selected from X282T, X282G, X282A, X282V, X282M, X282C, X282W, X282Q, X282S, X282T and X282R.

[0018] The present invention also relates to a method for the preparation of a compound having at least 80%, 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 Provided is a polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof comprising a polypeptide sequence having 9% or more sequence identity, said engineered carboxylesterase comprising at least one substitution or set of substitutions in said polypeptide sequence, and wherein amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the polynucleotide sequence is 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372, 65 / 320, 65 / 320 / 323, 68, 68 / 69 / 189 / 214, 68 / 69 / 189 / 214 / 215, 68 / 69 / 189 / 214 / 215 / 271, 68 / 69 / 1 89 / 214 / 215 / 271 / 281 / 282 / 343 / 381, 68 / 69 / 189 / 214 / 271 / 280, 68 / 69 / 189 / 214 / 372, 68 / 69 / 189 / 214 / 377 / 381, 68 / 69 / 189 / 271, 68 / 69 / 189 / 271 / 280 / 372 / 381, 68 / 69 / 189 / 280 / 281 / 282 / 372 / 377, 68 / 69 / 189 / 281 / 282 / 372 / 377 / 381, 68 / 69 / 1 89 / 343 / 381, 68 / 69 / 189 / 372, 68 / 69 / 189 / 381, 68 / 69 / 214 / 215 / 271, 68 / 69 / 214 / 343, 68 / 69 / 215, 68 / 69 / 271, 68 / 69 / 282 / 287, 68 / 69 / 343 / 372, 68 / 108 / 377, 68 / 184, 68 / 184 / 189, 68 / 189 / 271 / 372, 68 / 189 / 343, 68 / 214 / 215 / 271 / 281 / 282 / 372,68 / 215 / 271 / 343 / 372 / 381、68 / 215 / 377、68 / 271 / 372、68 / 377、69、69 / 70、69 / 70 / 331 / 372、69 / 70 / 372、69 / 70 / 459、69 / 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 282、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320、108 / 189 / 377、108 / 215、108 / 215 / 377、108 / 269 / 270、108 / 270、108 / 282 / 285 / 377、108 / 285、108 / 320 / 323、108 / 377、126、126 / 184 / 213 / 280 / 281 / 285 / 320、126 / 184 / 213 / 372、126 / 189 / 285 / 372、126 / 215、126 / 372、181 / 215、189、189 / 214、189 / 214 / 215 / 271 / 282、189 / 215、189 / 215 / 249 / 277、189 / 215 / 271 / 281 / 282 / 377、189 / 215 / 343 / 372、189 / 270 / 285、 , 189 / 270 / 372, 189 / 280 / 282, 189 / 320 / 377, 189 / 343, 189 / 343 / 377, 189 / 372 / 377, 189 / 377, 189 / 381, 213 / 215 / 320, 214 / 215 / 271, 214 / 215 / 271 / 377, 214 / 271, 214 / 280 / 282 / 343 / 377 / 38 1, 215, 215 / 249 / 280 / 281 / 285 / 372, 215 / 271 / 372, 215 / 280 / 281 / 285 / 372, 215 / 281 / 285 / 372, 215 / 281 / 285 / 373, 215 / 281 / 373, 215 / 285, 215 / 285 / 317, 215 / 285 / 346, 215 / 285 / 445, 215 / 3 20, 215 / 320 / 372, 215 / 323, 215 / 372, 215 / 372 / 377, 215 / 373, 215 / 377, 215 / 381, 249 / 377, 269 / 270 / 281 / 372 / 377, 270 / 377, 271, 271 / 343, 271 / 343 / 372, 271 / 343 / 372 / 381, 280 / 285 / 372, The present invention encodes an engineered carboxylesterase comprising at least one substitution or set of substitutions at positions selected from 281 / 372, 282 / 285 / 320 / 323, 285 / 323, 320, 343 / 372, 372, 372 / 377, 372 / 381, 373 and 377, wherein the amino acid positions are numbered with reference to SEQ ID NO:8. In some further embodiments, the polynucleotide sequence is 63A, 63A / 189A, 63A / 215R / 343V, 63R, 63R / 65G / 108G, 63R / 65G / 108G / 189L, 63R / 65G / 108G / 377I, 63R / 65G / 282A / 285L / 320W / 323I, 63R / 65G / 320W / 323I, 63R / 108G, 63R / 108G / 282A / 285L / 377L, 63R / 108G / 285L / 377I, 63R / 108G / 320W / 323C, 63R / 377I, 63T / 215R, 63Y, 63Y / 189L, 63Y / 212P / 215R, 63Y / 212P / 2 15R / 268A / 269N / 343V, 63Y / 215P / 269N, 63Y / 215R, 63Y / 215R / 270I / 271S, 63Y / 268A / 269N / 270I / 429V, 65G / 320W,<h2 style=";text-align:left;direction:ltr">65G / 320W / 323I, 65W / 69L / 372L, 65W / 69M / 70A / 281P / 372L, 65W / 69W / 70L / 372L, 65W / 70L / 372M, 68P, 68P / 69L / 189E / 214R / 271Y / 280G, 68P / 69L / 189E / 2 14R / 372L、68P / 69L / 189I / 214R / 215P / 271Y、68P / 69L / 189I / 281P / 282C / 37 2L / 377Y / 381L、68P / 69L / 189Q / 214R、68P / 69L / 189Q / 271Y / 280G / 372L / 381L 68P / 69L / 215P, 68P / 69L / 271Y, 68P / 69L / 282C / 287I, 68P / 69L / 343V / 372L, 68P / 69W / 189E / 214R / 215P / 271Y / 281P / 282G / 343V / 381L, 68P / 69W / 189E / 2 80G / 281P / 282A / 372L / 377Y、68P / 69W / 189E / 343V / 381L、68P / 69W / 189I / 21 4R / 215P、68P / 69W / 189I / 214R / 377Y / 381L、68P / 69W / 189I / 271Y、68P / 69W / 1 89I / 372L、68P / 69W / 189I / 381L、68P / 69W / 214R / 215P / 271Y、68P / 69W / 214R / 343V、68P / 69W / 215P、68P / 108G / 377L、68P / 184S、68P / 184S / 189E、68P / 189 I / 271Y / 372L, 68P / 189I / 343V, 68P / 214R / 215P / 271Y / 281P / 282A / 372L, 68P / 215P / 271Y / 343V / 372L / 381L, 68P / 215P / 377L, 68P / 271Y / 372L, 68P / 377L 69F / 108G / 270E / 372L / 377L, 69F / 189L, 69F / 215K, 69F / 215K / 269L / 270I / 377L, 69F / 215R, 69F / 285L / 373G, 69L, 69L / 70L / 331Q / 372M, 69L / 189E / 271Y / 281P / 282A、69L / 189I、69L / 189I / 214R / 271Y / 281P / 282A / 343V、69L / 189I / 271Y / 343V / 381L、69L / 189I / 280G / 282G / 343V / 372L / 381L、69L / 189I / 282A、69L / 、 <h2 style=";text-align:left;direction:ltr">189Q / 377Y、69L / 215P / 271Y / 280G / 281P / 282C、69L / 215P / 271Y / 282A、69L / 215P / 271Y / 372L、69L / 215P / 343V / 372L / 381L、69L / 215R / 285P / 317P、69L / 271Y、69L / 271Y / 372L、69L / 282C / 343V / 372L、69L / 372L、69M / 70A / 372M、69W、69W / 70L、69W / 70L / 372M、69W / 70L / 459R、69W / 108S、69W / 189E / 214R / 343 V / 372L、69W / 189E / 271Y / 343V、69W / 189E / 372L、69W / 189I、69W / 189I / 215P / 343V、69W / 189I / 271Y、69W / 189I / 343V / 381L、69W / 189Q / 372L / 377Y、69W / 212A / 213L / 215R / 280G / 281P、69W / 214R / 215P / 271Y / 372L / 377Y / 381L、69W / 214R / 271Y / 282A、69W / 214R / 271Y / 343V、69W / 215K / 343V、69W / 215P、69W / 215R、69W / 215R / 323Y、69W / 282A、69W / 372M、69Y / 108G / 281P / 285P、69Y / 11 0A / 215R / 281P、69Y / 189L / 281P / 373G、70L、70L / 212P、108G、108G / 189I / 28 2A / 285L / 320W, 108G / 189L, 108G / 189L / 320W, 108G / 189L / 377I, 108G / 215K, 108G / 215P / 377L, 108G / 269L / 270E, 108G / 270E, 108G / 282A / 285L / 377L, 1 08G / 285L、108G / 320W / 323I、108G / 377I、108G / 377L、126C、126C / 184S / 213 S / 280G / 281P / 285L / 320G、126C / 184S / 213S / 372L、126C / 189I / 285L / 372L、 126C / 215P, 126C / 372L, 181L / 215P, 189E / 372L / 377Y, 189I, 189I / 214R / 215P / 271Y / 282G, 189I / 215K, 189I / 215P / 343V / 372L, 189I / 215R / 249T / 277M,189I / 270E / 285L, 189I / 270E / 372L, 189I / 280G / 282A, 189, I / 320W / 377I, 189I / 343V, 189I / 377I, 189L, 189Q, 189Q / 214R, 189Q / 215P / 271Y / 281P / 282C / 377Y, 189Q / 343V, 189Q / 343 V / 377Y, 189Q / 381L, 213S / 215P / 320G, 214R / 215P / 271Y, 214R / 215P / 271Y / 377Y, 214R / 271Y, 214R / 280G / 282A / 343V / 377 Y / 381L, 215K, 215K / 281P / 285L / 372L, 215K / 281P / 373G, 215K / 285L / 317P, 215K / 285L / 445L, 215K / 323Y, 215K / 372L, 215 K / 372L / 377L, 215K / 373G, 215P, 215P / 271Y / 372L, 215P / 320G, 215P / 320G / 372L, 215P / 372L, 215P / 372L / 377L, 215P / 377 L, 215P / 381L, 215R, 215R / 249T / 280G / 281P / 285L / 372L, 215R / 280G / 281P / 285L / 372L, 215R / 281P / 285L / 373G, 215R / 285 P, 215R / 320G, 215R / 372L, 215W, 215W / 285L / 346S, 215W / 285P, 215W / 373G, 249T / 377L, 269L / 270E / 281P / 372L / 377L, 270 Operated stenosis comprising at least one substitution or set of substitutions selected from E / 377L, 271Y, 271Y / 343V, 271Y / 343V / 372L, 271Y / 343V / 372L / 381L, 280G / 285L / 372L, 281P / 372L, 282A / 285L / 320W / 323I, 285L / 323I, 320W, 343V / 372L, 372L, 372L / 377L, 372L / 381L, 372M, 373G, and 377L. In some further embodiments, the polynucleotide sequence encodes a carboxylesterase having a nucleotide sequence selected from the group consisting of P63A, P63A / M189A, P63A / N215R / A343V, P63R, P63R / Y65G / A108G, P63R / Y65G / A108G / M189L, P63R / Y65G / A108G / F377I, P63R / Y65G / T282A / A285L / Y320W / F323I, P63R / Y65G / Y320W / F323I, P63R / A108G, P63R / A108G / T282A / A285L / F377L, P63R / A108G / A285L / F377I, P63R / A108G / Y320W / F323C, P63R / F377I, P63T / N215R, P 63Y, P63Y / M189L, P63Y / G212P / N215R, P63Y / G212P / N215R / P268A / A269 N / A343V, P63Y / N215P / A269N, P63Y / N215R, P63Y / N215R / V270I / W271S , P63Y / P268A / A269N / V270I / A429V, Y65G / Y320W, Y65G / Y320W / F323I, Y 65W / I69L / I372L, Y65W / I69M / G70A / L281P / I372L, Y65W / I69W / G70L / I 372L, Y65W / G70L / I372M, A68P, A68P / I69L / M189E / G214R / W271Y / V280G , A68P / I69L / M189E / G214R / I372L, A68P / I69L / M189I / G214R / N215P / W 271Y, A68P / I69L / M189I / L281P / T282C / I372L / F377Y / A381L, A68P / I69 L / M189Q / G214R, A68P / I69L / M189Q / W271Y / V280G / I372L / A381L, A68P / I69L / N215P, A68P / I69L / W271Y, A68P / I69L / T282C / V287I, A68P / I69L / A343V / I372L, A68P / I69W / M189E / G214R / N215P / W271Y / L281P / T282G / A343V / A381L, A68P / I69W / M189E / V280G / L281P / T282A / I372L / F377Y,<h2 style=";text-align:left;direction:ltr">A68P / I69W / M189E / A343V / A381L、A68P / I69W / M189I / G214R / N215P、A68P / I69W / M189I / G214R / F377Y / A381L、A68P / I69W / M189I / W271Y、A68P / I69W / M 189I / I372L、A68P / I69W / M189I / A381L、A68P / I69W / G214R / N215P / W271Y、A 68P / I69W / G214R / A343V、A68P / I69W / N215P、A68P / A108G / F377L、A68P / E18 4S、A68P / E184S / M189E、A68P / M189I / W271Y / I372L、A68P / M189I / A343V、A6 8P / G214R / N215P / W271Y / L281P / T282A / I372L、A68P / N215P / W271Y / A343V / I372L / A381L, A68P / N215P / F377L, A68P / W271Y / I372L, A68P / F377L, I69F / A108G / V270E / I372L / F377L, I69F / M189L, I69F / N215K, I69F / N215K / A269L / V270I / F377L、I69F / N215R、I69F / A285L / T373G、I69L、I69L / G70L / P331Q / I372M、I69L / M189E / W271Y / L281P / T282A、I69L / M189I、I69L / M189I / G214 R / W271Y / L281P / T282A / A343V、I69L / M189I / W271Y / A343V / A381L、I69L / M189I / V280G / T282G / A343V / I372L / A381L、I69L / M189I / T282A、I69L / M189Q / F377Y, I69L / N215P / W271Y / V280G / L281P / T282C, I69L / N215P / W271Y / T282A, I69L / N215P / W271Y / I372L, I69L / N215P / A343V / I372L / A381L, I69L / N21 5R / A285P / T317P、I69L / W271Y、I69L / W271Y / I372L、I69L / T282C / A343V / I372L、I69L / I372L、I69M / G70A / I372M、I69W、I69W / G70L、I69W / G70L / I372M、I69W / G70L / G459R、I69W / A108S、I69、 W / M189E / G214R / A343V / I372L、I69W / M189E / W271Y / A343V、I69W / M189E / I372L、I69W / M189I、I69W / M189I / N215P / A343V、I69W / M189I / W271Y、I69W / M18 9I / A343V / A381L、I69W / M189Q / I372L / F377Y、I69W / G212A / A213L / N215R / V 280G / L281P、I69W / G214R / N215P / W271Y / I372L / F377Y / A381L、I69W / G214R / W271Y / T282A、I69W / G214R / W271Y / A343V、I69W / N215K / A343V、I69W / N215P 、I69W / N215R、I69W / N215R / F323Y、I69W / T282A、I69W / I372M、I69Y / A108G / L 281P / A285P、I69Y / T110A / N215R / L281P、I69Y / M189L / L281P / T373G、G70L、 G70L / G212P、A108G、A108G / M189I / T282A / A285L / Y320W、A108G / M189L、A108 G / M189L / Y320W、A108G / M189L / F377I、A108G / N215K、A108G / N215P / F377L、 A108G / A269L / V270E、A108G / V270E、A108G / T282A / A285L / F377L、A108G / A28 5L、A108G / Y320W / F323I、A108G / F377I、A108G / F377L、R126C、R126C / E184S / A213S / V280G / L281P / A285L / Y320G、R126C / E184S / A213S / I372L、R126C / M1 89I / A285L / I372L、R126C / N215P、R126C / I372L、V181L / N215P、M189E / I372 L / F377Y、M189I、M189I / G214R / N215P / W271Y / T282G、M189I / N215K、M189I / N 215P / A343V / I372L、M189I / N215R / A249T / G277M、M189I / V270E / A285L、M18 9I / V270E / I372L、M189I / V280G / T282A、M189I / Y320W / F377I、M189I / A343V、M189I / F377I, M189L, M189Q, M189Q / G214R, M189Q / N215P / W271Y / L281P / T282C / F377Y, M189Q / A343V, M189Q / A343V / F377Y, M189Q / A381L, A213S / N 215P / Y320G, G214R / N215P / W271Y, G214R / N215P / W271Y / F377Y, G214R / W2 71Y, G214R / V280G / T282A / A343V / F377Y / A381L, N215K, N215K / L281P / A28 5L / I372L, N215K / L281P / T373G, N215K / A285L / T317P, N215K / A285L / V44 5L, N215K / F323Y, N215K / I372L, N215K / I372L / F377L, N215K / T373G, N215 P, N215P / W271Y / I372L, N215P / Y320G, N215P / Y320G / I372L, N215P / I372L , N215P / I372L / F377L, N215P / F377L, N215P / A381L, N215R, N215R / A249T / V280G / L281P / A285L / I372L, N215R / V280G / L281P / A285L / I372L, N215R / L281P / A285L / T373G, N215R / A285P, N215R / Y320G, N215R / I372L, N215W, N 215W / A285L / G346S, N215W / A285P, N215W / T373G, A249T / F377L, A269L / V2 70E / L281P / I372L / F377L, V270E / F377L, W271Y, W271Y / A343V, W271Y / A34 3V / I372L, W271Y / A343V / I372L / A381L, V280G / A285L / I372L, L281P / I372L, T282A / A285L / Y320W / F323I, A285L / F323I, Y320W, A343V / I372L, I372L, I372L / F377L, I372L / A381L, I372M, T373G and F377L, wherein the amino acid positions are numbered with reference to SEQ ID NO:8.

[0019] The present invention also relates to a polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof, comprising SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 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, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 90, 101, 102, 103, 10 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0020] The present invention further provides polynucleotide sequences encoding the engineered carboxylesterases provided herein, operably linked to a regulatory sequence, hi some further embodiments, the polynucleotide sequences are codon optimized.

[0021] The present invention also provides expression vectors comprising at least one polynucleotide sequence encoding an engineered carboxylesterase provided herein. In addition, the present invention provides host cells comprising at least one expression vector provided herein. In some embodiments, the present invention also provides host cells comprising at least one polynucleotide sequence encoding at least one engineered carboxylesterase provided herein.

[0022] The invention also provides methods of producing an engineered carboxylesterase in a host cell, comprising culturing a host cell comprising an expression vector comprising at least one polynucleotide encoding at least one engineered carboxylesterase under suitable conditions, such that at least one engineered carboxylesterase is produced. In some embodiments, the method further comprises recovering the at least one engineered carboxylesterase from the culture and / or the host cell. In some further embodiments, the method further comprises purifying the at least one engineered carboxylesterase. [Brief description of the drawings]

[0023] [Figure 1-1] FIG. 1 illustrates each of the polynucleotide sequences encoding the engineered carboxylesterase sequences set forth in the Sequence Listing submitted herewith (SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 1 shows a sequence alignment of a polynucleotide sequence (SEQ ID NO: 1) encoding the E. coli codon optimized wild-type Thermobifida fusca (T. fusca) carboxylesterase enzyme to a polynucleotide sequence encoding the wild-type Thermobifida fusca (T. fusca) carboxylesterase enzyme (SEQ ID NO: 1) of the same sequence as the wild-type Thermobifida fusca (T. fusca) carboxylesterase enzyme (SEQ ID NO: 1). All of these disclosed polynucleotide sequences are 98.9-99.9% identical to each other. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14] Same as above. [Figure 1-15] Same as above. [Figure 1-16] Same as above. [Figure 1-17] Same as above. [Figure 1-18] Same as above. [Figure 1-19] Same as above. [Figure 1-20] Same as above. [Figure 1-21] Same as above. [Figure 1-22] Same as above. [Figure 2-1] FIG. 2 illustrates engineered polypeptide carboxylesterase sequences set forth in the Sequence Listing submitted herewith (SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 8, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136). All of these disclosed polypeptide sequences are 98.6-99.8% identical to each other. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Figure 2-7] Same as above. [Figure 2-8] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides engineered carboxylesterases (EC 3.1.1) having improved non-native properties compared to naturally occurring wild-type (WT) carboxylesterase enzymes, as well as polynucleotides encoding the engineered carboxylesterase enzymes, host cells capable of expressing the engineered carboxylesterase enzymes, and methods of applying the engineered carboxylesterase enzymes to amidation reactions.

[0025] It has been observed that enzyme active sites can catalyze several different chemical reactions via a single amino acid mutation, so switching of enzyme function and enzyme substrate range is feasible (see Rauwerdink and Kazluaskas, ACS Cat., 5:6153-6176

[2015] ). To improve the functionality and substrate range of carboxylesterases, wild-type carboxylesterases were subjected to directed evolution. The resulting variants had improved ability in the production of amide bonds using a diverse set of amine and carboxylester substrate pairs (see Scheme 1 below). The engineered carboxylesterases were engineered to produce amide bonds using a diverse set of amine and carboxylester substrate pairs (see Scheme 1 below). The engineered carboxylesterases were engineered to produce amide bonds using the methods described herein. Thus, not only were they active in aqueous systems, but they are also active in the presence of organic co-solvents and even near-complete organic solvent concentrations (e.g., about 98% v / v). Furthermore, immobilization of these engineered carboxylesterases facilitates continuous-flow operations for amide production, aids in purification of the final amide product, and improves the efficiency and overall cost of the amidation operation.

[0026] The present invention provides novel engineered carboxylesterase polypeptides along with corresponding polynucleotide sequences and application methods that demonstrate the formation of a general amide bond (see Scheme 1 below). In some embodiments, the engineered polypeptides have altered properties that expand the range of functionality and activity of these enzymes compared to the naturally occurring wild-type Thermobifida fusca (T. fusca) carboxylesterase (SEQ ID NO:2). Improved carboxylesterase properties include, but are not limited to, solvent stability, enzymatic activity, regiospecificity, stereoselectivity, reduced host cell toxicity, thermostability, pH stability, substrate range, and / or reduced substrate or product inhibition. The present invention also provides improved polynucleotides to facilitate expression of desired polypeptides in non-native host organisms (e.g., E. coli).

[0027] In some embodiments, the carboxylesterase polypeptides provided herein have altered properties that expand the range of functionality and activity of these enzymes as compared to the naturally occurring wild-type Geobacillus stearothermophilus carboxylesterase (SEQ ID NO: 138). In some cases, these polypeptides are enhanced carboxylesterase enzymes as compared to the wild-type Mycobacterium tuberculosis carboxylesterase (SEQ ID NO: 140). Additionally, in some embodiments, the invention provides improved polynucleotides to facilitate expression of a desired polypeptide in a non-native host organism (e.g., E. coli).

[0028] The improved properties of the presented carboxylesterase variants relate to engineered amidated polypeptides that contain residue differences at specific residue positions compared to a reference T. fusca carboxylesterase sequence or another engineered reference amidated polypeptide, such as the sequence of SEQ ID NO:8. In some embodiments, the residue difference is present at at least one of the following amino acid positions: X39, X62, X63, X64, X65, X66, X68, X69, X70, X71, X77, X103, X104, X105, X107, X108, X109, X110, X111, X113, X114, X115, X117, X118, X119, X126, X147, X153, X164, X174, X181, X183, X184, X185, X186, X187, X188, X189, X190, X209, X210, X211, X212, X213, X214, X215, X216, X218, X219, X220, X221, X222, X223, X224, X225, X226, X227, X228, X229, X230, X231, X232, X233, X234, X235, X236, X237, X238, X239, X240, X241, X242, X243, X244, X245, X246, X247, X248, X249, X250, X251, X252, X253, X254, X255, X256, X257, X258, X260, X261, X262, X263, X264, X265, X266, X267, X268, X269, X270, X271, X2 4, X215, X216, X217, X224, X231, X249, X263, X268, X269, X270, X271, X276, 277, X278, X279, X280, X281, X282, X283, X284, X285, X286, X287, X311, X320, X321, X323, X324, X331, X343, X345, X346, X349, X372, X373, X374, X376, X377, X381, X405, X416, X420, X427, X428, X429, X438, X445, X459 and X470.

[0029] In some embodiments, the engineered carboxylesterases provided herein are characterized by exhibiting increased thermostability compared to wild-type polypeptides under the same reaction conditions. The engineered carboxylesterases are able to mediate amidation conversions at higher temperatures and for longer periods of time than the WT carboxylesterase, as indicated by the continued formation of product (see Scheme 1 below). In some embodiments, the engineered carboxylesterase polypeptide ... concentrations of substrate ester (I) and / or amide (I). In some embodiments, the engineered carboxylesterase polypeptides maintain activity or have increased activity under conditions of various pH levels (e.g., pH 9.0) compared to WT carboxylesterase. In some embodiments, the engineered polypeptides with increased thermal stability, pH stability and / or substrate stability comprise an amino acid sequence that is at least 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the reference sequence of SEQ ID NOs: 2 and / or 8.

[0030] In some embodiments, the engineered carboxylesterases are capable of at least about a 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 150-fold, 500-fold or more improved biocatalytic activity for conversion of substrate compound(s) to product(s) compared to the activity of a wild-type carboxylesterase (SEQ ID NO:2) or an engineered reference carboxylesterase (SEQ ID NO:8) under suitable reaction conditions (see Scheme 1 below). In some embodiments, these improvements in enzyme activity range from associated increases in thermal stability, stereoselectivity, stereospecificity, regiospecificity, solvent stability, pH stability and / or substrate binding, or reduced substrate and / or product inhibition.

[0031] In some embodiments, the engineered carboxylesterases are characterized by activity towards a variety of structurally distinct carboxy ester (I) or amine (II) substrates. In some embodiments, the engineered polypeptides are capable of biocatalytically converting esters (N,N-diethylaminoglycine methyl ester, ethyl benzoate, ethyl acetate, pyrazine-2-carboxylic acid ethyl ester, 1H-indole-4-carboxylic acid ethyl ester, methyl phenylacetate) and amines (n-butylamine, isobutylamine, aniline, benzylamine, 2,6-dimethylaniline, t-butylamine, N'-t-butoxycarbonyl-benzhydrazide, 4-methylpiperidine, Ot-butylhydroxylamine, 2,6-dimethylaniline, or stereoselective conversion of (S)-(-)-α-methylbenzylamine, (R)-(+)-α-methylbenzylamine) to their corresponding amide products at a rate greater than the WT polypeptide of SEQ ID NO:2 and / or the engineered polypeptide of SEQ ID NO:8.

[0032] In some embodiments, the improved engineered variant polypeptide comprises an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 or 140. In some embodiments, the carboxylesterase enzymes provided herein are obtained by mutagenizing a gene encoding an engineered carboxylesterase polypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of a naturally occurring T. fusca carboxylesterase (SEQ ID NO:2).

[0033] In some further embodiments of the invention, the carboxylesterase polypeptide variant is encoded by a polynucleotide or polynucleotides that hybridize under highly stringent conditions to produce such a polynucleotide, as provided herein. In some embodiments, the polynucleotides include promoters and / or other regulatory elements useful for expression of the encoded engineered carboxylesterase and may utilize codons optimized for a specific expression system.

[0034] In some embodiments, the polynucleotide encoding the improved carboxylesterase enzyme comprises a sequence selected from SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, and 139.

[0035] In some further embodiments, the invention provides host cells comprising the polynucleotides and / or expression vectors provided herein. In some embodiments, the host cells are T. fusca, while in some alternative embodiments, they are other organisms (e.g., E. coli). The host cells are used in the expression of the encoded polynucleotides to produce engineered carboxylesterases and in the isolation of the engineered carboxylesterases described herein. In some embodiments, the host cells are used in the direct conversion of substrate(s) to desired product(s).

[0036] In some further embodiments, the invention provides methods for carrying out Reaction Scheme 1 (shown below) using any of the engineered carboxylesterase enzymes provided herein. In some embodiments, the methods comprise contacting or incubating carboxy ester (I) and amine (II) substrates with an engineered carboxylesterase polypeptide of the invention under suitable reaction conditions for conversion of the substrates to the corresponding amide products, thereby converting the substrates to product compounds. Whether the methods are carried out with whole cells, cell extracts, or purified carboxylesterase enzymes, a single carboxylesterase enzyme can be used, or alternatively a mixture of at least two carboxylesterase enzymes is used. [ka]

[0037] The engineered carboxylesterases of the invention are capable of converting a diverse set of carboxy ester (Scheme 1, I) and amine (II) substrates into their corresponding amide products (III). The range of available carboxy ester substrates is determined by the R 1 and R 2 In some embodiments, R 1 and R 2 The functional groups of the group include a variety of moieties ranging from hydrogen atoms to alkyl, alkenyl, alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl moieties, with the proviso that R 2 cannot be hydrogen. In the above state, R of amine (II) 3 and R 4The selected functional group at position consists of a hydrogen atom, an alkyl, an alkenyl, an alkynyl, an alkoxy, a carboxy, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a carboxyalkyl, an aminoalkyl, a haloalkyl, an alkylthioalkyl, a cycloalkyl, an aryl, an arylalkyl, a heterocycloalkyl, a heteroaryl, and a heteroarylalkyl. 1 and R 2 can be linked to form a 3- to 10-membered ring, with the proviso that R 3 and R 4 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, which may also be linked to form a 3- to 10-membered ring; optionally, R 1 or R 2 is connected via an alkyl, alkenyl, alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, or heteroarylalkyl tether to R 3 or R 4 The amidation process can be carried out under suitable reaction conditions to form compound (I). [ka] (In the formula, R 1 and R 2 is as defined above), and a compound of formula (II) [ka] (In the formula, R 3 and R 4is as defined above), and proceeds as an engineered polypeptide having amidation activity. In some embodiments of the reaction methods provided herein, WT T. fusca carboxylesterase (SEQ ID NO:2) or an engineered reference polypeptide (e.g., SEQ ID NO:8), a carboxylesterase derivative provided herein, is capable of producing primary and secondary amides conforming to the general formula (III). An engineered amidation enzyme provided herein (e.g., an engineered carboxylesterase polypeptide of an even-numbered sequence identifier of SEQ ID NOs:4-136) is used as a biocatalyst for the above reaction (Scheme 1). definition

[0038] In relation to the present invention, scientific and technical terms used in the description herein have the meanings commonly understood by those skilled in the art unless expressly defined otherwise. Thus, the following terms shall have the following meanings: All U.S. patents and published U.S. patent applications mentioned herein, including all sequences disclosed in such patents and patent applications, are expressly incorporated by reference. Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology and related fields known to those skilled in the art. Unless otherwise defined herein, all scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Although methods and materials that may be used in the present invention are described, preferred methods and materials are described. Indeed, it is intended that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, which may vary depending on the context in which they are used. The headings provided herein are not intended to limit the various aspects or embodiments of the present invention.

[0039] Nonetheless, to facilitate understanding of the present invention, certain terms are defined below. Numerical ranges are inclusive of the numerical values ​​defining the range. Thus, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were all expressly written herein.

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

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "host cell" includes a plurality of such host cells.

[0042] 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.

[0043] The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0044] As used herein, a "carboxylesterase" is defined as an enzyme that inherently has catalytic activity for the hydrolysis of carboxy esters resulting in the formation of organic acids and alcohols.

[0045] As used herein, "amidation" or amide synthesis refers to the process of creating an amide bond to yield a carboxamide (an organic amide).

[0046] As used herein, "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 modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). D- and L-amino acids and mixtures of D- and L-amino acids are included in this definition.

[0047] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. A polynucleotide can be composed entirely of ribonucleosides (i.e., RNA), entirely of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleosides and 2' deoxyribonucleosides. Nucleosides are typically linked together via standard phosphodiester linkages, although a polynucleotide can contain one or more non-standard linkages. A polynucleotide can be single-stranded or double-stranded, or can contain both single-stranded and double-stranded regions. Also, a polynucleotide typically contains naturally occurring coding nucleobases (i.e., adenine, guanine, argin ... The nucleic acid bases are generally composed of the amino acids 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0048] As used herein, "coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0049] As used herein, "naturally occurring," "wild type," and "WT" refer to forms found in nature. For example, a naturally occurring or wild type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a natural source, and has not been intentionally modified by human manipulation.

[0050] As used herein, "non-naturally occurring" or "engineered" or "recombinant" when used herein with respect to (e.g., a cell, a nucleic acid, or a polypeptide) refers to a material that is otherwise identical to, or corresponds to the natural or native form of, said material, but that has been modified in an otherwise non-naturally occurring manner or that has been produced and / or generated from synthetic material and / or by manipulation using recombinant techniques. Non-limiting examples include recombinant cells that express genes not found in the native (non-recombinant) form of the cell, among others, or that express native genes that are otherwise expressed at different levels.

[0051] As used herein, "percentage of sequence identity", "percentage of identity" and "percentage of identity" refer to a comparison between polynucleotide or polypeptide sequences, and are determined by comparing two sequences that are optimally aligned over a comparison region, and a portion of the polynucleotide or polypeptide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence of the optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue is present or where the nucleic acid base or amino acid residue aligns with a gap to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison region, and multiplying the result by 100 to determine the percentage of sequence identity. Optimal alignment and determination of percent sequence identity are performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol. 215:403-410

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

[1977] ). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0052] Briefly, BLAST analysis involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths in a query sequence that match or meet some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The cumulative alignment score is either reduced by an amount X from its maximum achieved value; accumulation of one or more negative scoring residue alignments causes the cumulative score to fall to 0 or below; or when the end of either sequence is reached, extension of the word hit in each direction is stopped. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc Natl. Soc. 1999, 14:1311-1323, 1999). (See Acad Sci USA 89:10915

[1989] ).

[0053] Many other algorithms that function similarly to BLAST in providing the percent identity of two sequences are available and known in the art. Optimal alignment of sequences for comparison can be performed using any suitable method known in the art (e.g., Smith and Waterman, Adv. Appl. Math. 2:482

[1981] local homology algorithm; Needleman and Wunsch, J. Mol. Biol. 48:443

[1970] homology alignment algorithm; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] similarity search method; and / or computer-controlled implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in GCG Wisconsin Software Package) or visual inspection using methods generally known in the art. In addition, sequence alignment and determination of sequence identity percentage can be performed using the BESTFIT or GAP program in GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0054] As used herein, a "reference sequence" refers to a defined sequence to which another sequence is compared. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides each may (1) contain similar sequences (i.e., portions of the complete sequence) between the two sequences, and (2) further contain sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides over a comparison region to identify and compare local regions of sequence similarity. The term "reference sequence" is not limited to wild-type sequences, but may include engineered or modified sequences. For example, in some embodiments, a "reference sequence" may be a previously engineered or modified amino acid sequence.

[0055] As used herein, a "comparison region" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison region can include 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison region can be longer than 20 contiguous residues, and optionally includes regions of 30, 40, 50, 100 or more.

[0056] As used herein, "corresponding to," "with reference to," or "compared to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues in a specified reference sequence when the given amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is not in accordance with the actual numbered position of the residue in the given amino acid sequence or polynucleotide sequence. In general, the number of residues in a given amino acid sequence, such as the amino acid sequence of an engineered carboxylesterase, is specified relative to the reference sequence, rather than to the amino acid sequence itself. For example, a given amino acid sequence, such as the amino acid sequence of an engineered carboxylesterase, may be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps are present, the numbering of residues in a given amino acid sequence or polynucleotide sequence is done relative to the reference sequence to which it is aligned. As used herein, references to residue positions such as "Xn", described further below, should be construed as references to "residues corresponding to" unless otherwise specifically indicated. Thus, for example, "X94" refers to any amino acid at position 94 of a polypeptide sequence (e.g., SEQ ID NO: 2, 4, 10, 26 or 42).

[0057] As used herein, "stereoselectivity" refers to the preferential formation of one stereoisomer over another or set of stereoisomers in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over another, or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as the enantioselectivity (the ratio of one enantiomer in the sum of both enantiomers (typically reported as a percentage)) or it is commonly reported in the art as the enantiomeric excess (ee) calculated therefrom (typically as a percentage) according to the formula [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is referred to as the diastereoselectivity (the ratio of one diastereomer in a mixture of two diastereomers (typically reported as a percentage)) or it is commonly reported as the diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess. It should also be understood that stereoselectivity is not limited to a single stereoisomer, but can be described for a set of stereoisomers.

[0058] As used herein, "highly stereoselective" refers to a chemical or enzymatic reaction that can convert a substrate to its corresponding chiral amide product in at least about 75% stereoisomeric excess.

[0059] As used herein, "increased enzyme activity" and "increased activity" refer to improved properties of an engineered enzyme, which may be expressed as an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the conversion rate of a substrate to product (e.g., the conversion rate of a starting amount of substrate to product in a specified period of time using a specified amount of carboxylesterase) compared to a reference enzyme. Exemplary methods for determining enzyme activity are provided in the Examples. m , V max or kcat Any characteristic related to the enzyme activity can be affected, including the classical enzyme characteristics of the enzyme, and these changes can result in an increase in enzyme activity. Carboxylesterase activity can be measured by any one of the standard assays used to measure carboxylesterases, such as changes in substrate or product concentration. Comparison of enzyme activity is performed using a defined preparation of the enzyme, a defined assay under set conditions, and one or more defined substrates, as described in more detail herein. In general, when comparing enzymes in cells, the number of cells and the amount of protein assayed are determined, and the same expression system and the same host cells are used to minimize variations in the amount of enzyme produced by the host cells and present in the lysate.

[0060] As used herein, "conversion" refers to the enzymatic conversion of a substrate to the corresponding product.

[0061] As used herein, "conversion rate" refers to the percent of a substrate that is converted to a product under specified conditions within a period of time. Thus, for example, a carboxylesterase polypeptide The "enzyme activity" or "activity" of a protease can be expressed as the "conversion rate" of a substrate to a product.

[0062] As used herein, "regiospecific" refers to a chemical reaction that produces only one structural isomer, where other isomers are theoretically possible.

[0063] As used herein, "thermostable" or "thermostable" are used interchangeably to refer to a polypeptide that is resistant to inactivation compared to the untreated enzyme when exposed to a range of temperature conditions (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hours) and thus retains a particular level of residual activity (e.g., greater than 60%-80%) after exposure to elevated temperatures.

[0064] As used herein, "solvent stable" refers to the ability of a polypeptide to maintain similar activity (e.g., greater than, for example, 60%-80%) compared to the untreated enzyme after exposure to various concentrations (e.g., 5-99%) of a solvent (e.g., isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours).

[0065] As used herein, an "amino acid difference" or "residue difference" refers to an amino acid residue difference at a position of a polypeptide sequence compared to an amino acid residue at a corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn" (where n refers to the corresponding position in the reference sequence on which the residue difference is based). For example, a "residue difference at position X40 compared to SEQ ID NO:2" refers to an amino acid residue difference at a polypeptide position corresponding to position 40 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a histidine at position 40, then a "residue difference at position X40 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than histidine at a polypeptide position corresponding to position 40 of SEQ ID NO:2. In most examples herein, a particular amino acid residue difference at a position is designated as "XnY" (where "Xn" designates the corresponding position as above, and "Y" is a single letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from in the reference polypeptide). In some cases, the present invention also provides specific amino acid differences represented by the conventional designation "AnB" (A is the single letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the engineered polypeptide sequence). In some cases, the polypeptides of the present invention may contain at least one amino acid residue difference compared to the reference sequence, as indicated by a list of the specific positions at which the residue difference exists compared to the reference sequence. In some embodiments, when more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., X192A / G). The present invention encompasses engineered polypeptide sequences that contain at least one amino acid difference, including either or both conservative and non-conservative amino acid substitutions. The amino acid sequences of certain recombinant carbonic anhydrase polypeptides included in the sequence listing of the present invention include an initial methionine (M) residue (i.e., M represents residue position 1).However, one of skill in the art will appreciate that, for example, in a host cell or in vitro translation system, biological processing mechanisms may remove this initial methionine residue to generate a mature protein that lacks the initial methionine residue but otherwise retains the properties of the enzyme. Consequently, the term "amino acid residue difference compared to SEQ ID NO:2 at position Xn" as used herein may refer to position "Xn" or a corresponding position (e.g., position (X-1)n) in a reference sequence that has been processed to lack an initial methionine.

[0066] As used herein, the phrase "conservative amino acid substitution" refers to the interchangeability of residues having similar side chains, and thus typically involves the replacement of an amino acid in a polypeptide with an amino acid within the same or a similar defined amino acid class. In the form, an amino acid having an aliphatic side chain is replaced with another aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, an amino acid having a hydroxyl side chain is replaced with another amino acid having a hydroxyl side chain, such as serine and threonine, an amino acid having an aromatic side chain is replaced with another amino acid having an aromatic side chain, such as phenylalanine, tyrosine, tryptophan, and histidine, an amino acid having a basic side chain is replaced with another amino acid having a basic side chain, such as lysine and arginine, an amino acid having an acidic side chain is replaced with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. The appropriate classification of any amino acid or residue will be apparent to one of skill in the art, especially in light of the detailed invention provided herein. Exemplary conservative substitutions are provided in Table 1. [Table 1]

[0067] As used herein, the phrase "non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within defined groups, affecting (a) the structure of the peptide backbone in the region of substitution (e.g., glycine to proline), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not of limitation, exemplary non-conservative substitutions may be an acidic amino acid replaced with a basic or aliphatic amino acid, an aromatic amino acid replaced with a small amino acid, and a hydrophilic amino acid replaced with a hydrophobic amino acid.

[0068] As used herein, "deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids that make up the polypeptide, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining improved properties of the engineered enzyme. Deletions may be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include contiguous segments. or may be discontinuous.

[0069] As used herein, "insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. In some embodiments, improved engineered carboxylesterase enzymes include the insertion of one or more amino acids into a naturally occurring carboxylesterase polypeptide and the insertion of one or more amino acids into an engineered carboxylesterase polypeptide. The insertion can be in an internal portion of the polypeptide or to the carboxy or amino terminus. As used herein, an insertion includes fusion proteins as known in the art. The insertion can be a contiguous amino acid segment in a naturally occurring polypeptide or can be separated by one or more amino acids.

[0070] The term "amino acid substitution set" or "substitution set" refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution set can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions present in any of the mutant carboxylesterases included in the tables provided in the Examples.

[0071] As used herein, a "fragment" refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. A fragment may typically have about 80%, about 90%, about 95%, about 98% or about 99% of a full-length carboxylesterase polypeptide, e.g., the polypeptide of SEQ ID NO: 4. In some embodiments, a fragment is "biologically active" (i.e., exhibits the same enzymatic activity as the full-length sequence).

[0072] A "functional fragment" or "biologically active fragment," as used interchangeably herein, refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or an internal deletion, but whose remaining amino acid sequence is identical to the corresponding positions in a sequence to which it is compared (e.g., a full-length engineered T. fusca enzyme of the invention), and which retains substantially all of the activity of the full-length polypeptide.

[0073] As used herein, an "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants that naturally accompany it, e.g., proteins, lipids, and polynucleotides. The term encompasses a polypeptide that has been recovered or purified from its naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). The improved carboxylesterase enzymes may be present intracellularly, in cell culture medium, or prepared in various forms (e.g., lysates or isolated preparations). As such, in some embodiments, the engineered carboxylesterase polypeptides of the invention may be isolated polypeptides.

[0074] As used herein, a "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and generally is a substantially purified composition when the species of interest constitutes at least about 50 percent of the macromolecular species present, by mole % or by weight. Generally, a substantially pure engineered carboxylesterase polypeptide composition will be about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, and about 92% or more, or about 93% or more, about 94% or more of all macromolecular species present in the composition. , about 95% or more, about 96% or more, about 97% or more, and about 98% or more, or about 99% or more, by mole percent or weight percent. Solvent species, small molecules (<500 Daltons) and elemental ion species are not considered to be macromolecular species. In some embodiments, the isolated improved carboxylesterase polypeptide is a substantially pure polypeptide composition.

[0075] As used herein, the term "heterologous" when used with respect to a nucleic acid or polypeptide refers to a sequence that is not normally expressed and secreted by an organism (e.g., a wild-type organism). In some embodiments, the term encompasses sequences that contain two or more subsequences that are not found in the same relationship to each other as they are normally found in nature, or that have been recombinantly engineered such that their expression levels or physical relationships with other nucleic acids or other molecules in a cell or structure are not normally found in nature. For example, heterologous nucleic acids having two or more sequences derived from unrelated genes arranged in a manner not found in nature are typically produced recombinantly (e.g., a nucleic acid open reading frame (ORF) of the invention operably linked to a promoter sequence inserted into an expression cassette such as a vector). In some embodiments, "heterologous polynucleotide" refers to any polynucleotide that is introduced into a host cell by laboratory techniques, including polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0076] As used herein, "codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. In some embodiments, a polynucleotide encoding a carboxylesterase enzyme may be codon optimized for optimal production from the host organism selected for expression.

[0077] As used herein, "control sequences" is defined herein to include all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present invention. Each control sequence may be native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators.

[0078] As used herein, "operably linked" is defined herein as a configuration in which a control sequence is suitably positioned relative to a polynucleotide of interest (i.e., in a functional relationship) such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0079] As used herein, "suitable reaction conditions" refers to conditions of a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a carboxylesterase polypeptide of the invention can convert a substrate compound to a product compound (e.g., convert one compound to another). Exemplary "suitable reaction conditions" are provided in the present disclosure and are illustrated by the Examples.

[0080] As used herein, "loading," such as "compound loading," "enzyme loading," or "cofactor loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0081] As used herein, "substrate" in the context of a biocatalyst-mediated process refers to the compound or molecule upon which the biocatalyst acts.

[0082] As used herein, "product" in the context of a biocatalyst-mediated process refers to a compound or molecule that results from the action of the biocatalyst.

[0083] As used herein, "equilibrium" refers to a process that results in steady-state concentrations of chemical species in a chemical or enzymatic reaction, including the interconversion of stereoisomers (e.g., the interconversion of two species A and B), as determined by the forward and reverse rate constants of the chemical or enzymatic reaction.

[0084] As used herein, "alkyl" refers to a saturated hydrocarbon group, either straight or branched, of 1 to 18 carbon atoms (inclusive), more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive). Alkyl groups having a specific number of carbon atoms are indicated in parentheses (e.g., (C1-C4)alkyl refers to alkyl of 1 to 4 carbon atoms).

[0085] As used herein, "alkenyl" refers to a group of 2 to 12 carbon atoms (inclusive) either straight or branched containing at least one double bond, but optionally containing more than one double bond.

[0086] As used herein, "alkynyl" refers to a group of 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one triple bond, but optionally containing more than one triple bond, and further optionally containing one or more double bond moieties.

[0087] As used herein, "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatom or heteroatom group. Heteroatoms and / or heteroatom groups that may replace a carbon atom include, but are not limited to, -O-, -S-, -SO-, -NRα-, -PH-, -S(O)-, -S(O)2-, -S(O)NRα-, -S(O)2NRα-, etc. (including combinations thereof), where each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0088] As used herein, "alkoxy" refers to the group -ORβ, where Rβ is an alkyl group as defined above, including optionally substituted alkyl groups also defined herein.

[0089] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group of 6 to 12 carbon atoms (inclusive) having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl, and the like.

[0090] As used herein, "amino" refers to the group -NH2. Substituted amino refers to the groups -NHR5, NR5R5 and NR5R5R5, where each R5 is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, and the like.

[0091] As used herein, "oxo" refers to =O.

[0092] As used herein, "oxy" refers to the divalent group --O-- which can have a variety of substituents to form different oxy groups, including ethers and esters.

[0093] As used herein, "carboxy" refers to --COOH.

[0094] As used herein, "carbonyl" refers to -C(O)-, which can have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0095] As used herein, "alkyloxycarbonyl" refers to -C(O)ORε, where Rε is an alkyl group, as defined herein, which can be optionally substituted.

[0096] As used herein, "aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NRδRδ, where the amino group NRδRδ is as defined herein.

[0097] As used herein, "halogen" and "halo" refer to fluoro, chloro, bromo and iodo.

[0098] As used herein, "hydroxy" refers to --OH.

[0099] As used herein, "cyano" refers to --CN.

[0100] As used herein, "heteroaryl" refers to an aromatic heterocyclic group containing from 1 to 10 carbon atoms (inclusive) and from 1 to 4 heteroatoms (inclusive) selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0101] As used herein, "heteroarylalkyl" refers to an alkyl substituted with a heteroaryl (i.e., a heteroaryl-alkyl group) preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 5 to 12 ring atoms (inclusive) in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl, and the like.

[0102] As used herein, "heteroarylalkenyl" refers to an alkenyl substituted with a heteroaryl (i.e., a heteroaryl-alkenyl- group) preferably having from 2 to 6 carbon atoms (inclusive) in the alkenyl moiety and from 5 to 12 ring atoms (inclusive) in the heteroaryl moiety.

[0103] As used herein, "heteroarylalkynyl" refers to an alkynyl substituted with a heteroaryl (i.e., a heteroaryl-alkynyl-group) preferably having from 2 to 6 carbon atoms (inclusive) in the alkynyl moiety and from 5 to 12 ring atoms (inclusive) in the heteroaryl moiety.

[0104] As used herein, "heterocycle", "heterocyclic" and, interchangeably, "heterocycloalkyl" refer to a heterocyclic ring having a single ring or multiple condensed rings and 2 to 10 carbon ring atoms (inclusive) and 1 to 4 ring atoms selected from nitrogen, sulfur or oxygen within the ring. Heterocyclic groups refer to saturated or unsaturated groups having heterocyclic ring atoms of (inclusive). Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocyclic rings include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

[0105] As used herein, "membered ring" is meant to include any cyclic structure. The number before the term "membered" indicates the number of skeletal atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are 6-membered rings, while cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0106] Unless otherwise specified, the position occupied by hydrogen in the aforementioned groups can be any of, but is not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amino, aryl, aryl, arylalkyl, arylamino ... It can be further substituted with substituents exemplified by amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that when open valences exist on these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, and when these open valences exist on carbon, they may be further substituted with halogens as well as oxygen, nitrogen or sulfur bonded substituents, and when multiple such open valences exist, these groups may be linked to form rings by direct formation of a bond or by formation of a bond to a new heteroatom, preferably oxygen, nitrogen or sulfur. It is further understood that the above substitutions may be made so long as the replacement of hydrogen by a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0107] As used herein, "optionally" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. Those of skill in the art will understand that with respect to any molecule described as containing one or more optional substituents, only sterically practical and / or synthetically feasible compounds are meant to be included.

[0108] As used herein, "optionally substituted" refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl," the "alkyl" and "aryl" portions of the molecule may be substituted or unsubstituted, and in the series "optionally substituted alkyl, cycloalkyl, aryl, and heteroaryl," the alkyl, cycloalkyl, aryl, and heteroaryl groups may be substituted or unsubstituted, independently of the others.

[0109] As used herein, a "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as desired during synthesis. Examples of protecting groups are well known in the art. Functional groups that can bear a protecting group include, but are not limited to, hydroxy, amino and carboxy groups. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitroveratryloxycarbonyl ("NVOC"), and the like. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (e.g., methyl and ethyl esters, acetate or propionate groups, or glycol esters) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups), and allyl ethers. Other protecting groups can be found in the references described herein. Engineered Carboxylesterase Polypeptides

[0110] The present invention provides engineered polypeptides with carboxylesterase activity (also referred to herein as "engineered carboxylesterase polypeptides") useful for amidation reactions. Thus, in one aspect, the present invention provides engineered polypeptides with carboxylesterase activity capable of converting substrate compound(s) into product compound(s), as shown in Table 3.1 of Example 3. Additionally, the present invention provides polynucleotides encoding the engineered polypeptides, associated vectors and host cells comprising said polynucleotides, methods for making the engineered polypeptides, and methods for using the engineered polypeptides, including suitable reaction conditions.

[0111] The engineered polypeptides of the invention are non-naturally occurring carboxylesterases engineered to have improved enzymatic properties (e.g., increased stereoselectivity) compared to the wild-type carboxylesterase polypeptide of T. fusca (GenBank Accession No. WP_011292850.1; SEQ ID NO: 2). In some embodiments, the various engineered carboxylesterase polypeptides provided herein exhibit improved enzymatic properties compared to other engineered reference carboxylesterase polypeptides provided herein. In some embodiments, the engineered polypeptides of the invention are non-naturally occurring carboxylesterases engineered to have improved enzymatic properties (e.g., increased thermostability) compared to the wild-type carboxylesterase polypeptide of G. stearothermophilus (GenBank Accession No. WP_033015113; SEQ ID NO: 138). In some further embodiments, the engineered polypeptides are engineered to have improved enzymatic properties (e.g., increased thermostability) compared to the wild-type carboxylesterase polypeptide of M. tuberculosis (GenBank Accession No. WP_003407276; SEQ ID NO: 140). It is a non-naturally occurring carboxylesterase that has been

[0112] In some embodiments, engineered carboxylesterase variants provided herein comprise a polypeptide sequence having at least 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, an engineered reference carboxylesterase (SEQ ID NO:8), or a functional fragment thereof, wherein the engineered carboxylesterase comprises at least one substitution or set of substitutions in the polypeptide sequence, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, engineered polypeptides having carboxylesterase activity include polypeptides having amino acid substitutions provided herein (see, e.g., Tables 8.1 and 11).

[0113] The present invention provides engineered carboxylesterases or functional fragments thereof comprising a polypeptide sequence having at least 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, comprising at least one substitution or set of substitutions in said polypeptide sequence, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 11 4, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372, 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 32 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of 39 / 323, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 153, 153 / 215, 164 / 271, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224 / 268 / 372, 249 / 284, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, 320 / 376 / 377, 321, 323, 324, 372, 372 / 37. 6, 373, 376, 377, 405, 420, 427, 428 and 429, where the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 39M / 323I, 62H / 117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H / 263R, 71P, 71R, 71V, 71Y, 77S / 184G, 103P, 103R ... 03R, 103T / 147S, 104P, 104Q / 429V, 105L, 107D / 185W, 107L, 107P, 107S, 108G, 108K, 108Q, 108R, 108S, 108W, 109G / 117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111V, 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F, 118G / 349V, 118I, 118N, 118N / 269T, 119G , 119P, 119S, 126C, 153H / 215P, 153L, 164R / 271T, 174D / 282V, 183P, 184F, 184G, 184P, 184S / 249T, 184Y, 185A, 185T, 186C, 186G, 186P, 186 R,186T,187P,188E,188G,190H,190K,190L,190M,190Q,190R,190W,209E,209G,209P,209S,209V,210P,210T,210W,211I,211L,211R,21 1V, 212A, 212P, 212R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q, 213R / 345G, 213S, 213T / 271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M, 2 15P, 215R, 215R / 271R, 215W, 216P, 217G, 217L, 217P, 217R, 217R / 231V, 217S, 217V, 217W, 224I / 268S / 372F, 249V / 284P, 269N, 269V, 270I,270I / 470M, 270R, 271A, 271K, 271L, 271P, 271Q / 416V, 271S, 271T, 276F, 277M, 278H, 278S, 279C, 279E, 279G, 279L / 280G / 282M, 279V, 280E, 280G, 280S, 281P, 2 81V, 281Y / 374N, 282A, 282C, 282Q, 282R, 282S, 282T, 282W, 283C, 283D, 283K, 2 83R / 429V, 283T, 283V, 283Y, 284C, 284T, 284T / 438T, 284V, 285L, 285M, 285P, 28 and 429L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of T39M / F323I, R62H / P117G, P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G, Y65S, Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W, I69Y, G70L, G70R, G70T, G70W, A71F, A71G, A71H / Q263R, A71 P, A71R, A71V, A71Y, F77S / E184G, W103P, W103R, W103T / P147S, I104P, I 104Q / A429V, H105L, G107D / S185W, G107L, G107P, G107S, A108G, A108K, A 108Q, A108R, A108S, A108W, F109G / P117M, T110A, T110H, T110P, T110S, N111L, N111M, N111R, N111S, N111V, N111W, S113P, G114A, G114H, G114Q,S115、 H、S115T、S115V、P117A、P117F、V118G / A349V、V118I、V118N、V118N / A269T、Y119G、Y119P、Y119S、R126C、R153H / N215P、R153L、W164R / W271T、G174D / L282V、G183P、E184F、E184G、E184P、E184S / A249T、E184Y、S185A、S185T、A186C、A186G、A186P、A186R、A186T、G187P、A188E、A188G、S190H、S190K、S190L、S190M、S190Q、S190R、S190W、L209E、L209G、L209P、L209S、L209V、Q210P、Q210T、Q210W、S211I、S211L、S211R、S211V、G212A、G212P、G212R、G212S、A213C、A213E、A213L、A213N、A213P、A213Q、A213R / S345G、A213S、A213T / W271K、A213V、G214K、G214L、G214T、G214V、N215K、N215M、N215P、N215R、N215R / W271R、N215W、M216P、A217G、A217L、A217P、A217R、A217R / A231V、A217S、A217V、A217W、T224I / P268S / I372F、A249V / F284P、A269N、A269V、V270I、V270I / V470M、V270R、W271A、W271K、W271L、W271P、W271Q / A416V、W271S、W271T、A276F、G277M、G278H、G278S、S279C、S279E、S279G、S279L / V280G / L282M、S279V、V280E、V280G、V280S、L281P、L281V、L281Y / D374N、L282A、L282C、L282Q、L282R、L282S、L282T、L282W、P283C、P283D、P283K、P283R / A429V、P283T、P283V、P283Y、F284C、F284T、F284T / P438T、F284V、A285L、A285M、A285P、P286V、L311I、T317C、T317P、Y320A、Y320F、Y320G、Y320G / F323S、Y320S、In some embodiments, the engineered carboxylesterase comprises a substitution selected from Y320S / F323S / I372A, Y320S / I372A / V376G, Y320S / V376G / F377V, Y320W, R321L, R321S, F323C, F323I, F323R, F323Y, L324A, I372A / V376A, I372L, T373G, V376A, V376G, V376L, V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V and A429L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the engineered carboxylesterase comprises a substitution at position 282, wherein the positions are numbered with reference to SEQ ID NO: 2. In some further embodiments, the substitution at position 282 is aliphatic, non-polar, basic, polar or aromatic. In still some further embodiments, the substitution is selected from X282T, X282G, X282A, X282V, X282M, X282C, X282W, X282Q, X282S, X282T and X282R.

[0114] The present invention provides an engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, wherein the engineered carboxylesterase or functional fragment thereof comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271、63 / 215 / 343、63 / 268 / 269 / 270 / 429、63 / 377、65 / 69 / 70 / 281 / 372、65 / 69 / 70 / 372、65 / 69 / 372、65 / 70 / 372、65 / 320、65 / 320 / 323、68、68 / 69 / 189 / 214、68 / 69 / 189 / 214 / 215、68 / 69 / 189 / 214 / 215 / 271、68 / 69 / 189 / 214 / 215 / 271 / 281 / 282 / 343 / 381、68 / 69 / 189 / 214 / 271 / 280、68 / 69 / 189 / 214 / 372、68 / 69 / 189 / 214 / 377 / 381、68 / 69 / 189 / 271、68 / 69 / 189 / 271 / 280 / 372 / 381、68 / 69 / 189 / 280 / 281 / 282 / 372 / 377、68 / 69 / 189 / 281 / 282 / 372 / 377 / 381、68 / 69 / 189 / 343 / 381、68 / 69 / 189 / 372、68 / 69 / 189 / 381、68 / 69 / 214 / 215 / 271、68 / 69 / 214 / 343、68 / 69 / 215、68 / 69 / 271、68 / 69 / 282 / 287、68 / 69 / 343 / 372、68 / 108 / 377、68 / 184、68 / 184 / 189、68 / 189 / 271 / 372、68 / 189 / 343、68 / 214 / 215 / 271 / 281 / 282 / 372、68 / 215 / 271 / 343 / 372 / 381、68 / 215 / 377、68 / 271 / 372、68 / 377、69、69 / 70、69 / 70 / 331 / 372、69 / 70 / 372、69 / 70 / 459、69 / 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 282、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320、108 / 189 / 377、108 / 215、108 / 215 / 377、108 / 269 / 270、108 / 270、108 / 282 / 285 / 377、108 / 285、108 / 320 / 323、108 / 377、126、126 / 184 / 213 / 280 / 281 / 285 / 320、126 / 184 / 213 / 372、126 / 189 / 285 / 372、126 / 215、126 / 372、181 / 215、189、189 / 214、189 / 214 / 215 / 271 / 282、189 / 215、189 / 215 / 249 / 277、189 / 215 / 271 / 281 / 282 / 377、189 / 215 / 343 / 372、189 / 270 / 285、189 / 270 / 372、189 / 280 / 282、189 / 320 / 377、189 / 343、189 / 343 / 377、189 / 372 / 377、189 / 377、189 / 381、213 / 215 / 320、214 / 215 / 271、214 / 215 / 271 / 377、214 / 271、214 / 280 / 282 / 343 / 377 / 381、215、215 / 249 / 280 / 281 / 285 / 372、215 / 271 / 372、215 / 280 / 281 / 285 / 372、215 / 281 / 285 / 372、215 / 281 / 285 / 373、215 / 281 / 373、215 / 285、215 / 285 / 317、215 / 285 / 346、215 / 285 / 445、215 / 320、215 / 320 / 372、215 / 323、215 / 372、215 / 372 / 377、215 / 373、215 / 377、215 / 381、249 / 377、269 / 270 / 281 / 372 / 377、270 / 377、 , 271, 271 / 343, 271 / 343 / 372, 271 / 343 / 372 / 381, 280 / 285 / 372, 281 / 372, 282 / 285 / 320 / 323, 285 / 323, 320, 343 / 372, 372, 372 / 377, 372 / 381, 373 and 377, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8. In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is 63A, 63A / 189A, 63A / 215R / 343V, 63R, 63R / 65G / 108G, 63R / 65G / 108G / 189L, 63R / 65G / 108G / 377I, 63R / 65G / 282A / 285L / 320W / 323I, 63R / 65G / 320W / 323I, 63R / 108G, 63R / 108G / 282A / 285L / 377 L, 63R / 108G / 285L / 377I, 63R / 108G / 320W / 323C, 63R / 377I, 63T / 215R, 63Y, 63Y / 189L, 63Y / 212P / 215R, 63Y / 212P / 215R / 268 A / 269N / 343V, 63Y / 215P / 269N, 63Y / 215R, 63Y / 215R / 270I / 271S, 63Y / 268A / 269N / 270I / 429V, 65G / 320W, 65G / 320W / 323I, 6 5W / 69L / 372L, 65W / 69M / 70A / 281P / 372L, 65W / 69W / 70L / 372L, 65W / 70L / 372M, 68P, 68P / 69L / 189E / 214R / 271Y / 280G, 68P / 6 9L / 189E / 214R / 372L, 68P / 69L / 189I / 214R / 215P / 271Y, 68P / 69L / 189I / 281P / 282C / 372L / 377Y / 381L, 68P / 69L / 189Q / 214R, 68P / 69L / 189Q / 271Y / 280G / 372L / 381L, 68P / 69L / 215P, 68P / 69L / 271Y, 68P / 69L / 282C / 287I, 68P / 69L / 343V / 372L, 68P / 69W / 189E / 214R / 215P / 271Y / 281P / 282G / 343V / 381L, 68P / 69W / 189E / 280G / 281P / 282A / 372L / 377Y, 68P / 69W / 189E / 343V / 381L,<h2 style=";text-align:left;direction:ltr">68P / 69W / 189I / 214R / 215P、68P / 69W / 189I / 214R / 377Y / 381L、68P / 69W / 18 9I / 271Y、68P / 69W / 189I / 372L、68P / 69W / 189I / 381L、68P / 69W / 214R / 215P / 271Y、68P / 69W / 214R / 343V、68P / 69W / 215P、68P / 108G / 377L、68P / 184S、6 8P / 184S / 189E、68P / 189I / 271Y / 372L、68P / 189I / 343V、68P / 214R / 215P / 27 1Y / 281P / 282A / 372L、68P / 215P / 271Y / 343V / 372L / 381L、68P / 215P / 377L、68P / 271Y / 372L、68P / 377L、69F / 108G / 270E / 372L / 377L、69F / 189L、69F / 2 15K, 69F / 215K / 269L / 270I / 377L, 69F / 215R, 69F / 285L / 373G, 69L, 69L / 70L / 331Q / 372M, 69L / 189E / 271Y / 281P / 282A, 69L / 189I, 69L / 189I / 214R / 271 Y / 281P / 282A / 343V、69L / 189I / 271Y / 343V / 381L、69L / 189I / 280G / 282G / 343V / 372L / 381L、69L / 189I / 282A、69L / 189Q / 377Y、69L / 215P / 271Y / 280G / 281P / 282C、69L / 215P / 271Y / 282A、69L / 215P / 271Y / 372L、69L / 215P / 343V / 372L / 381L、69L / 215R / 285P / 317P、69L / 271Y、69L / 271Y / 372L、69L / 282C / 343V / 372L、69L / 372L、69M / 70A / 372M、69W、69W / 70L、69W / 70L / 372M、69W / 70L / 459R、69W / 108S、69W / 189E / 214R / 343V / 372L、69W / 189E / 271Y / 343V、 69W / 189E / 372L, 69W / 189I, 69W / 189I / 215P / 343V, 69W / 189I / 271Y, 69W / 189I / 343V / 381L, 69W / 189Q / 372L / 377Y, 69W / 212A / 213L / 215R / 280G / 281P,<h2 style=";text-align:left;direction:ltr">69W / 214R / 215P / 271Y / 372L / 、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 377Y / 381L, 69W / 214R / 271Y / 282A, 69W / 214R / 271Y / 343V, 69W / 215K / 343V, 69W / 215P, 69W / 215R, 69W / 215R / 323Y, 69W / 282A, 69W / 372M, 69Y / 108G / 281P / 285P, 69Y / 110A / 215R / 281P, 69Y / 189L / 2 81P / 373G、70L、70L / 212P、108G、108G / 189I / 282A / 285L / 320W、108G / 189L、108G / 189L / 320W、108 G / 189L / 377I、108G / 215K、108G / 215P / 377L、108G / 269L / 270E、108G / 270E、108G / 282A / 285L / 377 L、108G / 285L、108G / 320W / 323I、108G / 377I、108G / 377L、126C、126C / 184S / 213S / 280G / 281P / 285 L / 320G、126C / 184S / 213S / 372L、126C / 189I / 285L / 372L、126C / 215P、126C / 372L、181L / 215P、189 E / 372L / 377Y,189I,189I / 214R / 215P / 271Y / 282G,189I / 215K,189I / 215P / 343V / 372L,189I / 215R / 249T / 277M,189I / 270E / 285L,189I / 270E / 372L,189I / 280G / 282A,189I / 320W / 377I,189I / 343V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> , 189I / 377I, 189L, 189Q, 189Q / 214R, 189Q / 215P / 271Y / 281P / 282C / 377Y, 189Q / 343V, 189Q / 343V / 377Y, 189Q / 381L, 213 S / 215P / 320G, 214R / 215P / 271Y, 214R / 215P / 271Y / 377Y, 214R / 271Y, 214R / 280G / 282A / 343V / 377Y / 381L, 215K, 215K / 281 P / 285L / 372L, 215K / 281P / 373G, 215K / 285L / 317P, 215K / 285L / 445L, 215K / 323Y, 215K / 372L, 215K / 372L / 377L, 215K / 37 3G, 215P, 215P / 271Y / 372L, 215P / 320G, 215P / 320G / 372L, 215P / 372L, 215P / 372L / 377L, 215P / 377L, 215P / 381L, 215R, 21 5R / 249T / 280G / 281P / 285L / 372L, 215R / 280G / 281P / 285L / 372L, 215R / 281P / 285L / 373G, 215R / 285P, 215R / 320G, 215R / 3 72L, 215W, 215W / 285L / 346S, 215W / 285P, 215W / 373G, 249T / 377L, 269L / 270E / 281P / 372L / 377L, 270E / 377L, 271Y, 271Y / 3 and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8.In some further embodiments, at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of P63A, P63A / M189A, P63A / N215R / A343V, P63R, P63R / Y65G / A108G, P63R / Y65G / A108G / M189L, P63R / Y65G / A108G / F377I, P63R / Y65G / T282A / A285L / Y320W / F323I, P63R / Y65G ... 3R / A108G, P63R / A108G / T282A / A285L / F377L, P63R / A108G / A285L / F377I, P63R / A108G / Y320W / F323C, P63R / F377I, P63T / N 215R, P63Y, P63Y / M189L, P63Y / G212P / N215R, P63Y / G212P / N215R / P268A / A269N / A343V, P63Y / N215P / A269N, P63Y / N215R,. P63Y / N215R / V270I / W271S、P63Y / P268A / A269N / V270I / A429V、Y65G / Y320W、Y65G / Y320W / F323I、Y65W / I69L / I372L、Y65W / I69M / G70A / L281P / I372L、Y65W / I69W / G70L / I372L、Y65W / G70L / I372M、A68P、A68P / I69L / M189E / G214R / W271Y / V280G、A68P / I69L / M189E / G214R / I372L、A68P / I69L / M189I / G214R / N215P / W271Y、A68P / I69L / M189I / L281P / T282C / I372L / F377Y / A381L、A68P / I69L / M189Q / G214R、A68P / I69L / M189Q / W271Y / V280G / I372L / A381L、A68P / I69L / N215P、A68P / I69L / W271Y、A68P / I69L / T282C / V287I、A68P / I69L / A343V / I372L、A68P / I69W / M189E / G214R / N215P / W271Y / L281P / T282G / A343V / A381L、A68P / I69W / M189E / V280G / L281P / T282A / I372L / F377Y、A68P / I69W / M189E / A343V / A381L、A68P / I69W / M189I / G214R / N215P、A68P / I69W / M189I / G214R / F377Y / A381L、A68P / I69W / M189I / W271Y、A68P / I69W / M189I / I372L、A68P / I69W / M189I / A381L、A68P / I69W / G214R / N215P / W271Y、A68P / I69W / G214R / A343V、A68P / I69W / N215P、A68P / A108G / F377L、A68P / E184S、A68P / E184S / M189E、A68P / M189I / W271Y / I372L、A68P / M189I / A343V、A68P / G214R / N215P / W271Y / L281P / T282A / I372L、A68P / N215P / W271Y / A343V / I372L / A381L、A68P / N215P / F377L、A68P / W271Y / I372L、A68P / F377L、<h2 style=";text-align:left;direction:ltr">I69F / A108G / V270E / I372L / F377L, I69F / M189L, I69F / N215K, I69F / N215K / A269L / V270I / F377L, I69F / N215R, I69F / A285L / T373G, I69L, I69L / G70L / P3 31Q / I372M、I69L / M189E / W271Y / L281P / T282A、I69L / M189I、I69L / M189I / G214R / W271Y / L281P / T282A / A343V、I69L / M189I / W271Y / A343V / A381L、I69L / M189I / V280G / T282G / A343V / I372L / A381L、I69L / M189I / T282A、I69L / M189Q / F377Y、I69L / N215P / W271Y / V280G / L281P / T282C、I69L / N215P / W271Y / T28 2A、I69L / N215P / W271Y / I372L、I69L / N215P / A343V / I372L / A381L、I69L / N215R / A285P / T317P、I69L / W271Y、I69L / W271Y / I372L、I69L / T282C / A343V / I3 72L、I69L / I372L、I69M / G70A / I372M、I69W、I69W / G70L、I69W / G70L / I372M、 I69W / G70L / G459R、I69W / A108S、I69W / M189E / G214R / A343V / I372L、I69W / M1 89E / W271Y / A343V、I69W / M189E / I372L、I69W / M189I、I69W / M189I / N215P / A 343V、I69W / M189I / W271Y、I69W / M189I / A343V / A381L、I69W / M189Q / I372L / F 377Y, I69W / G212A / A213L / N215R / V280G / L281P, I69W / G214R / N215P / W271Y / I372L / F377Y / A381L, I69W / G214R / W271Y / T282A, I69W / G214R / W271Y / A343 V、I69W / N215K / A343V、I69W / N215P、I69W / N215R、I69W / N215R / F323Y、I69W / T282A、I69W / I372M、I69Y / A108G / L281P / A285P、I69Y / T110A / N215R / L281P、I69Y / M189L / L281P / T373G、 <h2 style=";text-align:left;direction:ltr">、G70L、G70L / G212P、A108G、A108G / M189I / T282A / A285L / Y320W、A108G / M18 9L、A108G / M189L / Y320W、A108G / M189L / F377I、A108G / N215K、A108G / N215P / F377L, A108G / A269L / V270E, A108G / V270E, A108G / T282A, A285L / F377L, A108G / A285L, A108G / Y320W / F323I, A108G / F377I, A108G / F377L, R126C, R12 6C / E184S / A213S / V280G / L281P / A285L / Y320G、R126C / E184S / A213S / I372L 、R126C / M189I / A285L / I372L、R126C / N215P、R126C / I372L、V181L / N215P、M 189E / I372L / F377Y、M189I、M189I / G214R / N215P / W271Y / T282G、M189I / N215K、M189I / N215P / A343V / I372L、M189I / N215R / A249T / G277M、M189I / V270E / A285L, M189I / V270E / I372L, M189I / V280G / T282A, M189I / Y320W / F377I, M189I / A343V, M189I / F377I, M189L, M189Q, M189Q / G214R, M189Q / N215P / W271 Y / L281P / T282C / F377Y、M189Q / A343V、M189Q / A343V / F377Y、M189Q / A381L、A213S / N215P / Y320G、G214R / N215P / W271Y、G214R / N215P / W271Y / F377Y、G21 4R / W271Y、G214R / V280G / T282A / A343V / F377Y / A381L、N215K、N215K / L281P / A285L / I372L、N215K / L281P / T373G、N215K / A285L / T317P、N215K / A285L / V 445L, N215K / F323Y, N215K / I372L, N215K / I372L / F377L, N215K / T373G, N215P, N215P / W271Y / I372L, N215P / Y320G, N215P / Y320G / I372L, N215P / I372LN215P / I372L / F377L, N215P / F377L, N215P / A381L, N215R, N215R / A249T / V280 G / L281P / A285L / I372L, N215R / V280G / L281P / A285L / I372L, N215R / L281P / A28 5L / T373G, N215R / A285P, N215R / Y320G, N215R / I372L, N215W, N215W / A285L / G3 46S, N215W / A285P, N215W / T373G, A249T / F377L, A269L / V270E / L281P / I372L / F 377L, V270E / F377L, W271Y, W271Y / A343V, W271Y / A343V / I372L, W271Y / A343V / I372L / A381L, V280G / A285L / I372L, L281P / I372L, T282A / A285L / Y320W / F323I, A285L / F323I, Y320W, A343V / I372L, I372L, I372L / F377L, I372L / A381L, I372M, T373G and F377L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0115] In some embodiments, the invention also provides engineered carboxylesterase polypeptides, including fragments of any of the engineered carboxylesterase polypeptides described herein that retain functional carboxylesterase activity and / or improved properties of that engineered carboxylesterase polypeptide. Thus, in some embodiments, the invention provides polypeptide fragments that have carboxylesterase activity (e.g., are capable of converting a substrate to a product under appropriate reaction conditions) and that comprise at least about 80%, 90%, 95%, 98%, or 99% of the full-length amino acid sequence of an engineered polypeptide of the invention, such as the exemplary engineered polypeptides having the even-numbered sequence identifiers of SEQ ID NOs:2-136.

[0116] In some embodiments, the engineered carboxylesterase polypeptides of the invention , an amino acid sequence that includes at least one deletion, addition, and / or substitution compared to an engineered carboxylesterase polypeptide sequence described herein, e.g., any one of the exemplary engineered polypeptide sequences having even-numbered sequence identifiers SEQ ID NOs:2-136. Thus, for each and every embodiment of the engineered carboxylesterase polypeptides of the invention, the amino acid sequence can include deletions, additions, and / or substitutions of one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the carboxylesterase polypeptide, wherein the relevant functional activity and / or improved properties of the engineered carboxylesterases described herein are maintained. In some embodiments, the deletions, additions and / or substitutions may include additions and / or substitutions of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55 or 1 to 60 amino acid residues. In some embodiments, the number of deletions, additions and / or substitutions may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55 or 60 amino acid residues. In some embodiments the deletions, additions and / or substitutions may include deletions, additions and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25 or 30 amino acid residues.

[0117] In some embodiments, the invention provides engineered carboxylesterase polypeptides having an amino acid sequence that includes an insertion compared to an engineered carboxylesterase polypeptide sequence described herein, e.g., any one of the exemplary engineered polypeptide sequences having even-numbered sequence identifiers SEQ ID NOs:2-136. Thus, for each and every embodiment of the carboxylesterase polypeptides of the invention, the insertion can include one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, wherein the relevant functional activity and / or improved properties of the engineered carboxylesterases described herein are maintained. The insertion can be into the amino or carboxy terminus or an internal portion of the carboxylesterase polypeptide.

[0118] In some embodiments, the polypeptides of the invention are in the form of fusion polypeptides in which the engineered polypeptide is fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitopes), purification sequences (e.g., His tags for binding to metals), and cellular localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein may be used regardless of fusion to other polypeptides.

[0119] The engineered carboxylesterase polypeptides described herein are not limited to genetically encoded amino acids. Thus, in addition to genetically encoded amino acids, the polypeptides described herein can be composed in whole or in part of naturally occurring and / or synthetic non-encoded amino acids. Specific commonly encountered non-encoded amino acids that may be constructed include, but are not limited to, the D-stereoisomers of the genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cyclohexylglycine); Alanine (Cha);Norleucine (Nle);Naphthylalanine (Nal);2-Chlorophenylalanine (Ocf);3-Chlorophenylalanine (Mcf);4-Chlorophenylalanine (Pcf);2-Fluorophenylalanine (Off);3-Fluorophenylalanine (Mff);4-Fluorophenylalanine (Pff);2-Bromophenylalanine (Obf);3-Bromophenylalanine (Mbf);4-Bromophenylalanine (Pbf);2-Methylphenylalanine (Omf);3-Methylphenylalanine (Mmf);4 -Methylphenylalanine (Pmf);2-Nitrophenylalanine (Onf);3-Nitrophenylalanine (Mnf);4-Nitrophenylalanine (Pnf);2-Cyanophenylalanine (Ocf);3-Cyanophenylalanine (Mcf);4-Cyanophenylalanine (Pcf);2-Trifluoromethylphenylalanine (Otf);3-Trifluoromethylphenylalanine (Mtf);4-Trifluoromethylphenylalanine (Ptf);4-Aminophenylalanine (Paf);4-Iodophenylalanine (Pif);4-Aminophenylalanine (Pif); Aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); Pyrid-2-ylalanine (2pAla); Pyrid-3-ylalanine (3pAla); Pyrid-4-ylalanine (4pAla); Naphth-1-ylalanine (1nAla); Naphth-2-ylalanine (2nAla); Thiazolylalanine (taAla); Benzothienylalanine (bAla);Thienylalanine (tAla); Furylalanine (fAla); Homophenylalanine (hPhe); Homotyrosine (hTyr); Homotryptophan (hTrp); Pentafluorophenylalanine (5ff); Styrylkalanine (sAla); Authrylalanine (aAla); 3,3-Diphenylalanine (Dfa); 3-Amino-5-phenylpentanoic acid (Afp); Penicillamine (Pen); 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-Thienylalanine (Thi); Methionine sulfoxide (Mso); N(w)-Nitroarginine (nArg); Homolysine (hLys); Phosphonomethylphenylalanine (pmPhe); Phosphopserine (pSer); Phosphothreonine Homoaspartic acid (hAsp); homoglutamic acid (hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid (PA), azetidine-3-carboxylic acid (ACA), 1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine ​​(hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that may constitute the polypeptides described herein will be apparent to those skilled in the art. These amino acids may be in either the L- or D-configuration;

[0120] Those skilled in the art will recognize that amino acids or residues bearing side chain protecting groups may also be included in the polypeptides described herein. In this regard, non-limiting examples of such protected amino acids that belong to the aromatic category (protecting groups listed in parentheses) include, but are not limited to, Arg(tos), Cys(methylbenzyl), Cys(nitropyridine sulfenyl), ), Glu(δ-benzyl ester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0121] Structurally constrained non-coded amino acids that may comprise the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

[0122] As will be apparent to one of skill in the art, the aforementioned residue positions and specific amino acid residues at each residue position may be used individually or in various combinations to synthesize carboxylesterase polypeptides with desired improved properties including, inter alia, enzymatic activity, substrate / product preference, stereoselectivity, substrate / product tolerance, and stability under various conditions such as high temperature, solvents and / or pH.

[0123] The engineered carboxylesterase polypeptides of the invention, generated by directed evolution of SEQ ID NO:2 for efficient amidation of a substrate of interest to a product of interest under certain industrially relevant conditions, have one or more residue differences compared to a reference carboxylesterase polypeptide. These residue differences are associated with improvements in various enzyme properties, particularly increased activity, increased solvent tolerance, and reduced toxicity to host cells (e.g., E. coli). In some further embodiments, the mutant carboxylesterases also exhibited increased stereoselectivity, increased stability, and tolerance to increased substrate and / or product concentrations (e.g., reduced product inhibition). Thus, in some embodiments, an engineered polypeptide having carboxylesterase activity can convert substrate compound(s) into product(s) with at least about a 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more increased activity compared to the activity of a wild-type T. fusca carboxylesterase polypeptide (e.g., SEQ ID NO: 2) under appropriate reaction conditions. In some embodiments, an engineered polypeptide with carboxylesterase activity can convert a substrate to a product with a conversion of at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or 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%, or at least about 99% in a reaction time of about 48 hours, about 36 hours, about 24 hours, or even less, under appropriate reaction conditions. In some embodiments, an engineered polypeptide with carboxylesterase activity can convert a substrate to a product with a diastereomeric excess of at least 90%, 95%, 97%, 98%, 99% or more, under appropriate reaction conditions.

[0124] In some embodiments, an engineered polypeptide having carboxylesterase activity is capable of converting a substrate to a product under appropriate reaction conditions with increased tolerance to the presence of a substrate compared to the substrate tolerance of a reference polypeptide (e.g., SEQ ID NO: 2). Thus, in some embodiments, an engineered polypeptide is capable of converting a substrate to a product under appropriate reaction conditions with at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 94%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% conversion in about 72 hours, about 48 hours, about 36 hours, about 24 hours, or even shorter periods of reaction time. The enzyme can convert substrate to product in the presence of substrate loading concentrations of about 1 g / L, 5 g / L, 10 g / L, 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 70 g / L, about 75 g / L, or about 100 g / L.

[0125] Some suitable reaction conditions, with respect to polypeptide concentration or amount, substrate, buffer, co-solvent, pH and / or conditions (including temperature and reaction time), under which the above-mentioned improved properties of the engineered polypeptide may be determined, are provided herein. In some embodiments, suitable reaction conditions include the assay conditions described below and in the Examples.

[0126] In some embodiments, the engineered polypeptides may be provided on a solid support, such as a membrane, resin, solid carrier or other solid phase material. The solid support is composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide, as well as copolymers and grafts thereof. The solid support may also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or metal, such as gold or platinum. The shape of the solid support may be in the form of beads, spheres, particles, granules, gels, membranes or surfaces. The surfaces may be planar, substantially planar or non-planar. The solid support may be porous or non-porous, and may have swelling or non-swelling properties. The solid support may be formed in the form of wells, recesses or other containers, vessels, features or locations.

[0127] In some embodiments, engineered polypeptides having carboxylesterase activity are bound or immobilized on a solid support such that they retain at least some of their improved properties compared to a reference polypeptide (e.g., SEQ ID NO:2). In such embodiments, the immobilized polypeptides may facilitate biocatalytic conversion of substrate compounds to desired products, and are easily retained after completion of the reaction (e.g., by retaining the beads to which the polypeptide is immobilized) and then reused or recycled in subsequent reactions. Such immobilized enzyme processes allow for further efficiencies and cost reductions. Thus, it is further contemplated that any of the methods using the engineered carboxylesterase polypeptides of the invention may be performed using the same carboxylesterase polypeptides bound or immobilized on a solid support.

[0128] The engineered carboxylesterase polypeptides can be non-covalently or covalently bound. A variety of methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art. Other methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (see, e.g., Yi et al., Proc. Biochem., 42:895-898

[2007] ; Martin et al., Appl. Microbiol. Biotechnol., 76:843-851

[2007] ; Koszelewski et al., J. Mol. Cat. B: Enz., 63:39-44

[2010] ; Truppo et al., Org. Proc. Res. Develop., published online:dx.doi.org / 10.1021 / op200157c; and Mateo et al., Biotechnol. Prog., 18:629-34

[2002] , etc.). Useful solid supports for immobilizing the engineered carboxylesterase polypeptides of the invention include, but are not limited to, beads or resins comprising polymethacrylates with epoxide functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers, or polymethacrylates with octadecyl functional groups. Exemplary solid supports useful for immobilizing the engineered carboxylesterases of the invention include, but are not limited to, chitosan beads including the following different types of SEPABEADs: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120, Eupergit C, and SEPABEADs (Mitsubishi). can be.

[0129] In some embodiments, the engineered carboxylesterase polypeptides can be provided in the form of an array in which the polypeptides are arranged at different positional locations. In some embodiments, the different positional locations are wells in a solid support, such as a 96-well plate. Multiple supports can be arranged on the array at different locations that can be addressed by robotic delivery of reagents or by detection methods and / or instruments. Such arrays can be used to test different substrate compounds for conversion by the polypeptides.

[0130] In some embodiments, the engineered polypeptides described herein may be provided in the form of a kit. The polypeptides in the kit may be present individually or as multiple polypeptides. The kit may further include reagents for performing the enzymatic reaction, substrates for assessing the activity of the polypeptide, and reagents for detecting the product. The kit may also include a reagent dispenser and instructions for use of the kit. In some embodiments, the kit of the invention includes an array comprising multiple different engineered carboxylesterase polypeptides at different addressable locations, the different polypeptides being different variants of a reference sequence, each having at least one different improved enzymatic property. Such arrays comprising multiple engineered polypeptides and methods for their use are known (see, e.g., WO 2009 / 008908). Polynucleotides encoding engineered carboxylesterases

[0131] In another aspect, the invention provides a polynucleotide encoding an engineered carboxylesterase enzyme. The polynucleotide may be operably linked to one or more heterologous regulatory sequences that control gene expression to generate a recombinant polynucleotide capable of expressing said polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered carboxylesterase may be introduced into a suitable host cell to express the corresponding carboxylesterase polypeptide.

[0132] With knowledge of the codons corresponding to the various amino acids, the availability of the protein sequence provides a description of all polynucleotides that can code for the subject. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for a very large number of nucleic acids to be created, all of which code for the improved carboxylesterase enzymes disclosed herein. Thus, once a particular amino acid sequence is identified, one of skill in the art may create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that can be created by selecting combinations based on possible codon choices, and all such variations should be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequence shown in the table of examples. In various embodiments, the codons are preferably selected to be compatible with the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express genes in bacteria; preferred codons used in yeast are used for expression in yeast, and preferred codons used in mammals are used for expression in mammalian cells. As an example, the polynucleotide of SEQ ID NO:1 has been codon-optimized for expression in E. coli, but otherwise encodes the naturally occurring carboxylesterase of T. fusca.

[0133] In some embodiments, the polynucleotide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, In one embodiment, the present invention encodes an engineered carboxylesterase polypeptide comprising an amino acid sequence selected from the group consisting of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, or 136.

[0134] In some embodiments, a polynucleotide encoding an engineered carboxylesterase or functional fragment thereof is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0135] In some embodiments, the polynucleotide is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0136] In some embodiments, the polynucleotide encodes a polypeptide as described herein, but has about 80% or more sequence identity, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered carboxylesterase. In some embodiments, the reference polynucleotide comprises SEQ ID NO:1, and in some other embodiments, the reference polynucleotide comprises SEQ ID NO:137. In some further embodiments, the reference polynucleotide sequence comprises SEQ ID NO:139. In some further embodiments, the engineered carboxylesterase sequence comprises a sequence that includes positions identified as beneficial, as described in the Examples.

[0137] The present invention also provides polynucleotide sequences encoding at least one engineered carboxylesterase provided herein. In some embodiments, the polynucleotide sequence encodes at least one engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 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, wherein the engineered carboxylesterase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the polynucleotide sequence is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 118 / 269, 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372, 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, 320 / 376 / 377, 321, 323, 324, 345, 349, 372, 372 / 376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some further embodiments, the polynucleotide sequence is 39 / 323, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 11 8 / 269, 118 / 349, 119, 126, 153, 153 / 215, 164 / 271, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224 / 268 / 3 72, 249 / 284, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, and encoding at least one engineered carboxylesterase comprising at least one substitution or set of substitutions selected from 320 / 376 / 377, 321, 323, 324, 372, 372 / 376, 373, 376, 377, 405, 420, 427, 428 and 429, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.In some further embodiments, the polynucleotide sequence is 39M / 323I, 62H / 117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H / 263R, 71P, 71R, 71V, 71Y, 77S / 18 4G, 103P, 103R, 103T / 147S, 104P, 104Q / 429V, 105L, 107D / 185W, 107L, 107P, 107S, 108G, 108K, 108Q, 108R, 108S, 108W, 10 9G / 117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111V, 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117 F, 118G / 349V, 118I, 118N, 118N / 269T, 119G, 119P, 119S, 126C, 153H / 215P, 153L, 164R / 271T, 174D / 282V, 183P, 184F, 184 G, 184P, 184S / 249T, 184Y, 185A, 185T, 186C, 186G, 186P, 186R, 186T, 187P, 188E, 188G, 190H, 190K, 190L, 190M, 190Q, 190R , 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T, 210W, 211I, 211L, 211R, 211V, 212A, 212P, 212R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q, 213R / 345G, 213S, 213T / 271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M, 215P, 215R, 215R / 271R, 215W. 216P, 217G, 217L, 217P, 217R, 217R / 231V, 217S, 217V, 217W, 224I / 268S / 372F, 249V / 284P, 269N, 2 69V, 270I, 270I / 470M, 270R, 271A, 271K, 271L, 271P, 271Q / 416V, 271S, 271T, 276F, 277M, 278H, 27 8S, 279C, 279E, 279G, 279L / 280G / 282M, 279V, 280E, 280G, 280S, 281P, 281V, 281Y / 374N, 282A, 282 C, 282Q, 282R, 282S, 282T, 282W, 283C, 283D, 283K, 283R / 429V, 283T, 283V, 283Y, 284C, 284T, 284T / 438T, 284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F, 320G, 320G / 323S, 320S, 320S / 3 23S / 372A, 320S / 372A / 376G, 320S / 376G / 377V, 320W, 321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372 and / or 376A, 372L, 373G, 376A, 376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V and 429L, wherein the amino acid positions are numbered with reference to SEQ ID NO:2. In some further embodiments, the polynucleotide sequence is selected from the group consisting of T39M / F323I, R62H / P117G, P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G, Y65S, Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W, I69Y , G70L, G70R, G70T, G70W, A71F, A71G, A71H / Q263R, A71P, A71R, A71V, A71Y, F77S / E184G, W103P, W103R, W1 03T / P147S, I104P, I104Q / A429V, H105L, G107D / S185W, G107L, G107P, G107S, A108G, A108K, A108Q, A108R,A108S、A108W、F109G / P117M、T110A、T110H、T110P、T110S、N111L、N111M、N111R、N111S、N111V、N111W、S113P、G114A、G114H、G114Q、S115H、S115T、S115V、P117A、P117F、V118G / A349V、V118I、V118N、V118N / A269T、Y119G、Y119P、Y119S、R126C、R153H / N215P、R153L、W164R / W271T、G174D / L282V、G183P、E184F、E184G、E184P、E184S / A249T、E184Y、S185A、S185T、A186C、A186G、A186P、A186R、A186T、G187P、A188E、A188G、S190H、S190K、S190L、S190M、S190Q、S190R、S190W、L209E、L209G、L209P、L209S、L209V、Q210P、Q210T、Q210W、S211I、S211L、S211R、S211V、G212A、G212P、G212R、G212S、A213C、A213E、A213L、A213N、A213P、A213Q、A213R / S345G、A213S、A213T / W271K、A213V、G214K、G214L、G214T、G214V、N215K、N215M、N215P、N215R、N215R / W271R、N215W、M216P、A217G、A217L、A217P、A217R、A217R / A231V、A217S、A217V、A217W、T224I / P268S / I372F、A249V / F284P、A269N、A269V、V270I、V270I / V470M、V270R、W271A、W271K、W271L、W271P、W271Q / A416V、W271S、W271T、A276F、G277M、G278H、G278S、S279C、S279E、S279G、S279L / V280G / L282M、S279V、V280E、V280G、V280S、L281P、L281V、L281Y / D374N、L282A、L282C、L282Q、L282R、L282S、L282T、L282W、P283C、P283D、P283K、P283R / A429V、P283T、P283V、P283Y、F284C、F284、 T, F284T / P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P, Y320A, Y320F, Y320G, Y320G / F323S, Y32 0S, Y320S / F323S / I372A, Y320S / I372A / V376G, Y320S / V376G / F377V, Y320W, R321L, R321S, F323C, F323I, F323R, F3 In some embodiments, the polynucleotide sequence encodes at least one engineered carboxylesterase comprising at least one substitution or set of substitutions selected from 23Y, L324A, I372A / V376A, I372L, T373G, V376A, V376G, V376L, V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V and A429L, where the amino acids are numbered with reference to SEQ ID NO: 2. In some embodiments, the polynucleotide sequence encodes at least one engineered carboxylesterase comprising a substitution at position 282, where the positions are numbered with reference to SEQ ID NO: 2. In some further embodiments, the substitution at position 282 is aliphatic, non-polar, basic, polar or aromatic. In still some further embodiments, the substitutions are selected from X282T, X282G, X282A, X282V, X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

[0138] The present invention also provides a polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in its polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the polynucleotide sequence is 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 2 12 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372, 65 / 320, 65 / 320 / 323, 68, 68 / 69 / 189 / 214, 68 / 69 / 189 / 214 / 215, 68 / 69 / 189 / 214 / 215 / 271, 68 / 69 / 189 / 214 / 215 / 271 / 281 / 282 / 343 / 381, 68 / 69 / 189 / 214 / 271 / 280, 68 / 69 / 189 / 214 / 372, 68 / 69 / 189 / 214 / 377 / 381, 68 / 69 / 189 / 271, 68 / 69 / 189 / 271 / 280 / 372 / 381, 68 / 69 / 189 / 280 / 281 / 282 / 372 / 377, 68 / 69 / 189 / 281 / 282 / 372 / 377 / 381, 68 / 69 / 189 / 343 / 381, 68 / 69 / 189 / 372, 68 / 69 / 189 / 381, 68 / 69 / 214 / 215 / 271, 68 / 69 / 214 / 343, 68 / 69 / 215, 68 / 69 / 271, 68 / 69 / 282 / 287, 68 / 69 / 343 / 372,68 / 108 / 377、68 / 184、68 / 184 / 189、68 / 189 / 271 / 372、68 / 189 / 343、68 / 214 / 215 / 271 / 281 / 282 / 372、68 / 215 / 271 / 343 / 372 / 381、68 / 215 / 377、68 / 271 / 372、68 / 377、69、69 / 70、69 / 70 / 331 / 372、69 / 70 / 372、69 / 70 / 459、69 / 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 2、 82、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320、108 / 189 / 377、108 / 215、108 / 215 / 377、108 / 269 / 270、108 / 270、108 / 282 / 285 / 377、108 / 285、108 / 320 / 323、108 / 377、126、126 / 184 / 213 / 280 / 281 / 285 / 320、126 / 184 / 213 / 372、126 / 189 / 285 / 372、126 / 215、126 / 372、181 / 215、189、189 / 214、189 / 214 / 215 / 271 / 282、189 / 215、189 / 215 / 249 / 277、189 / 215 / 271 / 281 / 282 / 377、189 / 215 / 343 / 372、189 / 270 / 285、189 / 270 / 372、189 / 280 / 282、189 / 320 / 377、189 / 343、189 / 343 / 377、189 / 372 / 377、189 / 377、189 / 381、213 / 215 / 320、214 / 215 / 271、214 / 215 / 271 / 377、214 / 271、214 / 280 / 282 / 343 / 377 / 381、215、215 / 249 / 280 / 281 / 285 / 372、215 / 271 / 372、215 / 280 / 281 / 285 / 372、215 / 281 / 285 / 372、215 / 281 / 285 / 373, 215 / 281 / 373, 215 / 285, 215 / 285 / 317, 215 / 285 / 346, 215 / 285 / 445, 215 / 320, 215 / 320 / 372, 215 / 323, 215 / 372, 215 / 372 / 377, 215 / 373, 215 / 377, 215 / 381, 249 / 377, 269 / 270 / 281 / 372 / 377, 270 / 377, 271, 271 / 343, 27 and 377, wherein the amino acid positions are numbered with reference to SEQ ID NO:8. In some further embodiments, the polynucleotide sequence is 63A, 63A / 189A, 63A / 215R / 343V, 63R, 63R / 65G / 108G, 63R / 65G / 108G / 189L, 63R / 65G / 108G / 377I, 63R / 65G / 282A / 285L / 320W / 323I, 63R / 65G / 320W / 323I, 63R / 108G, 63R / 108G / 282A / 285L / 377L, 63R / 108G / 285L / 377I, 63R / 108G / 320W / 32 3C, 63R / 377I, 63T / 215R, 63Y, 63Y / 189L, 63Y / 212P / 215R, 63Y / 212P / 215R / 268A / 269N / 34 3V, 63Y / 215P / 269N, 63Y / 215R, 63Y / 215R / 270I / 271S, 63Y / 268A / 269N / 270I / 429V, 65G / 3 20W, 65G / 320W / 323I, 65W / 69L / 372L, 65W / 69M / 70A / 281P / 372L, 65W / 69W / 70L / 372L, 65W / 70L / 372M, 68P, 68P / 69L / 189E / 214R / 271Y / 280G, 68P / 69L / 189E / 214R / 372L, 68P / 69L / 18 9I / 214R / 215P / 271Y, 68P / 69L / 189I / 281P / 282C / 372L / 377Y / 381L, 68P / 69L / 189Q / 214R,68P / 69L / 189Q / 271Y / 280G / 372L、 <h2 style=";text-align:left;direction:ltr"> / 381L、68P / 69L / 215P、68P / 69L / 271Y、68P / 69L / 282C / 287I、68P / 69L / 343V / 372L、68P / 69W / 189E / 214R / 215P / 271Y / 281P / 282G / 343V / 381L、68P / 69W / 189E / 280G / 281P / 282A / 372L / 377Y、68P / 69W / 189E / 343V / 381L、68P / 69W / 189I / 214R / 215P、68P / 69W / 189I / 214R / 377Y / 381L、68P / 69W / 189I / 271Y、6 8P / 69W / 189I / 372L、68P / 69W / 189I / 381L、68P / 69W / 214R / 215P / 271Y、68P / 69W / 214R / 343V、68P / 69W / 215P、68P / 108G / 377L、68P / 184S、68P / 184S / 189 E、68P / 189I / 271Y / 372L、68P / 189I / 343V、68P / 214R / 215P / 271Y / 281P / 282A / 372L、68P / 215P / 271Y / 343V / 372L / 381L、68P / 215P / 377L、68P / 271Y / 372 L, 68P / 377L, 69F / 108G / 270E / 372L / 377L, 69F / 189L, 69F / 215K, 69F / 215K / 269L / 270I / 377L, 69F / 215R, 69F / 285L / 373G, 69L, 69L / 70L / 331Q / 372M, 69 L / 189E / 271Y / 281P / 282A、69L / 189I、69L / 189I / 214R / 271Y / 281P / 282A / 343V、69L / 189I / 271Y / 343V / 381L、69L / 189I / 280G / 282G / 343V / 372L / 381L、6 9L / 189I / 282A、69L / 189Q / 377Y、69L / 215P / 271Y / 280G / 281P / 282C、69L / 215P / 271Y / 282A、69L / 215P / 271Y / 372L、69L / 215P / 343V / 372L / 381L、69L / 21 5R / 285P / 317P、69L / 271Y、69L / 271Y / 372L、69L / 282C / 343V / 372L、69L / 372L、69M / 70A / 372M、69W、69W / 70L、69W / 70L / 372M、69W / 70L / 459R、69W / 108S、69W / 189E / 214R / 343V / 372L、69W / 189E / 271Y / 343V、69W / 189E / 372L、69W / 189I、69W / 189I / 215P / 343V、69W / 189I / 271Y、69W / 189I / 343V / 381L、69W / 189Q / 372L / 377Y、69W / 212A / 213L / 215R / 280G / 281P、69W / 214R / 215P / 271Y / 372L / 377Y / 381L、69W / 214R / 271Y / 282A、69W / 214R / 271Y / 343V、69W / 215K / 343V、69W / 215P、69W / 215R、69W / 215R / 323Y、69W / 282A、69W / 372M、69Y / 108G / 281P / 285P、69Y / 110A / 215R / 281P、69Y / 189L / 281P / 373G、70L、70L / 212P、108G、108G / 189I / 282A / 285L / 320W、108G / 189L、108G / 189L / 320W、108G / 189L / 377I、108G / 215K、108G / 215P / 377L、108G / 269L / 270E、108G / 270E、108G / 282A / 285L / 377L、108G / 285L、108G / 320W / 323I、108G / 377I、108G / 377L、126C、126C / 184S / 213S / 280G / 281P / 285L / 320G、126C / 184S / 213S / 372L、126C / 189I / 285L / 372L、126C / 215P、126C / 372L、181L / 215P、189E / 372L / 377Y、189I、189I / 214R / 215P / 271Y / 282G、189I / 215K、189I / 215P / 343V / 372L、189I / 215R / 249T / 277M、189I / 270E / 285L、189I / 270E / 372L、189I / 280G / 282A、189、 I / 320W / 377I、189I / 343V、189I / 377I、189L、189 Q, 189Q / 214R, 189Q / 215P / 271Y / 281P / 282C / 377Y, 189Q / 343V, 189Q / 343V / 377Y, 189Q / 381L, 213S / 215P / 320G, 214R / 215 P / 271Y, 214R / 215P / 271Y / 377Y, 214R / 271Y, 214R / 280G / 282A / 343V / 377Y / 381L, 215K, 215K / 281P / 285L / 372L, 215K / 281P / 373G, 215K / 285L / 317P, 215K / 285L / 445L, 215K / 323Y, 215K / 372L, 215K / 372L / 377L, 215K / 373G, 215P, 215P / 271Y / 372L , 215P / 320G, 215P / 320G / 372L, 215P / 372L, 215P / 372L / 377L, 215P / 377L, 215P / 381L, 215R, 215R / 249T / 280G / 281P / 285L / 372L, 215R / 280G / 281P / 285L / 372L, 215R / 281P / 285L / 373G, 215R / 285P, 215R / 320G, 215R / 372L, 215W, 215W / 285L / 346S, 215W / 285P, 215W / 373G, 249T / 377L, 269L / 270E / 281P / 372L / 377L, 270E / 377L, 271Y, 271Y / 343V, 271Y / 343V / 372L, 271Y / 3 and 377L, wherein the amino acid positions are numbered with reference to SEQ ID NO:8. In some further embodiments, the polynucleotide sequence is P63A, P63A / M189A, P63A / N215R / A343V, P63R, P63R / Y65G / A108G, P63R / Y65G / A108G / M189L, P63R / Y65G / A108G / F377I, P63R / Y65G / T282A / A285L / Y320W / F323I, P63R / Y65G / Y320W / F323I, P63R / A108G,P63R / A108G / T282A / A285L / F377L、P63R / A108G / A285L / F377I、P63R / A108G / Y320W / F323C、P63R / F377I、P63T / N215R、P63Y、P63Y / M189L、P63Y / G212P / N215R、P63Y / G212P / N215R / P268A / A269N / A343V、P63Y / N215P / A269N、P63Y / N215R、P63Y / N215R / V270I / W271S、P63Y / P268A / A269N / V270I / A429V、Y65G / Y320W、Y65G / Y320W / F323I、Y65W / I69L / I372L、Y65W / I69M / G70A / L281P / I372L、Y65W / I69W / G70L / I372L、Y65W / G70L / I372M、A68P、A68P / I69L / M189E / G214R / W271Y / V280G、A68P / I69L / M189E / G214R / I372L、A68P / I69L / M189I / G214R / N215P / W271Y、A68P / I69L / M189I / L281P / T282C / I372L / F377Y / A381L、A68P / I69L / M189Q / G214R、A68P / I69L / M189Q / W271Y / V280G / I372L / A381L、A68P / I69L / N215P、A68P / I69L / W271Y、A68P / I69L / T282C / V287I、A68P / I69L / A343V / I372L、A68P / I69W / M189E / G214R / N215P / W271Y / L281P / T282G / A343V / A381L、A68P / I69W / M189E / V280G / L281P / T282A / I372L / F377Y、A68P / I69W / M189E / A343V / A381L、A68P / I69W / M189I / G214R / N215P、A68P / I69W / M189I / G214R / F377Y / A381L、A68P / I69W / M189I / W271Y、A68P / I69W / M189I / I372L、A68P / I69W / M189I / A381L、A68P / I69W / G214R / N215P / W271Y、A68P / I69W / G214R / A343V、A68P / I69W / N215P、A68P / A108G / F377L、A68P / E184S、A68P / E184S、 <h2 style=";text-align:left;direction:ltr"> / M189E、A68P / M189I / W271Y / I372L、A68P / M189I / A343V、A68P / G214R / N215 P / W271Y / L281P / T282A / I372L、A68P / N215P / W271Y / A343V / I372L / A381L、A 68P / N215P / F377L、A68P / W271Y / I372L、A68P / F377L、I69F / A108G / V270E / I 372L / F377L、I69F / M189L、I69F / N215K、I69F / N215K / A269L / V270I / F377L、I 69F / N215R, I69F / A285L / T373G, I69L, I69L / G70L / P331Q / I372M, I69L / M189E / W271Y / L281P / T282A, I69L / M189I, I69L / M189I / G214R / W271Y / L281P / T2 82A / A343V、I69L / M189I / W271Y / A343V / A381L、I69L / M189I / V280G / T282G / A343V / I372L / A381L、I69L / M189I / T282A、I69L / M189Q / F377Y、I69L / N215P / W271Y / V280G / L281P / T282C、I69L / N215P / W271Y / T282A、I69L / N215P / W271Y / I372L、I69L / N215P / A343V / I372L / A381L、I69L / N215R / A285P / T317P、I69 L / W271Y、I69L / W271Y / I372L、I69L / T282C / A343V / I372L、I69L / I372L、I69M / G70A / I372M、I69W、I69W / G70L、I69W / G70L / I372M、I69W / G70L / G459R、I69 W / A108S、I69W / M189E / G214R / A343V / I372L、I69W / M189E / W271Y / A343V、I6 9W / M189E / I372L、I69W / M189I、I69W / M189I / N215P / A343V、I69W / M189I / W27 1Y、I69W / M189I / A343V / A381L、I69W / M189Q / I372L / F377Y、I69W / G212A / A213L / N215R / V280G / L281P、I69W / G214R / N215P / W271Y / I372L / F377Y / A381L、<h2 style=";text-align:left;direction:ltr">I69W / G214R / W271Y / T282A、I69W / G214R / W271Y / A343V、I69W / N215K / A343 V、I69W / N215P、I69W / N215R、I69W / N215R / F323Y、I69W / T282A、I69W / I372M 、I69Y / A108G / L281P / A285P、I69Y / T110A / N215R / L281P、I69Y / M189L / L281 P / T373G、G70L、G70L / G212P、A108G、A108G / M189I / T282A / A285L / Y320W、A1 08G / M189L、A108G / M189L / Y320W、A108G / M189L / F377I、A108G / N215K、A10 8G / N215P / F377L、A108G / A269L / V270E、A108G / V270E、A108G / T282A / A285L / F377L、A108G / A285L、A108G / Y320W / F323I、A108G / F377I、A108G / F377L、R 126C、R126C / E184S / A213S / V280G / L281P / A285L / Y320G、R126C / E184S / A21 3S / I372L,R126C / M189I / A285L / I372L,R126C / N215P,R126C / I372L,V181L / N215P,M189E / I372L / F377Y,M189I,M189I / G214R / N215P / W271Y / T282G M189I / N215K, M189I / N215P / A343V / I372L, M189I / N215R / A249T / G277M, M189I / V270E / A285L, M189I / V270E / I372L, M189I / V280G / T282A, M189I / Y320 W / F377I、M189I / A343V、M189I / F377I、M189L、M189Q、M189Q / G214R、M189Q / N215P / W271Y / L281P / T282C / F377Y、M189Q / A343V、M189Q / A343V / F377Y、M1 89Q / A381L, A213S / N215P / Y320G, G214R / N215P / W271Y, G214R / N215P / W271Y / F377Y, G214R / W271Y, G214R / V280G / T282A / A343V / F377Y / A381L, N215K,N215K / L281P / , A285L / I372L, N215K / L281P / T373G, N215K / A285L / T317P, N215K / A285L / V445L, N215K / F323Y, N215K / I372L, N 215K / I372L / F377L, N215K / T373G, N215P, N215P / W271Y / I372L, N215P / Y320G, N215P / Y320G / I372L, N215P / I37 2L, N215P / I372L / F377L, N215P / F377L, N215P / A381L, N215R, N215R / A249T / V280G / L281P / A285L / I372L, N215 R / V280G / L281P / A285L / I372L, N215R / L281P / A285L / T373G, N215R / A285P, N215R / Y320G, N215R / I372L, N215W, N215W / A285L / G346S, N215W / A285P, N215W / T373G, A249T / F377L, A269L / V270E / L281P / I372L / F377L, V270E / F 377L, W271Y, W271Y / A343V, W271Y / A343V / I372L, W271Y / A343V / I372L / A381L, V280G / A285L / I372L, L281P / I37 2L, T282A / A285L / Y320W / F323I, A285L / F323I, Y320W, A343V / I372L, I372L, I372L / F377L, I372L / A381L, I372M, T373G and F377L, and the amino acid positions are numbered with reference to SEQ ID NO:8.

[0139] An isolated polynucleotide encoding an improved carboxylesterase polypeptide may be manipulated in a variety of ways to provide for expression of the polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide prior to insertion into the vector may be desirable or necessary. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0140] In bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the invention include promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA, 1999). 75:3727-3731

[1978] ), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25

[1983] ). Further suitable promoters will be known to those of skill in the art.

[0141] In filamentous fungal host cells, suitable promoters for directing transcription of the nucleic acid constructs of the invention include those derived from Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like proteinase Examples of such promoters include the promoter obtained from the gene for Aspergillus niger neutral α-amylase (WO 96 / 00787), and the NA2-tpi promoter (a hybrid of a promoter derived from the gene for Aspergillus niger neutral α-amylase and a promoter derived from the gene for Aspergillus oryzae triosephosphate isomerase), as well as mutant promoters, truncated promoters and hybrid promoters thereof.

[0142] In yeast hosts, useful promoters include, but are not limited to, Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces Examples of useful promoters for yeast host cells include promoters derived from the genes for Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) and Saccharomyces cerevisiae 3-phosphoglycerate kinase, as well as other useful promoters for yeast host cells (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0143] The control sequence may also be a suitable transcription terminator sequence (a sequence recognized by a host cell to terminate transcription). The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0144] For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0145] Exemplary terminators for yeast host cells can be those obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase, as well as other useful terminators for yeast host cells known in the art (see, for example, Romanos et al., supra).

[0146] The control sequence may also be a suitable leader sequence, which is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. An exemplary leader for filamentous fungal host cells is the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase genes. Suitable leaders for yeast host cells are obtained from the genes for 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).

[0147] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of a nucleic acid sequence which is recognized by a host cell during transcription as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. An exemplary polyadenylation sequence for a filamentous fungal host cell is the Aspergillus oryzae TAKA amylase polyadenylation sequence. , Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase genes, as well as additional useful polyadenylation sequences for yeast host cells known in the art (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0148] The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide, which directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may naturally contain a signal peptide coding region that is naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region may be required when the coding sequence does not naturally contain a signal peptide coding region.

[0149] Alternatively, to enhance secretion of the polypeptide, the foreign signal peptide coding region may simply replace the native signal peptide coding region, However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

[0150] Useful signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA, as well as additional signal peptides known in the art (see, e.g., Simonen et al., Microbiol. Rev., 57:109-137

[1993] ).

[0151] Useful signal peptide coding regions for filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells can be those from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase, as well as additional useful signal peptide coding regions (see, e.g., Romanos et al., 1992, supra).

[0152] The control sequence may also be a propeptide coding region, which codes for an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or in some cases, a zymogen). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions include those encoding Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase. The gene for this enzyme can be obtained from WO 95 / 33836.

[0153] When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region.

[0154] It may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that turn the expression of the gene on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include the lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include, by way of example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA α-amylase promoter, the Aspergillus niger glucoamylase promoter and the Aspergillus oryzae glucoamylase promoter.

[0155] Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the carboxylesterase polypeptide of the invention is operably linked to the regulatory sequence.

[0156] Thus, in some embodiments, the present invention is also directed to recombinant expression vectors that include a polynucleotide encoding an engineered carboxylesterase polypeptide or variant thereof, and one or more expression control regions, such as promoters and terminators, an origin of replication, and the like, depending on the type of host into which it will be introduced. The various nucleic acids and control sequences can be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a polypeptide at such sites. Alternatively, the nucleic acid sequences of the present invention can be expressed by inserting the nucleic acid sequence, or a nucleic acid construct comprising the sequence, into an appropriate vector for expression. In making an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

[0157] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause the expression of polynucleotide sequence.The selection of vector typically depends on the compatibility of the vector with the host cell into which the vector is introduced.The vector can be a linear or closed circular plasmid.

[0158] The expression vector can be an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity) whose replication is independent of chromosomal replication (e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector can contain any means for ensuring self-replication. Alternatively, the vector can be one that is integrated into the genome when introduced into a host cell and replicates together with the chromosome(s) into which it is integrated. Furthermore, one vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell or into a transposon can be used.

[0159] The expression vectors of the invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

[0160] 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. Embodiments for use in Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.

[0161] The expression vector of the present invention may contain an element(s) that allows the vector to be integrated into the genome of the host cell or to autonomously replicate in the cell, independent of the genome. For integration into the genome of the host cell, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or non-homologous recombination.

[0162] Alternatively, the expression vector may contain additional nucleic acid sequences that direct integration into the genome of the host cell by homologous recombination. The additional nucleic acid sequences allow the vector to be integrated into the genome of the host cell at a precise location(s) of the chromosome(s). To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids that are highly homologous to the corresponding target sequence to enhance the probability of homologous recombination, such as preferably 100-10,000 base pairs, preferably 400-10,000 base pairs, and most preferably 800-10,000 base pairs. The integration element may be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integration element may be a non-coding or coding nucleic acid sequence. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0163] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. A non-limiting example of a bacterial origin of replication is P15A. ori or the origin of replication of plasmids pBR322, pUC19, pACYC177 (plasmids with P15A ori), or pACYC184 which allows replication in E. coli and pUB110, pE194 or pTA1060 which allows replication in Bacillus. Examples of replication origins for use in yeast host cells are the 2 micron replication origin, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The replication origin may be one which has a mutation that makes it functional temperature sensitive in the host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433

[1978] ).

[0164] To increase the production of the gene product, more than one copy of the nucleic acid sequence of the present invention can be inserted into the host cell. The increase in copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including an amplifiable selectable marker gene together with the nucleic acid sequence, in which case the cells containing the amplified copy of the selectable marker gene and thus the additional copy of the nucleic acid sequence can be selected by culturing the cells in the presence of an appropriate selectable agent.

[0165] Many expression vectors for use in the present invention are commercially available. Suitable commercially available expression vectors include, but are not limited to, the p3xFLAG™ expression vector (Sigma-Aldrich), which contains a CMV promoter, an hGH polyadenylation site for expression in mammalian host cells, a pBR322 origin of replication, and an ampicillin resistance marker for amplification in E. coli. Other commercially available suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV vectors (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (see, for example, Lathe et al., Gene 57:193-201

[1987] ).

[0166] Those skilled in the art will recognize that during enzyme production, post-translational modifications may occur, particularly depending on the cell line used and the specific amino acid sequence of the enzyme.For example, such post-translational modifications may include cleavage of specific leader sequences, addition of various sugar moieties with different glycosylation and phosphorylation patterns, deamidation, oxidation, disulfide bond scrambling, isomerization, C-terminal lysine clipping and N-terminal glutamine cyclization.The present invention encompasses the use of engineered carboxylesterase enzymes that have been subjected to or have been subjected to one or more post-translational modifications.Thus, the engineered carboxylesterases of the present invention include those that have been subjected to, for example, the post-translational modifications described herein.

[0167] Deamidation is an enzymatic reaction that primarily converts asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in a ratio of approximately 3:1. This deamidation reaction therefore involves the isomerization of aspartate (D) to isoaspartate. Both the deamidation of asparagine and the isomerization of aspartate involve the intermediate succinimide. To a much lesser extent, deamidation can occur at glutamine residues as well.

[0168] Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and results in the covalent modification of proteins induced directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation occurs primarily at methionine residues, but can also occur at tryptophan and free cysteine ​​residues.

[0169] Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can be broken or formed incorrectly resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0170] The N-terminal glutamine (Q) and glutamate (E) in engineered carboxylesterases can form pyroglutamate (pGlu) via cyclization. Although most pGlu formation occurs during production, it can also be formed nonenzymatically, depending on the pH and temperature of processing and storage conditions.

[0171] C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in enzymes. A variation of this process involves the removal of lysines from enzymes derived from recombinant host cells.

[0172] In the present invention, the above-mentioned post-translational modifications and changes in the primary amino acid sequence do not result in a significant change in the activity of the engineered carboxylesterase enzyme. Host Cells for Expression of Carboxylesterase Polypeptides

[0173] In another aspect, the invention provides a host cell comprising a polynucleotide encoding an improved carboxylesterase polypeptide of the invention, operably linked to one or more control sequences for expressing the carboxylesterase enzyme in the host cell. Host cells for use in expressing the carboxylesterase polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells, such as E. coli, Geobacillus stearothermophilus, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus minor, Mycobacterium tuberculosis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells, such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells, such as CHO cells, COS cells, BHK cells, 293 cells and Bowes melanoma cells; and plant cells. Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0174] Polynucleotides for expression of carboxylesterases can be introduced into cells by a variety of methods known in the art, including electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. A variety of methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0175] E. coli W3110 is the host strain used in the present invention, but the present invention is not limited to this particular host strain. An expression vector was generated by operably linking the polynucleotide encoding the improved carboxylesterase to plasmid pCK110900, which is operably linked to a lac promoter under the control of the lacI repressor. The expression vector also contained a P15a origin of replication and a chloramphenicol resistance gene. Cells containing the polynucleotide of interest in E. coli W3110 can be isolated by subjecting the cells to chloramphenicol selection. Methods for Producing Engineered Carboxylesterase Polypeptides.

[0176] In some embodiments, to generate the improved carboxylesterase polynucleotides and polypeptides of the invention, the naturally occurring carboxylesterase enzyme that catalyzes the amidation reaction is obtained (or derived) from T. fusca. In some embodiments, the parent polynucleotide sequence is codon-optimized to enhance the expression of the carboxylesterase in a particular host cell. By way of illustration, a parent polynucleotide sequence encoding a wild-type carboxylesterase polypeptide of T. fusca was constructed from oligonucleotides prepared based on the known polypeptide sequence of the T. fusca carboxylesterase sequence available in the Genbank database (Genbank accession number WP_011292850.1). The parent polynucleotide sequence designated as SEQ ID NO: 1 was codon-optimized for expression in E. coli, and the codon-optimized polynucleotide was cloned into an expression vector, placing the expression of the carboxylesterase gene under the control of the lac promoter and lacI repressor gene. Clones expressing active carboxylesterase in E. coli were identified, and the genes were sequenced to confirm their identity. The codon-optimized polynucleotide sequence designated as SEQ ID NO:1 was the parent sequence utilized as the starting point for most of the experiments and library construction of engineered carboxylesterases evolved from the original wild-type carboxylesterase.

[0177] In other embodiments, to generate the improved carboxylesterase polynucleotides and polypeptides of the invention, the naturally occurring carboxylesterase enzyme that catalyzes the amidation reaction is obtained (or derived) from G. stearothermophilus. In some embodiments, the parent polynucleotide sequence is codon-optimized to enhance expression of the carboxylesterase in a particular host cell. By way of illustration, a parent polynucleotide sequence encoding a wild-type carboxylesterase polypeptide of G. stearothermophilus was constructed from oligonucleotides prepared based on the known polypeptide sequence of the G. stearothermophilus carboxylesterase sequence available in the Genbank database (Genbank accession number WP_033015113). The parent polynucleotide sequence designated as SEQ ID NO: 137 was codon-optimized for expression in E. coli, and the codon-optimized polynucleotide was cloned into an expression vector, placing expression of the carboxylesterase gene under the control of the lac promoter and lacI repressor gene. Clones expressing active carboxylesterase in E. coli were identified, and the genes were sequenced to confirm their identity. The polynucleotide sequence designated as SEQ ID NO:137 was the parent sequence utilized as the starting point for most of the experiments and library construction of engineered carboxylesterases evolved from the original wild-type carboxylesterase.

[0178] In some embodiments, to generate the improved carboxylesterase polynucleotides and polypeptides of the invention, the naturally occurring carboxylesterase enzyme catalyzing the amidation reaction was obtained or derived from M. tuberculosis. In some embodiments, the parent polynucleotide sequence was codon-optimized to enhance the expression of the carboxylesterase in a particular host cell. By way of illustration, a parent polynucleotide sequence encoding a wild-type carboxylesterase polypeptide of M. tuberculosis was constructed from oligonucleotides prepared based on the known polypeptide sequence of the M. tuberculosis carboxylesterase sequence available in the Genbank database (Genbank accession number WP_003407276). The parent polynucleotide sequence designated as SEQ ID NO: 139 was codon-optimized for expression in E. coli, and the codon-optimized polynucleotide was cloned into an expression vector, placing the expression of the carboxylesterase gene under the control of the lac promoter and lacI repressor gene. Clones expressing active carboxylesterase in E. coli were identified, and the genes were sequenced to confirm their identity. The codon-optimized polynucleotide sequence designated as SEQ ID NO: 139 was the parent sequence utilized as the starting point for most of the experiments and library construction of engineered carboxylesterases evolved from the original wild-type carboxylesterase as described herein.

[0179] In some embodiments, the engineered carboxylesterase is obtained by subjecting a polynucleotide encoding a naturally occurring carboxylesterase or a codon-optimized version of a polynucleotide encoding a naturally occurring carboxylesterase to mutagenesis and / or directed evolution methods, as described above. Mutagenesis can be performed according to any of the techniques known in the art, including random mutagenesis and site-directed mutagenesis. Directed evolution can be performed using any of the techniques known in the art for screening for improved promoter variants, including shuffling. Mutagenesis and directed evolution methods are well known in the art (see, for example, 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, U.S. Pat. Nos. 5,821, ... No. 6,132,970, U.S. Patent No. 6,165,793, U.S. Patent No. 6,180,406, U.S. Patent No. 6,251,674, U.S. Patent No. 6,265,201, U.S. Patent No. 6,277,638, U.S. Patent No. 6,287,861, U.S. Patent No. 6,287,862, U.S. Patent No. 6,291,242, U.S. Patent No. 6,297,053, U.S. Patent No. 6,303,344, U.S. Patent No. 6,309,883, U.S. Patent No. 6,319,713, U.S. Patent No. 6,319,714, U.S. Patent No. 6,323,030, U.S. Patent No. 6,326,20 4, U.S. Patent No. 6,335,160, U.S. Patent No. 6,335,198, U.S. Patent No. 6,344,356, U.S. Patent No. 6,352,859, U.S. Patent No. 6,355,484, U.S. Patent No. 6,358,740, U.S. Patent No. 6,358,742, U.S. Patent No. 6,365,377, U.S. Patent No. 6,365,408, U.S. Patent No. 6,368,861, U.S. Patent No. 6,372,497, U.S. Patent No. 6,337,186, U.S. Patent No. 6,376,246, U.S. Patent No. 6,379,964, U.S. Patent No. 6,387,702, U.S. Patent No. 6, 391,552, U.S. Patent No. 6,391,640, U.S. Patent No. 6,395,547, U.S. Patent No. 6,406,855, U.S. Patent No. 6,406,910, U.S. Patent No. 6,413,745, U.S. Patent No. 6,413,774, U.S. Patent No. 6,420,175, U.S. Patent No. 6,423,542, U.S. Patent No. 6,426,224, U.S. Patent No. 6,436,675, U.S. Patent No. 6,444,468, U.S. Patent No. 6,455,253, U.S. Patent No. 6,479,652, U.S. Patent No. 6,482,647, U.S. Patent No. 6,483,011, U.S. Patent No. US Patent No. 6,484,105, US Patent No. 6,489,146, US Patent No. 6,500,617, US Patent No. 6,500,639, US Patent No. 6,506,602, US Patent No. 6,506,603, US Patent No. 6,518,065, US Patent No. 6,519,065, US Patent No. 6,521,453, US Patent No. 6,528,311, US Patent No. 6,537,746, US Patent No. 6,573,098, US Patent No. 6,576,467, US Patent No. 6,579,678, US Patent No. 6,586,182, US Patent No. 6,602,986, U.S. Patent No. 6,605,430, U.S. Patent No. 6,613,514, U.S. Patent No. 6,653,072, U.S. Patent No. 6,686,515, U.S. Patent No. 6,703,240, U.S. Patent No. 6,716,631, U.S. Patent No. 6,825,001, U.S. Patent No. 6,902,922, U.S. Patent No. 6,917,882, U.S. Patent No. 6,946,296, U.S. Patent No. 6,961,664, U.S. Patent No. 6,995,017, U.S. Patent No. 7,024,312, U.S. Patent No. 7,058,515, U.S. Patent No. 7,105,297, U.S. Patent No. US Patent No. 7,148,054, US Patent No. 7,220,566, US Patent No. 7,288,375, US Patent No. 7,384,387, US Patent No. 7,421,347, US Patent No. 7,430,477, US Patent No. 7,462,469, US Patent No. 7,534,564, US Patent No. 7,620,500, US Patent No. 7,620,502, US Patent No. 7,629,170, US Patent No. 7,702,464, US Patent No. 7,747,391, US Patent No. 7,747,393, US Patent No. 7,751,986, US Patent No. 7, Nos. 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,13 No. 8, U.S. Pat. No. 8,108,150, U.S. Pat. No. 8,170,806, U.S. Pat. No. 8,224,580, U.S. Pat. No. 8,377,681, U.S. Pat. No. 8,383,346, U.S. Pat. No. 8,457,903, U.S. Pat. No. 8,504,498, U.S. Pat. No. 8,589,085, U.S. Pat. No. 8,762,066, U.S. Pat. No. 8,768,871, U.S. Pat. No. 9,593,326, U.S. Pat. No. 9,665,694, U.S. Pat. No. 9,684,771 and all related non-U.S. counterparts; Ling et al., Anal. Biochem.,254(2):157-78

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[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and WO 2009 / 152336). It is not intended that the present invention be limited to any particular method, as a variety of methods are used in the art.

[0180] In some embodiments where the desired improved enzyme property is thermostability, the enzyme activity may be measured after subjecting the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after heat treatment. Clones containing the polynucleotide encoding the carboxylesterase are then isolated and sequenced to identify nucleotide sequence changes, if any, and used to express the enzyme in a host cell.

[0181] If the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then linked (e.g., by enzymatic or chemical ligation or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present invention can be prepared by chemical synthesis (typically performed by automated synthesis methods, for example, using the classical phosphoramidite method described in Beaucage et al., Tet. Lett., 22:1859-69

[1981] , or the method described in Matthes et al., EMBO J., 3:801-05

[1984] ). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into a suitable vector. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources (e.g., The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA, etc.).

[0182] The engineered carboxylesterase enzyme expressed in the host cells can be recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation and chromatography, among others. A suitable solution for lysis and highly efficient extraction of proteins from bacteria such as E. coli is commercially available under the trade name CelLytic B (Sigma-Aldrich).

[0183] As a chromatographic technique for isolating carboxylesterase polypeptides These include, among others, reverse phase chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to one of skill in the art.

[0184] In some embodiments, affinity techniques are used to isolate improved carboxylesterase enzymes. For affinity chromatography purification, any antibody that specifically binds to the carboxylesterase polypeptide may be used. For production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with the carboxylesterase. The carboxylesterase polypeptide may be attached to a suitable carrier, such as BSA, by a side chain functional group or by a linker attached to a side chain functional group. To increase the immunological response, depending on the host species, various carriers may be used, including but not limited to Freund's (complete and incomplete), inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and carboxylesterases such as BCG (Bacillus Calmette Guerin) and Corynebacterium A variety of adjuvants may be used, including potentially useful human adjuvants such as parvum.

[0185] The carboxylesterase may be prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. The carboxylesterase may be prepared as a lyophilizate, in the form of a powder (e.g., an acetone powder), or as an enzyme solution. In some embodiments, the carboxylesterase may be in the form of a substantially pure preparation.

[0186] In some embodiments, the carboxylesterase polypeptides may be attached to a solid substrate. The substrate may be a solid phase, a surface and / or a membrane. The solid support is composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide, as well as copolymers and grafts thereof. The solid support may also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or metal, such as gold or platinum. The shape of the substrate may be in the form of beads, spheres, particles, granules, gels, membranes or surfaces. The surfaces may be planar, substantially planar or non-planar. The solid support may be porous or non-porous and may have swelling or non-swelling properties. The solid support may be formed in the form of a well, recess or other container, vessel, feature or location. A plurality of supports may be arranged in an array, at various locations that can be addressed by robotic delivery of reagents or by detection methods and / or instruments. Methods of using engineered carboxylesterase enzymes and compounds prepared therewith - Patents.com

[0187] Whole cells transformed with a gene(s) encoding an engineered carboxylesterase enzyme and / or an optional cofactor regenerating enzyme, or cell extracts and / or lysates thereof, may be used in a variety of forms, including solid (e.g., freeze-dried, spray-dried, etc.) or semi-solid (e.g., a crude paste).

[0188] Cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.) followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before lyophilization. Any cell preparation may be stabilized by cross-linking, for example using known cross-linking agents such as glutaraldehyde, or fixation to a solid phase (e.g., Eupergit C, etc.).

[0189] Solid reactants (e.g., enzymes, salts, etc.) may be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray dried, etc.), solutions, emulsions, suspensions, etc. Reactants may be readily lyophilized or spray dried using methods and equipment known to those of skill in the art. For example, a small volume of protein solution may be frozen at -80°C and then added to a pre-freezing drying chamber, followed by application of a vacuum. After removal of water from the sample, the temperature is typically raised to 4°C for 2 hours before releasing the vacuum and recovering the lyophilized sample.

[0190] The amount of reactants used in the amidation reaction generally varies depending on the amount of desired product and the amount of carboxylesterase substrate used concomitantly. The following guidelines may be used to determine the amount of carboxylesterase and / or amine. In general, the ester and amine substrates may be used at a concentration of about 5 to 200 grams / liter, using about 50 mg to about 5 g of carboxylesterase.

[0191] Those skilled in the art will readily understand how to vary these amounts to adapt them to desired productivity levels and production scales.

[0192] The order of addition of the reactants is not important. The reactants can be added together at the same time to the solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or alternatively, some of the reactants can be added separately and some can be added together at different times. For example, the carboxylesterase and the carboxylesterase substrate can be added to the solvent first.

[0193] To improve mixing efficiency when using an aqueous co-solvent system, the carboxylesterase and amine may be added to the aqueous phase and mixed first. The organic phase may then be added and mixed, followed by the addition of the carboxylesterase substrate. Alternatively, the carboxylesterase substrate may be premixed in the organic phase prior to addition to the aqueous phase.

[0194] Suitable conditions for carrying out the carboxylesterase-catalyzed amidation reactions described herein include a wide variety of conditions that can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered carboxylesterase enzyme and substrate at the experimental pH and temperature, and detecting the product using, for example, the methods described in the Examples provided herein.

[0195] Carboxylesterase-catalyzed amidation is typically carried out at a temperature ranging from about 15° C. to about 75° C. In some embodiments, the reaction is carried out at a temperature ranging from about 20° C. to about 55° C. In yet other embodiments, it is carried out at a temperature ranging from about 20° C. to about 45° C. The reaction can also be carried out under ambient conditions.

[0196] The amidation reaction is generally allowed to proceed until the coupling of the substrate is essentially complete or nearly complete. The amide formation (product) can be monitored using known methods by detecting the substrate and / or product. Suitable methods include, but are not limited to, gas chromatography, HPLC, and the like. The conversion yield of the amide product produced in the reaction mixture is generally greater than about 50%, can be greater than about 60%, can be greater than about 70%, can be greater than about 80%, can be greater than 90%, and often is greater than about 97%. EXAMPLES

[0197] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting.

[0198] In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); R T (room temperature); MWD (multiple wavelength detector); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); RP-HPLC (reverse-phase high performance liquid chromatography); FIOPC (fold improvement over positive control); HTP (high throughput); LB (Luria Broth); TFA (trifluoroacetic acid); MeCN (acetonitrile); TEoA (triethanolamine); THF (tetrahydrofuran); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Millipore (Millipore, Corp., Billerica Difco(Difco Laboratories,BD Diagnostic Systems,Detroit,MI);Daicel(Daicel,West Chester,PA);Genetix(Genetix USA,Inc.,Beaverton,OR);Molecular Devices(Molecular Devices,LLC,Sunnyvale,CA);Applied Biosystems(Applied Biosystems,part of Life Technologies,Corp.,Grand Island, NY); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA); Corning (Corning, Inc.Bio-Rad (Bio-Rad Laboratories, Hercules, CA); Phenomenex (Phenomenex, Inc., Torrence, CA); Epicentre (Epicentre, Madison, WI). . Example 1 Obtaining wild-type carboxylesterase genes and constructing expression vectors

[0199] This example describes obtaining codon-optimized recombinant polynucleotides encoding wild-type carboxylesterases (SEQ ID NOs: 2, 138 and 140) from which the genes encoding the engineered carboxylesterases in the following examples were derived, as well as suitable expression vectors and host cells for such engineering.

[0200] Codon-optimized versions of wild-type genes (SEQ ID NO: 1, 137 and 139) encoding wild-type carboxylesterases (SEQ ID NO: 2, 138 and 140) of T. fusca, G. stearothermophilus and M. tuberculosis, respectively, were synthesized for expression in E. coli. The codon-optimized genes were cloned into expression vector pCK110900 under the control of the lac promoter (see, e.g., U.S. Pat. No. 9,714,437 and U.S. Patent Application Publication No. 2006 / 0195947, both of which are incorporated by reference in their entirety for all purposes). The expression vectors also contained a P15a origin of replication and a chloramphenicol resistance gene. These sequence-verified vectors were transformed into E. coli strain W3110 for expression. Polynucleotides (odd numbered SEQ ID NOs: 1-135) encoding the engineered carboxylesterases of the invention (even numbered SEQ ID NOs: 2-136) were also cloned into the vector pCK110900 for expression in derivatives of the E. coli W3110 strain. Directed evolution techniques commonly known to those skilled in the art were used to generate a library of engineered carboxylesterases. Example 2 HTP production and analysis of wild-type carboxylesterase polypeptides

[0201] HTP lysates were prepared by using the codon-optimized carboxylesterase gene (described in Example 1), which was then transformed into E. coli W3110 and plated on Luria-Bertani Broth (LB) agar containing 1% glucose and 30 μg / mL chloramphenicol (CAM). After at least 16 hours of incubation at 30°C, colonies were picked using a Q-botobotic colony picker (Genetix) into 96-well shallow-well microtiter plates containing 200 μL of LB, 1% glucose and 30 μg / mL CAM. Cells were grown for 18-20 hours at 30°C with shaking at 200 rpm. 20 μL of these cultures were then transferred to 360 μL of Terrific Broth (TB) and 30 μg / mL CAM in deep-well plates. After 2.5 h incubation at 30 °C with shaking at 250 rpm (OD600 0.6-0.8), recombinant gene expression was induced by adding isopropyl thioglycoside (IPTG) to a final concentration of 1 mM. The plates were then incubated for 18-21 h at 30 °C with shaking at 250 rpm.

[0202] The cell culture was pelleted at 3500×g for 20 min and the medium was discarded. The cell pellet was dissolved in 200 μL of 0.2 M TEoA, pH 7.5, containing 1 g / L lysozyme, 0.5 g / L polymyxin B sulfate and 0.5 μL OmniCleave™ endonuclease (Epicentre) by shaking at room temperature for 2 h. The sample was centrifuged at 3500×g for 20 min to clear the cell debris and the supernatant was used to carry out the transformation described in Examples 3 and 4. Example 3 Activity of wild-type carboxylesterases on substrate set #1

[0203] Wild-type carboxylesterase polypeptides were produced as described in Example 2. To analyze the amidation activity towards substrate set #1 (Table 3.1), 20 μL of the supernatant was added to a mixture of 10 μL aniline, 10 μL ethyl acetate, and 100 μL 0.1 M sodium phosphate buffer, pH 7.0. The reactions were incubated at 20° C. and shaken at 300 rpm for 18 hours. The samples were quenched by diluting 2-fold with MeCN. Analysis of the reactions was performed by RP-HPLC as described in Example 12.1. [Table 3] Example 4 Activity of wild-type carboxylesterases toward substrate sets #2-7

[0204] Wild-type carboxylesterase polypeptides were produced according to the method described in Example 2. To analyze the amidation activity towards substrate set #2 (see Table 3.1), 100 μL of the supernatant was added to a mixture of 10 μL of methyl phenylacetate, 10 μL of n-butylamine, 10 μL of dimethylsulfoxide (DMSO) and 20 μL of 0.1 M sodium phosphate buffer, pH 7.0. The reaction was incubated at room temperature and shaken at 300 rpm for 18 hours. The sample was quenched by dilution with an equal volume of MeCN. Substrate and product analysis was performed by RP-HPLC using the method described in Example 12.1.

[0205] To analyze the amidation activity for substrate sets #3-7 (see Table 3.1), 120 μL of a mixture of 75-100 g / L amine and 15 g / L ethyl benzoate in 0.1 M potassium phosphate buffer, pH 7.5, with or without 50% isopropanol, was added to 100 μL of supernatant prepared as described in Example 2. The mixture was incubated at 55°C and shaken at 400 rpm for 18 hours. Samples were quenched by 3-fold dilution with MeCN and then centrifuged at 4000×g for 5-8 minutes. The resulting supernatants were analyzed by RP-HPLC using the method described in Example 12.2. In one experiment using Substrate Set #3, 120 μL of a mixture of 5 g / L isobutylamine and 15 g / L ethyl benzoate in 0.1 M potassium phosphate buffer, pH 7.5, was added to 100 μL of the supernatant made as described in Example 2. The results for all of these substrate sets are provided in Table 4.1, with the substrate sets indicated by the "reaction" number (i.e., "R1", "R2", "R3", etc.). [Table 4] Example 5 Preparation of wild-type carboxylesterase shake flask powder

[0206] Wild-type carboxylesterases (SEQ ID NOs: 2, 138 and 140) were produced in shake flasks for further characterization. E. coli transformants containing the plasmid encoding the WT carboxylesterase were grown on Luria-Bertani Broth (LB) agar medium containing 1% glucose and 30 μg / mL chloramphenicol (CAM). After incubation at 30 °C for at least 16 h, single colonies were picked into 6 mL of LB, 1% glucose and 30 μg / mL CAM. Cells were grown at 30 °C for 18-20 h with shaking at 250 rpm. The culture was then cultured at 37 °C for 18-20 h until a final OD of 0.2 was reached. 600 and transferred to Terrific Broth (TB) and 30 μg / mL CAM for a final volume of 250 mL. The flasks were incubated at 25° C. or 30° C. with shaking at 250 rpm for 2.5 h (OD 600Expression of the recombinant genes was induced with isopropyl thioglycoside (IPTG) to a final concentration of 1 mM (pH 0.6-0.8). The flasks were then incubated at 30 °C for 18-21 h with shaking at 250 rpm. The cells were pelleted at 3500 × g for 20 min and the supernatant was discarded. The cell pellet was washed three times with 30 mL of ice-cold 50 mM sodium phosphate pH 7.5, resuspended in 12 mL of the same buffer and lysed using a cell disrupter at 18-20 kpsi. The lysate was clarified at 10000 × g for 30 min and the clarified supernatant was lyophilized to an off-white powder. [Table 5] Example 6 Evaluation of the solvent tolerance of wild-type carboxylesterase polypeptides

[0207] This example describes experiments performed to determine the solvent tolerance of wild-type carboxylesterases. In these experiments, the organic solvent tolerance of the lyophilized shake flask powders prepared in Example 5 was determined by testing the activity against substrate sets #3 and #5. Reactions were performed in 96-well plates (220 μL reaction volume) containing 10 g / L enzyme powder, 30-34 g / L amine, 43 g / L ester and 10% DMSO or 50% isopropanol or 15-25% MeCN or 25% THF in 0.1 M potassium phosphate buffer, pH 7.5. Reactions were heated to 50° C. with shaking at 400 rpm for 18-21 hours. Reactions were quenched by 3-fold dilution with MeCN. Reaction samples were analyzed by RP-HPLC using the method described in Example 12.2. [Table 6] Example 7 Generation and analysis of an engineered T. fusca carboxylesterase polypeptide library

[0208] A plasmid library containing the evolved T. fusca carboxylesterase genes was transformed into E. coli as described in Example 1 and produced as described in Example 2. Cell lysates were used for activity assays as described in Examples 8 and 9. Example 8 Amidation activity of engineered T. fusca carboxylesterase polypeptides toward substrate sets #3, 8–12

[0209] T. fusca carboxylesterase variants were produced according to Example 7. To analyze the amidation activity towards substrate set #3 (see Table 3.1), 20 μL of the supernatant produced as described in Example 7 was added to a mixture of 25 μL MeCN containing 3 M ethyl benzoate, 110 μL 0.6 M isobutylamine in 0.2 M TEoA (pH adjusted to 9.5) and 65 μL 0.2 M TEoA, pH 8.5. The reaction was incubated at 50° C. and shaken at 300 rpm for 18 hours. The sample was quenched by diluting 4-fold with MeCN. Analysis of the reaction was performed by RP-HPLC using the method described in Example 12.2.

[0210] To evaluate the amidation activity for substrate sets #3, 8-12 (see Table 3.1), 20-50 μL of supernatant prepared as described in Example 7 was added to a mixture of 25 μL MeCN containing 2-3.2 M amine, 110 μL of 0.4-0.6 M ester suspension in 0.2 M TEoA (pH adjusted to 8) and 35-65 μL of 0.2 M TEoA, pH 8.5-9.0. The reactions were incubated at 50 °C and shaken at 300 rpm for 16-18 h. Samples were quenched by diluting 4-fold with MeCN. Analysis of the reactions was performed by RP-HPLC as described in Examples 13.3, 13.4 and 13.5. In one experiment using Substrate Set #8, 50 μL of supernatant made as described in Example 7 was added to a mixture of 3M glycine methyl ester in 25 μL MeCN, 110 μL 0.6M aniline in 0.2M TEoA (pH adjusted to 8) and 35 μL 0.2M TEoA, pH 8.5. The reaction was incubated at 50° C. and shaken at 300 rpm for 18 hours. The sample was quenched by diluting 4-fold with MeCN. Analysis of the reaction was performed by RP-HPLC using the method described in Example 12.3. The amino acid substitutions of these mutants are shown relative to SEQ ID NO:2. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] Example 9 Evaluation of shake flask powders of engineered T. fusca carboxylesterase polypeptides.

[0211] Powder of evolved T. fusca carboxylesterase was prepared in shake flask scale quantities according to the method described in Example 5. The amidation activity of the lyophilized shake flask powder was evaluated by testing the activity against substrate set #3. First, a 1.0 mL reaction mixture was prepared containing 10 g / L enzyme powder, 0.3 M isobutylamine, 0.3 M ethyl benzoate in toluene, followed by the addition of 20 μL of 0.2 M TEoA buffer, pH 8.5. The reaction was heated to 50° C. with shaking at 500 rpm for 60 hours. The reaction was quenched by diluting 100 μL of the reaction mixture with 1.4 mL of acetone. The reaction samples were analyzed by RP-HPLC using the method provided in Table 12.2. [Table 9-1] [Table 9-2] Example 10 Generation and analysis of a combinatorial T. fusca carboxylesterase library

[0212] The plasmid library obtained by combinatorial shuffling of T. fusca carboxylesterase mutants (SEQ ID NO: 8) was transformed into E. coli W3110 according to the method described in Example 1. Activity assays described in Example 11 were performed using HTP lysates prepared according to the method described in Example 2. Example 11 Activity of engineered T. fusca carboxylesterase polypeptides against selected substrate sets 3, 5, 8, 12-15

[0213] T. fusca carboxylesterase variants generated from the combinatorial library were analyzed for amidation activity towards substrate sets #3, 5, 8 and 12-15 (see Table 3.1). To analyze the amidation activity towards substrate set #3 (see Table 3.1), 10 μL of the supernatant made as described in Example 7 was added to a mixture of 25 μL MeCN containing 3 M ethyl benzoate, 110 μL 0.6 M isobutylamine in 0.2 M TEoA (pH adjusted to 8) and 75 μL 0.2 M TEoA, pH 8.5. The reaction was incubated at 50 °C and shaken at 300 rpm for 18 h. The sample was quenched by diluting 4-fold with MeCN. To analyze for amidation activity towards substrate sets #5, 8 and 12-15, 20-50 μL of supernatant was prepared as described in Example 7 and added to a mixture of 25 μL MeCN containing 2-3.2 M amine, 110 μL of 0.4-0.6 M ester suspension in 0.2 M TEoA (pH adjusted to 8) and 35-65 μL of 0.2 M TEoA, pH 8.5-9. Reactions were incubated at 50 °C and shaken at 300 rpm for 16-18 h. Samples were quenched by diluting 4-fold with MeCN. Analysis of reactions was performed by RP-HPLC using the methods described in Examples 12.2 (Set 3), 12.3 (Set 8), 12.5 (Sets 12 and 13), 12.6 (Sets 14) and 12.7 (Sets 5 and 15). In one experiment using Substrate Set #8, 20 μL of the supernatant made as described in Example 7 was added to a mixture of 3 M glycine methyl ester in 25 μL MeCN, 110 μL 0.6 M aniline in 0.2 M TEoA (pH adjusted to 9.5) and 65 μL 0.2 M TEoA, pH 8.5. The reaction was incubated at 50° C. and shaken at 300 rpm for 18 hours. The sample was quenched by diluting 4-fold with MeCN. Analysis of the reaction was performed by RP-HPLC using the method described in Example 12.3. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] [Table 11-9] Example 12 Analytical detection of generated amides and precursor substrates

[0214] The analytical methods in Tables 12.1-12.7 were used to collect the data described in the above examples. All methods provided herein are used in the analysis of products from T. fusca carboxylesterase variants produced using the present invention. However, the present invention is not limited to the methods described herein for the analysis of products provided herein and / or produced using the methods provided herein. Indeed, any suitable method is used in the present invention. Product peak elution was confirmed by confirmation with a commercially available standard or by LC / MS / MS analysis. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0215] While various specific embodiments have been illustrated and described, it will be appreciated that various modifications can be made without departing from the spirit and scope of the invention. The present invention also provides the following: (Item 1) 1. An engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 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, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 2) The at least one substitution or set of substitutions in the polypeptide sequence is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 117, 118 , 118 / 269, 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 2 68 / 372, 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 2. The engineered carboxylesterase of claim 1, comprising a substitution at a position selected from among 376, 320 / 376 / 377, 321, 323, 324, 345, 349, 372, 372 / 376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 3) 1. An engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. (Item 4) The at least one substitution or set of substitutions in the polypeptide sequence is selected from the group consisting of 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372, 65 / 320, 65 / 320 / 323, 68, 68 / 69 / 189 / 214, 68 / 69 / 189 / 214 / 215, 68 / 69 / 18 9 / 214 / 215 / 271, 68 / 69 / 189 / 214 / 215 / 271 / 281 / 282 / 343 / 381, 68 / 69 / 189 / 214 / 271 / 280, 68 / 69 / 189 / 214 / 372, 68 / 69 / 189 / 214 / 377 / 381, 68 / 69 / 189 / 271, 68 / 69 / 189 / 271 / 280 / 372 / 381、68 / 69 / 189 / 280 / 281 / 282 / 372 / 377、68 / 69 / 189 / 281 / 282 / 372 / 377 / 381、68 / 69 / 189 / 343 / 381、68 / 69 / 189 / 372、68 / 69 / 189 / 381、68 / 69 / 214 / 215 / 271、68 / 69 / 214 / 343、68 / 69 / 215、68 / 69 / 271、68 / 69 / 282 / 287、68 / 69 / 343 / 372、68 / 108 / 377、68 / 184、68 / 184 / 189、68 / 189 / 271 / 372、68 / 189 / 343、68 / 214 / 215 / 271 / 281 / 282 / 372、68 / 215 / 271 / 343 / 372 / 381、68 / 215 / 377、68 / 271 / 372、68 / 377、69、69 / 70、69 / 70 / 331 / 372、69 / 70 / 372、69 / 70 / 459、69 / 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 282、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320, 108 / 189 / 377, 108 / 215, 108 / 215 / 377, 108 / 269 / 270, 108 / 270, 108 / 282 / 285 / 377, 108 / 285, 108 / 320 / 323, 108 / 377, 126, 126 / 184 / 213 / 280 / 281 / 285 / 320, 126 / 184 / 213 / 372, 126 / 189 / 285 / 372, 126 / 215, 126 / 372, 181 / 215, 189, 189 / 214, 189 / 214 / 215 / 271 / 282, 18 9 / 215, 189 / 215 / 249 / 277, 189 / 215 / 271 / 281 / 282 / 377, 189 / 215 / 343 / 372, 189 / 270 / 285, 189 / 270 / 372, 189 / 280 / 282, 189 / 320 / 377, 189 / 343, 189 / 343 / 377, 189 / 372 / 377, 189 / 377, 189 / 381, 213 / 215 / 320, 214 / 215 / 271, 214 / 215 / 271 / 377, 214 / 271, 214 / 280 / 282 / 343 / 377 / 381, 2 15, 215 / 249 / 280 / 281 / 285 / 372, 215 / 271 / 372, 215 / 280 / 281 / 285 / 372, 215 / 281 / 285 / 372, 215 / 281 / 285 / 373, 215 / 281 / 373, 215 / 285, 215 / 285 / 317, 215 / 285 / 346, 215 / 285 / 445, 215 / 320, 215 / 320 / 372, 215 / 323, 215 / 372, 215 / 372 / 377, 215 / 373, 215 / 377, 215 / 381, 249 / 377, 269 4. The engineered carboxylesterase of item 3, comprising a substitution at a position selected from: 270 / 281 / 372 / 377, 270 / 377, 271, 271 / 343, 271 / 343 / 372, 271 / 343 / 372 / 381, 280 / 285 / 372, 281 / 372, 282 / 285 / 320 / 323, 285 / 323, 320, 343 / 372, 372, 372 / 377, 372 / 381, 373 and 377, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8. (Item 5) SEQ ID NO: 138 and at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an engineered carboxylesterase or functional fragment thereof, wherein the engineered carboxylesterase comprises a polypeptide sequence having at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 138. (Item 6) 1. An engineered carboxylesterase or functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:140, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:140. (Item 7) 2. The engineered carboxylesterase of item 1, comprising an amino acid substitution at position 282, said positions being numbered with reference to SEQ ID NO:2. (Item 8) 8. The engineered carboxylesterase of item 7, wherein the amino acid at position 282 is selected from an aliphatic amino acid, a nonpolar amino acid, a basic amino acid, a polar amino acid and an aromatic amino acid. (Item 9) 8. The engineered carboxylesterase of item 7, comprising an amino acid substitution selected from X282T, X282G, X282A, X282V, X282M, X282C, X282W, X282Q, X282S, X282T and X282R. (Item 10) 2. The engineered carboxylesterase of item 1, wherein the polypeptide sequence is selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136. (Item 11) 11. The engineered carboxylesterase according to any of items 1 to 4 and 7 to 10, comprising at least one improved property compared to the wild-type T. fusca carboxylesterase of SEQ ID NO:2. (Item 12) 12. The engineered carboxylesterase of item 11, wherein the improved property is selected from improved amidation activity, solvent tolerance, thermostability, pH stability, regioselectivity, stereoselectivity, substrate range, and / or reduced substrate or product inhibition, and reduced toxicity to a bacterial host cell producing the engineered carboxylesterase. (Item 13) 13. The engineered carboxylesterase of item 12, wherein the bacterial host cell comprises Escherichia coli. (Item 14) 13. The engineered carboxylesterase of claim 12, wherein the solvent is selected from acetone, acetonitrile, toluene, tetrahydrofuran, isopropanol, isopropyl acetate, dimethylsulfoxide and methyl ethyl ketone. (Item 15) 15. The engineered carboxylesterase of any of items 1 to 14, which exhibits greater activity than wild-type T. fusca carboxylesterase towards at least one substrate selected from aniline; isobutylamine; n-butylamine; t-butylamine; N'-t-butoxycarbonyl-benzhydrazide; 4-methylpiperidine; Ot-butylhydroxylamine; benzylamine; 2,6-dimethylaniline; (S)-(-)-α-methylbenzylamine; (R)-(+)-α-methylbenzylamine; methyl phenylacetate; ethyl acetate; ethyl benzoate; 2-pyrazinylethyl ester; 4-ethyl-1H-indole ester; and N,N-diethylglycylmethyl ester. (Item 16) 16. The engineered carboxylesterase of any of items 1 to 15, which exhibits greater activity than a wild-type T. fusca carboxylesterase for at least one substrate or set of substrates selected from: aniline; isobutylamine; n-butylamine; t-butylamine; N'-t-butoxycarbonyl-benzhydrazide; 4-methylpiperidine; Ot-butylhydroxylamine; benzylamine; 2,6-dimethylaniline; (S)-(-)-α-methylbenzylamine; (R)-(+)-α-methylbenzylamine; methyl phenylacetate; ethyl acetate; ethyl benzoate; 2-pyrazinylethyl ester; 4-ethyl-1H-indole ester; and N,N-diethylglycylmethyl ester. (Item 17) Acetanilide;Nn-Butyl-benzylacetamide;N-[(S)-1-Phenylethyl]-pyrazinylamide;N-[(S)-1-Phenylethyl]-benzamide;N-[(R)-1-Phenylethyl]-benzamide;N'-t-Butoxycarbonyl-benzhydrazide;1-Benzoyl-4-methylpiperidine;2-Pyrazinyl-4-methylpiperidine;N-Isobutyl-benzamide;Nt-Butyl-benzamide;Nt-Butylhydroxyl-benzamide 17. The engineered carboxylesterase of any of items 1 to 16, which exhibits greater activity than wild-type T. fusca carboxylesterase in producing at least one product selected from: N-isobutyl-1H-indole-4-amide; N',N'-(diethylamino)-N-phenylacetamide; N',N'-(diethylamino)-N-benzylacetamide; and N',N'-(diethylamino)-N-2,6-dimethylphenylacetamide. (Item 18) 2. The engineered carboxylesterase of item 1, comprising at least one substitution selected from X343V, X372L, X320W / G, X214R, X282C, X271Y, X65G, and exhibiting greater activity towards hindered amines for the formation of N',N'-(diethylamino)-N-2,6-dimethylphenylacetamide from ethyl benzoate; and 2,6-dimethylaniline than wild-type T. fusca carboxylesterase. (Item 19) 2. The engineered carboxylesterase of item 1, comprising at least one substitution selected from X268A, X63A / R, X189Q / I / E, X214R, X282G / C, X381L, X69W, and exhibiting greater activity towards secondary amines for the formation of 1-benzoyl-4-methyl-piperidine from ethyl benzoate and 4-methylpiperidine than the wild-type T. fusca carboxylesterase. (Item 20) 20. The engineered carboxylesterase according to any of items 1 to 19, which is purified. (Item 21) 21. The engineered carboxylesterase according to any of items 1 to 20, which is immobilized. (Item 22) 22. A composition comprising at least one engineered carboxylesterase according to any of items 1 to 21. (Item 23) 20. A polynucleotide sequence encoding at least one engineered carboxylesterase according to any one of items 1 to 19. (Item 24) 1. A polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof comprising a polypeptide sequence having at least 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, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 25) The encoded at least one engineered carboxylesterase is 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 11 8 / 269, 118 / 349, 119, 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 174 / 282, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372 , 231, 249, 249 / 284, 263, 268, 269, 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280 / 282, 280, 281, 281 / 374, 282, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, 320 / 25. The polynucleotide sequence according to item 24, comprising at least one substitution or set of substitutions at a position selected from 376 / 377, 321, 323, 324, 345, 349, 372, 372 / 376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 26) 1. A polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, wherein said engineered carboxylesterase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. (Item 27) The encoded engineered carboxylesterase is selected from the group consisting of 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 282 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 282 / 285 / 377, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372 、65 / 320、65 / 320 / 323、68、68 / 69 / 189 / 214、68 / 69 / 189 / 214 / 215、68 / 69 / 189 / 214 / 215 / 271、68 / 69 / 189 / 214 / 215 / 271 / 281 / 282 / 343 / 381、68 / 69 / 189 / 214 / 271 / 280、68 / 69 / 189 / 214 / 372、68 / 69 / 189 / 214 / 377 / 381、68 / 69 / 189 / 271、68 / 69 / 189 / 271 / 280 / 372 / 381、68 / 69 / 189 / 280 / 281 / 282 / 372 / 377、68 / 69 / 189 / 281 / 282 / 372 / 377 / 381、68 / 69 / 189 / 343 / 381、68 / 69 / 189 / 372、68 / 69 / 189 / 381、68 / 69 / 214 / 215 / 271、68 / 69 / 214 / 343、68 / 69 / 215、68 / 69 / 271、68 / 69 / 282 / 287、68 / 69 / 343 / 372、68 / 108 / 377、68 / 184、68 / 184 / 189、68 / 189 / 271 / 372、68 / 189 / 343、68 / 214 / 215 / 271 / 281 / 282 / 372、68 / 215 / 271 / 343 / 372 / 381、68 / 215 / 377、68 / 271 / 372、68 / 377、69、69 / 70、69 / 70 / 331 / 372、69 / 70 / 372、69 / 70 / 459、69 / 108、69 / 108 / 270 / 372 / 377、69 / 108 / 281 / 285、69 / 110 / 215 / 281、69 / 189、69 / 189 / 214 / 271 / 281 / 282 / 343、69 / 189 / 214 / 343 / 372、69 / 189 / 215 / 343、69 / 189 / 271、69 / 189 / 271 / 281 / 282、69 / 189 / 271 / 343、69 / 189 / 271 / 343 / 381、69 / 189 / 280 / 282 / 343 / 372 / 381、69 / 189 / 281 / 373、69 / 189 / 282、69 / 189 / 343 / 381、69 / 189 / 372、69 / 189 / 372 / 377、69 / 189 / 377、69 / 212 / 213 / 215 / 280 / 281、69 / 214 / 215 / 271 / 372 / 377 / 381、69 / 214 / 271 / 282、69 / 214 / 271 / 343、69 / 215、69 / 215 / 269 / 270 / 377、69 / 215 / 271 / 280 / 281 / 282、69 / 215 / 271 / 282、69 / 215 / 271 / 372、69 / 215 / 285 / 317、69 / 215 / 323、69 / 215 / 343、69 / 215 / 343 / 372 / 381、69 / 271、69 / 271 / 372、69 / 282、69 / 282 / 343 / 372、69 / 285 / 373、69 / 372、70、70 / 212、108、108 / 189、108 / 189 / 282 / 285 / 320、108 / 189 / 320、108 / 189 / 377、108 / 215、108 / 215 / 377、108 / 269 / 270、108 / 270、108 / 282 / 285 / 377、108 / 285、108 / 320 / 323、108 / 377、126、126 / 184 / 213 / 280 / 281 / 285 / 320、126 / 184 / 213 / 372、126 / 189 / 285 / 372、126 / 215、126 / 372、181 / 215、189、189 / 214、189 / 214 / 215 / 271 / 282、189 / 215、189 / 215 / 249 / 277、189 / 215 / 271 / 281 / 282 / 377、189 / 215 / 343 / 372、189 / 270 / 285、189 / 270 / 372、189 / 280 / 282、189 / 320 / 377、189 / 343、189 / 343 / 377、189 / 372 / 377、189 / 377、189 / 381、213 / 215 / 320、214 / 215 / 271、214 / 215 / 271 / 377、214 / 271、214 / 280 / 282 / 343 / 377 / 381、215、215 / 249 / 280 / 281 / 285 / 372、215 / 271 / 372、215 / 280 / 281 / 285 / 372、215 / 281 / 285 / 372、215 / 281 / 285 / 373、215 / 281 / 373、215 / 285、215 / 285 / 317、215 / 285 / 346、215 / 285 / 445、215 / 320、215 / 320 / 372、215 / 323、215 / 372、215 / 372 / 377、215 / 373、215 / 377、215 / 381、249 / 377、269 / 270 / 281 / 372 / 377、270 / 377、271、271 / 343、271 / 343 / 372、271 / 343 / 372 / 381、280 / 285 / 372、281 / 372、282 / 285 / 320 / 323、285 / 323、320、343 / 372、3 27. The polynucleotide sequence according to item 26, comprising at least one substitution or set of substitutions in the polypeptide sequence at a position selected from among 72, 372 / 377, 372 / 381, 373 and 377, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8. (Item 28) 1. A polynucleotide sequence encoding at least one engineered carboxylesterase or a functional fragment thereof, comprising: , 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139. (Item 29) 29. The polynucleotide sequence according to any of items 23 to 28, operably linked to a regulatory sequence. (Item 30) 30. The polynucleotide sequence according to any one of items 23 to 29, which is codon-optimized. (Item 31) An expression vector comprising at least one polynucleotide sequence according to any one of items 23 to 30. (Item 32) A host cell comprising at least one expression vector according to item 31. (Item 33) A host cell comprising at least one polynucleotide sequence according to any one of items 23 to 30. (Item 34) 34. The host cell according to items 32 and / or 33, which is Escherichia coli. (Item 35) 35. A method for producing an engineered carboxylesterase in a host cell, comprising culturing a host cell according to any of items 32 to 34 under suitable conditions such that at least one engineered carboxylesterase is produced. (Item 36) 36. The method of claim 35, further comprising recovering the at least one engineered carboxylesterase from the culture and / or host cell. (Item 37) 37. The method of claim 35, further comprising a step of purifying the at least one engineered carboxylesterase.

Claims

1. 1. An engineered carboxylesterase comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, said engineered carboxylesterase comprising a substitution at position 282, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2; The engineered carboxylesterase comprises at least increased amidation activity compared to a wild-type T. fusca carboxylesterase of SEQ ID NO:2, and the engineered carboxylesterase comprises an amino acid substitution selected from L282T, L282G, L282A, L282V, L282M, L282C, L282W, L282Q, L282S and L282R.

2. The engineered carboxylesterases are 39, 39 / 323, 62, 62 / 117, 63, 64, 65, 66, 68, 69, 70, 71, 71 / 263, 77, 77 / 184, 103, 103 / 147, 104, 104 / 429, 105, 107, 107 / 185, 108, 109, 109 / 117, 110, 111, 113, 114, 115, 117, 118, 118 / 269, 118 / 349, 119 , 126, 147, 153, 153 / 215, 164, 164 / 271, 174, 183, 184, 184 / 249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213 / 271, 213 / 345, 214, 215, 215 / 271, 216, 217, 217 / 231, 224, 224 / 268 / 372, 231, 249, 249 / 284, 263, 268, 269 , 270, 270 / 470, 271, 271 / 416, 276, 277, 278, 279, 279 / 280, 280, 281, 281 / 374, 283, 283 / 429, 284, 284 / 438, 285, 286, 311, 317, 320, 320 / 323, 320 / 323 / 372, 320 / 372 / 376, 320 / 376 / 377, 321, 323, 324, 345, 349, 372, 372 / 376 2. The engineered carboxylesterase of claim 1, further comprising at least one substitution or set of substitutions in the polypeptide sequence comprising substitutions at positions selected from among 370, 371, 372, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438 and 470, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2 and the " / " means "and".

3. The polypeptide sequence is 63, 63 / 65 / 108, 63 / 65 / 108 / 189, 63 / 65 / 108 / 377, 63 / 65 / 285 / 320 / 323, 63 / 65 / 320 / 323, 63 / 108, 63 / 108 / 285 / 377, 63 / 108 / 320 / 323, 63 / 189, 63 / 212 / 215, 63 / 212 / 215 / 268 / 269 / 343, 63 / 215, 63 / 215 / 269, 63 / 215 / 270 / 271, 63 / 215 / 343, 63 / 268 / 269 / 270 / 429, 63 / 377, 65 / 69 / 70 / 281 / 372, 65 / 69 / 70 / 372, 65 / 69 / 372, 65 / 70 / 372, 65 / 320, 65 / 320 / 323, 68, 68 / 69 / 189 / 214, 68 / 69 / 189 / 214 / 215, 68 / 69 / 189 / 214 / 215 / 271, 68 / 69 / 189 / 2 14 / 215 / 271 / 281 / 343 / 381, 68 / 69 / 189 / 214 / 271 / 280, 68 / 69 / 189 / 214 / 372, 68 / 69 / 189 / 214 / 377 / 381, 68 / 69 / 189 / 271, 68 / 69 / 189 / 271 / 280 / 372 / 381 , 68 / 69 / 189 / 280 / 281 / 372 / 377, 68 / 69 / 189 / 281 / 372 / 377 / 381, 68 / 69 / 189 / 343 / 381, 68 / 69 / 189 / 372, 68 / 69 / 189 / 381, 68 / 69 / 214 / 215 / 271, 68 / 69 / 214 / 343, 68 / 69 / 215, 68 / 69 / 271, 68 / 69 / 287, 68 / 69 / 343 / 372, 68 / 108 / 377, 68 / 184, 68 / 184 / 189, 68 / 189 / 271 / 372, 68 / 189 / 343, 68 / 214 / 215 / 271 / 28 1 / 372, 68 / 215 / 271 / 343 / 372 / 381, 68 / 215 / 377, 68 / 271 / 372, 68 / 377, 69, 69 / 70, 69 / 70 / 331 / 372, 69 / 70 / 372, 69 / 70 / 459, 69 / 108, 69 / 108 / 270 / 372 / 377, 69 / 108 / 281 / 285, 69 / 110 / 215 / 281, 69 / 189, 69 / 189 / 214 / 271 / 281 / 343, 69 / 189 / 214 / 343 / 372, 69 / 189 / 215 / 343, 69 / 189 / 271, 69 / 189 / 271 / 281,６９／１８９／２７１／３４３、６９／１８９／２７１／３４３／３８１、６９／１８９／２８０／３４３／３７２／３８１、６９／１８９／２８１／３７３、６９／１８９／３４３／３８１、６９／１８９／３７２、６９／１８９／３７２／３７７、６９／１８９／３７７、６９／２１２／２１３／２１５／２８０／２８１、６９／２１４／２１５／２７１／３７２／３７７／３８１、６９／２１４／２７１、６９／２１４／２７１／３４３、６９／２１５、６９／２１５／２６９／２７０／３７７、６９／２１５／２７１／２８０／２８１、６９／２１５／２７１、６９／２１５／２７１／３７２、６９／２１５／２８５／３１７、６９／２１５／３２３、６９／２１５／３４３、６９／２１５／３４３／３７２／３８１、６９／２７１、６９／２７１／３７２、６９／３４３／３７２、６９／２８５／３７３、６９／３７２、７０、７０／２１２、１０８、１０８／１８９、１０８／１８９／２８５／３２０、１０８／１８９／３２０、１０８／１８９／３７７、１０８／２１５、１０８／２１５／３７７、１０８／２６９／２７０、１０８／２７０、１０８／２８５／３７７、１０８／２８５、１０８／３２０／３２３、１０８／３７７、１２６、１２６／１８４／２１３／２８０／２８１／２８５／３２０、１２６／１８４／２１３／３７２、１２６／１８９／２８５／３７２、１２６／２１５、１２６／３７２、１８１／２１５、１８９、１８９／２１４、１８９／２１４／２１５／２７１、１８９／２１５、１８９／２１５／２４９／２７７、１８９／２１５／２７１／２８１／３７７、１８９／２１５／３４３／３７２、１８９／２７０／２８５、１８９／２７０／３７２、１８９／２８０、１８９／３２０／３７７、１８９／３４３、１８９／３４３／３７７、１８９／３７２／３７７、１８９／３７７、１８９／３８１、２１３／２１５／３２０、２１４／２１５／２７１、２１４／２１５／２７１／３７７、２１４／２７１、２１４／２８０／３４３／３７７／３８１、２１５、２１５／２４９／２８０／２８１／２８５／３７２、２１５／２７１／３７２、２１５／２８０／２８１／２８５／３７２、２１５／２８１／２８５／３７２、２１５／２８１／２８５／３７３、２１５／２８１／３７３、２１５／２８５、２１５／２８５／３１７、２１５／２８５／３４６、２１５／２８５／４４５、215 / 320, 215 / 320 / 372, 215 / 323, 215 / 372, 215 / 372 / 377, 215 / 373, 215 / 377, 215 / 381, 249 / 377, 269 / 270 / 281 / 372 / 377, 270 / 377, 271, 271 / 343, 271 / 343 / 372, 271 / 343 / 372 / 381, 280 / 285 / 372, 281 / 372, 285 2. The engineered carboxylesterase of claim 1, further comprising one or more substitutions at positions selected from: 1) 320 / 323, 285 / 323, 320, 343 / 372, 372, 372 / 377, 372 / 381, 373 and 377, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:8, and wherein the " / " means "and".

4. 2. The engineered carboxylesterase of claim 1, wherein the polypeptide sequence is selected from SEQ ID NOs: 4, 6, 8, 12, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, and 134.

5. Aniline; isobutylamine; n-butylamine; t-butylamine; N'-t-butoxycarbonyl-benzhydrazide; 4-methylpiperidine; O-t-butylhydroxylamine; benzylamine; 2,6-Dimethylaniline; (S)-(-)-α-Methylbenzylamine; (R)-(+)-α-Methylbenzylamine; Methyl phenylacetate; Ethyl acetate; Ethyl benzoate; 5. The engineered carboxylesterase of any one of claims 1 to 4, which exhibits greater activity than wild-type T. fusca carboxylesterase towards at least one substrate selected from 2-pyrazinylethyl ester; 4-ethyl-1H-indole ester; and N,N-diethylglycylmethyl ester.

6. and N',N'-(diethylamino)-N-phenylacetamide; N',N'-(diethylamino)-N-benzylacetamide; and N',N'-(diethylamino)-N-2,6-dimethylphenylacetamide.

6. The engineered carboxylesterase of any one of claims 1 to 5, which exhibits greater activity than the A. fusca carboxylesterase.

7. 2. The engineered carboxylesterase of claim 1, further comprising at least one substitution selected from A343V, I372L, Y320W / G, G214R, W271Y and Y65G, wherein " / " means "or".

8. 2. The engineered carboxylesterase of claim 1, further comprising at least one substitution selected from P268A, P63A / R, M189Q / I / E, G214R, A381L and I69W, wherein the " / " means "or".

9. 9. The engineered carboxylesterase of any one of claims 1 to 8, which is purified.

10. 10. The engineered carboxylesterase of any one of claims 1 to 9 which is immobilized.

11. A composition comprising at least one engineered carboxylesterase according to any one of claims 1 to 10.

12. A polynucleotide encoding at least one engineered carboxylesterase according to any one of claims 1 to 8.

13. 13. The polynucleotide of claim 12, comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 3, 5, 7, 11, 25, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.

14. 14. The polynucleotide of claim 12 or 13, operably linked to a control sequence.

15. The polynucleotide according to any one of claims 12 to 14, which is codon-optimized.

16. An expression vector comprising at least one polynucleotide according to any one of claims 12 to 15.

17. A host cell comprising at least one expression vector according to claim 16.

18. A host cell comprising at least one polynucleotide according to any one of claims 12 to 15.

19. 19. The host cell of claim 17 or 18, which is Escherichia coli.

20. 20. A method for producing an engineered carboxylesterase in a host cell, comprising culturing a host cell according to any one of claims 17 to 19 under suitable conditions of culture such that at least one engineered carboxylesterase is produced.

21. 21. The method of claim 20, further comprising recovering the at least one engineered carboxylesterase from the culture and / or host cell.

22. 22. The method of claim 20 or 21, further comprising the step of purifying the at least one engineered carboxylesterase.

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