Engineered monoamine oxidases for the preparation of stereoisomerically pure fused bicyclic proline compounds

Abstract

The present invention provides a novel biocatalyst and related method that is useful for the oxidative desymmetrization of fused bicyclic proline analogs to produce APIs and intermediates.The novel biocatalyst of the present disclosure is an engineered monoamine oxidase enzyme that has improved solubility, thermal stability and activity towards fused bicyclic proline compounds compared to reference monoamine oxidase.In particular, the engineered monoamine oxidase enzyme of the present disclosure requires reduced enzyme loading in the production of APIs and intermediates compared to reference monoamine oxidase enzyme.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 319,135, filed March 11, 2022, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Technical Field The present disclosure relates to engineered monoamine oxidase enzymes and biocatalytic processes for the preparation of substantially stereoisomerically pure fused bicyclic proline compounds.

[0003] Reference to a sequence listing, table or computer program An official copy of the Sequence Listing has been filed contemporaneously herewith as an XML file with the filename "CX2-228US1 ST26.xml", a creation date of March 8, 2023, and a size of 2.32 megabytes. This Sequence Listing as filed is a part of the present specification and is incorporated by reference in its entirety. [Background technology]

[0004] background Bicyclic proline analogs are used in the discovery and development of peptidomimetic drugs. (A. Trabocchi et al. (2008) Amino Acids (2008) 34: 1-24). The hepatitis C virus protease inhibitor boceprevir (SCH 505034; ((1R,2S,5S)-N-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-3-((S)-2-(3-tert- ブチルウレイド )-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide) (Malcolm et al. (2006) Antimicrob. Agents Chemother. 50(3): 1013 20) is shown below: [ka]

[0005] Boceprevir is prepared from the ester of the cis-fused bicyclic L-proline analog (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid shown below: [ka]

[0006] WO2000 / 20824 and WO2002 / 18369 describe numerous other Hepatitis C protease inhibitors, including a variety of fused bicyclic L-proline analogues.

[0007] Recently, the global SARS-CoV-2 (Covid-19) pandemic has led to the discovery of a major coronavirus protease, M Pro This has inspired the evaluation of the efficacy of existing peptidomimetics and small molecule protease inhibitors, including boceprevir, against Covid-19 (Citarella et al. (2021) Biomolecules 2021;11(4):607). Various fused bicyclic L-proline analogues have been investigated for their antiviral activity against Covid-19 in mouse models (Qiao et al. (2021) Science. 2021 Mar 26;371(6536):1374-1378). Indeed, a promising coronavirus M Pro A recent review of inhibitors identified numerous fused bicyclic L-proline analogues among the most potent covalent inhibitors (Macip et al. (2021) Int J Mol Sci. 2021;23(1):259). Thus, fused bicyclic L-proline analogues have demonstrated potential antiviral activity across the spectrum of viral pathogens.

[0008] Therefore, efficient methods for the synthesis of fused bicyclic L-proline analogs are essential to enable the development of novel compounds and treatments against existing and newly emerging viral threats.

[0009] Although the syntheses of such complex molecules using organic chemistry methods and tools have been reported, these syntheses are generally multi-step, involved, expensive, and inefficient processes with low overall yields.

[0010] Wu et al. (WO 2007 / 075790) disclose the preparation of the methyl ester of the bicyclic proline analog (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid from the corresponding symmetric (achiral) bicyclic amine of the structural formula (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane, which is [ka] The process begins with its oxidation to the corresponding racemic imine.

[0011] The racemic imine is then reacted with cyanide to give the racemic aminonitrile of the following structure: [ka] Obtained,

[0012] It is then reacted with acid and methanol to give the racemic amino acid methyl ester of the following structure: [ka] get.

[0013] Finally, these (1R,2S,5S) (undesired) and (1S,2R,5R) (desired) stereoisomeric methyl esters are separated by diastereomeric salt resolution, forming either the di-p-toluoyl-D-tartrate salt of the former enantiomer or the di-p-toluoyl-L-tartrate salt of the latter enantiomer.

[0014] Tanoury et al. (WO 2007 / 022459) describe the synthesis of racemic (t-butoxycarbonyl)octahydrocyclopenta[c]pyrrole-1-carboxylic acid from the corresponding symmetric (achiral) bicyclic amine to produce the N-Boc derivative, which is reacted with a pyrophoric agent, sec-butyllithium, in the presence of a stoichiometric amount or more of a bulky diamine chelate, followed by carbon dioxide, all at less than -70°C, to give the racemic N-Boc amine acid depicted below: [ka] The present invention discloses the synthesis of

[0015] The (1R,2S,5S) (undesired) and (1S,2R,5R) (desired) stereoisomers of these racemic Boc-acids are then separated by diastereomeric salt resolution using a single enantiomer chiral base such as S-1-aminotetralin.

[0016] Although the desired stereoisomers of amino acid derivatives can be obtained by these methods, resolution of the mixture of enantiomers of these bicyclic proline analogs inherently involves the waste of at least one-half of all materials (e.g., raw materials, reagents, solvents, catalysts) used to produce the racemic mixture.

[0017] Monoamine oxidase enzymes have been used for the resolution and deracemization of racemic chiral amines by stereospecific oxidation of one enantiomer to the corresponding imine using oxygen. A derivative of the flavin-dependent monoamine oxidase from Aspergillus niger (MAO N) (Shilling et al. (1995) Biochim. Biophys. Acta. 1243: 529 37), in combination with a nonspecific chemical reducing agent, has been reported to be useful for the deracemization of (d / l) α-methylbenzylamine to give enantiomerically pure (93% ee) (d) α-methylbenzylamine (Alexeeva et al. (2002), Angew. Chem. Int. Ed. 41: 3177-3180). Derivatives of the flavin-dependent monoamine oxidase from Aspergillus niger were also used to deracemize (R / S)-2-phenylpyrrolidine to give enantiomerically pure (98% ee) (R)-2-phenylpyrrolidine (Carr et al. (2005), ChemBioChem 6: 637 39;Gotor et al. “Enantioselective Enzymatic Desymmetrization in Organic Synthesis,” Chem. Rev. (2005) 105: 313-54). Previously, an efficient chemoenzymatic process for the production of boceprevir bicyclic [3.1.0] proline intermediate using engineered monoamine oxidases was reported. Monoamine oxidases from A. niger and A. oryzae were engineered to improve solubility, thermal stability, and activity towards 6,6-dimethyl-3-azabicyclo[3.1.0]hexane substrate (Li et al. (2012), J. Am. Chem. Soc. 134, 6467-6472; WO2010008828). The optimized process proceeds, in this case, via sulfonation to a chiral aminosulfonate ester and cyanation to the trans-nitrile, before conversion to the methyl ester under pina-conditions (Li et al.). However, the large scale production of substantially enantiomerically pure chiral compounds is limited by the enzyme loading required for the synthesis.Therefore, there is a need to provide improved engineered enzymes with reduced enzyme loading (wt / wt) requirements for producing APIs and intermediates. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] International Publication No. 2000 / 20824 [Patent Document 2] International Publication No. 2002 / 18369 [Patent Document 3] International Publication No. 2007 / 075790 [Patent Document 4] International Publication No. 2007 / 022459 [Patent Document 5] International Publication No. 2010 / 008828 [Non-patent literature]

[0019] [Non-Patent Document 1] A. Trabocchi et al. (2008) Amino Acids (2008) 34: 1-24 [Non-Patent Document 2] Malcolm et al. (2006) Antimicrob. Agents Chemother. 50(3): 1013 20 [Non-Patent Document 3] Citarella et al. (2021) Biomolecules 2021;11(4):607 [Non-Patent Document 4] Qiao et al. (2021) Science. 2021 Mar 26;371(6536):1374-1378 [Non-Patent Document 5] Macip et al. (2021) Int J Mol Sci. 2021;23(1):259 [Non-Patent Document 6] Shilling et al. (1995) Biochim. Biophys. Acta. 1243: 529 37 [Non-Patent Document 7] Alexeeva et al. (2002), Angew. Chem. Int. Ed. 41: 3177-3180 [Non-Patent Document 8] Carr et al. (2005), ChemBioChem 6: 637 39 [Non-Patent Document 9] Gotor et al. “Enantioselective Enzymatic Desymmetrization in Organic Synthesis,” Chem. Rev. (2005) 105: 313-54 [Non-Patent Document 10] Li et al. (2012), J. Am. Chem. Soc. 134, 6467-6472 Summary of the Invention [Means for solving the problem]

[0020] Abstract The present invention provides novel biocatalysts and related methods useful for the enzymatic oxidative desymmetrization of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane and related bicyclic proline analogs to produce APIs and intermediates, including peptidomimetic antiviral compounds. The monoamine oxidase biocatalysts of the present disclosure are engineered polypeptide variants of a previously engineered monoamine oxidase (SEQ ID NO:6) derived from a fusion polypeptide of an Aspergillus niger monoamine oxidase homolog (SEQ ID NO:2) and an Aspergillus oryzae monoamine oxidase homolog (SEQ ID NO:4). These engineered monoamine oxidase polypeptides can catalyze the conversion of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane and related bicyclic proline analogs to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene and related compounds.

[0021] The present invention is 13, 32 / 65 / 135 / 246 / 284, 32 / 135 / 284, 32 / 284, 47 / 197, 55 / 347 / 348, 61, 65 / 135, 65 / 190, 65 / 246, 65 / 284, 79, 80, 82, 85, 87, 87 / 185, 93, 100, 100 / 144 / 421 / 447, 100 / 144 / 447, 104, 107, 113, 114, 115, 117, 127, 135, 136, 144, 144 / 447, 145, 146, 147, 149, 149 / 157, 153, 155, 161, 162, 166, 167, 169, 170, 174, 177, 181, 184, 185, 187 / 242, 193, 196, 198, 199, 208, 211, 218, 219, 219 / 332, 221, 229, 232, 234, 238, 239, 240, 242, 246, 250, 252, 254, 260, 262 / 266 / 421, 26 2 / 421 / 447, 266 / 447, 277, 277 / 347, 284, 290, 292, 294, 298, 300, 306, 310, 323, 332, 338, 347, 349, 359, 361, 366, 381, 385, 386, 387, 392, 393, 400, 407, 411 / 445, 413, 418, 421, 425, 429, 433, 434, 441, 444, 445, 447, 449, 452, 475, and 4 Provided is an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NO:6, comprising at least one substitution or set of substitutions at one or more positions selected from 83, wherein the positions are numbered with reference to SEQ ID NO:6. In some additional embodiments, the engineered polypeptide is 13G, 32G / 65V / 135Q / 246M / 284D, 32G / 135Q / 284D, 32G / 284D, 47A / 197N, 55A / 347D / 348K, 61C, 65V / 135Q, 65V / 190D, 65V / 246M, 65V / 284D, 79A, 79D, 79Q, 79S, 79V, 79Y, 80H, 80I, 80N, 80T, 80V, 82F, 82L, 82R, 82W, 85D, 85S, 87G, 87H, 87L,87Q / 185F, 87S, 87W, 87Y, 93G, 100P, 100P / 144E / 421S / 447A, 100P / 144E / 4 47A, 104K, 107W, 113G, 113W, 114G, 115M, 117A, 117C, 117I, 117K, 117L, 11 7R, 117S, 117T, 127D, 127G, 127R, 135G, 136R, 144E / 447A, 144M, 145G, 145 L, 145S, 146A, 146F, 147A, 149M, 149M / 157A, 149V, 153T, 155C, 155L, 155Q, 155R, 161D, 161L, 161R, 161Y, 162Q, 166E, 166G, 166L, 167C, 169G, 170K, 1 74G, 174S, 174T, 174V, 177G, 177R, 177W, 181C, 181G, 181L, 181P, 184F, 184 R, 185G, 185K, 185L, 185R, 187P / 242I, 193A, 196G, 196M, 198D, 199I, 199T , 199V, 208S, 211G, 218G, 219P / 332L, 219S, 221C, 221R, 221V, 229E, 232T, 2 34V, 238K, 238R, 239G, 240A, 242I, 246M, 250V, 252A, 252E, 252G, 252L, 25 4A, 260F, 260S, 260V, 262F / 266L / 421S, 262F / 421S / 447A, 266L / 447A, 277T , 277T / 347D, 284D, 290Q, 290Y, 292N, 294V, 298C, 300R, 306V, 310S, 323R, 332G, 332L, 332S, 338A, 338G, 338S, 347D, 347N, 349E, 359S, 361L, 366G, 38 1A, 385G, 386E, 386G, 387I, 387Q, 387W, 392G, 392S, 393G, 393K, 393M, 400D, 407G, 411V / 445R, 413R, 418A, 418G, 421S, 425L, 429G, 433N, 433S, 434E, 441H, 444D, 444L, 444R, 444S, 445D, 445R, 447A, 449R, 449S, 452M, 475E, 475I, 475L, 475S, and 483R,Numbered with reference to SEQ ID NO: 6. In some embodiments, the engineered polypeptide is selected from the group consisting of T13G, E32G / T65V / T135Q / I246M / N284D, E32G / T135Q / N284D, E32G / N284D, G47A / D197N, T55A / C347D / P348K, A61C, T65V / T135Q, T65V / E190D, T65V / I246M, T65V / N284D, W79A, W79D, W79Q, W79S, W79V, W79Y, S80H, S80I, S80N, S80T, S80V, N82F, N82L, N82R, N82W, G85D, G85S , P87G, P87H, P87L, P87Q / E185F, P87S, P87W, P87Y, T93G, S100P, S100P / A1 44E / C421S / C447A, S100P / A144E / C447A, R104K, T107W, N113G, N113W, A114 G, L115M, P117A, P117C, P117I, P117K, P117L, P117R, P117S, P117T, H127D , H127G, H127R, T135G, T136R, A144E / C447A, A144M, E145G, E145L, E145S, D146A, D146F, E147A, L149M, L149M / T157A, L149V, L153T, K155C, K155L, K 155Q, K155R, G161D, G161L, G161R, G161Y, T162Q, T166E, T166G, T166L, V16 7C, P169G, F170K, M174G, M174S, M174T, M174V, V177G, V177R, V177W, R181 C, R181G, R181L, R181P, D184F, D184R, E185G, E185K, E185L, E185R, S187P / M242I, D193A, R196G, R196M, E198D, L199I, L199T, L199V, E208S, I211G, T 218G, L219P / I332L, L219S, N221C, N221R, N221V, H229E, A232T, S234V, Y23 8K, Y238R, Q239G, G240A, M242I, I246M, F250V, D252A, D252E, D252G, D252 L, Q254A, R260F, R260S, R260V, W262F / A266L / C421S, W262F / C421S / C447A,A266L / C447A, C277T, C277T / C347D, N284D, R290Q, R290Y, T292N, R294 V, E298C, A300R, C306V, L310S, S323R, I332G, I332L, I332S, C338A, C33 8G, C338S, C347D, C347N, D349E, P359S, N361L, A366G, C381A, A385G, H3 86E, H386G, N387I, N387Q, N387W, E392G, E392S, E393G, E393K, E393M, M In some further embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: 400D, P407G, D411V / L445R, K413R, H418A, H418G, C421S, F425L, A429G, A433N, A433S, P434E, S441H, E444D, E444L, E444R, E444S, L445D, L445R, C447A, H449R, H449S, V452M, T475E, T475I, T475L, T475S, and E483R, where positions are numbered with reference to SEQ ID NO:6. In some further embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any of the even-numbered sequences set forth in SEQ ID NOs:8-414.

[0022] The present invention relates to an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence of SEQ ID NO: 6, including at least one substitution or set of substitutions selected from 79, 80, 87, 146, 149, 219 / 332, 238, 260, 262 / 266 / 421, 338, and 429, where positions are numbered with reference to SEQ ID NO: 6. In some embodiments, engineered polypeptides are also provided, in which the engineered polypeptide comprises at least one substitution or set of substitutions selected from 79D, 80V, 87G, 146A, 149M, 219P / 332L, 238R, 260S, 262F / 266L / 421S, 338G, and 429G, where the positions are numbered with reference to SEQ ID NO: 6. In some further embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from W79D, S80V, P87G, D146A, L149M, L219P / I332L, Y238R, R260S, W262F / A266L / C421S, C338G, and A429G, where the positions are numbered with reference to SEQ ID NO: 6.

[0023] The present invention is directed to 79, 79 / 80, 79 / 80 / 332 / 429, 79 / 93, 79 / 93 / 238 / 386 / 429, 79 / 93 / 386, 79 / 183 / 284 / 332, 79 / 238, 79 / 238 / 332 / 429, 79 / 238 / 429, 80, 80 / 183 / 284 / 332 / 381 / 386 / 429, 80 / 188 / 190 / 332 / 386, 80 / 237 / 332, 80 / 238 / 332, 80 / 238 / 386 / 429, 80 / 284, 80 / 284 / 386, 80 / 332, 80 / 332 / 381 / 386, 80 / 332 / 386, 80 / 381, 80 / 386, 80 / 386 / 429, 80 / 429, 82, 82 / 87, 82 / 87 / 113, 82 / 87 / 113 / 117 / 218, 82 / 87 / 113 / 117 / 219, 82 / 87 / 113 / 117 / 219 / 418, 82 / 87 / 113 / 117 / 260 / 338, 82 / 87 / 113 / 117 / 338 / 418, 82 / 87 / 113 / 421, 82 / 87 / 117, 82 / 87 / 117 / 218 / 421, 82 / 87 / 117 / 260 / 338 / 421, 82 / 87 / 117 / 338 / 418, 82 / 87 / 218, 82 / 87 / 218 / 219 / 338, 82 / 87 / 218 / 219 / 421, 82 / 87 / 218 / 338, 82 / 87 / 219, 82 / 87 / 219 / 421, 82 / 87 / 338, 82 / 87 / 338 / 418, 82 / 87 / 338 / 421, 82 / 113 / 218 / 338, 82 / 113 / 218 / 338 / 418, 82 / 113 / 219 / 338, 82 / 113 / 338, 82 / 218 / 219 / 421, 82 / 260 / 338 / 421, 82 / 338 / 418 / 421, 82 / 418, 87, 87 / 113, 87 / 113 / 117 / 219 / 338 / 41 8 / 421, 87 / 113 / 117 / 260 / 338 / 421, 87 / 113 / 117 / 418 / 421, 87 / 113 / 218 / 219 / 260 / 338, 87 / 113 / 219 / 421, 87 / 113 / 260, 87 / 117, 87 / 117 / 218 / 219, 87 / 117 / 219 / 310 / 338, 87 / 218, 87 / 218 / 219, 87 / 218 / 219 / 338, 87 / 218 / 418, 87 / 219, 87 / 219 / 338, 87 / 219 / 418, 87 / 260, 87 / 310, 87 / 310 / 338, 87 / 338, 87 / 418,87 / 421、93 / 184 / 429、93 / 238、93 / 238 / 284、93 / 238 / 386、93 / 238 / 386 / 429、100、100 / 143 / 145 / 146 / 149 / 198 / 242 / 347 / 348 / 445 / 447、100 / 143 / 145 / 149 / 347 / 447、100 / 145 / 146 / 149、100 / 145 / 146 / 149 / 154 / 348 / 445、100 / 145 / 146 / 149 / 198、100 / 145 / 146 / 149 / 198 / 242 / 250 / 252 / 347 / 348 / 447、100 / 145 / 146 / 149 / 205 / 347 / 445 / 447、100 / 145 / 146 / 149 / 242 / 445、100 / 145 / 146 / 149 / 250 / 277 / 347 / 348 / 447、100 / 145 / 149 / 154 / 246 / 277、100 / 145 / 149 / 198 / 242 / 246 / 277 / 348 / 447、100 / 145 / 149 / 198 / 242 / 246 / 445 / 447、100 / 145 / 149 / 198 / 242 / 277 / 347 / 348 / 447、100 / 145 / 149 / 198 / 252 / 348 / 445、100 / 145 / 149 / 205、100 / 145 / 149 / 242 / 246 / 277 / 347 / 445、100 / 145 / 149 / 242 / 246 / 347 / 348 / 445 / 447、100 / 145 / 149 / 242 / 445 / 447、100 / 145 / 149 / 246 / 252 / 277 / 347 / 445 / 447、100 / 145 / 149 / 246 / 347 / 445、100 / 145 / 149 / 277 / 348 / 445、100 / 145 / 149 / 347 / 445 / 447、100 / 145 / 198 / 242 / 246 / 348、100 / 145 / 198 / 277 / 348 / 445、100 / 145 / 198 / 300、100 / 145 / 205 / 246 / 277 / 348 / 445、100 / 145 / 205 / 277 / 348、100 / 145 / 242 / 246 / 277 / 445 / 447、100 / 145 / 246 / 347 / 348 / 445、100 / 145 / 277 / 347、100 / 145 / 348、100 / 146 / 149 / 198 / 242 / 246 / 277 / 347 / 348 / 445 / 447、100 / 146 / 198 / 347 / 348 / 447、100 / 146 / 198 / 445、100 / 146 / 242 / 277 / 447、100 / 146 / 250 / 447、100 / 146 / 347 / 348 / 445 / 447、100 / 149 / 198、100 / 149 / 242 / 246 / 277 / 347 / 348 / 445、100 / 149 / 242 / 246 / 277 / 347 / 348 / 447、100 / 149 / 242 / 347 / 348 / 447、100 / 149 / 246 / 277 / 348 / 445、100 / 149 / 246 / 447、100 / 149 / 250 / 347 / 447、100 / 198 / 205 / 348、100 / 198 / 277 / 347 / 348、100 / 198 / 347 / 348、100 / 198 / 347 / 348 / 445、100 / 198 / 347 / 445、100 / 198 / 447、100 / 242 / 246 / 277、100 / 242 / 246 / 347、100 / 242 / 277 / 348、100 / 242 / 347 / 348 / 445、100 / 242 / 347 / 348 / 445 / 447、100 / 242 / 348 / 445、100 / 246 / 277 / 348 / 445、100 / 277 / 347 / 445 / 447、100 / 277 / 348、100 / 277 / 348 / 447、100 / 347 / 348 / 445、100 / 347 / 447、100 / 445 / 447、110 / 338 / 418、113 / 218 / 338 / 421、113 / 338、113 / 338 / 421、117 / 219、117 / 260、121 / 198 / 347 / 445 / 447、145 / 146 / 149 / 154 / 246 / 277 / 347 / 348、145 / 146 / 149 / 198 / 242 / 252 / 277 / 347、145 / 146 / 149 / 198 / 277 / 445 / 447、145 / 146 / 149 / 205 / 348 / 447、145 / 146 / 149 / 250 / 277 / 347 / 348 / 447、145 / 146 / 149 / 277 / 347、145 / 146 / 149 / 347 / 348、145 / 146 / 198 / 205 / 242 / 277 / 348 / 447、145 / 146 / 198 / 445 / 447、145 / 146 / 246 / 277 / 447、145 / 146 / 246 / 347 / 348 / 447、145 / 146 / 277 / 347、145 / 146 / 347 / 447、145 / 149 / 198 / 205 / 348、145 / 149 / 198 / 242 / 246 / 277 / 347 / 447、145 / 149 / 198 / 242 / 246 / 348、145 / 149 / 198 / 242 / 277 / 347、145 / 149 / 198 / 246 / 447、145 / 149 / 198 / 347 / 447、145 / 149 / 205 / 242 / 347 / 348 / 445、145 / 149 / 205 / 246 / 250 / 252、145 / 149 / 205 / 277 / 445、145 / 149 / 242、145 / 149 / 242 / 348、145 / 149 / 246、145 / 149 / 246 / 347 / 445、145 / 149 / 246 / 447、145 / 149 / 347 / 348 / 445、145 / 149 / 347 / 447、145 / 149 / 447、145 / 155 / 445 / 447、145 / 198 / 205 / 348、145 / 246、145 / 246 / 252 / 347 / 447、145 / 250 / 252 / 348、145 / 252 / 348 / 447、145 / 255、145 / 277 / 347 / 445、145 / 277 / 348 / 447、145 / 347 / 348 / 445 / 447 / 491、145 / 445 / 447、146 / 149 / 151 / 246 / 348、146 / 149 / 242 / 246 / 445、146 / 149 / 246 / 252 / 447、146 / 149 / 277 / 348 / 447、146 / 198 / 246 / 277、146 / 198 / 277 / 348 / 447、146 / 242、146 / 242 / 246 / 277 / 347 / 348 / 445 / 447、146 / 246 / 277 / 348 / 445 / 447、146 / 277、146 / 277 / 347 / 348、146 / 277 / 347 / 445 / 447、146 / 277 / 445 / 447、146 / 347 / 348 / 447、146 / 348、146 / 445 / 447、149、149 / 198 / 277 / 347 / 348、149 / 198 / 348、149 / 198 / 445、149 / 242 / 246、149 / 242 / 246 / 348 / 445、149 / 347 / 348 / 445 / 447、149 / 445、184、184 / 284、184 / 284 / 429、184 / 386、198 / 242 / 348、198 / 246、198 / 250、198 / 277 / 348 / 447、198 / 445、205、205 / 246 / 347、205 / 252 / 347 / 445 / 447、205 / 347 / 348 / 445 / 447、218、218 / 260 / 338、219、219 / 338 / 418, 219 / 421, 238, 238 / 242 / 284, 238 / 284 / 386 / 429, 238 / 332 / 429, 238 / 381 / 429, 238 / 386, 238 / 429, 242, 242 / 277 / 347 / 348, 242 / 284 / 381 / 429, 242 / 348, 242 / 445 / 447, 246, 246 / 277 / 348 / 447, 246 / 445 / 447, 260, 260 / 338, 260 / 338 / 418 / 421, 277 / 347 / 348 / 445, 277 / 347 / 447, 284, 284 / 332, 284 / 386, 284 / 386 / 429, 310 / 338 / 418, 310 / 418 / 4 Further provided is an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NO:6, including at least one substitution or set of substitutions selected from: 21, 332, 332 / 386, 332 / 429, 338, 338 / 418, 347 / 348 / 445, 347 / 348 / 447, 348, 348 / 445 / 447, 348 / 447, 386, 386 / 429, 418, 429, and 447, wherein the positions are numbered with reference to SEQ ID NO:6. In some additional embodiments, the engineered polypeptide is selected from the group consisting of 79A / 80H, 79A / 93G / 238R / 386E / 429G, 79A / 93G / 386E, 79A / 238R, 79A / 238R / 332G / 429G, 79A / 238R / 429G, 79D, 79D / 80V, 79D / 80V / 332G / 429G, 79D / 93G, 79D / 183F / 284D / 332G, 80H, 80H / 183F / 284D / 332G / 381A / 386 E / 429G, 80H / 188F / 190D / 332G / 386E, 80H / 238R / 386E / 429G, 80H / 332G / 381A / 386E, 80H / 381A, 80H / 386E, 80H / 429G, 80V, 80V / 237S / 332G, 80V / 238R / 332G, 80V / 284D, 80V / 284D / 386E, 80V / 332G, 80V / 332G / 386E, 80V / 386E, 80V / 386E / 429G, 82F,82F / 87G / 113G / 117K / 219S、82F / 87G / 113G / 117K / 260S / 338G、82F / 87G / 117K、82F / 87G / 218G、82F / 87G / 218G / 219S / 421S、82F / 87G / 218G / 338S、82F / 87G / 338S / 418G、82F / 87L、82F / 87L / 113G / 117K / 218G、82F / 87L / 11、 <h2 style=";text-align:left;direction:ltr">3G / 117K / 219S / 418G, 82F / 87L / 113G / 117K / 338G / 418G, 82F / 87L / 117K, 82F / 87L / 219S / 421S, 82F / 87L / 338S, 82F / 113G / 218G / 338S / 418G, 82F / 41 8G、82R / 87G、82R / 87G / 113G / 421S、82R / 87G / 117K / 218G / 421S、82R / 87G / 117K / 260S / 338S / 421S、82R / 87G / 117K / 338A / 418G、82R / 87L、82R / 87L / 11 3G, 82R / 87L / 218G / 219S / 338S, 82R / 87L / 219S, 82R / 87L / 338G / 421S, 82R / 113G / 218G / 338A, 82R / 113G / 219S / 338S, 82R / 113G / 338S, 82R / 218G / 21 9S / 421S、82R / 260S / 338A / 421S、82R / 338A / 418G / 421S、87G、87G / 113G、8 7G / 113G / 117K / 418G / 421S、87G / 113G / 260S、87G / 117K / 219S / 310S / 338S、 87G / 218G / 219S / 338S、87G / 219S、87G / 260S、87G / 310S、87G / 310S / 338S、 87G / 338A、87G / 418G、87L、87L / 113G、87L / 113G / 117K / 219S / 338G / 418G / 421S、87L / 113G / 117K / 260S / 338S / 421S、87L / 113G / 218G / 219S / 260S / 33 8A、87L / 113G / 219S / 421S、87L / 117K、87L / 117K / 218G / 219S、87L / 218G、87 L / 218G / 219S、87L / 218G / 418G、87L / 219S、87L / 219S / 338G、87L / 219S / 41 8G、87L / 338A、87L / 421S、93G / 184R / 429G、93G / 238R、93G / 238R / 284D、93 G / 238R / 386E、93G / 238R / 386E / 429G、100P、100P / 143D / 145S / 146A / 149M / 198D / 242I / 347N / 348K / 445R / 447A、100P / 143V / 145G / 149M / 347N / 447A、100P / 145G / 146A / 149M / 250V / 277T / 347N / 348K / 447A、100P / 145G / 149M / 154L / 246M / 277T、100P / 145G / 149M / 198D / 242I / 246M / 445R / 447A、100P / 145G / 149M / 198D / 252G / 348K / 445R、100P / 145G / 149M / 205T、100P / 145G / 149M / 242I / 246M / 347D / 348K / 445R / 447A、100P / 145G / 149M / 242I / 445R / 447A、100P / 145G / 198D / 242I / 246M / 348K、100P / 145G / 198D / 277T / 348K / 445R、100P / 145G / 198D / 300V、100P / 145G / 205T / 246M / 277T / 348K / 445R、100P / 145G / 242I / 246M / 277T / 445R / 447A、100P / 145G / 348K、100P / 145S / 146A / 149M、100P / 145S / 146A / 149M / 154L / 348K / 445R、100P / 145S / 146A / 149M / 198D、100P / 145S / 146A / 149M / 198D / 242I / 250V / 252G / 347N / 348K / 447A、100P / 145S / 146A / 149M / 205T / 347D / 445R / 447A、100P / 145S / 146A / 149M / 242I / 445R、100P / 145S / 149M / 198D / 242I / 246M / 277T / 348K / 447A、100P / 145S / 149M / 198D / 242I / 277T / 347N / 348K / 447A、100P / 145S / 149M / 242I / 246M / 277T / 347N / 445R、100P / 145S / 149M / 246M / 252G / 277T / 347N / 445R / 447A、100P / 145S / 149M / 246M / 347N / 445R、100P / 145S / 149M / 277T / 348K / 445R、100P / 145S / 149M / 347N / 445R / 447A、100P / 145S / 205T / 277T / 348K、100P / 145S / 246M / 347N / 348K / 445R、100P / 145S / 277T / 347N、100P / 146A / 149M / 198D / 242I / 246M / 277T / 347D / 348K / 445R / 447A、100P / 146A / 198D / 347D / 348K / 447A、100P / 146A / 198D / 445R、100P / 146A / 242I / 277T / 447A、100P / 146A / 250V / 447A、100P / 146A / 347D / 348K / 445R / 447A、100P / 149M / 198D、100P / 149M / 242I / 246M / 277T / 347N / 348K / 445R、100P / 149M / 242I / 246M / 277T / 347N / 348K / 447A、100P / 149M / 242I / 347N / 348K / 447A、100P / 149M / 246M / 277T / 348K / 445R、100P / 149M / 246M / 447A、100P / 149M / 250V / 347N / 447A、100P / 198D / 205T / 348K、100P / 198D / 277T / 347N / 348K、100P / 198D / 347D / 348K、100P / 198D / 347D / 348K / 445R、100P / 198D / 347N / 445R、100P / 198D / 447A、100P / 242I / 246M / 277T、100P / 242I / 246M / 347N、100P / 242I / 277T / 348K、100P / 242I / 347D / 348K / 445R、100P / 242I / 347D / 348K / 445R / 447A、100P / 242I / 348K / 445R、100P / 246M / 277T / 348K / 445R、100P / 277T / 347D / 445R / 447A、100P / 277T / 348K、100P / 277T / 348K / 447A、100P / 347N / 348K / 445R、100P / 347N / 447A、100P / 445R / 447A、110N / 338G / 418G、113G / 218G / 338S / 421S、113G / 338A / 421S、113G / 338G、117K / 219S、117K / 260S、121C / 198D / 347N / 445R / 447A、145G / 146A / 149M / 277T / 347D、145G / 146A / 149M / 347D / 348K、145G / 146A / 277T / 347N、145G / 146A / 347D / 447A、145G / 149M / 198D / 347N / 447A、145G / 149M / 205T / 242I / 347N / 348K / 445R、145G / 149M / 205T / 246M / 250V / 252G、145G / 149M / 242I / 348K、145G / 149M / 246M、145G / 155I / 445R / 447A、145G / 198D / 205T / 348K、145G / 250V / 252G / 348K、145G / 255R、145G / 277T / 347N / 445R、145S / 146A / 149M / 154L / 246M / 277T / 347N / 348K、145S / 146A / 149M / 198D / 242I / 252G / 277T / 347N、145S / 146A / 149M / 198D / 277T / 445R / 447A、145S / 146A / 149M / 205T / 348K / 447A、145S / 146A / 149M / 250V / 277T / 347D / 348K / 447A、145S / 146A / 198D / 205T / 242I / 277T / 348K / 447A、145S / 146A / 198D / 445R / 447A、145S / 146A / 246M / 277T / 447A、145S / 146A / 246M / 347N / 348K / 447A、145S / 149M / 198D / 205T / 348K、145S / 149M / 198D / 242I / 246M / 277T / 347N / 447A、145S / 149M / 198D / 242I / 246M / 348K、145S / 149M / 198D / 242I / 277T / 347D、145S / 149M / 198D / 246M / 447A、145S / 149M / 205T / 277T / 445R、145S / 149M / 242I、145S / 149M / 246M / 347D / 445R、145S / 149M / 246M / 447A、145S / 149M / 347N / 348K / 445R、145S / 149M / 347N / 447A、145S / 149M / 447A、145S / 246M、145S / 246M / 252G / 347D / 447A、145S / 252G / 348K / 447A、145S / 277T / 348K / 447A、145S / 347D / 348K / 445R / 447A / 491A、145S / 445R / 447A、146A / 149M / 151C / 246M / 348K、146A / 149M / 242I / 246M / 445R、146A / 149M / 246M / 252G / 447A、146A / 149M / 277T / 348K / 447A、146A / 198D / 246M / 277T、146A / 198D / 277T / 348K / 447A、146A / 242I、146A / 242I / 246M / 277T / 347N / 348K / 445R / 447A、146A / 246M / 277T / 348K / 445R / 447A、146A / 277T、146A / 277T / 347N / 348K、146A / 277T / 347N / 445R / 447A、146A / 277T / 445R / 447A、146A / 347D / 348K / 447A、146A / 348K、146A / 445R / 447A、149M、149M / 198D / 277T / 347N / 348K、149M / 198D / 348K、149M / 198D / 445R、149M / 242I / 246M、149M / 242I / 246M / 348K / 445R、149M / 347N / 348K / 445R / 447A、149M / 445R、184R、184R / 284D、184R / 284D / 429G、184R / 386E、198D / 242I / 348K、198D / 246M、198D / 250V、198D / 277T / 348K / 447A、198D / 445R、205T、205T / 246M / 347N、205T / 252G / 347D / 445R / 447A、205T / 347D / 348K / 445R / 447A、218G、218G / 260S / 338A、219S、219S / 338A / 418G、219S / 421S、238R、238R / 242L / 284D、238R / 284D / 386E / 429G、238R / 332G / 429G、238R / 381A / 429G、238R / 386E、238R / 429G、242I、242I / 277T / 347D / 348K、242I / 348K、242I / 445R / 447A、242L / 284D / 381A / 429G、246M、246M / 277T / 348K / 447A、246M / 445R / 447A、260S、260S / 338G、260S / 338G / 418G / 421S、277T / 347N / 348K / 445R、277T / 347N / 447A、284D、284D / 332G、284D / 386E、284D / 386E / 429G、310S / 338S / 418G、310S / 418G / 421S、332G、332G / 386E、332G / 429G、338G、In some further embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from 338G / 418G, 347N / 348K / 445R, 347N / 348K / 447A, 348K, 348K / 445R / 447A, 348K / 447A, 386E, 386E / 429G, 418G, 429G, and 447A, where positions are numbered with reference to SEQ ID NO: 6. In some further embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from W79A / S80H, W79A / T93G / Y238R / H386E / A, 429G、W79A / T93G / H386E、W79A / Y238R、W79A / Y238R / I332G / A429G、W79A / Y238R / A429G、W79D、W79D / S80V、W79D / S80V / I332G / A429G、W79D / T93G、W79D / Y183F / N284D / I332G、S80H、S80H / Y183F / N284D / I332G / C381A / H386E / A429G、S80H / Y188F / E190D / I332G / H386E、S80H / Y238R / H386E / A429G、S80H / I332G / C381A / H386E、S80H / C381A、S80H / H386E、S80H / A429G、S80V、S80V / T237S / I332G、S80V / Y238R / I332G、S80V / N284D、S80V / N284D / H386E、S80V / I332G、S80V / I332G / H386E、S80V / H386E、S80V / H386E / A429G、N82F、N82F / P87G / N113G / P117K / L219S、N82F / P87G / N113G / P117K / R260S / C338G、N82F / P87G / P117K、N82F / P87G / T218G、N82F / P87G / T218G / L219S / C421S、N82F / P87G / T218G / C338S、N82F / P87G / C338S / H418G、N82F / P87L、N82F / P87L / N113G / P117K / T218G、N82F / P87L / N113G / P117K / L219S / H418G、N82F / P87L / N113G / P117K / C338G / H418G、N82F / P87L / P117K、N82F / P87L / L219S / C421S、N82F / P87L / C338S、N82F / N113G / T218G / C338S / H418G、N82F / H418G、N82R / P87G、N82R / P87G / N113G / C421S、N82R / P87G / P117K / T218G / C421S、N82R / P87G / P117K / R260S / C338S / C421S、N82R / P87G / P117K / C338A / H418G、N82R / P87L、N82R / P87L / N113G、N82R / P87L / T218G / L219S / C338S、N82R / P87L / L219S、N82R / P87L / C338G / C421S、N82R / N113G / T218G / C338A、N82R / N113G / L219S / C338S、N82R / N113G / C338S、N82R / T218G / L219S / C421S、N82R / R260S / C338A / C421S、N82R / C338A / H418G / C421S、P87G、P87G / N113G、P87G / N113G / P117K / H418G / C421S、P87G / N113G / R260S、P87G / P117K / L219S / L310S / C338S、P87G / T218G / L219S / C338S、P87G / L219S、P87G / R260S、P87G / L310S、P87G / L310S / C338S、P87G / C338A、P87G / H418G、P87L、P87L / N113G、P87L / N113G / P117K / L219S / C338G / H418G / C421S、P87L / N113G / P117K / R260S / C338S / C421S、P87L / N113G / T218G / L219S / R260S / C338A、P87L / N113G / L219S / C421S、P87L / P117K、P87L / P117K / T218G / L219S、P87L / T218G、P87L / T218G / L219S、P87L / T218G / H418G、P87L / L219S、P87L / L219S / C338G、P87L / L219S / H418G、P87L / C338A、P87L / C421S、T93G / D184R / A429G、T93G / Y238R、T93G / Y238R / N284D、T93G / Y238R / H386E、T93G / Y238R / H386E / A429G、S100P、S100P / E143D / E145S / D146A / L149M / E198D / M242I / C347N / P348K / L445R / C447A、S100P / E143V / E145G / L149M / C347N / C447A、S100P / E145G / D146A / L149M / F250V / C277T / C347N / P348K / C447A、S100P / E145G / L149M / H154L / I246M / C277T、S100P / E145G / L149M / E198D / M242I / I246M / L445R / C447A、S100P / E145G / L149M / E198D / D252G / P348K / L445R、S100P / E145G / L149M / S205T、S100P / E145G / L149M / M242I / I246M / C347D / P348K / L445R / C447A、S100P / E145G / L149M / M242I / L445R / C447A、S100P / E145G / E198D / M242I / I246M / P348K、S100P / E145G / E198D / C277T / P348K / L445R、S100P / E145G / E198D / A300V、S100P / E145G / S205T / I246M / C277T / P348K / L445R、S100P / E145G / M242I / I246M / C277T / L445R / C447A、S100P / E145G / P348K、S100P / E145S / D146A / L149M、S100P / E145S / D146A / L149M / H154L / P348K / L445R、S100P / E145S / D146A / L149M / E198D、S100P / E145S / D146A / L149M / E198D / M242I / F250V / D252G / C347N / P348K / C447A、S100P / E145S / D146A / L149M / S205T / C347D / L445R / C447A、S100P / E145S / D146A / L149M / M242I / L445R、S100P / E145S / L149M / E198D / M242I / I246M / C277T / P348K / C447A、S100P / E145S / L149M / E198D / M242I / C277T / C347N / P348K / C447A、S100P / E145S / L149M / M242I / I246M / C277T / C347N / L445R、S100P / E145S / L149M / I246M / D252G / C277T / C347N / L445R / C447A、S100P / E145S / L149M / I246M / C347N / L445R、S100P / E145S / L149M / C277T / P348K / L445R、S100P / E145S / L149M / C347N / L445R / C447A、S100P / E145S / S205T / C277T / P348K、S100P / E145S / I246M / C347N / P348K / L445R、S100P / E145S / C277T / C347N、S100P / D146A / L149M / E198D / M242I / I246M / C277T / C347D / P348K / L445R / C447A、S100P / D146A / E198D / C347D / P348K / C447A、S100P / D146A / E198D / L445R、S100P / D146A / M242I / C277T / C447A、S100P / D146A / F250V / C447A、S100P / D146A / C347D / P348K / L445R / C447A、S100P / L149M / E198D、S100P / L149M / M242I / I246M / C277T / C347N / P348K / L445R、S100P / L149M / M242I / I246M / C277T / C347N / P348K / C447A、S100P / L149M / M242I / C347N / P348K / C447A、S100P / L149M / I246M / C277T / P348K / L445R、S100P / L149M / I246M / C447A、S100P / L149M / F250V / C347N / C447A、S100P / E198D / S205T / P348K、S100P / E198D / C277T / C347N / P348K、S100P / E198D / C347D / P348K、S100P / E198D / C347D / P348K / L445R、S100P / E198D / C347N / L445R、S100P / E198D / C447A、S100P / M242I / I246M / C277T、S100P / M242I / I246M / C347N、S100P / M242I / C277T / P348K、S100P / M242I / C347D / P348K / L445R、S100P / M242I / C347D / P348K / L445R / C447A、S100P / M242I / P348K / L445R、S100P / I246M / C277T / P348K / L445R、S100P / C277T / C347D / L445R / C447A、S100P / C277T / P348K、S100P / C277T / P348K / C447A、S100P / C347N / P348K / L445R、S100P / C347N / C447A、S100P / L445R / C447A、K110N / C338G / H418G、N113G / T218G / C338S / C421S、N113G / C338A / C421S、N113G / C338G、P117K / L219S、P117K / R260S、F121C / E198D / C347N / L445R / C447A、E145G / D146A / L149M / C277T / C347D、E145G / D146A / L149M / C347D / P348K、E145G / D146A / C277T / C347N、E145G / D146A / C347D / C447A、E145G / L149M / E198D / C347N / C447A、E145G / L149M / S205T / M242I / C347N / P348K / L445R、E145G / L149M / S205T / I246M / F250V / D252G、E145G / L149M / M242I / P348K、E145G / L149M / I246M、E145G / K155I / L445R / C447A、E145G / E198D / S205T / P348K、E145G / F250V / D252G / P348K、E145G / S255R、E145G / C277T / C347N / L445R、E145S / D146A / L149M / H154L / I246M / C277T / C347N / P348K、E145S / D146A / L149M / E198D / M242I / D252G / C277T / C347N、E145S / D146A / L149M / E198D / C277T / L445R / C447A、E145S / D146A / L149M / S205T / P348K / C447A、E145S / D146A / L149M / F250V / C277T / C347D / P348K / C447A、E145S / D146A / E198D / S205T / M242I / C277T / P348K / C447A、E145S / D146A / E198D / L445R / C447A、E145S / D146A / I246M / C277T / C447A、E145S / D146A / I246M / C347N / P348K / C447A、E145S / L149M / E198D / S205T / P348K、E145S / L149M / E198D / M242I / I246M / C277T / C347N / C447A、E145S / L149M / E198D / M242I / I246M / P348K、E145S / L149M / E198D / M242I / C277T / C347D、E145S / L149M / E198D / I246M / C447A、E145S / L149M / S205T / C277T / L445R、E145S / L149M / M242I、E145S / L149M / I246M / C347D / L445R、E145S / L149M / I246M / C447A、E145S / L149M / C347N / P348K / L445R、E145S / L149M / C347N / C447A、E145S / L149M / C447A、E145S / I246M、E145S / I246、 M / D252G / C347D / C447A、E145S / D252G / P348K / C447A、E145S / C277T / P348K / C447A、E145S / C347D / P348K / L445R / C447A / V491A、E145S / L445R / C447A、D146A / L149M / S151C / I246M / P348K、D146A / L149M / M242I / I246M / L445R、D146A / L149M / I246M / D252G / C447A、D146A / L149M / C277T / P348K / C447A、D146A / E198D / I246M / C277T、D146A / E198D / C277T / P348K / C447A、D146A / M242I、D146A / M242I / I246M / C277T / C347N / P348K / L445R / C447A、D146A / I246M / C277T / P348K / L445R / C447A、D146A / C277T、D146A / C277T / C347N / P348K、D146A / C277T / C347N / L445R / C447A、D146A / C277T / L445R / C447A、D146A / C347D / P348K / C447A、D146A / P348K、D146A / L445R / C447A、L149M、L149M / E198D / C277T / C347N / P348K、L149M / E198D / P348K、L149M / E198D / L445R、L149M / M242I / I246M、L149M / M242I / I246M / P348K / L445R、L149M / C347N / P348K / L445R / C447A、L149M / L445R、D184R、D184R / N284D、D184R / N284D / A429G、D184R / H386E、E198D / M242I / P348K、E198D / I246M、E198D / F250V、E198D / C277T / P348K / C447A、E198D / L445R、S205T、S205T / I246M / C347N、S205T / D252G / C347D / L445R / C447A、S205T / C347D / P348K / L445R / C447A、T218G、T218G / R260S / C338A、L219S、L219S / C338A / H418G、L219S / C421S、Y238R、Y238R / M242L / N284D、Y238R / N284D / H386E / A429G, Y238R / I332G / A429G, Y238R / C381A / A429G, Y238R / H386E, Y23 8R / A429G, M242I, M242I / C277T / C347D / P348K, M242I / P348K, M242I / L445R / C447A, M242L / N284D / C381A / A429G, I246M, I246M / C277T / P348K / C447A, I246M / L445R / C447A, R260S, R26 0S / C338G, R260S / C338G / H418G / C421S, C277T / C347N / P348K / L445R, C277T / C347N / C447A, N 284D, N284D / I332G, N284D / H386E, N284D / H386E / A429G, L310S / C338S / H418G, L310S / H418 G / C421S, I332G, I332G / H386E, I332G / A429G, C338G, C338G / H418G, C347N / P348K / L445R, C In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from 347N / P348K / C447A, P348K, P348K / L445R / C447A, P348K / C447A, H386E, H386E / A429G, H418G, A429G, and C447A, where the positions are numbered with reference to SEQ ID NO:6. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any of the even-numbered sequences set forth in SEQ ID NOs:416-994.

[0024] The present invention is 80 / 429, 87 / 113 / 117 / 260 / 338 / 421, 87 / 421, 100 / 145 / 146 / 149 / 198 / 242 / 250 / 252 / 347 / 348 / 447, 100 / 145 / 149 / 198 / 242 / 277 / 347 / 348 / 447, 110 / 338 / 418, 145 / 149 / 205 / 246 / 250 / 252, 145 / 246 / 252 / 347 / 447, 145 / 252 / 348 / 447, 238 / 386 Also provided is an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NO:6, comprising at least one substitution or set of substitutions selected from 242 / 284 / 381 / 429, and 242 / 284 / 381 / 429, wherein positions are numbered with reference to SEQ ID NO:6. In some embodiments, the engineered polypeptide is 80H / 429G, 87L / 113G / 117K / 260S / 338S / 421S, 87L / 421S, 100P / 145S / 146A / 149M / 198D / 242I / 250V / 252G / 347N / 348K / 447A, 100P / 145S / 149M / 198D / 242I / 277T / 347N / 348K / 447A, 110N / 338 G / 418G, 145G / 149M / 205T / 246M / 250V / 252G, 145S / 246M / 252G / 347D / 447A, 145S / 252G / 348K / 447A, 238R / 386E / 429G, and 242L / 284D / 381A / 429G, where positions are numbered with reference to SEQ ID NO:6.In some further embodiments, the engineered polypeptide is selected from the group consisting of S80H / A429G, P87L / N113G / P117K / R260S / C338S / C421S, P87L / C421S, S100P / E145S / D146A / L149M / E198D / M242I / F250V / D252G / C347N / P348K / C447A, S100P / E145S / L149M / E198D / M242I / C277T / C347N / P348K / C447A, K110 N / C338G / H418G, E145G / L149M / S205T / I246M / F250V / D252G, E145S / I246M / D252G / C347D / C447A, E145S / D252G / P348K / C447A, Y238R / H386E / A429G, and M242L / N284D / C381A / A429G, wherein positions are numbered with reference to SEQ ID NO:6.

[0025] The present invention is directed to 87 / 113 / 145 / 198 / 242 / 246 / 262 / 266, 87 / 117 / 149 / 242, 87 / 117 / 242 / 260 / 266, 87 / 145 / 149 / 198 / 447, 87 / 149 / 242 / 260 / 266, 87 / 149 / 260 / 262 / 266 / 381, 87 / 198, 87 / 242 / 246 / 260 / 262 / 266 / 338, 87 / 284 / 338 / 347, 113 / 117 / 146 / 149 / 260 / 262, 113 / 117 / 266 / 381, 113 / 1 17 / 447, 113 / 145 / 146 / 149 / 242 / 447, 113 / 242 / 347, 113 / 242 / 381, 117 / 145 / 146 / 262 / 284, 117 / 145 / 149 / 284 / 447, 117 / 149, 117 / 198, 117 / 198 / 262, 117 / 242 / 246 / 260 / 266 / 284 / 381, 117 / 242 / 260 / 262, 117 / 246 / 260 / 262 / 381, 117 / 246 / 284, 117 / 266, 145 / 146 / 149 / 260 / 262 / 338 / 347, 145 / 146 / 242 / 260 / 262 / 338 / 347 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 145 / 149 / 260 / 266 / 338 / 347 / 381 / 447, 146 / 149 / 260 / 262 / 347, 149 / 198 / 246 / 260 / 266 / 421, 198 / 447, 242, 242 / 246, 242 / 246 / 338 / 347, 242 / 260 / 262 / 338, 242 / 381, 246, 260 / 262 / 277 / 284 / Also provided is an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NO: 414, comprising at least one substitution or set of substitutions at one or more positions selected from 447, 260 / 266, 262, 347 / 381, and 447, wherein the positions are numbered with reference to SEQ ID NO: 414. In some additional embodiments, the engineered polypeptide comprises 87G / 113G / 145L / 198D / 242I / 246M / 262F / 266L, 87G / 117K / 149M / 242I,87G / 145L / 149M / 198D / 447A, 87G / 149M / 242I / 260S / 266L, 87L / 117K / 242I / 260S / 266L, 87L / 149M / 260S / 262F / 266L / 381A, 87L / 198D, 87L / 242I / 246M / 260S / 262F / 266L / 338G, 87L / 284D / 338G / 347N, 113G / 117K / 146A / 149M / 260S / 262F, 113G / 117K / 266L / 381A, 113G / 11 7K / 447A, 113G / 145L / 146A / 149M / 242I / 447A, 113G / 242I / 347N, 113G / 242I / 381A, 117K / 145L / 146A / 262F / 284D, 117K / 145L / 149M / 28 4D / 447A, 117K / 149M, 117K / 198D, 117K / 198D / 262F, 117K / 242I / 246M / 260S / 266L / 284D / 381A, 117K / 242I / 260S / 262F, 117K / 246M / 260 S / 262F / 381A, 117K / 246M / 284D, 117K / 266L, 145L / 146A / 149M / 260S / 262F / 338G / 347N, 145L / 146A / 242I / 260S / 262F / 338G / 347N / 447 A, 145L / 149M / 198D / 260S / 266L / 338G / 347N, 145L / 149M / 260S / 266L / 338G / 347N / 381A / 447A, 146A / 149M / 260S / 262F / 347N, 149M / 198 D / 246M / 260S / 266L / 421S, 198D / 447A, 242I, 242I / 246M, 242I / 246M / 338G / 347N, 242I / 260S / 262F / 338G, 242I / 381A, 246M, 260S / 262F / 277T / 284D / 447A, 260S / 266L, 262F, 347N / 381A, and 447A, wherein the positions are numbered with reference to SEQ ID NO:414. In some embodiments, the engineered polypeptide is P87G / N113G / E145L / E198D / M242I / I246M / W262F / A266L, P87G / P117K / L149M / M242I,P87G / E145L / L149M / E198D / C447A、P87G / L149M / M242I / R260S / A266L、P87L / P117K / M242I / R260S / A266L、P87L / L149M / R260S / W262F / A266L / C381A、P87L / E198D、P87L / M242I / I246M / R260S / W262F / A266L / C338G、P87L / N284D / C338G / C347N、N113G / P117K / D146A / L149M / R260S / W262F、N113G / P117K / A266L / C381A、N113G / P117K / C447A、N113G / E145L / D146A / L149M / M242I / C447A、N113G / M242I / C347N、N113G / M242I / C381A、P117K / E145L / D146A / W262F / N284D、P117K / E145L / L149M / N284D / C447A、P117K / L149M、P117K / E198D、P117K / E198D / W262F、P117K / M242I / I246M / R260S / A266L / N284D / C381A、P117K / M242I / R260S / W262F、P117K / I246M / R260S / W262F / C381A、P117K / I246M / N284D、P117K / A266L、E145L / D146A / L149M / R260S / W262F / C338G / C347N、E145L / D146A / M242I / R260S / W262F / C338G / C347N / C447A、E145L / L149M / E198D / R260S / A266L / C338G / C347N、E145L / L149M / R260S / A266L / C338G / C347N / C381A / C447A、D146A / L149M / R260S / W262F / C347N、L149M / E198D / I246M / R260S / A266L / C421S、E198D / C447A、M242I、M242I / I246M、M242I / I246M / C338G / C347N、M242I / R260S / W262F / C338G、M242I / C381A、I246M、R260S / W262F / C277T / N284D / C447A、R260S / A266L、W262F、C347N / C381A、and C447A, where positions are numbered with reference to SEQ ID NO: 414. In some further embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any of the even-numbered sequences set forth in SEQ ID NOs: 1000 to 1084.

[0026] The present invention relates to at least one of the following: 87 / 145 / 149 / 198 / 447, 87 / 149 / 260 / 262 / 266 / 381, 113 / 117 / 146 / 149 / 260 / 262, 117 / 145 / 149 / 284 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 146 / 149 / 260 / 262 / 347, and 149 / 198 / 246 / 260 / 266 / 421. Also provided is an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NO:414, including at least one substitution or set of substitutions, wherein positions are numbered with reference to SEQ ID NO:414. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from 87G / 145L / 149M / 198D / 447A, 87L / 149M / 260S / 262F / 266L / 381A, 113G / 117K / 146A / 149M / 260S / 262F, 117K / 145L / 149M / 284D / 447A, 145L / 149M / 198D / 260S / 266L / 338G / 347N, 146A / 149M / 260S / 262F / 347N, and 149M / 198D / 246M / 260S / 266L / 421S, where the positions are numbered with reference to SEQ ID NO:414. In some further embodiments, the engineered polypeptide is P87G / E145L / L149M / E198D / C447A, P87L / L149M / R260S / W262F / A266L / C381A, N113G / P117K / D146A / L149M / R260S / W262F, P117K / E145L / L149M / N284D / C447A, E145L / L and at least one substitution or set of substitutions selected from 149M / E198D / R260S / A266L / C338G / C347N, D146A / L149M / R260S / W262F / C347N, and L149M / E198D / I246M / R260S / A266L / C421S, where positions are numbered with reference to SEQ ID NO:414.

[0027] In some embodiments, the engineered monoamine oxidase polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution or set of substitutions selected from 429, 149, 198, 246, 260, 266, and 421, where the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:6. In some embodiments, the engineered monoamine oxidase polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution selected from 429G, 149M, 198D, 246M, 260S, 266L, and 421S, where the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:6. In some embodiments, the engineered monoamine oxidase polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution selected from A429G, L149M, E198D, I246M, R260S, A266L, and C421S, where the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:6.

[0028] The present invention also provides engineered polynucleotides encoding at least one engineered polypeptide described in the paragraph above. In some embodiments, the engineered polynucleotide comprises the odd-numbered sequences set forth in SEQ ID NO:7 through SEQ ID NO:1083.

[0029] The present invention further provides a vector comprising at least one engineered polynucleotide as described above. In some embodiments, the vector further comprises at least one regulatory sequence.

[0030] The present invention also provides host cells comprising the vectors provided herein. In some embodiments, the host cells produce at least one engineered polypeptide provided herein.

[0031] The invention further provides a method of producing an engineered monoamine oxidase comprising culturing a host cell provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced, in some embodiments, the method further comprises recovering the engineered polypeptide.

[0032] The present invention further provides methods of using the engineered monoamine oxidases to produce various fused bicyclic proline analogs, in some embodiments, the methods further comprise steps for isolating and recovering the fused bicyclic proline analogs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Description of the Invention Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In general, the nomenclature used herein and the experimental procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are well known and commonly used in the art. Such techniques are well known and described in numerous textbooks and references well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis. All patents, patent applications, papers and publications mentioned herein both above and below are hereby expressly incorporated by reference into this specification.

[0034] Any suitable method and material similar or equivalent to those described herein can be used in the practice of the present invention, and some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodology, protocols and reagents described, since these methodologies, protocols and reagents can vary depending on the context in which they are used by those skilled in the art.Therefore, the terms defined immediately below are more fully explained by referring to the present invention in general.

[0035] It should be understood that both the summary above and the detailed description below are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and should not be considered as limiting the subject matter described. Numerical ranges are inclusive of the numbers that define 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 limit disclosed herein is also intended to include every lower (upper) numerical limit, as if such lower (upper) numerical limit were expressly written herein.

[0036] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a polypeptide" includes more than one polypeptide. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0037] It will be understood by those skilled in the art that when the description of various embodiments uses the term "comprising", in some specific instances, the embodiment may instead be described using the words "consisting essentially of" or "consisting of". When the description of various embodiments uses the terms "optionally" and "optionally", it is further understood that the event or circumstance described thereafter may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the present disclosure. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0038] Abbreviation: The abbreviations used for the genetically encoded amino acids are conventional and are as follows: [Table A]

[0039] When three-letter abbreviations are used, the amino acid may be in the L or D configuration about the alpha carbon (Cα), unless specifically preceded by "L" or "D" or otherwise clear from the context in which the abbreviation is used. For example, "Ala" indicates alanine with no designation of configuration about the alpha carbon, while "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively.

[0040] When one-letter abbreviations are used, an upper case letter indicates an amino acid in the L-configuration about the alpha carbon, and a lower case letter indicates an amino acid in the D-configuration about the alpha carbon. For example, "A" indicates L-alanine and "a" indicates D-alanine. When a polypeptide sequence is presented as a series of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxy (C) direction, according to common convention.

[0041] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless otherwise specified, the abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as either ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is presented as a series of one-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, with no phosphate indicated.

[0042] definition With respect to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by those skilled in the art unless otherwise defined. Accordingly, the following terms are intended to have the following meanings:

[0043] "EC" numbers refer to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0044] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0045] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0046] "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, myristoylation, ubiquitination, etc.). This definition includes D-amino acids, L-amino acids, mixtures of D- and L-amino acids, polymers comprising D-amino acids, polymers comprising L-amino acids, and polymers comprising mixtures of D- and L-amino acids.

[0047] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter codes.

[0048] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides that are covalently linked together. A polynucleotide may be composed entirely of ribonucleotides (i.e., RNA), entirely of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2' deoxyribonucleotides. Nucleosides are typically linked together by standard phosphodiester linkages, although a polynucleotide may contain one or more non-standard linkages. A polynucleotide may be single-stranded or double-stranded, or may contain both single-stranded and double-stranded regions. Additionally, a polynucleotide is typically composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), but may also contain one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, and the like. In some embodiments, such modified or synthetic nucleobases are nucleobases that code for an amino acid sequence.

[0049] "Monoamine oxidase" or "MAO" as used herein refers to a wild-type or engineered enzyme (EC 1.4.3.4) with monoamine oxidase activity that naturally catalyzes the oxidation of primary amines (or sometimes secondary or tertiary amines) to the corresponding imines. In one embodiment, the monoamine oxidase polypeptide variant catalyzes the asymmetric oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene. The definition of monoamine oxidase activity is intended to be non-limiting with respect to substrates and products, and various examples of known substrates and products are presented in this disclosure. The monoamine oxidase of the present disclosure is a flavoprotein that requires a FAD cofactor. The monoamine oxidase enzymes of the present disclosure are derived from Aspergillus niger and Aspergillus oryzae, although the disclosure is not so limited and the monoamine oxidase enzymes may be derived from any suitable organism, or the monoamine oxidase enzymes may be synthetically produced. Monoamine oxidase, as used herein, includes naturally occurring (wild-type) monoamine oxidases as well as non-naturally occurring engineered polypeptides produced by human manipulation.

[0050] "Coding sequence" refers to that portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

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

[0052] As used herein, "recombinant," "engineered," and "non-naturally occurring" when used with respect to a cell, nucleic acid, or polypeptide refer to a material that has been modified in a manner that would not otherwise occur in nature, or that corresponds to the natural or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived 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, or that express native genes that would otherwise be expressed at different levels, among others.

[0053] The terms "percent sequence identity" and "percent homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to optimal alignment of these two sequences. The percentage can be calculated by determining the number of positions where the identical nucleic acid base or amino acid residue appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions where either the identical nucleic acid base or amino acid residue appears in both sequences or the number of positions where the nucleic acid base or amino acid residue is aligned with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art understand that there are many established algorithms available for aligning two sequences.The optimal alignment of sequences for comparison can be performed, for example, by Smith and Waterman's local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482

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

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

[1988] ), by computer implementation of these algorithms (for example, GAP, BESTFIT, FASTA and TFASTA in GCG Wisconsin Software Package), or by visual inspection, as known in the art.Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215: 403-410

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

[1977] ). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a matching residue pair, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

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

[0054] "Reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. In general, 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 a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) further contain a sequence that differs between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, in which case the reference sequence is a sequence that may have one or more changes in the primary sequence. For example, a "reference sequence based on SEQ ID NO:4 with a valine at the residue corresponding to X14," or X14V, refers to a reference sequence in which the corresponding residue at X14 in SEQ ID NO:4, which is a tyrosine, has been changed to a valine.

[0055] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, including windows of 30, 40, 50, 100 or more, as appropriate.

[0056] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80 percent sequence identity, at least 85 percent identity, between at least 89-95 percent sequence identity, or more usually at least 99 percent sequence identity, relative to a reference sequence, over a comparison window of at least 20 residue positions, often over a window of at least 30-50 residues, where the sequence identity percentage is calculated by comparing the reference sequence over the comparison window to a sequence that contains deletions or additions totaling 20 percent or less of the reference sequence. In some specific embodiments as applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or higher sequence identity (e.g., 99 percent sequence identity), when optimally aligned, e.g., optimally aligned by the programs GAP or BESTFIT using default gap weights. In some embodiments, residue positions that are not identical in the compared sequences differ by conservative amino acid substitutions.

[0057] "Corresponding to," "to," and "compared to" when used in the context of numbering a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are given relative to the reference sequence, not according to the actual number positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, e.g., an engineered monoamine oxidase, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although gaps are present, the numbering of the residues in the given amino acid or polynucleotide sequence is done relative to the reference sequence to which it is aligned.

[0058] An "amino acid difference" or "residue difference" refers to a change in an amino acid residue at a position of a polypeptide sequence compared to an amino acid residue at a corresponding position in a reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering is maintained compared to an equivalent reference sequence without the histidine tag. 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 to which the residue difference is based. For example, a "residue difference at position X25 compared to SEQ ID NO:2" refers to a change in the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a valine at position 25, then a "residue difference at position X25 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than valine at the polypeptide position corresponding to position 25 of SEQ ID NO:2. In most instances herein, a particular amino acid residue difference at a position is indicated as "XnY", where "Xn" designates the corresponding position as described above, and "Y" is a one-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from that in the reference polypeptide). In some embodiments, more than one amino acid may appear at a specified residue position (i.e., alternative amino acids may be listed in the form XnY / Z, where Y and Z represent alternative amino acid residues). In some cases (e.g., in Tables 4.2, 5.1, 6.1, 7.1, 8.1 and 9.1), the invention also provides specific amino acid differences, designated by the conventional designation "AnB," where A is a one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is a one-letter identifier of the residue substitution in the engineered polypeptide sequence. Additionally, in some cases, the polypeptides of the invention contain one or more amino acid residue differences compared to the reference sequence, as indicated by a list of the specific positions that are changed compared to the reference sequence. In some additional embodiments, the invention provides engineered polypeptide sequences that include both conservative and non-conservative amino acid substitutions. As used herein, "conservative amino acid substitution" refers to the substitution of a residue with a different residue that has a similar side chain, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acid.By way of example, and without limitation, an amino acid with an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine and isoleucine); an amino acid with a hydroxyl side chain is replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan and histidine); an amino acid with a basic side chain is replaced with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced with another amino acid with 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. Exemplary conservative substitutions are provided in Table 1 below. [Table 1]

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

[0060] "Deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletion can 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, or up to 20% of the total number of amino acids, constituting the reference enzyme, while retaining the enzymatic activity and / or improving properties of the engineered monoamine oxidase enzyme. Deletion can concern internal and / or terminal parts of the polypeptide. In various embodiments, deletion can include a continuous segment or can be discontinuous.

[0061] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered monoamine oxidase enzyme includes the insertion of one or more amino acids into a naturally occurring monoamine oxidase polypeptide as well as into other improved monoamine oxidase polypeptides. The insertion may be in an internal portion of the polypeptide or at the carboxy or amino terminus. An insertion as used herein includes fusion proteins as known in the art. An insertion may be a contiguous segment of amino acids in a naturally occurring polypeptide or may be separated by one or more amino acids.

[0062] "Fragment" is used herein to refer to a polypeptide having an amino- and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or more, and can be up to 70%, 80%, 90%, 95%, 98% and 99% of a full-length monoamine oxidase polypeptide, e.g., the polypeptide of SEQ ID NO: 6, or an engineered monoamine oxidase provided in the even-numbered sequences of SEQ ID NOs: 6-1084.

[0063] An "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cell or in vitro synthesis). Engineered monoamine oxidase enzymes may be present within a cell, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, an engineered monoamine oxidase enzyme may be an isolated polypeptide.

[0064] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis), and is generally a substantially purified composition when the target species constitutes at least about 50 percent, by molar or weight percent, of the macromolecular species present. Generally, a substantially pure monoamine oxidase composition constitutes about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more, by molar or weight percent, of the total macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity, such that the composition consists essentially of a single macromolecular species (i.e., contaminants cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated engineered monoamine oxidase enzyme is a substantially pure polypeptide composition.

[0065] "Stereoselective" refers to the preference for the formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, when one stereoisomer is preferred over the other, or complete, when only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity, which is the fraction (typically reported as a percentage) of one enantiomer in the sum of both enantiomers. This is generally instead reported in the art as the enantiomeric excess (ee) (typically as a percentage), which is calculated therefrom according to the formula [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, which is the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is generally reported instead as diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0066] "Highly stereoselective" refers to a chemical or enzymatic reaction that can convert a substrate or substrates (e.g., the substrate compound 6,6-dimethyl-3-azabicyclo[3.1.0]hexane) to the corresponding imine product (e.g., to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene) in at least about 85% stereoisomeric excess.

[0067] As used herein, "improved enzymatic property" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered monoamine oxidase polypeptides that exhibit an improvement in any enzymatic property compared to a reference monoamine oxidase polypeptide or another engineered monoamine oxidase polypeptide. For the engineered monoamine oxidase polypeptides described herein, the comparison is generally made with the parent enzyme from which the monoamine oxidase is derived, although in some embodiments, the reference enzyme may be another improved engineered monoamine oxidase. Thus, the level of "improvement" may be determined and compared between various monoamine oxidase polypeptides, including wild-type as well as engineered monoamine oxidases. Improved properties include, but are not limited to, properties such as enzyme activity (which may be expressed in terms of percent conversion of substrate), thermostability, solvent stability, pH activity profile, cofactor requirements, recalcitrance to inhibitors (e.g., substrate or product inhibition), stereospecificity, and / or stereoselectivity (including enantioselectivity).

[0068] "Increased enzymatic activity" refers to an improved property of an engineered monoamine oxidase polypeptide, which may be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percent conversion of substrate to product (e.g., the percent conversion of a starting amount of substrate to product within a specified period of time using a specified amount of monoamine oxidase) compared to a reference monoamine oxidase enzyme. Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max or k catAny property related to enzyme activity can be affected, including classical enzyme properties (these changes can lead to increased enzyme activity). The improvement in enzyme activity can be from about 1.2 times the enzyme activity of the corresponding parent enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold or more of the enzyme activity of the naturally occurring or another engineered monoamine oxidase from which the monoamine oxidase polypeptide is derived. Monoamine oxidase activity can be measured by any one of the standard assays, for example, by monitoring changes in the properties of the substrate, cofactor or product. In some embodiments, the amount of product produced can be measured by liquid chromatography-mass spectrometry (LC-MS). The 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 further described in detail herein. In general, when lysates are compared, the number of cells and the amount of protein to be assayed are determined, as well as the use of the same expression system and the same host cells to minimize the variation in the amount of enzyme produced by the host cells and present in the lysate.

[0069] "Conversion" refers to the enzymatic conversion of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product under specified conditions and within a period of time. Thus, the "enzyme activity" or "activity" of a monoamine oxidase peptide can be expressed as the "percent conversion" of a substrate to a product.

[0070] "Thermostable" refers to a monoamine oxidase polypeptide that, after exposure to elevated temperatures (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hours), maintains similar activity (e.g., 60%-80% greater) compared to the parent enzyme exposed to the same elevated temperature.

[0071] "Solvent-stable" refers to a monoamine oxidase polypeptide that maintains similar activity (e.g., 60%-80% greater) after exposure to various concentrations (e.g., 5-99%) of a solvent (e.g., ethanol, isopropyl alcohol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours) compared to the parent enzyme exposed to the same solvent at the same concentration.

[0072] "Heat and solvent stable" refers to a monoamine oxidase polypeptide that is both heat stable and solvent stable.

[0073] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, hybrid stability is determined by the melting temperature (T m In general, hybrid stability is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. mValues ​​can be calculated using known methods for predicting melting temperatures (e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ; Freier et al., 1990, Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ; Kierzek et al., Biochem., 25:7840-7846

[1986] ; Rychlik et al., Nucl. Acids Res., 18:6409-6412

[1990] ). (See errata, Nucl. Acids Res., 19:698

[1991] ; Sambrook et al., supra); Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown et al. [eds.], pp. 683-693, Academic Press, Cambridge, MA

[1981] ; and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-259

[1991] .) In some embodiments, the polynucleotides encode the polypeptides disclosed herein and hybridize under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered monoamine oxidase enzyme of the invention.

[0074] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, during nucleic acid hybridization. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washing of various but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow the target DNA to bind to complementary nucleic acids with about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, and more than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are those that correspond to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency ... m Generally, high stringency conditions refer to conditions that are about 10°C or less from 0.018M NaCl. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 65°C in 0.018M NaCl (i.e., if a hybrid is not stable at 65°C in 0.018M NaCl, it will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization in 5x SSC containing 0.1% (w / v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other highly stringent hybridization conditions, and moderately stringent conditions, are described in the references above.

[0075] A "heterologous" polynucleotide is any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that have been removed from the host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0076] "Codon-optimized" refers to the change of codons in a polynucleotide encoding a protein to those preferentially used in that particular organism such that the encoded protein is efficiently expressed in that organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons, called "synonymous" or "synonymous" codons, it is well known that the codon usage by a particular organism is not random, but is biased toward certain codon triplets. This bias in codon usage may be more severe for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of the organism's genome. In some embodiments, a polynucleotide encoding a monoamine oxidase enzyme may be codon-optimized for optimal production from the host organism selected for expression.

[0077] As used herein, "preferred, optimal, high codon usage bias codons" refer synonymously to codons that are used in protein coding regions at a higher frequency than other codons that code for the same amino acid. Preferred codons may be determined with respect to codon usage frequency in a single gene, a set of genes of common function or origin, highly expressed genes, with respect to codon frequency in aggregate protein coding regions across organisms, with respect to codon frequency in aggregate protein coding regions of related organisms, or with respect to a combination thereof. Codons whose frequency increases with gene expression levels are typically optimal codons for expression. A variety of methods are known for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, including multivariate analyses, for example using cluster analysis or correspondence analysis, as well as for determining the effective number of codons used in a gene (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

[1998] ; Stenico et al., Nucl. Acids Res., 222437-46

[1994] ; Wright, Gene 87:23-29

[1990] ). Codon usage tables are available for many different organisms (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret, et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington DC, p. 2047-2066

[1996] ).The data source for obtaining codon usage frequency can be based on any available nucleotide sequence that can code for a protein. These data sets include nucleic acid sequences that are actually known to code for expressed proteins (e.g., complete protein coding sequences-CDS), to code for expressed sequence tags (ESTS), or to code for predicted coding regions of genomic sequences (see, for example, Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0078] "Control sequences" are 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 nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequence includes a promoter, and transcriptional and translational stop signals. The control sequence may also be provided with linkers to introduce specific restriction sites that facilitate ligation of the control sequence with the coding region of the nucleic acid sequence encoding the polypeptide.

[0079] "Operably linked" is defined herein as a configuration in which a control sequence is positioned relative to a polynucleotide of interest such that the control sequence is suitable (i.e., in a functional relationship) to direct or regulate expression of the polynucleotide and / or polypeptide of interest.

[0080] "Promoter sequence" refers to a nucleic acid sequence, e.g., a coding sequence, that is recognized by a host cell for expression of a polynucleotide of interest. The promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and the promoter can be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0081] "Suitable reaction conditions" refers to conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, cosolvents, etc.) that allow a monoamine oxidase polypeptide of the invention to convert one or more substrate compounds to product compounds (e.g., conversion of a compound of Formula I to a compound of Formula II as shown in Scheme 1). Exemplary "suitable reaction conditions" are provided herein and illustrated by the Examples.

[0082] "Composition" refers to a mixture or combination of one or more substances, where each substance or component of the composition retains its individual properties. As used herein, a biocatalyst composition refers to a combination of one or more substances useful for biocatalysis.

[0083] "Loading" as in "compound loading" or "enzyme loading" or "cofactor loading" refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0084] "Substrate," in the context of a biocatalyst-mediated process, refers to a compound or molecule that is acted upon by the biocatalyst. For example, 6,6-dimethyl-3-azabicyclo[3.1.0]hexane is a substrate for monoamine oxidase.

[0085] "Product" in the context of a biocatalyst-mediated process refers to a compound or molecule that results from the action of a biocatalyst. For example, (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene is the product of monoamine oxidase-mediated conversion of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0086] "Alkyl" refers to a saturated hydrocarbon group, either linear or branched, having from 1 to 18 carbon atoms inclusive, more preferably from 1 to 8 carbon atoms inclusive, and most preferably from 1 to 6 carbon atoms inclusive. Alkyl groups having a specified number of carbon atoms are shown in parentheses (e.g., (C1-C6)alkyl refers to alkyl having 1 to 6 carbon atoms).

[0087] "Alkenyl" refers to a hydrocarbon group of from 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one double bond, but optionally containing more than one double bond.

[0088] "Alkynyl" refers to a hydrocarbon group of from 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one triple bond, but optionally containing more than one triple bond.

[0089] "Heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl, respectively, 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 can replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR α -, -PH-, -S(O)-, -S(O)2-, -S(O)NR α -, -S(O)NR α -, etc., including combinations thereof, where each R αis independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0090] "Aryl" refers to an unsaturated aromatic carbocyclic group having from 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.

[0091] "Arylalkyl" refers to an aryl-substituted alkyl (i.e., aryl-alkyl) group preferably having 1 to 6 (inclusive) carbon atoms in the alkyl moiety and 6 to 12 (inclusive) carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl, and the like.

[0092] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 12 carbon atoms (inclusive) having a single cyclic ring or multiple fused rings, which may be optionally substituted with 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, single ring structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures, including bridged ring systems, such as adamantyl, and the like.

[0093] "Cycloalkylalkyl" refers to a cycloalkyl-substituted alkyl (i.e., a cycloalkyl-alkyl group) preferably having 1 to 6 (inclusive) carbon atoms in the alkyl portion and 3 to 12 (inclusive) carbon atoms in the cycloalkyl portion. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl, and the like.

[0094] "Amino" refers to the group -NH2. Substituted amino refers to the group -NHR δ , N.R.δ R δ and N.R. δ R δ R δ (Each R in the formula δ is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, etc. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, and the like.

[0095] "Aminoalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.

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

[0097] "Oxy" refers to the divalent group -O-, which can have a variety of substituents to form different oxy groups including ethers and esters.

[0098] The terms "alkoxy" and "alkyloxy" are used interchangeably herein to refer to the group -ORβ, where Rβ is an alkyl group, including an optionally substituted alkyl group.

[0099] "Carboxy" refers to --COOH.

[0100] "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.

[0101] "Halogen" or "halo" refers to fluoro, chloro, bromo and iodo.

[0102] "Haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with halogen. Thus, the term "haloalkyl" is intended to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc. up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0103] "Hydroxy" refers to --OH.

[0104] "Hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms is replaced with one or more hydroxy groups.

[0105] A "thiol" or "sulfanyl" refers to -SH. A substituted thiol or sulfanyl is -SR η (R in the formula η is an alkyl, aryl or other suitable substituent.

[0106] "Alkylthio" is -SR ζ (R in the formula ζ is an optionally substituted alkyl. Typical alkylthio groups include, but are not limited to, methylthio, ethylthio, n-propylthio, and the like.

[0107] An "alkylthioalkyl" is an alkylthio group -SR ζ (R in the formula ζ refers to alkyl, substituted with (which is optionally substituted alkyl).

[0108] "Sulfonyl" refers to -SO2-. Substituted sulfonyl is -SO2-R η (R in the formula η is an alkyl, aryl or other suitable substituent.

[0109] "Alkylsulfonyl" is -SO2-R ζ (R in the formula ζ is an optionally substituted alkyl. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0110] "Alkylsulfonylalkyl" refers to the alkylsulfonyl group -SO2-R ζ (R in the formula ζ refers to alkyl, substituted with (which is optionally substituted alkyl).

[0111] "Heteroaryl" refers to an aromatic heterocyclic group having from 1 to 10 (inclusive) carbon atoms and from 1 to 4 (inclusive) heteroatoms selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0112] "Heteroarylalkyl" refers to a heteroaryl-substituted alkyl (i.e., heteroaryl-alkyl) group preferably having 1 to 6 (inclusive) carbon atoms in the alkyl portion and 5 to 12 (inclusive) ring atoms in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl, and the like.

[0113] "Heterocycle", "heterocyclic", and equivalently "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings containing from 2 to 10 carbon ring atoms (inclusive) and from 1 to 4 heterocyclic atoms selected from nitrogen, sulfur, or oxygen in the ring (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 heterocycles 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.

[0114] "Membered ring" is intended to encompass 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 six-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are five-membered rings.

[0115] "Fused bicyclic ring," as used herein, refers to both unsubstituted and substituted carbocyclic and / or heterocyclic ring moieties having from 3 to 8 atoms in each ring and where the rings have two atoms in common.

[0116] "Optionally substituted," as used herein with respect to the chemical groups described above, means that the position of the chemical group occupied by a hydrogen is replaced with another atom (unless otherwise specified), exemplified but not limited to, carbon, oxygen, nitrogen, or sulfur, or with another atom, such as 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 amine, or the like. wherein the heteroatoms may be optionally substituted with chemical groups exemplified by, but not limited to, no, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, 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, with preferred heteroatoms being oxygen, nitrogen, and sulfur.Furthermore, when free valences exist on the chemical groups of these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, when these free valences exist on carbon, they may be further substituted with halogens, and with oxygen-, nitrogen- or sulfur-linked substituents, and when there are multiple such free valences, these groups may be connected to form rings, either by direct formation of a bond or by formation of a bond to a new heteroatom, preferably oxygen, nitrogen or sulfur. It is further contemplated that the above substitutions may be made provided that the replacement of hydrogen with a substituent does not introduce unacceptable instability to the molecules of the invention and is otherwise chemically reasonable. For any chemical group described as being multiple optionally substituted, those skilled in the art will understand that only sterically realistic and / or synthetically feasible chemical groups are intended to be included.As used herein, "optionally substituted" refers to all subsequent modifiers in a chemical group term or series of chemical groups.For example, in the term "optionally substituted arylalkyl", the "alkyl" and "aryl" portions of the molecule can be substituted or unsubstituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups can be substituted or unsubstituted independently of each other.

[0117] "Reaction," as used herein, refers to a process or processes by which one or more substances or compounds or substrates are converted into one or more different substances, compounds.

[0118] Oxidation of Bicyclic Proline Analog Amines to Imines The present invention provides a novel biocatalyst and related method that is useful for the oxidative desymmetrization of fused bicyclic proline analogs to produce APIs and intermediates.The novel biocatalyst of the present disclosure is an engineered monoamine oxidase enzyme that has improved solubility, thermal stability, and activity towards fused bicyclic proline compounds compared to reference monoamine oxidases.In particular, the engineered monoamine oxidase enzyme of the present disclosure requires a reduced enzyme loading (wt / wt) in the production of APIs and intermediates compared to reference monoamine oxidase enzymes.

[0119] The monoamine oxidase biocatalysts of the present disclosure are engineered polypeptide variants of previously engineered monoamine oxidases (SEQ ID NO:6) derived from Aspergillus niger monoamine oxidase (SEQ ID NO:2) and Aspergillus oryzae monoamine oxidase (SEQ ID NO:4). WO2010 / 008828A2. These engineered polypeptides can catalyze the asymmetric oxidation of various fused bicyclic proline analog amine compounds to imine compounds. In one embodiment, the monoamine oxidase polypeptide variant catalyzes the conversion of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene. In other embodiments, the engineered monoamine oxidase enzymes of the present disclosure are used to produce various bicyclic proline analogs.

[0120] Thus, in specific embodiments, the present disclosure provides engineered monoamine oxidase enzymes for the preparation of the following fused bicyclic proline compounds, which are useful as intermediates in the synthesis of one or more therapeutic agents: [ka]

[0121] In certain other specific embodiments, the present disclosure provides a biocatalytic process for the synthesis of the following fused bicyclic proline compounds, useful as intermediates in the synthesis of one or more therapeutic agents: [ka]

[0122] Thus, the present disclosure provides engineered monoamine oxidase enzymes for the synthesis of fused bicyclic proline compounds useful as intermediates in the synthesis of one or more therapeutic agents, and biocatalytic processes for the synthesis of these fused bicyclic proline compounds. The engineered monoamine oxidase enzymes of the present disclosure are useful in the biocatalytic processes disclosed below. [ka]

[0123] Scheme 1 depicts the reaction catalyzed by an engineered monoamine oxidase enzyme of the present disclosure in which a secondary amine, i.e., a heterobicyclic compound according to structural formula I, is oxidized to the corresponding imine compound of structural formula II.

[0124] In some embodiments, the present disclosure provides a substantially stereoisomerically pure compound according to structural formula II: [ka] (Wherein, A is O, CR 1 R 2 , -C=C-, or -CH-CH-, where R 1 and R 2 are each independently selected from -H, -COOH, -X, -NH2, -CH2NHC(NH)NH2, -CX3, -CH3, -CH2CH3, where X is selected from F, Cl and Br. including its salts and hydrates, Amine compounds according to structural formula I [ka] wherein A is as defined for structural formula II. with oxygen in the presence of an engineered monoamine oxidase enzyme with a cofactor under conditions whereby the engineered monoamine oxidase enzyme oxidizes the amine compound of structural formula I to the corresponding imine compound of structural formula II.

[0125] In certain embodiments, the oxidized imine compound of formula II can be isolated from the reaction mixture, purified, and characterized. In other embodiments, described below, the imine compound of formula II can be converted to another adduct or intermediate and used in a subsequent step without isolation or purification. [ka]

[0126] In some embodiments, the present disclosure provides a mixture comprising a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b): [ka] (Wherein, A is O, CR 1 R 2 , -C=C-, or -CH-CH-, where R 1 and R 2 are each independently selected from -H, -COOH, -X, -NH2, -CH2NHC(NH)NH2, -CX3, -CH3, -CH2CH3, where X is selected from F, Cl and Br. including its salts and hydrates, Amine compounds according to structural formula I [ka] wherein A is as defined for structural formula III. with oxygen in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite under conditions to produce a mixture comprising a substantially enantiomerically aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically aminosulfonic acid compound according to structural formula III(b). Includes.

[0127] In certain embodiments, the engineered monoamine oxidases of the present disclosure may be inhibited by the product imine of structural formula II. Thus, in certain embodiments, the reaction in which a compound of structural formula I is oxidized to a compound of structural formula II further includes an agent that reacts with the imine compound of structural formula II as depicted in Scheme 2 to form an adduct that has reduced or eliminated ability to inhibit the engineered monoamine oxidases of the present disclosure.

[0128] In one aspect of this embodiment, the agent is added at the beginning of the reaction or intermittently or continuously in an amount large enough to prevent accumulation of inhibitory amounts of the imine compound of formula II, but small enough to avoid accumulation of enzyme-inhibitory amounts of the agent. In one embodiment, the agent is sodium bisulfite, conveniently provided as sodium metabisulfite, which hydrolyzes to sodium bisulfite in water. Reaction of the bisulfite salt with the imine compound of formula II results in the sulfite adduct of formula III. In certain embodiments, sodium bisulfite is added continuously to the reaction at a rate such that the reagent is "instantly" consumed and the potentially inhibitory imine product of formula II is "trapped" as a less inhibitory or non-inhibitory sulfite adduct of formula III.

[0129] The addition of bisulfite to react with the imine product whether or not the engineered monoamine oxidase is inhibited by the imine product also provides a practical process operation option. Certain imines of the present invention are highly volatile, some of which are malodorous and / or toxic. Trapping them as free bases requires a closed reaction without gas flow, or sufficient condensation of chemical trapping (e.g., by bisulfite solution). Their in situ reaction to bisulfite adducts (aminosulfonates) obviates operational constraints, yet also provides the option of performing the subsequent reaction with cyanide in the same reaction vessel.

[0130] The formation of the bisulfite addition compound, formula III, can be reversed at high pH, ​​thereby generating again the corresponding imine of formula II. Thus, in certain embodiments, the reaction of Scheme 2 is quenched by adding a base, e.g., 10N NaOH, to raise the pH to about 13, thereby generating again the imine of formula II, which can be extracted, e.g., with methyl t-butyl ether ("MBTE"), and, in certain embodiments, isolated by distillation to obtain the imine of formula II as a colorless oil.

[0131] In certain embodiments, the rates of addition of the substrate of structural formula I, the sequestering agent (e.g., sodium bisulfite), and the pH control agent (e.g., NaOH) are monitored and controlled, in part, to minimize or obviate both substrate and product inhibition of the engineered monoamine oxidase used as a biocatalyst for the conversion of a compound of structural formula I to a compound of structural formula II. [ka]

[0132] In some embodiments, the present disclosure provides a substantially enantiomerically pure aminonitrile compound according to structural formula IV(a): [ka] (Wherein, A is O, CR 1 R 2 , -C=C-, or -CH-CH-, where R 1 and R 2 are each independently selected from -H, -COOH, -X, -NH2, -CH2NHC(NH)NH2, -CX3, -CH3, -CH2CH3, where X is selected from F, Cl and Br. including its salts and hydrates, Amine compounds according to structural formula I [ka] wherein A is as defined for structural formula IV. in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite to produce a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b). [ka] with oxygen under conditions to produce a mixture comprising: contacting the compounds according to structural formula III(a) and III(b) with cyanide under conditions to yield a substantially stereoisomerically pure aminonitrile compound according to structural formula IV(a); Includes.

[0133] In another embodiment, as depicted in Scheme 3, NaCN is added to the reaction of Scheme 2 to raise the pH to about pH 10, thereby stereoselectively converting the sulfite adduct of formula III to the trans aminonitrile compound of formula IV(a). In aspects of this embodiment, about 1 to about 3 equivalents, about 1 to about 2, about 1.05 to about 1.5 equivalents, or about 1.1 to about 1.2 equivalents of NaCN relative to the imine compound of formula II are added to the reaction to convert the sulfite adduct of formula III to the trans aminonitrile compound of formula IV(a). In addition, reaction of the compound of formula III produces a cis aminonitrile compound of formula IV(b).

[0134] The trans aminonitrile compound of formula IV(a) can be extracted from the reaction mixture (1:1 organic solvent:aqueous reaction mixture) using, for example, 2-methyltetrahydrofuran, MTBE, or isopropyl acetate. The trans aminonitrile compound can be recovered from the organic solvent extract, for example the organic extract, and, with further clarification of the intermediate if necessary, can be concentrated under reduced pressure to provide the aminonitrile compound of formula IV.

[0135] In another embodiment, the trans aminonitrile compound of formula IV(a), prepared, for example, according to Scheme 3, can be contacted with an aqueous acid (e.g., HCl or H2SO4) to provide an amino acid. In yet another embodiment, the corresponding t-butyl ester can be prepared by contacting the amine compound with an acid (e.g., methanesulfonic acid) and isobutylene or a t-butyl ester (e.g., t-butyl acetate).

[0136] In another embodiment, the trans aminonitrile compound of formula IV(a), prepared, for example, according to Scheme 3, can be contacted with HCl and methanol in a Piner reaction to provide a methyl ester. [ka]

[0137] Scheme 4 depicts an overall process for the preparation of compounds according to formula VII from secondary amines of formula I.

[0138] In some embodiments, the present disclosure provides a substantially stereoisomerically pure amino amide compound according to structural formula VII: [ka] (Wherein, A is O, CR 1 R 2 , -C=C-, or -CH-CH-, where R 1 and R 2 are each independently selected from -H, -COOH, -X, -NH2, -CH2NHC(NH)NH2, -CX3, -CH3, -CH2CH3, where X is selected from F, Cl and Br. and a method for preparing the compound, including salts thereof, Amine compounds according to structural formula I [ka] wherein A is as defined for structural formula VII. in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite to produce a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b). [ka] with oxygen under conditions to produce a mixture comprising: a stereoisomerically pure amine bisulfite adduct according to structural formula III(a) or a substantially stereoisomerically pure amine bisulfite adduct according to structural formula III(b), or a mixture thereof, [ka] wherein A is as defined for the amino compounds of formula VII. with cyanide under conditions to produce contacting an aminonitrile compound of structural formula IV(a) with an acid under conditions whereby the aminonitrile compound is converted to a substantially stereoisomerically pure aminoamide compound according to structural formula VII; Includes.

[0139] In another embodiment, any of the above processes involving a trans-aminonitrile compound of formula IV(a) can be carried out with a cis-aminonitrile compound of formula IV(b) to form the corresponding cis amino acid and amide products.

[0140] Engineered monoamine oxidase polypeptides The present disclosure provides engineered monoamine polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods of using the polypeptides. Where a description refers to a polypeptide, it is understood that the description may also describe the polynucleotide that encodes the polypeptide.

[0141] Suitable reaction conditions under which the desired reaction occurs due to the above-mentioned improved properties of the engineered polypeptide can be determined as further explained below and in the Examples, with respect to conditions including the concentration or amount of the polypeptide, substrate, co-substrate, buffer, solvent, pH, temperature and reaction time, and / or conditions involving the polypeptide immobilized on a solid support.

[0142] In some embodiments, exemplary engineered monoamine oxidase polypeptides having improved properties, particularly with respect to the conversion of bicyclic proline analogs of formula I to the corresponding imines of formula II, comprise amino acid sequences having one or more residue differences at the residue positions set forth in Tables 4.2, 5.1, 6.1, 7.1, 8.1 and 9.1 compared to SEQ ID NOs:6 and / or 414.

[0143] The structural and functional information of exemplary non-naturally occurring (or engineered) polypeptides of the invention is based on the conversion of bicyclic proline analogs of formula I to the corresponding imines of formula II, the results of which are shown in Tables 4.2, 5.1, 6.1, 7.1, 8.1 and 9.1 below, as further described in the Examples. The odd-numbered sequence identifiers (i.e., SEQ ID NOs) in these tables refer to nucleotide sequences that encode the amino acid sequences provided by the even-numbered SEQ ID NOs in these tables. Exemplary sequences are provided in the electronic format of the sequence listing file accompanying the invention, which is hereby incorporated by reference herein. Amino acid residue differences are based on comparison to the reference sequences of SEQ ID NOs: 6 and / or 414, as previously described.

[0144] Previously engineered monoamine oxidases (SEQ ID NO:6) derived from Aspergillus niger (SEQ ID NO:2) and Aspergillus oryzae (SEQ ID NO:4) were selected based on the conversion of bicyclic proline analogs of formula I to the corresponding imines of formula II. SEQ ID NO:5 is a codon-optimized polynucleotide for expression in Escherichia coli that was synthesized based on the polypeptide sequence of SEQ ID NO:6.

[0145] The activity of each engineered monoamine oxidase polypeptide relative to the reference polypeptide of SEQ ID NO: 6 and / or 414 was determined as the conversion of substrate as described in the Examples herein. In some embodiments, shake flask powder (SFP) is used as a secondary screen to evaluate the properties of the engineered monoamine oxidase polypeptide, the results of which are provided in the Examples. In some embodiments, the SFP form provides a more purified powder preparation of the engineered polypeptide and may contain up to 30% of the engineered polypeptide total protein.

[0146] In some embodiments, the particular enzymatic property is associated with a residue difference at a residue position set forth herein when compared to SEQ ID NO: 6 and / or 414. In some embodiments, residue differences that affect polypeptide expression can be used to increase expression of an engineered monoamine oxidase polypeptide.

[0147] Given the guidance provided herein, any of the exemplary engineered polypeptides comprising SEQ ID NO:6 and / or the even-numbered sequences of 414 are used as starting amino acid sequences for synthesizing other engineered monoamine oxidase polypeptides, for example, by subsequent rounds of evolution that incorporate new combinations of various amino acid differences from other polypeptides at residue positions in Tables 4.2, 5.1, 6.1, 7.1, 8.1 and 9.1, as well as other residue positions described herein. Further improvements can be made by including amino acid differences at residue positions that were maintained as invariant through prior rounds of evolution.

[0148] In some embodiments, the engineered monoamine oxidase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 13, 32 / 65 / 135 / 246 / 284, 32 / 135 / 284, 32 / 284, 47 / 197, 55 / 347 / 348, 61, 65 / 135, 65 / 190, 65 / 246, 65 / 284, 79, 80, 82, 85, 87, 87 / 185, 93, 100, 100 / 144 / 421 / 447, 100 / 144 / 447, 104, 107, 113, 114, 115, 117, 127, 135, 136, 144, 144 / 447, 145, 146, 147, 149, 149 / 157, 153, 155, 161, 162, 166, 167, 1 69, 170, 174, 177, 181, 184, 185, 187 / 242, 193, 196, 198, 199, 208, 211, 218, 219, 219 / 332, 221, 229, 232, 234, 238, 239, 240, 242, 246, 250, 252, 254, 260, 262 / 266 / 421, 262 / 421 / 447, 266 / 447, 277, 277 / 347, 284, 290, 292, 294, 298 , 300, 306, 310, 323, 332, 338, 347, 349, 359, 361, 366, 381, 385, 386, 387, 392, 393, 400, 407, 411 / 445, 413, 418, 421, 425, 429, 433, 434, 441, 444, 445, 447, 449, 452, 475, and 483. In some embodiments, the engineered monoamine oxidase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 13G, 32G / 65V / 135Q / 246M / 284D, 32G / 135Q / 284D, 32G / 284D, 47A / 197N, 55A / 347D / 348K, 61C, 65V / 135Q, 65V / 190D, 65V / 246M,65V / 284D、79A、79D、79Q、79S、79V、79Y、80H、80I、80N、80T、80V、82F、82L、82R、82W、85D、85S、87G、87H、87L、87Q / 185F、87S、87W、87Y、93G、100P、100P / 144E / 421S / 447A、100P / 144E / 447A、104K、107W、113G、113W、114G、115M、117A、117C、117I、117K、117L、117R、117S、117T、127D、127G、127R、135G、136R、144E / 447A、144M、145G、145L、145S、146A、146F、147A、149M、149M / 157A、149V、153T、155C、155L、155Q、155R、161D、161L、161R、161Y、162Q、166E、166G、166L、167C、169G、170K、174G、174S、174T、174V、177G、177R、177W、181C、181G、181L、181P、184F、184R、185G、185K、185L、185R、187P / 242I、193A、196G、196M、198D、199I、199T、199V、208S、211G、218G、219P / 332L、219S、221C、221R、221V、229E、232T、234V、238K、238R、239G、240A、242I、246M、250V、252A、252E、252G、252L、254A、260F、260S、260V、262F / 266L / 421S、262F / 421S / 447A、266L / 447A、277T、277T / 347D、284D、290Q、290Y、292N、294V、298C、300R、306V、310S、323R、332G、332L、332S、338A、338G、338S、347D、347N、349E、359S、361L、366G、381A、385G、386E、386G、387I、387Q、387W、392G、392S、393G、393K、393M、400D、407G、411V / 445R、413R、418A、418G、421S、425L、429G、433N、433S、434E、441H、444D、444L、444R、444S、445D、445R、447A、449R、449S、In some embodiments, the engineered monoamine oxidase polypeptide comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO:6 selected from 452M, 475E, 475I, 475L, 475S, and 483R. In some embodiments, the engineered monoamine oxidase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as any of the following: T13G, E32G / T65V / T135Q / I246M / N284D, E32G / T135Q / N284D, E32G / N284D, G47A / D197N, T55A / C347D / P348K, A61 C, T65V / T135Q, T65V / E190D, T65V / I246M, T65V / N284D, W79A, W79D, W79Q, W79S, W79V, W79Y, S80H, S80I, S80N, S80T, S80V, N82F, N82L, N82 R, N82W, G85D, G85S, P87G, P87H, P87L, P87Q / E185F, P87S, P87W, P87Y, T93G, S100P, S100P / A144E / C421S / C447A, S100P / A144E / C447A, R104 K, T107W, N113G, N113W, A114G, L115M, P117A, P117C, P117I, P117K, P117L, P117R, P117S, P117T, H127D, H127G, H127R, T135G, T136R, A144 E / C447A, A144M, E145G, E145L, E145S, D146A, D146F, E147A, L149M, L149M / T157A, L149V, L153T, K155C, K155L, K155Q, K155R, G161D, G161L , G161R, G161Y, T162Q, T166E, T166G, T166L, V167C, P169G, F170K, M174G, M174S, M174T, M174V, V177G, V177R, V177W, R181C, R181G, R181L , R181P, D184F, D184R, E185G, E185K, E185L, E185R, S187P / M242I, D193A, R196G, R196M, E198D, L199I, L199T, L199V, E208S, I211G, T218G,L219P / I332L, L219S, N221C, N221R, N221V, H229E, A232T, S234V, Y238K, Y238R, Q239G, G240A, M 242I, I246M, F250V, D252A, D252E, D252G, D252L, Q254A, R260F, R260S, R260V, W262F / A266L / C4 21S, W262F / C421S / C447A, A266L / C447A, C277T, C277T / C347D, N284D, R290Q, R290Y, T292N, R29 4V, E298C, A300R, C306V, L310S, S323R, I332G, I332L, I332S, C338A, C338G, C338S, C347D, C347 N, D349E, P359S, N361L, A366G, C381A, A385G, H386E, H386G, N387I, N387Q, N387W, E392G, E392S , E393G, E393K, E393M, M400D, P407G, D411V / L445R, K413R, H418A, H418G, C421S, F425L, A429G, The amino acid sequence has one or more residue differences compared to SEQ ID NO:6 selected from A433N, A433S, P434E, S441H, E444D, E444L, E444R, E444S, L445D, L445R, C447A, H449R, H449S, V452M, T475E, T475I, T475L, T475S, and E483R.

[0149] In some embodiments, the engineered monoamine oxidase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, and one or more residue differences compared to SEQ ID NO:6 selected from 79, 80, 87, 146, 149, 219 / 332, 238, 260, 262 / 266 / 421, 338, and 429. In some embodiments, the engineered monoamine oxidase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, and one or more residue differences compared to SEQ ID NO:6 selected from 79D, 80V, 87G, 146A, 149M, 219P / 332L, 238R, 260S, 262F / 266L / 421S, 338G, and 429G. In some embodiments, the engineered monoamine oxidase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, and one or more residue differences compared to SEQ ID NO:6 selected from W79D, S80V, P87G, D146A, L149M, L219P / I332L, Y238R, R260S, W262F / A266L / C421S, C338G, and A429G.

[0150] In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 79, 79 / 80, 79 / 80 / 332 / 429, 79 / 93, 79 / 93 / 238 / 386 / 429, 79 / 93 / 386, 79 / 183 / 284 / 332, 79 / 238, 79 / 238 / 332 / 429, 79 / 238 / 429, 80, 80 / 183 / 28 4 / 332 / 381 / 386 / 429, 80 / 188 / 190 / 332 / 386, 80 / 237 / 332, 80 / 238 / 332, 80 / 238 / 386 / 429, 80 / 284, 80 / 284 / 386, 80 / 332, 80 / 332 / 381 / 386, 80 / 332 / 386 , 80 / 381, 80 / 386, 80 / 386 / 429, 80 / 429, 82, 82 / 87, 82 / 87 / 113, 82 / 87 / 113 / 117 / 218, 82 / 87 / 113 / 117 / 219, 82 / 87 / 113 / 117 / 219 / 418, 82 / 87 / 113 / 117 / 260 / 338, 82 / 87 / 113 / 117 / 338 / 418, 82 / 87 / 113 / 421, 82 / 87 / 117, 82 / 87 / 117 / 218 / 421, 82 / 87 / 117 / 260 / 338 / 421, 82 / 87 / 117 / 338 / 418, 82 / 87 / 218, 82 / 87 / 218 / 219 / 338, 82 / 87 / 218 / 219 / 421, 82 / 87 / 218 / 338, 82 / 87 / 219, 82 / 87 / 219 / 421, 82 / 87 / 338, 82 / 87 / 338 / 418, 82 / 87 / 338 / 421, 82 / 113 / 218 / 338 , 82 / 113 / 218 / 338 / 418, 82 / 113 / 219 / 338, 82 / 113 / 338, 82 / 218 / 219 / 421, 82 / 260 / 338 / 421, 82 / 338 / 418 / 421, 82 / 418, 87, 87 / 113, 87 / 113 / 117 / 219 / 3 38 / 418 / 421, 87 / 113 / 117 / 260 / 338 / 421, 87 / 113 / 117 / 418 / 421, 87 / 113 / 218 / 219 / 260 / 338, 87 / 113 / 219 / 421, 87 / 113 / 260, 87 / 117, 87 / 117 / 218 / 219,87 / 117 / 219 / 310 / 338、87 / 218、87 / 218 / 219、87 / 218 / 219 / 338、87 / 218 / 418、87 / 219、87 / 219 / 338、87 / 219 / 418、87 / 260、87 / 310、87 / 310 / 338、87 / 338、87 / 418、87 / 421、93 / 184 / 429、93 / 238、93 / 238 / 284、93 / 238 / 386、93 / 238 / 386 / 429、100、100 / 143 / 145 / 146 / 149 / 198 / 242 / 347 / 348 / 445 / 447、100 / 143 / 145 / 149 / 347 / 447、100 / 145 / 146 / 149、100 / 145 / 146 / 149 / 154 / 348 / 445、100 / 145 / 146 / 149 / 198、100 / 145 / 146 / 149 / 198 / 242 / 250 / 252 / 347 / 348 / 447、100 / 145 / 146 / 149 / 205 / 347 / 445 / 447、100 / 145 / 146 / 149 / 242 / 445、100 / 145 / 146 / 149 / 250 / 277 / 347 / 348 / 447、100 / 145 / 149 / 154 / 246 / 277、100 / 145 / 149 / 198 / 242 / 246 / 277 / 348 / 447、100 / 145 / 149 / 198 / 242 / 246 / 445 / 447、100 / 145 / 149 / 198 / 242 / 277 / 347 / 348 / 447、100 / 145 / 149 / 198 / 252 / 348 / 445、100 / 145 / 149 / 205、100 / 145 / 149 / 242 / 246 / 277 / 347 / 445、100 / 145 / 149 / 242 / 246 / 347 / 348 / 445 / 447、100 / 145 / 149 / 242 / 445 / 447、100 / 145 / 149 / 246 / 252 / 277 / 347 / 445 / 447、100 / 145 / 149 / 246 / 347 / 445、100 / 145 / 149 / 277 / 348 / 445、100 / 145 / 149 / 347 / 445 / 447、100 / 145 / 198 / 242 / 246 / 348、100 / 145 / 198 / 277 / 348 / 445、100 / 145 / 198 / 300、100 / 145 / 205 / 246 / 277 / 348 / 445、100 / 145 / 205 / 277 / 348、100 / 145 / 242 / 246 / 277 / 445 / 447、100 / 145 / 246 / 347 / 348 / 445、100 / 145 / 277 / 347、100 / 145 / 348、100 / 146 / 149 / 198 / 242 / 246 / 277 / 347 / 348 / 445 / 447、100 / 146 / 198 / 347 / 348 / 447、100 / 146 / 198 / 445、100 / 146 / 242 / 277 / 447、100 / 146 / 250 / 447、100 / 146 / 347 / 348 / 445 / 447、100 / 149 / 198、100 / 149 / 242 / 246 / 277 / 347 / 348 / 445、100 / 149 / 242 / 246 / 277 / 347 / 348 / 447、100 / 149 / 242 / 347 / 348 / 447、100 / 149 / 246 / 277 / 348 / 445、100 / 149 / 246 / 447、100 / 149 / 250 / 347 / 447、100 / 198 / 205 / 348、100 / 198 / 277 / 347 / 348、100 / 198 / 347 / 348、100 / 198 / 347 / 348 / 445、100 / 198 / 347 / 445、100 / 198 / 447、100 / 242 / 246 / 277、100 / 242 / 246 / 347、100 / 242 / 277 / 348、100 / 242 / 347 / 348 / 445、100 / 242 / 347 / 348 / 445 / 447、100 / 242 / 348 / 445、100 / 246 / 277 / 348 / 445、100 / 277 / 347 / 445 / 447、100 / 277 / 348、100 / 277 / 348 / 447、100 / 347 / 348 / 445、100 / 347 / 447、100 / 445 / 447、110 / 338 / 418、113 / 218 / 338 / 421、113 / 338、113 / 338 / 421、117 / 219、117 / 260、121 / 198 / 347 / 445 / 447、145 / 146 / 149 / 154 / 246 / 277 / 347 / 348、145 / 146 / 149 / 198 / 242 / 252 / 277 / 347、145 / 146 / 149 / 198 / 277 / 445 / 447、145 / 146 / 149 / 205 / 348 / 447、145 / 146 / 149 / 250 / 277 / 347 / 348 / 447、145 / 146 / 149 / 277 / 347、145 / 146 / 149 / 347 / 348、145 / 146 / 198 / 205 / 242 / 277 / 348 / 447、145 / 146 / 198 / 445 / 447、145 / 146 / 246 / 277 / 447、145 / 146 / 246 / 347 / 348 / 447、145 / 146 / 277 / 347、145 / 146 / 347 / 447、145 / 149 / 198 / 205 / 348、145 / 149 / 198 / 242 / 246 / 277 / 347 / 447、145 / 149 / 198 / 242 / 246 / 348、145 / 149 / 198 / 242 / 277 / 347、145 / 149 / 198 / 246 / 447、145 / 149 / 198 / 347 / 447、145 / 149 / 205 / 242 / 347 / 348 / 445、145 / 149 / 205 / 246 / 250 / 252、145 / 149 / 205 / 277 / 445、145 / 149 / 242、145 / 149 / 242 / 348、145 / 149 / 246、145 / 149 / 246 / 347 / 445、145 / 149 / 246 / 447、145 / 149 / 347 / 348 / 445、145 / 149 / 347 / 447、145 / 149 / 447、145 / 155 / 445 / 447、145 / 198 / 205 / 348、145 / 246、145 / 246 / 252 / 347 / 447、145 / 250 / 252 / 348、145 / 252 / 348 / 447、145 / 255、145 / 277 / 347 / 445、145 / 277 / 348 / 447、145 / 347 / 348 / 445 / 447 / 491、145 / 445 / 447、146 / 149 / 151 / 246 / 348、146 / 149 / 242 / 246 / 445、146 / 149 / 246 / 252 / 447、146 / 149 / 277 / 348 / 447、146 / 198 / 246 / 277、146 / 198 / 277 / 348 / 447、146 / 242、146 / 242 / 246 / 277 / 347 / 348 / 445 / 447、146 / 246 / 277 / 348 / 445 / 447、146 / 277、146 / 277 / 347 / 348、146 / 277 / 347 / 445 / 447、146 / 277 / 445 / 447、146 / 347 / 348 / 447、146 / 348、146 / 445 / 447、149、149 / 198 / 277 / 347 / 348、149 / 198 / 348、149 / 198 / 445、149 / 242 / 246、149 / 242 / 246 / 348 / 445、149 / 347 / 348 / 445 / 447、149 / 445、184、184 / 284、184 / 284 / 429、184 / 386、198 / 242 / 348、198 / 246、198 / 250, 198 / 277 / 348 / 447, 198 / 445, 205, 205 / 246 / 347, 205 / 252 / 347 / 445 / 447, 205 / 347 / 348 / 445 / 447, 218, 218 / 260 / 338, 219, 219 / 338 / 418, 219 / 421, 238, 238 / 2 42 / 284, 238 / 284 / 386 / 429, 238 / 332 / 429, 238 / 381 / 429, 238 / 386, 238 / 429, 242, 242 / 277 / 347 / 348, 242 / 284 / 381 / 429, 242 / 348, 242 / 445 / 447, 246, 246 / 277 / 348 / 44 7, 246 / 445 / 447, 260, 260 / 338, 260 / 338 / 418 / 421, 277 / 347 / 348 / 445, 277 / 347 / 447, 284, 284 / 332, 284 / 386, 284 / 386 / 429, 310 / 338 / 418, 310 / 418 / 421, 332, 332 / 386, 332 / 429, 338, 338 / 418, 347 / 348 / 445, 347 / 348 / 447, 348, 348 / 445 / 447, 348 / 447, 386, 386 / 429, 418, 429, and 447. In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 79A / 80H, 79A / 93G / 238R / 386E / 429G, 79A / 93G / 386E, 79A / 238R, 79A / 238R / 332G / 429G, 79A / 238R / 429G, 79D, 79D / 80V, 79D / 80V / 332G / 429G, 79D / 93G, 79D / 183F / 284D / 332G, 80H, 80H / 183F / 284D / 332G / 381A / 386E / 429G, 80H / 188F / 190D / 332G / 386E, 80H / 238R / 386E / 429G, 80H / 332G / 381A / 386E, 80H / 381A, 80H / 386E, 80H / 429G, 80V, 80V / 237S / 332G, 80V / 238R / 332G, 80V / 284D,80V / 284D / 386E、80V / 332G、80V / 332G / 386E、80V / 386E、80V / 386E / 429G、82F、82F / 87G / 113G / 117K / 219S、82F / 87G / 113G / 117K / 260S / 338G、82F / 87G / 117K、82F / 87G / 218G、82F / 87G、 / 218G / 219S / 421S、82F / 87G / 218G / 338S、82F / 87G / 338S / 418G、82F / 87L、82F / 87L / 113G / 117K / 218G、82F / 87L / 113G / 117K / 219S / 418G、82F / 87L / 113G / 117K / 338G / 418G、82F / 87L / 117K、82F / 87L / 219S / 421S、82F / 87L / 338S、82F / 113G / 218G / 338S / 418G、82F / 418G、82R / 87G、82R / 87G / 113G / 421S、82R / 87G / 117K / 218G / 421S、82R / 87G / 117K / 260S / 338S / 421S、82R / 87G / 117K / 338A / 418G、82R / 87L、82R / 87L / 113G、82R / 87L / 218G / 219S / 338S、82R / 87L / 219S、82R / 87L / 338G / 421S、82R / 113G / 218G / 338A、82R / 113G / 219S / 338S、82R / 113G / 338S、82R / 218G / 219S / 421S、82R / 260S / 338A / 421S、82R / 338A / 418G / 421S、87G、87G / 113G、87G / 113G / 117K / 418G / 421S、87G / 113G / 260S、87G / 117K / 219S / 310S / 338S、87G / 218G / 219S / 338S、87G / 219S、87G / 260S、87G / 310S、87G / 310S / 338S、87G / 338A、87G / 418G、87L、87L / 113G、87L / 113G / 117K / 219S / 338G / 418G / 421S、87L / 113G / 117K / 260S / 338S / 421S、87L / 113G / 218G / 219S / 260S / 338A、87L / 113G / 219S / 421S、87L / 117K、87L / 117K / 218G / 219S、87L / 218G、87L / 218G / 219S、87L / 218G / 418G、87L / 219S、87L / 219S / 338G、87L / 219S / 418G、87L / 338A、87L / 421S、93G / 184R / 429G、93G / 238R、93G / 238R / 284D、93G / 238R / 386E、93G / 238R / 386E / 429G、100P、100P / 143D / 145S / 146A / 149M / 198D / 242I / 347N / 348K / 445R / 447A、100P / 143V / 145G / 149M / 347N / 447A、100P / 145G / 146A / 149M / 250V / 277T / 347N / 348K / 447A、100P / 145G / 149M / 154L / 246M / 277T、100P / 145G / 149M / 198D / 242I / 246M / 445R / 447A、100P / 145G / 149M / 198D / 252G / 348K / 445R、100P / 145G / 149M / 205T、100P / 145G / 149M / 242I / 246M / 347D / 348K / 445R / 447A、100P / 145G / 149M / 242I / 445R / 447A、100P / 145G / 198D / 242I / 246M / 348K、100P / 145G / 198D / 277T / 348K / 445R、100P / 145G / 198D / 300V、100P / 145G / 205T / 246M / 277T / 348K / 445R、100P / 145G / 242I / 246M / 277T / 445R / 447A、100P / 145G / 348K、100P / 145S / 146A / 149M、100P / 145S / 146A / 149M / 154L / 348K / 445R、100P / 145S / 146A / 149M / 198D、100P / 145S / 146A / 149M / 198D / 242I / 250V / 252G / 347N / 348K / 447A、100P / 145S / 146A / 149M / 205T / 347D / 445R / 447A、100P / 145S / 146A / 149M / 242I / 445R、100P / 145S / 149M / 198D / 242I / 246M / 277T / 348K / 447A、100P / 145S / 149M / 198D / 242I / 277T / 347N / 348K / 447A、100P / 145S / 149M / 242I / 246M / 277T / 347N / 445R、100P / 145S / 149M / 246M / 252G / 277T / 347N / 445R / 447A、100P / 145S / 149M / 246M / 347N / 445R、100P / 145S / 149M / 277T / 348K / 445R、100P / 145S / 149M / 347N / 445R / 447A、100P / 145S / 205T / 277T / 348K、100P / 145S / 246M / 347N / 348K / 445R、100P / 145S / 277T / 347N、100P / 146A / 149M / 198D / 242I / 246M / 277T / 347D / 348K / 445R / 447A、100P / 146A / 198D / 347D / 348K / 447A、100P / 146A / 198D / 445R、100P / 146A / 242I / 277T / 447A、100P / 146A / 250V / 447A、100P / 146A / 347D / 348K / 445R / 447A、100P / 149M / 198D、100P / 149M / 242I / 246M / 277T / 347N / 348K / 445R、100P / 149M / 242I / 246M / 277T / 347N / 348K / 447A、100P / 149M / 242I / 347N / 348K / 447A、100P / 149M / 246M / 277T / 348K / 445R、100P / 149M / 246M / 447A、100P / 149M / 250V / 347N / 447A、100P / 198D / 205T / 348K、100P / 198D / 277T / 347N / 348K、100P / 198D / 347D / 348K、100P / 198D / 347D / 348K / 445R、100P / 198D / 347N / 445R、100P / 198D / 447A、100P / 242I / 246M / 277T、100P / 242I / 246M / 347N、100P / 242I / 277T / 348K、100P / 242I / 347D / 348K / 445R、100P / 242I / 347D / 348K / 445R / 447A、100P / 242I / 348K / 445R、100P / 246M / 277T / 348K / 445R、100P / 277T / 347D / 445R / 447A、100P / 277T / 348K、100P / 277T / 348K / 447A、100P / 347N / 348K / 445R、100P / 347N / 447A、100P / 445R / 447A、110N / 338G / 418G、113G / 218G / 338S / 421S、113G / 338A / 421S、113G / 338G、117K / 219S、117K / 260S、121C / 198D / 347N / 445R / 447A、145G / 146A / 149M / 277T / 347D、145G / 146A / 149M / 347D / 348K、145G / 146A / 277T / 347N、145G / 146A / 347D / 447A、145G / 149M / 198D / 347N / 447A、145G / 149M / 205T / 242I / 347N / 348K / 445R、145G / 149M / 205T / 246M / 250V / 252G、145G / 149M / 242I / 348K、145G / 149M / 246M、145G / 155I / 445R / 447A、145G / 198D / 205T / 348K、145G / 250V / 252G / 348K、145G / 255R、145G / 277T / 347N / 445R、145S / 146A / 149M / 154L / 246M / 277T / 347N / 348K、145S / 146A / 149M / 198D / 242I / 252G / 277T / 347N、145S / 146A / 149M / 198D / 277T / 445R / 447A、145S / 146A / 149M / 205T / 348K / 447A、145S / 146A / 149M / 250V / 277T / 347D / 348K / 447A、145S / 146A / 198D / 205T / 242I / 277T / 348K / 447A、145S / 146A / 198D / 445R / 447A、145S / 146A / 246M / 277T / 447A、145S / 146A / 246M / 347N / 348K / 447A、145S / 149M / 198D / 205T / 348K、145S / 149M / 198D / 242I / 246M / 277T / 347N / 447A、145S / 149M / 198D / 242I / 246M / 348K、145S / 149M / 198D / 242I / 277T / 347D、145S / 149M / 198D / 246M / 447A、145S / 149M / 205T / 277T / 445R、145S / 149M / 242I、145S / 149M / 246M / 347D / 445R、145S / 149M / 246M / 447A、145S / 149M / 347N / 348K / 445R、145S / 149M / 347N / 447A、145S / 149M / 447A、145S / 246M、145S / 246M / 252G / 347D / 447A、145S / 252G / 348K / 447A、145S / 277T / 348K / 447A、145S / 347D / 348K / 445R / 447A / 491A、145S / 445R / 447A、146A / 149M / 151C / 246M / 348K、146A / 149M / 242I / 246M / 445R、146A / 149M / 246M / 252G / 447A、146A / 149M / 277T / 348K / 447A、146A / 198D / 246M / 277T、146A / 198D / 277T / 348K / 447A、146A / 242I、146A / 242I / 246M / 277T / 347N / 348K / 445R / 447A、146A / 246M / 277T / 348K / 445R / 447A、146A / 277T、146A / 277T / 347N / 348K、146A / 277T / 347N / 445R / 447A、146A / 277T / 445R / 447A、146A / 347D / 348K / 447A、146A / 348K、146A / 445R / 447A、149M、149M / 198D / 277T / 347N / 348K、149M / 198D / 348K、149M / 198D / 445R、149M / 242I / 246M、149M / 242I / 246M / 348K / 445R、149M / 347N / 348K / 445R / 447A、149M / 445R、184R、184R / 284D、184R / 284D / 429G、184R / 386E、198D / 242I / 348K、198D / 246M、198D / 250V、198D / 277T / 348K / 447A、198D / 445R、205T、205T / 246M / 347N、205T / 252G / 347D / 445R / 447A、205T / 347D / 348K / 445R / 447A、218G、218G / 260S / 338A、219S、219S / 338A / 418G、219S / 421S、238R、238R / 242L / 284D、238R / 284D / 386E / 429G、238R / 332G / 429G、238R / 381A / 429G、238R / 386E、238R / 429G、242I、242I / 277T / 347D / 348K、242I / 348K、242I / 445R / 447A、242L / 284D / 381A / 429G、246M、246M / 277T / 348K / 447A、246M / 445R / 447A、260S、260S / 338G、260S / 338G / 418G / 421S、277T / 347N / 348K / 445R、277T / 347N / 447A、284D、284D / 332G、284D / 386E、284D / 386E / 429G、one or more residues compared to SEQ ID NO:6 selected from 310S / 338S / 418G, 310S / 418G / 421S, 332G, 332G / 386E, 332G / 429G, 338G, 338G / 418G, 347N / 348K / 445R, 347N / 348K / 447A, 348K, 348K / 445R / 447A, 348K / 447A, 386E, 386E / 429G, 418G, 429G, and 447A; In some embodiments, the engineered monoamine oxidase polypeptides include those that have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as W79A / S80H, W79A / T93G / Y238R / H386E / A429G, W79A / T93G / H386E, W79A / Y238R, W79A / Y238R / I332G / A429G, W79A / Y238R / A429G, W79D, W79D / S80V, W79D / S80V / I332G / A429G, W79D / T93G, W79D / Y183F / N2 84D / I332G, S80H, S80H / Y183F / N284D / I332G / C381A / H386E / A429G, S80H / Y188F / E190D / I332G / H386E, S80H / Y238R / H386E / A429G, S80H / I332G / C38 1A / H386E, S80H / C381A, S80H / H386E, S80H / A429G, S80V, S80V / T237S / I33 2G, S80V / Y238R / I332G, S80V / N284D, S80V / N284D / H386E, S80V / I332G, S 80V / I332G / H386E, S80V / H386E, S80V / H386E / A429G, N82F, N82F / P87G / N1 13G / P117K / L219S, N82F / P87G / N113G / P117K / R260S / C338G, N82F / P87G / P117K, N82F / P87G / T218G, N82F / P87G / T218G / L219S / C421S, N82F / P87G / T 218G / C338S, N82F / P87G / C338S / H418G, N82F / P87L, N82F / P87L / N113G / P 117K / T218G, N82F / P87L / N113G / P117K / L219S / H418G, N82F / P87L / N113G / P117K / C338G / H418G, N82F / P87L / P117K, N82F / P87L / L219S / C421S, N82F / P87L / C338S, N82F / N113G / T218G / C338S / H418G, N82F / H418G, N82R / P87G,N82R / P87G / N113G / C421S、N82R / P87G / P117K / T218G / C421S、N82R / P87G / P117K / R260S / C338S / C421S、N82R / P87G / P117K / C338A / H418G、N82R / P87L、N82R / P87L / N113G、N82R / P87L / T218G / L219S / C338S、N82R / P87L / L219S、N82R / P87L / C338G / C421S、N82R / N113G / T218G / C338A、N82R / N113G / L219S / C338S、N82R / N113G / C338S、N82R / T218G / L219S / C421S、N82R / R260S / C338A / C421S、N82R / C338A / H418G / C421S、P87G、P87G / N113G、P87G / N113G / P117K / H418G / C421S、P87G / N113G / R260S、P87G / P117K / L219S / L310S / C338S、P87G / T218G / L219S / C338S、P87G / L219S、P87G / R260S、P87G / L310S、P87G / L310S / C338S、P87G / C338A、P87G / H418G、P87L、P87L / N113G、P87L / N113G / P117K / L219S / C338G / H418G / C421S、P87L / N113G / P117K / R260S / C338S / C421S、P87L / N113G / T218G / L219S / R260S / C338A、P87L / N113G / L219S / C421S、P87L / P117K、P87L / P117K / T218G / L219S、P87L / T218G、P87L / T218G / L219S、P87L / T218G / H418G、P87L / L219S、P87L / L219S / C338G、P87L / L219S / H418G、P87L / C338A、P87L / C421S、T93G / D184R / A429G、T93G / Y238R、T93G / Y238R / N284D、T93G / Y238R / H386E、T93G / Y238R / H386E / A429G、S100P、S100P / E143D / E145S / D146A / L149M / E198D / M242I / C347N / P348K / L445R / C447A、S100P / E143V / E145G / L149M / C347N / C447A、S100P / E145G / D146A / L149M / F250V / C277T / C347N / P348K / C447A、S100P / E145G / L149M / H154L / I246M / C277T、S100P / E145G / L149M / E198D / M242I / I246M / L445R / C447A、S100P / E145G / L149M / E198D / D252G / P348K / L445R、S100P / E145G / L149M / S205T、S100P / E145G / L149M / M242I / I246M / C347D / P348K / L445R / C447A、S100P / E145G / L149M / M242I / L445R / C447A、S100P / E145G / E198D / M242I / I246M / P348K、S100P / E145G / E198D / C277T / P348K / L445R、S100P / E145G / E198D / A300V、S100P / E145G / S205T / I246M / C277T / P348K / L445R、S100P / E145G / M242I / I246M / C277T / L445R / C447A、S100P / E145G / P348K、S100P / E145S / D146A / L149M、S100P / E145S / D146A / L149M / H154L / P348K / L445R、S100P / E145S / D146A / L149M / E198D、S100P / E145S / D146A / L149M / E198D / M242I / F250V / D252G / C347N / P348K / C447A、S100P / E145S / D146A / L149M / S205T / C347D / L445R / C447A、S100P / E145S / D146A / L149M / M242I / L445R、S100P / E145S / L149M / E198D / M242I / I246M / C277T / P348K / C447A、S100P / E145S / L149M / E198D / M242I / C277T / C347N / P348K / C447A、S100P / E145S / L149M / M242I / I246M / C277T / C347N / L445R、S100P / E145S / L149M / I246M / D252G / C277T / C347N / L445R / C447A、S100P / E145S / L149M / I246M / C347N / L445R、S100P / E145S / L149M / C277T / P348K / L445R、S100P / E145S / L149M / C347N / L445R / C447A、S100P / E145S / S205T / C277T / P348K、S100P / E145S / I246M / C347N / P348K / L445R、S100P / E145S / C277T / C347N、S100P / D146A / L149M / E198D / M242I / I246M / C277T / C347D / P348K / L445R / C447A、S100P / D146A / E198D / C347D / P348K / C447A、S100P / D146A / E198D / L445R、S100P / D146A / M242I / C277T / C447A、S100P / D146A / F250V / C447A、S100P / D146A / C347D / P348K / L445R / C447A、S100P / L149M / E198D、S100P / L149M / M242I / I246M / C277T / C347N / P348K / L445R、S100P / L149M / M242I / I246M / C277T / C347N / P348K / C447A、S100P / L149M / M242I / C347N / P348K / C447A、S100P / L149M / I246M / C277T / P348K / L445R、S100P / L149M / I246M / C447A、S100P / L149M / F250V / C347N / C447A、S100P / E198D / S205T / P348K、S100P / E198D / C277T / C347N / P348K、S100P / E198D / C347D / P348K、S100P / E198D / C347D / P348K / L445R、S100P / E198D / C347N / L445R、S100P / E198D / C447A、S100P / M242I / I246M / C277T、S100P / M242I / I246M / C347N、S100P / M242I / C277T / P348K、S100P / M242I / C347D / P348K / L445R、S100P / M242I / C347D / P348K / L445R / C447A、S100P / M242I / P348K / L445R、S100P / I246M / C277T / P348K / L445R、S100P / C277T / C347D / L445R / C447A、S100P / C277T / P348K、S100P / C277T / P348K / C447A、S100P / C347N / P348K / L445R、S100P / C347N / C447A、S100P / L445R / C447A、K110N / C338G / H418G、N113G / T218G / C338S / C421S、N113G / C338A / C421S、N113G / C338G、P117K / L219S、P117K / R260S、F121C / E198D / C347N / L445R / C447A、E145G / D146A / L149M / C277T / C347D、E145G / D146A / L149M / C347D / P348K、E145G / D146A / C277T / C347N、E145G / D146A / C347D / C447A、E145G / L149M / E198D / C347N / C447A、E145G / L149M / S205T / M242I / C347N / P348K / L445R、E145G / L149M / S205T / I246M / F250V / D252G、E145G / L149M / M242I / P348K、E145G / L149M / I246M、E145G / K155I / L445R / C447A、E145G / E198D / S205T / P348K、E145G / F250V / D252G / P348K、E145G / S255R、E145G / C277T / C347N / L445R、E145S / D146A / L149M / H154L / I246M / C277T / C347N / P348K、E145S / D146A / L149M / E198D / M242I / D252G / C277T / C347N、E145S / D146A / L149M / E198D / C277T / L445R / C447A、E145S / D146A / L149M / S205T / P348K / C447A、E145S / D146A / L149M / F250V / C277T / C347D / P348K / C447A、E145S / D146A / E198D / S205T / M242I / C277T / P348K / C447A、E145S / D146A / E198D / L445R / C447A、E145S / D146A / I246M / C277T / C447A、E145S / D146A / I246M / C347N / P348K / C447A、E145S / L149M / E198D / S205T / P348K、E145S / L149M / E198D / M242I / I246M / C277T / C347N / C447A、E145S / L149M / E198D / M242I / I246M / P348K、E145S / L149M / E198D / M242I / C277T / C347D、E145S / L149M / E198D / I246M / C447A、E145S / L149、 M / S205T / C277T / L445R、E145S / L149M / M242I、E145S / L149M / I246M / C347D / L445R、E145S / L149M / I246M / C447A、E145S / L149M / C347N / P348K / L445R、E145S / L149M / C347N / C447A、E145S / L149M / C447A、E145S / I246M、E145S / I246M / D252G / C347D / C447A、E145S / D252G / P348K / C447A、E145S / C277T / P348K / C447A、E145S / C347D / P348K / L445R / C447A / V491A、E145S / L445R / C447A、D146A / L149M / S151C / I246M / P348K、D146A / L149M / M242I / I246M / L445R、D146A / L149M / I246M / D252G / C447A、D146A / L149M / C277T / P348K / C447A、D146A / E198D / I246M / C277T、D146A / E198D / C277T / P348K / C447A、D146A / M242I、D146A / M242I / I246M / C277T / C347N / P348K / L445R / C447A、D146A / I246M / C277T / P348K / L445R / C447A、D146A / C277T、D146A / C277T / C347N / P348K、D146A / C277T / C347N / L445R / C447A、D146A / C277T / L445R / C447A、D146A / C347D / P348K / C447A、D146A / P348K、D146A / L445R / C447A、L149M、L149M / E198D / C277T / C347N / P348K、L149M / E198D / P348K、L149M / E198D / L445R、L149M / M242I / I246M、L149M / M242I / I246M / P348K / L445R、L149M / C347N / P348K / L445R / C447A、L149M / L445R、D184R、D184R / N284D、D184R / N284D / A429G、D184R / H386E、E198D / M242I / P348K、E198D / I246M、E198D / F250V、E198D / C277T / P348K / C447A、E198D / L445R, S205T, S205T / I246M / C347N, S205T / D252G / C347D / L445R / C447A, S205T / C347D / P348K / L445R / C447A , T218G, T218G / R260S / C338A, L219S, L219S / C338A / H418G, L219S / C421S, Y238R, Y238R / M242L / N284D, Y238R / N284D / H386E / A429G, Y238R / I332G / A429G, Y238R / C381A / A429G, Y238R / H386E, Y238R / A429G, M242I, M242I / C277T / C347 D / P348K, M242I / P348K, M242I / L445R / C447A, M242L / N284D / C381A / A429G, I246M, I246M / C277T / P348K / C447A, I246 M / L445R / C447A, R260S, R260S / C338G, R260S / C338G / H418G / C421S, C277T / C347N / P348K / L445R, C277T / C347N / C44 7A, N284D, N284D / I332G, N284D / H386E, N284D / H386E / A429G, L310S / C338S / H418G, L310S / H418G / C421S, I332G, I33 2G / H386E, I332G / A429G, C338G, C338G / H418G, C347N / P348K / L445R, C347N / P348K / C447A, P348K, P348K / L445R / C447A, P348K / C447A, H386E, H386E / A429G, H418G, A429G, and C447A.

[0151] In some embodiments, the engineered monoamine oxidase polypeptides have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 80 / 429, 87 / 113 / 117 / 260 / 338 / 421, 87 / 421, 100 / 145 / 146 / 149 / 198 / 242 / 2 and 242 / 284 / 381 / 429. In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as 80H / 429G, 87L / 113G / 117K / 260S / 338S / 421S, 87L / 421S, 100P / 145S / 146A / 149M / 198D / 242I / 250V / 252G / 347N / 3 The amino acid sequence has one or more residue differences compared to SEQ ID NO:6 selected from: 48K / 447A, 100P / 145S / 149M / 198D / 242I / 277T / 347N / 348K / 447A, 110N / 338G / 418G, 145G / 149M / 205T / 246M / 250V / 252G, 145S / 246M / 252G / 347D / 447A, 145S / 252G / 348K / 447A, 238R / 386E / 429G, and 242L / 284D / 381A / 429G.In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:6, as well as S80H / A429G, P87L / N113G / P117K / R260S / C338S / C421S, P87L / C421S, S100P / E145S / D146A / L149M / E198D / M242I / F250V / D252G / C347N / P348K / C44 7A, S100P / E145S / L149M / E198D / M242I / C277T / C347N / P348K / C447A, K110N / C338G / H418G, E145G / L149M / S205T / I246M / F250V / D252G, E145S / I246M / D252G / C347D / C447A, E145S / D252G / P348K / C447A, Y238R / H386E / A429G, and M242L / N284D / C381A / A429G.

[0152] In some embodiments, the engineered monoamine oxidase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:414, as well as 87 / 113 / 145 / 198 / 242 / 246 / 262 / 266, 87 / 117 / 149 / 242, 87 / 117 / 242 / 260 / 266, 87 / 145 / 149 / 198 / 44 7, 87 / 149 / 242 / 260 / 266, 87 / 149 / 260 / 262 / 266 / 381, 87 / 198, 87 / 242 / 246 / 260 / 262 / 266 / 338, 87 / 284 / 338 / 347, 113 / 117 / 146 / 149 / 260 / 262, 113 / 117 / 266 / 381, 113 / 117 / 447, 113 / 145 / 146 / 149 / 242 / 447, 113 / 242 / 347, 113 / 242 / 381, 117 / 145 / 146 / 262 / 284, 117 / 145 / 149 / 284 / 447, 117 / 149, 117 / 198, 117 / 198 / 262, 117 / 242 / 246 / 260 / 266 / 284 / 381, 117 / 242 / 260 / 262, 117 / 246 / 260 / 262 / 381, 117 / 246 / 284, 117 / 266, 145 / 146 / 149 / 260 / 262 / 338 / 347, 145 / 146 / 242 / 260 / 262 / 338 / 347 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 145 / 149 / 2 and 447. In some embodiments, the engineered monoamine oxidase has a sequence identity that is 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%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 199% or greater sequence identity, as well as 87G / 113G / 145L / 198D / 242I / 246M / 262F / 266L, 87G / 117K / 149M / 242I, 87G / 145L / 149M / 198D / 447A, 87G / 149M / 242I / 260S / 266L, 87L / 117K / 242I / 260S / 266L, 87L / 149M / 260S / 262F / 266L / 381A, 87L / 198D, 87L / 242I / 246M / 260S / 262F / 266L / 338G, 87L / 284D / 338 G / 347N, 113G / 117K / 146A / 149M / 260S / 262F, 113G / 117K / 266L / 381A, 11 3G / 117K / 447A, 113G / 145L / 146A / 149M / 242I / 447A, 113G / 242I / 347N, 1 13G / 242I / 381A, 117K / 145L / 146A / 262F / 284D, 117K / 145L / 149M / 284D / 447A, 117K / 149M, 117K / 198D, 117K / 198D / 262F, 117K / 242I / 246M / 260S / 266L / 284D / 381A, 117K / 242I / 260S / 262F, 117K / 246M / 260S / 262F / 381 A, 117K / 246M / 284D, 117K / 266L, 145L / 146A / 149M / 260S / 262F / 338G / 34 7N, 145L / 146A / 242I / 260S / 262F / 338G / 347N / 447A, 145L / 149M / 198D / 2 60S / 266L / 338G / 347N, 145L / 149M / 260S / 266L / 338G / 347N / 381A / 447A, In some embodiments, the engineered monoamine oxidase comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO:414 selected from: 146A / 149M / 260S / 262F / 347N, 149M / 198D / 246M / 260S / 266L / 421S, 198D / 447A, 242I, 242I / 246M, 242I / 246M / 338G / 347N, 242I / 260S / 262F / 338G, 242I / 381A, 246M, 260S / 262F / 277T / 284D / 447A, 260S / 266L, 262F, 347N / 381A, and 447A. In some embodiments, the engineered monoamine oxidase comprises an amino acid sequence having one or more residue differences compared to the reference sequence SEQ ID NO:414 selected from: 146A / 149M / 260S / 262F / 347N, 149M / 198D / 246M / 260S / 266L / 421S, 198D / 447A, 242I, 242I / 246M, 242I / 246M / 338G / 347N, 242I / 260S / 262F / 338G, 242I / 381A, 246M,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, as well as P87G / N113G / E145L / E198D / M242I / I246M / W262F / A266L, P87G / P117K / L149M / M242I, P87G / E145L / L149M / E198D / C447A, P87G / L149M / M242I / R260S / A266L, P87L / P117K / M242I / R260S / A266L, P87L / L149M / R26 0S / W262F / A266L / C381A, P87L / E198D, P87L / M242I / I246M / R260S / W262F / A 266L / C338G, P87L / N284D / C338G / C347N, N113G / P117K / D146A / L149M / R260S / W262F, N113G / P117K / A266L / C381A, N113G / P117K / C447A, N113G / E145L / D 146A / L149M / M242I / C447A, N113G / M242I / C347N, N113G / M242I / C381A, P117 K / E145L / D146A / W262F / N284D, P117K / E145L / L149M / N284D / C447A, P117K / L149M, P117K / E198D, P117K / E198D / W262F, P117K / M242I / I246M / R260S / A26 6L / N284D / C381A, P117K / M242I / R260S / W262F, P117K / I246M / R260S / W262F / C381A, P117K / I246M / N284D, P117K / A266L, E145L / D146A / L149M / R260S / W2 62F / C338G / C347N, E145L / D146A / M242I / R260S / W262F / C338G / C347N / C447 A, E145L / L149M / E198D / R260S / A266L / C338G / C347N, E145L / L149M / R260S / A 266L / C338G / C347N / C381A / C447A, D146A / L149M / R260S / W262F / C347N, L14 9M / E198D / I246M / R260S / A266L / C421S, E198D / C447A, M242I, M242I / I246M,The amino acid sequence has one or more residue differences compared to SEQ ID NO: 414 selected from M242I / I246M / C338G / C347N, M242I / R260S / W262F / C338G, M242I / C381A, I246M, R260S / W262F / C277T / N284D / C447A, R260S / A266L, W262F, C347N / C381A, and C447A.

[0153] In some embodiments, the engineered monoamine oxidase polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:414, as well as 87 / 145 / 149 / 198 / 447, 87 / 149 / 260 / 262 414, 113 / 117 / 146 / 149 / 260 / 262, 117 / 145 / 149 / 284 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 146 / 149 / 260 / 262 / 347, and 149 / 198 / 246 / 260 / 266 / 421. In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:414, as well as 87G / 145L / 149M / 198D / 447A, 87L / 149M / 260S / 262F / 266L / 381A, 113 and 149M / 198D / 260S / 266L / 421S.In some embodiments, the engineered monoamine oxidase has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the reference sequence SEQ ID NO:414, as well as P87G / E145L / L149M / E198D / C447A, P87L / L149M / R260S / W262F / A266L / C381A, N113G / P117K / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 414 selected from D146A / L149M / R260S / W262F, P117K / E145L / L149M / N284D / C447A, E145L / L149M / E198D / R260S / A266L / C338G / C347N, D146A / L149M / R260S / W262F / C347N, and L149M / E198D / I246M / R260S / A266L / C421S.

[0154] As will be appreciated by those of skill in the art, in some embodiments, one or a combination of the above selected residue differences may be held constant (i.e., maintained) as a basic feature in the engineered monoamine oxidase, or additional residue differences at other residue positions may be incorporated into the sequence to generate additional engineered monoamine oxidase polypeptides with improved properties. Thus, for any engineered monoamine oxidase containing one or a subset of the above residue differences, it is understood that the invention contemplates other engineered monoamine oxidases that include one or a subset of the residue differences and additionally include one or more residue differences at other residue positions disclosed herein.

[0155] As discussed above, the engineered monoamine oxidase polypeptides can also convert a substrate (e.g., Formula I or Compound 1) to a product (e.g., Formula II or Compound 2). In some embodiments, the engineered monoamine oxidase polypeptides can convert a substrate compound to a product compound with at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more activity compared to the activity of a reference polypeptide of SEQ ID NO:6 and / or 414.

[0156] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting a substrate compound to a product compound with at least twice the activity as compared to SEQ ID NO:6 and / or 414 comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NOs:6-1084.

[0157] In some embodiments, the engineered monoamine oxidase has an amino acid sequence that includes one or more residue differences compared to SEQ ID NO: 6 or 414, and increases expression of the engineered monoamine oxidase activity in a bacterial host cell, particularly in E. coli.

[0158] In some embodiments, the engineered monoamine oxidase has an amino acid sequence that contains one or more residue differences compared to SEQ ID NO:6 or 414, and reduces the enzyme load required for conversion of substrate to product.

[0159] In some embodiments, the engineered monoamine oxidase has an amino acid sequence that contains one or more residue differences compared to SEQ ID NO:6 or 414 and reduces the enzyme load required for conversion of substrate to product by about 10%, 25%, 33%, 50%, 75% or more compared to the reference sequence of SEQ ID NO:6 or 414.

[0160] In some embodiments, the engineered monoamine oxidase has an amino acid sequence that includes one or more residue differences compared to SEQ ID NO: 6 or 414, and the enzyme loading of the engineered monoamine oxidase required for conversion of substrate to product is about 0.5% (w / w), 1% (w / w), 1.5% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 10% (w / w), or less of the substrate compound loading.

[0161] In some embodiments, the engineered monoamine oxidase polypeptide with improved properties has an amino acid sequence comprising a sequence selected from the even numbered sequences within the range of SEQ ID NOs: 6-1084.

[0162] In some embodiments, the engineered monoamine oxidase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to one of the even numbered sequences in the range of SEQ ID NOs: 6-1084, as provided in the Examples, and an amino acid residue difference present in any one of the even numbered sequences in the range of SEQ ID NOs: 6-1084 compared to SEQ ID NOs: 6 or 414.

[0163] In some embodiments, an engineered monoamine oxidase polypeptide of the disclosure comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 429 is a glycine, and the residues are numbered with reference to the amino acid sequence of SEQ ID NO: 6 or 414. Modifications can include substitutions, deletions and insertions. Substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.

[0164] In some embodiments, the engineered monoamine oxidase polypeptides of the present disclosure comprise an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 347 is a cysteine ​​or asparagine and the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with reference to the amino acid sequence of SEQ ID NO: 6 or 414. Modifications can include substitutions, deletions and insertions. Substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.

[0165] In some embodiments, an engineered monoamine oxidase polypeptide of the disclosure comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 421 is a cysteine, the amino acid residue corresponding to residue position 347 is a cysteine ​​or an asparagine, and the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with reference to the amino acid sequence of SEQ ID NO: 6 or 414. Modifications can include substitutions, deletions and insertions. Substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.

[0166] In some embodiments, an engineered monoamine oxidase polypeptide of the disclosure comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 145 is leucine, the amino acid residue corresponding to residue position 149 is methionine, the amino acid residue corresponding to residue position 198 is aspartic acid, the amino acid residue corresponding to residue position 260 is serine, the amino acid residue corresponding to residue position 266 is leucine, the amino acid residue corresponding to residue position 338 is glycine, the amino acid residue corresponding to residue position 347 is asparagine, and the amino acid residue corresponding to residue position 429 is glycine, wherein the residues are numbered with reference to the amino acid sequence of SEQ ID NO: 6 or 414. Modifications may include substitutions, deletions, and insertions. The substitutions may be non-conservative, may be conservative, or may be a combination of non-conservative and conservative substitutions.

[0167] In some embodiments, an engineered monoamine oxidase polypeptide of the disclosure comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 87 is a leucine, the amino acid residue corresponding to residue position 113 is a glycine, the amino acid residue corresponding to residue position 117 is a lysine, the amino acid residue corresponding to residue position 260 is a serine, the amino acid residue corresponding to residue position 338 is a serine, and the amino acid residue corresponding to residue position 421 is a serine, wherein the residues are numbered with reference to the amino acid sequence of SEQ ID NO: 6 or 414. Modifications can include substitutions, deletions and insertions. Substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.

[0168] In addition to the residue positions specified above, any of the engineered monoamine oxidase polypeptides disclosed herein may further comprise other residue differences at other residue positions (i.e., residue positions other than those included herein) compared to SEQ ID NO:6 and / or 414. Residue differences at these other residue positions may result in additional variation in the amino acid sequence without adversely affecting the ability of the polypeptide to convert a substrate to a product. Thus, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered monoamine oxidase polypeptides selected from the even-numbered sequences within the range of SEQ ID NOs: 6-1084, the sequence may further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other amino acid residue positions compared to SEQ ID NOs: 6 and / or 414. In some embodiments, the number of amino acid residue differences compared to the reference sequence can 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 or 50 residue positions. In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 residue positions. Residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, residue differences can include conservative and non-conservative substitutions compared to the monoamine oxidase polypeptide of SEQ ID NO: 6 or 414.

[0169] In some embodiments, the present invention also provides engineered polypeptides, including fragments of any of the engineered monoamine oxidase polypeptides described herein, that retain the functional activity and / or improved properties of the engineered monoamine oxidase. Thus, in some embodiments, the present invention provides polypeptide fragments that are capable of converting a substrate to a product under suitable reaction conditions, the fragments comprising at least about 90%, 95%, 96%, 97%, 98% or 99% of the full-length amino acid sequence of an engineered monoamine oxidase of the invention, such as the exemplary engineered monoamine oxidase polypeptides selected from the even-numbered sequences within the range of SEQ ID NOs: 6-1084. In some embodiments, an engineered monoamine oxidase may have an amino acid sequence that includes a deletion in any one of the monoamine oxidase polypeptide sequences described herein, such as the exemplary engineered polypeptides of the even-numbered sequences within the range of SEQ ID NOs: 6-1084.

[0170] Thus, for any and all embodiments of the engineered monoamine oxidase polypeptides of the invention, the amino acid sequence may include deletions 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 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the monoamine oxidase polypeptide, provided that the relevant functional activity and / or improved properties of the engineered monoamine oxidases described herein are maintained. In some embodiments, the deletions may comprise 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues. In some embodiments, the number of deletions 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, or 50 amino acid residues. In some embodiments, the deletion may comprise the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residues.

[0171] In some embodiments, the engineered monoamine oxidase polypeptides described herein may have an amino acid sequence that includes an insertion compared to any one of the engineered monoamine oxidase polypeptides described herein, such as the exemplary engineered polypeptides of the even-numbered sequences within the range of SEQ ID NOs: 6-1084. Thus, for any and all embodiments of the monoamine oxidase polypeptides of the present disclosure, the insertion may 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, twenty or more amino acids, thirty or more amino acids, forty or more amino acids, or fifty or more amino acids, provided that the relevant functional activity and / or improved properties of the engineered monoamine oxidases described herein are maintained. The insertion may be at the amino or carboxy terminus of the monoamine oxidase polypeptide, or may be an internal insertion.

[0172] In some embodiments, the engineered monoamine oxidases herein may have an amino acid sequence selected from the even-numbered sequences in the range of SEQ ID NOs: 6-1084, and optionally including one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the number of amino acid residues in the amino acid sequence is 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, or 50 amino acid residue deletions, insertions, and / or substitutions, as appropriate. In some embodiments, the amino acid sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions, and / or substitutions, as appropriate. In some embodiments, the substitutions may be conservative or non-conservative.

[0173] In the above embodiments, suitable reaction conditions for engineered polypeptides are provided in the Examples herein.

[0174] In some embodiments, the polypeptides of the invention are 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 can be used with or without being fused to other polypeptides.

[0175] It is understood that the polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein can be composed in whole or in part of naturally occurring amino acids and / or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids that may comprise the polypeptides described herein include, but are not limited to, the D stereoisomers of 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 (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 (Bf); 2-fluoro ... phenylalanine (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 2-Trifluoromethylphenylalanine (Otf); 3-Trifluoromethylphenylalanine (Mtf); 4-Trifluoromethylphenylalanine (Ptf); 4-Aminophenylalanine (Paf); 4-Iodophenylalanine (Pif); 4-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);Styrylalanine (sAla);Anthrylalanine (aAla);3,3-Diphenylalanine (Dfa);3-Amino-5-phenylpentanoic acid (phenypentanoic 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);phosphoserine (pSer);phosphothreonine (pThr);homoaspartic acid (hAsp);homoglutanic acid (homoglutanic 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 (aGly);propargylglycine (pgGly);homoalanine (hAla);norvaline (nVal);homoleucine (hLeu), homovaline (hVal);homoisoleucine (hIle);homoarginine (hArg);N-acetyllysine (AcLys);2,4-diaminobutyric acid (Dbu);2,3-diaminobutyric acid (Dab);N-methylvaline (MeVal);homocysteine ​​(hCys);homoserine (hSer);These include hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that may comprise the polypeptides described herein will be apparent to one of skill in the art (see, e.g., the various amino acids provided in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70

[1989] , and the references cited therein, all of which are incorporated herein by reference). These amino acids may be in either the L- or D-configuration;

[0176] Those skilled in the art will appreciate that amino acids or residues with side chain protecting groups can also be included in the polypeptides described herein.Non-limiting examples of such protected amino acids in the aromatic category in this case 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) (protecting groups are listed in parentheses).

[0177] Conformationally constrained non-coded amino acids that may be comprised in 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.

[0178] In some embodiments, the engineered polypeptide may be in various forms, such as, for example, a substantially purified enzyme, an isolated preparation such as a whole cell transformed with a gene encoding the enzyme, and / or a cell extract and / or lysate of such a cell. The enzyme may be lyophilized, spray dried, precipitated, or in the form of a crude paste, as discussed further below.

[0179] In some embodiments, the engineered polypeptide may be in the form of a biocatalyst composition. In some embodiments, the biocatalyst composition comprises (a) a means for converting a fused bicyclic proline analog compound of formula I to the corresponding imine of formula II by contact with a monoamine oxidase polypeptide, and (b) a suitable cofactor in the presence of oxygen. In some embodiments, the biocatalyst composition comprises a FAD cofactor. In some embodiments, the biocatalyst composition comprises catalase. In some embodiments, the composition further comprises NaHSO3. In some embodiments, the composition further comprises HCN.

[0180] In some embodiments, the engineered polypeptides can be provided on a solid support, such as a membrane, resin, solid carrier, or other solid phase material. The solid support can be composed of organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or metal, such as gold or platinum. The configuration of the solid support can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. The surfaces can be planar, substantially planar, or non-planar. The solid support can be porous or non-porous, and can have swelling or non-swelling characteristics. The solid support can also be composed in the form of wells, depressions, or other containers, vessels, features, or locations.

[0181] In some embodiments, the engineered monoamine oxidase polypeptides of the invention can be immobilized on a solid support so that they retain their improved activity and / or other improved properties compared to the reference polypeptides of SEQ ID NO: 6 or 414. In such embodiments, the immobilized polypeptides can facilitate the biocatalytic conversion of substrate compounds or other suitable substrates to products, and can be easily retained after the reaction is completed (e.g., by retaining the beads to which the polypeptide is immobilized), and thus reused or recycled in subsequent reactions. Such immobilized enzyme processes allow for further efficiency and cost reduction. Thus, it is further contemplated that any of the methods using the monoamine oxidase polypeptides of the invention can be performed using monoamine oxidase polypeptides bound or immobilized on a solid support.

[0182] Methods of enzyme immobilization are well known in the art. Engineered polypeptides can be non-covalently or covalently bound. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (e.g., Yi et al., Proc. Biochem., 42(5): 895-898

[2007] ; Martin et al., Appl. Microbiol. Biotechnol., 76(4): 843-851

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

[2010] ; Truppo et al., Org. Proc. Res. Dev., published online at: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques, 2 nded., Academic Press, Cambridge, MA

[2008] ; Mateo et al., Biotechnol. Prog., 18(3):629-34

[2002] ; and "Bioconjugation Protocols: Strategies and Methods," In Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY

[2004] , the disclosures of each of which are incorporated herein by reference.) Solid supports useful for immobilizing the engineered monoamine oxidases 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 monoamine oxidase polypeptides of the invention include, but are not limited to, chitosan beads, Eupergit C, and SEPABEAD (Mitsubishi Chemical Corporation), which includes the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0183] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzymes in the kit may be present individually or as multiple enzymes. The kit may further include reagents for performing the enzymatic reaction, substrates for evaluating the activity of the enzyme, and reagents for detecting the product. The kit may also include a reagent dispenser and instructions for using the kit.

[0184] In some embodiments, the kit of the present invention comprises an array comprising a plurality of different monoamine oxidase polypeptides at different addressable positions, the different polypeptides being different variants of a reference sequence, each having at least one different improved enzymatic property. In some embodiments, the plurality of polypeptides immobilized on the solid support are configured on the array at different positions that are addressable by robotic delivery of reagents or by detection methods and / or instruments. The array can be used to test a variety of substrate compounds for conversion by the polypeptide. Such arrays comprising a plurality of engineered polypeptides and their methods of use are known in the art (see, for example, WO2009 / 008908A2).

[0185] Polynucleotides encoding engineered monoamine oxidases, expression vectors and host cells In another aspect, the present invention provides a polynucleotide encoding an engineered monoamine oxidase polypeptide as described herein. The polynucleotide may be operatively linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing at least one heterologous polynucleotide encoding an engineered monoamine oxidase is introduced into a suitable host cell to express the corresponding monoamine oxidase polypeptide.

[0186] As will be apparent to one of skill in the art, having a protein sequence and knowledge of the codons corresponding to the various amino acids provides a description of all polynucleotides that can encode the subject polypeptide. The degeneracy of the genetic code, in which the same amino acids are coded for by alternative or synonymous codons, allows for the creation of a very large number of nucleic acids, all of which code for improved monoamine oxidase enzymes. Thus, with knowledge of a particular amino acid sequence, one of skill in the art could 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 any and all possible variations of polynucleotides that can be made to encode the polypeptides described herein by selecting combinations based on possible codon choices, and any such variations should be considered specifically disclosed for any of the polypeptides disclosed as even-numbered sequences within the range of SEQ ID NOs: 6-1084 in the Sequence Listing described herein, including the amino acid sequences presented in Tables 4.2, 5.1, 6.1, 7.1, 8.1 and 9.1, and incorporated herein by reference.

[0187] In various embodiments, the codons are preferably selected to suit the host cell in which the protein will be produced. For example, the preferred codons used in bacteria are used to express the gene in bacteria, the preferred codons used in yeast are used for expression in yeast, and the preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, it is not necessary to replace all codons to optimize the codon usage of monoamine oxidase, since the native sequence will likely contain preferred codons, and the use of preferred codons may not be required for all amino acid residues. Thus, the codon-optimized polynucleotide encoding the monoamine oxidase enzyme may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or more than 90% of the codon positions of the full-length coding region.

[0188] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a monoamine oxidase polypeptide amino acid sequence represented by SEQ ID NO: 5 and / or 413. In some embodiments, the polynucleotide has a nucleic acid sequence that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence encoding an even-numbered sequence within the range of SEQ ID NOs: 6-1084. In some embodiments, the polynucleotide has a nucleic acid sequence that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence within the range of SEQ ID NOs: 5-1083. In some embodiments, codon-optimized sequences of odd-numbered sequences within the range of SEQ ID NOs: 5-1083 enhance expression of the encoded monoamine oxidase, thus resulting in a preparation of the enzyme capable of converting a substrate into a product.

[0189] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference sequence selected from the odd-numbered sequences of SEQ ID NOs: 5-1083, or its complementary sequence, and encodes a monoamine oxidase polypeptide.

[0190] In some embodiments, as described above, the polynucleotide encodes an engineered monoamine oxidase polypeptide having improved properties compared to SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to a reference sequence selected from SEQ ID NO: 6 or 414 and one or more residue differences compared to SEQ ID NO: 6 or 414, wherein the sequence is selected from even numbered sequences in the range of SEQ ID NO: 6-1084. In some embodiments, the reference amino acid sequence is selected from even numbered sequences in the range of SEQ ID NO: 6-1084. In some embodiments, the reference amino acid sequence is SEQ ID NO: 6, while in some other embodiments, the reference sequence is SEQ ID NO: 414.

[0191] In some embodiments, the polynucleotide encodes a monoamine oxidase polypeptide capable of converting one or more substrates into products having improved properties compared to SEQ ID NO: 6 or 414, wherein the polypeptide comprises an amino acid 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 the reference sequence SEQ ID NO: 6 or 414.

[0192] In some embodiments, the polynucleotide encoding the engineered monoamine oxidase comprises a polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NOs: 5-1083.

[0193] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from the odd-numbered sequences in the range of SEQ ID NOs: 5-1083, or its complement, and encodes a monoamine oxidase polypeptide having one or more of the improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a monoamine oxidase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any of the even-numbered sequences of SEQ ID NOs: 6-1084, having an amino acid sequence containing one or more residue differences compared to SEQ ID NOs: 6 and / or 414.

[0194] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered monoamine oxidase with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6 or 414. In some embodiments, the polynucleotide encodes a polypeptide as described herein but has at least about 80%, 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 monoamine oxidase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5-1083.

[0195] In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered monoamine oxidase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encodes a polypeptide as described herein, but has at least about 80%, 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 monoamine oxidase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5-993.

[0196] In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered monoamine oxidase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 414. In some embodiments, the polynucleotide encodes a polypeptide as described herein but has at least about 80%, 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 monoamine oxidase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 995-1083.

[0197] In some embodiments, the isolated polynucleotide encoding any of the engineered monoamine oxidase polypeptides provided herein is engineered in various ways to facilitate expression of the enzyme polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector, in which one or more control sequences are present to regulate expression of the polynucleotide and / or the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0198] In some embodiments, control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As is known in the art, suitable promoters can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application include, but are not limited to, promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 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] ).Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a promoter from the gene for Aspergillus niger neutral alpha-amylase and the Aspergillus oryzae triosephosphate isomerase gene), as well as mutants, truncations and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0199] In some embodiments, the control sequence is 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 host cell selected can be used in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., supra).

[0200] In some embodiments, the control sequence is a suitable leader sequence, a non-translated 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. Exemplary leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) genes. The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of a nucleic acid sequence, which, when transcribed, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0201] In some embodiments, the control sequence is a coding region that encodes a signal peptide, an amino acid sequence linked to the amino terminus of a polypeptide that directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region that is naturally linked in translation reading frame 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. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in expressing the engineered monoamine oxidase polypeptides provided herein. Useful signal peptide coding regions for bacterial host cells include, but are not limited to, those from the genes of Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). 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 include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0202] In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is sometimes referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions include, but are not limited to, the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, for example, WO95 / 33836). When both a signal peptide region and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0203] In some embodiments, regulatory sequences are also utilized. These sequences aid in regulating the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned 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, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory sequences include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0204] The present invention also includes recombinant expression vectors that include a polynucleotide encoding an engineered monoamine oxidase polypeptide and, depending on the type of host to be introduced, one or more expression control regions, such as a promoter and terminator, an origin of replication, etc. In some embodiments, the various nucleic acids and control sequences described above are combined together to generate a recombinant expression vector that includes one or more convenient restriction sites to allow for the insertion or substitution of a nucleic acid sequence encoding a variant monoamine oxidase polypeptide at such sites. Alternatively, the polynucleotide sequences of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct containing the polynucleotide sequence into an appropriate vector for expression. In creating an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

[0205] The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the variant monoamine oxidase polynucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector will be introduced. The vector can be a linear or closed circular plasmid.

[0206] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is not dependent on chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means to ensure self-replication. In some alternative embodiments, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell or into a transposon, may be used.

[0207] In some embodiments, the expression vector preferably contains one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to, 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 include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one engineered monoamine oxidase of the invention, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered monoamine oxidase(s) in the host cell.Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Accession No. 201178]); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Exemplary host cells are Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).

[0208] Thus, in another aspect, the invention provides a method of producing an engineered monoamine oxidase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered monoamine oxidase under conditions suitable for expression of the polypeptide, in some embodiments the method further comprises isolating and / or purifying the monoamine oxidase polypeptide as described herein.

[0209] Suitable culture media and growth conditions for the above host cells are well known in the art. Polynucleotides for the expression of monoamine oxidase polypeptides can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0210] The engineered monoamine oxidase with the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered monoamine oxidase polypeptide to mutagenesis and / or directed evolution as known in the art and described herein.Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

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

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

[1996] ).

[0211] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened and assayed. Mutagenesis and directed evolution methods are well known in the art (e.g., U.S. Patents 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,3 44, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,3 95,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453 , 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 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, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all related U.S. patents, as well as PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

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

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

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

[1985] ;Carter, Biochem.

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

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

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

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

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

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

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

[1994] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; and WO2009 / 152336, all of which are incorporated herein by reference).

[0212] In some embodiments, the enzyme clones obtained after the mutagenesis treatment are screened by subjecting the enzyme to a defined temperature (or other assay conditions, e.g., testing the activity of the enzyme on a wide range of substrates) and measuring the amount of enzyme activity remaining after the heat treatment or other assay conditions. Clones containing polynucleotides encoding monoamine oxidase polypeptides are then sequenced to identify nucleotide sequence changes (if any) and used to express the enzyme in a host cell. Measurement of enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0213] In some embodiments, the clones obtained after the mutagenesis treatment can be screened for engineered monoamine oxidases with one or more desired improved enzymatic properties (e.g., improved regioselectivity). Measurement of enzyme activity from an expression library can be performed using standard biochemical techniques, such as GC analysis, HPLC analysis, and / or derivatization of the products (before or after separation), for example with dansyl chloride or OPA (see, e.g., Yaegaki et al., J Chromatogr. 356(1):163-70

[1986] ).

[0214] When 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 connected (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides encoding portions of monoamine oxidase can be prepared by chemical synthesis known in the art (e.g., the classical phosphoramidite method of Beaucage et al., Tet. Lett. 22:1859-69

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

[1984] ), typically performed in an automated synthesis method. 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. In some embodiments, additional variations can be created by synthesizing oligonucleotides containing deletions, insertions and / or substitutions, and combining oligonucleotides in various permutations to create engineered monoamine oxidases with improved properties.

[0215] Thus, in some embodiments, a method of preparing an engineered monoamine oxidase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 6-1084, wherein the polypeptide has one or more residue differences compared to SEQ ID NO: 6 or 414; and (b) expressing the monoamine oxidase polypeptide encoded by the polynucleotide.

[0216] In some embodiments of the methods, the polynucleotide encodes an engineered monoamine oxidase, optionally having one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 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, or 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the substitutions may be conservative or non-conservative.

[0217] In some embodiments, any of the engineered monoamine oxidase enzymes 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. Solutions suitable for lysis of bacteria such as E. coli and highly efficient extraction of proteins from bacteria are commercially available (e.g., CelLytic B™, Sigma-Aldrich, St. Louis MO).

[0218] Chromatographic techniques for isolation of monoamine oxidase polypeptides include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography, among others. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those of skill in the art.

[0219] In some embodiments, affinity techniques may be used to isolate improved monoamine oxidase enzymes. Affinity chromatography purification may use any antibody that specifically binds to a monoamine oxidase polypeptide. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a monoamine oxidase polypeptide or a fragment thereof. The monoamine oxidase polypeptide or fragment may be bound to a suitable carrier, such as BSA, by a side chain functional group or by a linker that is attached to the side chain functional group. In some embodiments, affinity purification may use a specific ligand that the monoamine oxidase binds, or a dye affinity column (see, e.g., EP0641862; Stellwagen, "Dye Affinity Chromatography," In Current Protocols in Protein Science, Unit 9.2-9.2.16

[2001] ).

[0220] Methods of Using Monoamine Oxidase Enzymes In some embodiments, the monoamine oxidase enzymes described herein are used in a process for the conversion of one or more suitable substrates to products. In some embodiments, the monoamine oxidase enzymes described herein are used in a process for the conversion of one or more suitable amine substrates to one or more imine products. In some embodiments, the imine products are fused bicyclic proline analogs.

[0221] In another aspect, the engineered monoamine oxidase polypeptides disclosed herein can be used in a process for the conversion of a substrate of formula 1 to a product of formula II as depicted in Scheme 1 above. [ka]

[0222] Scheme 5 depicts three elementary reactions that together provide the overall net reaction depicted in Scheme 1. In the first reaction of Scheme 5, a secondary amine, which is a heterobicyclic compound according to structural formula I, is enantioselectively oxidized to the corresponding imine compound of structural formula II by the engineered monoamine oxidase of the present disclosure (complexed with flavin adenine nucleotide cofactor (FAD)) to yield the corresponding substantially enantiomerically pure imine of structural formula II, and a reduced monoamine oxidase FAD complex (MAO-FAD-H2). In the second step, the reduced monoamine oxidase (MAO-FAD-H2 complex) is reoxidized by molecular oxygen, thereby generating hydrogen peroxide (H2O2) as a by-product. In the third reaction (not catalyzed by the engineered monoamine oxidase), hydrogen peroxide (H2O2) is decomposed to water and oxygen.

[0223] In certain embodiments, the disclosure provides engineered monoamine oxidases capable of converting an amine compound of formula I to an imine compound of formula II. In one embodiment, the engineered monoamine oxidase polypeptides are capable of catalyzing the conversion of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane (compound 1) to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene (compound 2), as depicted in Scheme 6 below. [ka]

[0224] In some embodiments, the disclosure provides an engineered monoamine oxidase enzyme having improved activity compared to a reference enzyme in producing compound 2. In some embodiments, the engineered monoamine oxidase enzyme has an activity of Scheme 6.

[0225] In some embodiments, the monoamine oxidase enzymes described herein are used in a process for the conversion of one or more substrates to a product.

[0226] In another aspect, the disclosure provides a process for the conversion of a substrate of formula I to a product of formula II using one or more engineered monoamine oxidases. In one embodiment, the disclosure provides a process for the conversion of a substrate of compound 1 to a product of compound 2 using one or more engineered monoamine oxidases.

[0227] In some embodiments, the present disclosure provides a product of formula II [ka] A process for preparing a substrate of formula I [ka] under suitable conditions such that a product of formula II is prepared.

[0228] In some embodiments, the present disclosure provides compound 2 [ka] A process for preparing a substrate of compound 1 [ka] with an engineered monoamine oxidase polypeptide disclosed herein under suitable conditions such that the product compound 2 is prepared.

[0229] In some embodiments, the process of preparing compound 2 from compound 1 comprises contacting compound 1 with an engineered monoamine oxidase comprising 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity to a polypeptide sequence selected from the even-numbered sequences of SEQ ID NOs: 6-1084. In some embodiments, the process of preparing compound 2 from compound 1 comprises contacting compound 1 with an engineered monoamine oxidase comprising a polypeptide sequence selected from the even-numbered sequences of SEQ ID NOs: 6-1084.

[0230] In certain embodiments, the present disclosure provides engineered monoamine oxidases capable of converting amine compounds of formula I to imine compounds of formula II, which may be further converted to aminosulfonic acid products of formula III as depicted above in Scheme 2. In another embodiment, the aminosulfonic acid products of formula III are further converted to amino acids in the presence of acid and water.

[0231] In one embodiment, compound 2 is converted to compound 3 to sequester the oxidation product, as depicted below in Scheme 7. Compound 3 is then quenched with NaOH to isolate and purify the compound 2 product. [ka]

[0232] In the embodiments provided herein and illustrated in the Examples, a range of suitable reaction conditions that may be used in the process include, but are not limited to, substrate loading, cofactor loading, pH, temperature, buffer, solvent system, polypeptide loading, oxygen sparging, antifoam and reaction time. Additional reaction conditions suitable for carrying out a biocatalytic conversion process of substrate compounds to product compounds using the engineered monoamines described herein can be readily optimized by routine experimentation in light of the guidance provided herein, including, but not limited to, contacting the engineered monoamine polypeptide with one or more substrate compounds under experimental reaction conditions for concentration, pH, temperature and solvent conditions, and detecting the product compounds.

[0233] Substrate secondary amines of structural formula I are either commercially available or readily synthesized using methods and reagents known in the art or readily adapted in light of the present disclosure from methods and reagents known in the art (see, e.g., Delalu et al. (1999) J. Heterocyclic Chem. 36, 681; WO2007 / 022459; and WO2007 / 075790, and references cited therein).

[0234] Hydrogen peroxide is a strong oxidizing agent that can irreversibly inactivate monoamine oxidase enzymes. Thus, in certain embodiments, a component useful for facilitating the disproportionation reaction depicted in step 3 of Scheme 5 above, in which hydrogen peroxide (H2O2) is decomposed into molecular oxygen and water. In certain embodiments, the component is selected from chemical agents such as, but not limited to, Pd, Fe, and the like, while in other embodiments, the component is an enzyme, such as the enzyme catalase. In certain embodiments, the reaction mixture further comprises the enzyme catalase to catalyze the disproportionation reaction in step 3 of Scheme 5, in which two hydrogen peroxide molecules are decomposed to yield two water molecules and one oxygen molecule. In certain embodiments, the catalase is Aspergillus niger catalase, which is included in the reaction at a concentration of about 0.01% to about 1% (w / v), about 0.05% to about 0.5% (w / v), or about 0.1% to about 0.2% (w / v). In certain other embodiments, the catalase is bovine catalase.

[0235] In cases where the compound of formula I is a volatile liquid, the compound of formula I may be provided as a salt for ease of handling. In one aspect of this embodiment, the amine substrate is converted to the acetate salt, for example, by adding one equivalent of acetic acid to a 10% solution of the free base dissolved in heptane. The precipitated salt is collected, washed with a solvent (e.g., the solvent in which the salt was precipitated, e.g., heptane), and dried under reduced pressure at room temperature (about 21°C).

[0236] As shown in Scheme 1, the ultimate oxidant is molecular oxygen. In view of the limited solubility of oxygen in water, and its decreasing solubility with increasing temperature and salinity (solute concentration), molecular oxygen in the solution for the reaction can be replenished by gas-liquid mass transfer from the gas phase. Typically, the activity and amount of the preferred monoamine oxidase provided for the actual reaction rate is sufficient activity and amount for the reaction to be limited by the gas-liquid mass transfer rate. As is well known, the rate of gas-liquid mass transfer depends on the partial pressure of the gas in the liquid, the solubility of the gas in the liquid, and the gas-liquid interfacial area. Therefore, the rate of gas-liquid mass transfer limited reactions can be increased by operating environments that provide active gas-liquid mixing, including those by sparging, hollow shaft impeller suction, countercurrent gas-liquid circulation, vertical serpentine tubular flow, etc. Additionally, in any operating environment, the rate of oxygen gas-liquid mass transfer limited reactions can be increased by increasing the partial pressure of oxygen, either by increasing the total pressure of the gas, increasing the oxygen fraction in the gas (e.g., enriching air with purified oxygen or substituting purified oxygen for air, for both). In certain embodiments, the concentration of dissolved oxygen and / or the rate of oxygen consumption from the gas phase are continuously monitored, and the oxygen supply rate, partial pressure, mixing efficiency, or a combination thereof, are adjusted to obtain a beneficial reaction rate or total reaction time to completion.

[0237] The substrate compounds in the reaction mixture can be varied, for example, taking into consideration the desired amount of product compounds, the effect of each substrate concentration on enzyme activity, the stability of the enzyme under the reaction conditions, and the percent conversion of substrate to product. In some embodiments, suitable reaction conditions include substrate compound loadings of at least about 0.5 to about 25 g / L, 1 to about 25 g / L, 5 to about 25 g / L, about 10 to about 25 g / L, or 20 to about 25 g / L. In some embodiments, suitable reaction conditions include substrate compound loadings of at least about 0.5 g / L, at least about 1 g / L, at least about 3 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, or even higher.

[0238] In carrying out the monoamine oxidase-mediated processes described herein, the engineered polypeptides can be added to the reaction mixture in the form of purified enzymes, partially purified enzymes, whole cells transformed with genes encoding the enzymes, as cell extracts and / or lysates of such cells, and / or as enzymes immobilized on solid supports. Whole cells or cell extracts transformed with genes encoding engineered monoamine oxidase enzymes, their lysates, and isolated enzymes can be utilized in a variety of different forms, including solids (e.g., lyophilized, spray-dried, etc.) or semi-solids (e.g., crude pastes). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.), followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before lyophilization. Any of the enzyme preparations (including whole cell preparations) may be stabilized by cross-linking, for example using known cross-linking agents such as glutaraldehyde, or by immobilization on a solid phase (e.g., Eupergit C, etc.).

[0239] Genes encoding monoamine oxidase polypeptides can be transformed into host cells separately or can be transformed together into the same host cell. For example, in some embodiments, one set of host cells can be transformed with a gene encoding one engineered monoamine oxidase polypeptide, and another set can be transformed with a gene encoding another engineered monoamine oxidase polypeptide. Both sets of transformed cells can be used together in the reaction mixture, either in the form of whole cells or in the form of lysates or extracts obtained therefrom. In other embodiments, host cells can be transformed with genes encoding multiple engineered monoamine oxidase polypeptides. In some embodiments, engineered polypeptides can be expressed in the form of secreted polypeptides, and the culture medium containing the secreted polypeptides can be used for monoamine oxidase reaction.

[0240] In some embodiments, the improved activity of the engineered monoamine oxidase polypeptides disclosed herein results in a process that can obtain a higher conversion percentage at a lower concentration of engineered polypeptide. In some embodiments of the process, suitable reaction conditions include an engineered polypeptide amount of about 0.5% (w / w), 1% (w / w), 1.5% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of the substrate compound load.

[0241] In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. In some embodiments, the monoamine oxidase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L.

[0242] During the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range. This can be done by adding an acid or base (such as NaOH) before and / or during the reaction. Alternatively, the pH can be controlled by using a buffering agent. Thus, in some embodiments, the reaction conditions include a buffering agent. Suitable buffering agents for maintaining a desired pH range are known in the art and include, by way of example and without limitation, sodium phosphate, borate, phosphoric acid, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. In some embodiments, the reaction conditions include water as a suitable solvent in the absence of a buffering agent.

[0243] In process embodiments, the reaction conditions include a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base (such as NaOH), a suitable buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled prior to and / or during the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 11, a pH of about 5 to about 10, a pH of about 5 to about 9, a pH of about 6 to about 9, or a pH of about 6 to about 8. In some embodiments, the reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or 11.

[0244] In embodiments of the process herein, suitable temperatures are used for the reaction conditions, for example, taking into account the increased reaction rate at higher temperatures and the activity of the enzyme during the reaction. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 60°C, about 10°C to about 55°C, about 15°C to about 60°C, about 20°C to about 60°C, about 20°C to about 55°C, about 25°C to about 55°C, or about 30°C to about 50°C. In some embodiments, suitable reaction conditions include temperatures of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzyme reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzyme reaction can be adjusted by a temperature profile during the reaction.

[0245] In some embodiments, the process of the invention is carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or co-solvent systems, with the co-solvent system generally comprising an aqueous solvent, an organic solvent, and / or a polymeric solvent. The aqueous solvent (water or aqueous co-solvent system) may or may not be pH buffered. In some embodiments, the process using the engineered monoamine oxidase polypeptide may be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IPA), dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), an ionic or polar solvent (e.g., 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, the co-solvent may be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or a lower alcohol. The non-aqueous co-solvent component of the aqueous co-solvent system may be miscible with the aqueous component, thus resulting in a single liquid phase, or may be partially miscible or immiscible with the aqueous component, thus resulting in two liquid phases. An exemplary aqueous co-solvent system may include water and one or more co-solvents selected from organic solvents, polar solvents, and polyol solvents. In general, the co-solvent component of the aqueous co-solvent system is selected such that it does not deleteriously inactivate the monoamine oxidase enzyme under the reaction conditions. A suitable co-solvent system can be readily identified by utilizing an enzyme activity assay, such as those described herein, to measure the enzyme activity of a designated engineered monoamine oxidase enzyme with a defined substrate of interest in a candidate solvent system.

[0246] In some embodiments of the process, suitable reaction conditions include an aqueous co-solvent that includes about 1% to about 50% (v / v), about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v), or about 10% to about 20% (v / v) DMSO. In some embodiments of the process, suitable reaction conditions may include an aqueous co-solvent that includes about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v) ethanol.

[0247] In some embodiments, the reaction conditions include a surfactant to stabilize or enhance the reaction. The surfactant may include nonionic, cationic, anionic and / or amphiphilic surfactants. Exemplary surfactants include, by way of example and without limitation, nonylphenoxypolyethoxyethanol (NP40), TRITON® X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamide sulfate, polyoxyethylene-sorbitan monostearate, hexadecyldimethylamine, and the like. Any surfactant that can stabilize or enhance the reaction can be utilized. The concentration of the surfactant utilized in the reaction can generally be 0.1-50 mg / ml, particularly 1-20 mg / ml.

[0248] In some embodiments, the reaction conditions may include an antifoaming agent to help reduce or prevent the formation of bubbles in the reaction solution, for example when the reaction solution is mixed or sparged. Antifoaming agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary antifoaming agents include Antifoam-204 or Antifoam-Y 30 (Sigma, St. Louis MO), Y-30® (Dow Corning), polyglycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the antifoaming agent may be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the antifoaming agent may be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v), or higher, as desired to facilitate the reaction.

[0249] The amounts of reactants used in the monoamine oxidase reaction will generally vary depending on the amount of product desired, and concomitantly, the amount of monoamine oxidase substrate utilized. Those of skill in the art will readily understand how to vary these amounts to suit the desired productivity levels and scale of production.

[0250] In some embodiments, the order of addition of the reactants is not important. The reactants may be added together and at the same time to a solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or alternatively, some of the reactants may be added separately and some may be added together at different times. For example, a cofactor and a substrate may be added to the solvent first.

[0251] Solid reactants (e.g., enzymes, salts, etc.) can be supplied to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray dried, etc.), solutions, oil emulsions, suspensions, etc. Reactants can be readily lyophilized or spray dried using methods and equipment known to those of skill in the art. For example, a protein solution can be frozen in small portions at -80°C and then added to a pre-cooled lyophilization chamber followed by application of a vacuum.

[0252] When using an aqueous co-solvent system, the monoamine oxidase and cofactors can be added and mixed first in the aqueous phase to improve mixing efficiency. The monoamine oxidase substrate can be added and mixed followed by the organic layer, or the substrate can be dissolved in the organic layer and mixed. Alternatively, the monoamine oxidase substrate can be premixed in the organic phase prior to addition to the aqueous phase.

[0253] The process of the present invention is generally allowed to proceed until the further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate is converted, or less than 5% of substrate is converted).In some embodiments, the reaction is allowed to proceed until the substrate is completely or nearly completely converted to product.The conversion of substrate to product can be monitored using known methods by detection of substrate and / or product, with or without derivatization.Suitable analytical methods include gas chromatography, HPLC, MS, etc.

[0254] In some embodiments of the process, suitable reaction conditions include a substrate loading for each of one or more substrates of at least about 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, or more, and the method results in at least about 50%, 60%, 70%, 80%, 90%, 95%, or more conversion of substrate compounds to product compounds in about 24 hours or less, in about 12 hours or less, in about 6 hours or less, or in about 4 hours or less.

[0255] In some further embodiments of the process for converting one or more substrate compounds to product compounds using engineered monoamine oxidase polypeptides, suitable reaction conditions include an initial substrate loading to the reaction solution so that the polypeptide is contacted. The reaction solution is then further supplemented with additional substrate compound over time as continuous or batchwise addition at a rate of at least about 3 g / L / h, at least about 5 g / L / h, at least about 8 g / L / h, at least about 10 g / L / h, or higher for the substrate compound. Thus, according to these suitable reaction conditions, the polypeptide is added to a solution having an initial substrate loading of at least about 10 g / L, 20 g / L, or 30 g / L. This addition of the polypeptide is then followed by continuous addition of additional substrate to the solution at a rate of at least about 3 g / L / h, at least about 5 g / L / h, at least about 8 g / L / h, at least about 10 g / L / h, or higher, until a much higher final substrate loading of at least about 60 g / L or higher is reached. Thus, in some embodiments of the process, suitable reaction conditions include adding the polypeptide to a solution having an initial substrate loading of at least about 10 g / L, 20 g / L, or 30 g / L, followed by adding additional substrate to the solution at a rate of 3 g / L / h, at least about 5 g / L / h, at least about 8 g / L / h, at least about 10 g / L / h or faster, to a final substrate loading of at least about 60 g / L or more. This substrate-replenishment reaction condition allows for a higher substrate loading to be achieved while maintaining a high conversion rate of substrate to product of at least about 5%, 25%, 50%, 75%, 90% or more conversion of substrate.

[0256] In some embodiments of the process, the suitable reaction conditions include a cofactor. In some embodiments, the cofactor is selected from the group consisting of FAD, FMN, NADP, and NAD. In certain embodiments, the cofactor is FAD. In some embodiments, the suitable reaction conditions include about 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, or 0.4 mM of FDA cofactor.

[0257] In some embodiments of the process, the suitable reaction conditions include a component useful for promoting the disproportionation of the hydrogen peroxide by-product of the monoamine oxidase-catalyzed reaction to molecular oxygen and water. In certain embodiments, the component is Pd, Fe, or catalase. In certain embodiments, the suitable reaction conditions include catalase. In some embodiments, the suitable reaction conditions include about 0.0005 g / L, 0.001 g / L, 0.01 g / L, 0.1 g / L, 0.5 g / L, or 1 g / L of catalase.

[0258] In certain embodiments of the processes disclosed herein, the suitable reaction conditions include a monoamine oxidase enzyme together with a cofactor and together with bisulfite under conditions that result in a mixture that includes an aminosulfonate. In some embodiments, the bisulfite is added to the reaction before or simultaneously with the engineered monoamine oxidase. In some embodiments, the bisulfate is added after the addition of the monoamine oxidase. In certain other embodiments, the amine substrate is contacted with oxygen and the monoamine oxidase enzyme in association with a cofactor prior to the addition of bisulfite. In certain further embodiments, the suitable reaction conditions further include cyanide such that the aminosulfonate is further converted to an aminonitrile. In yet further embodiments, the aminonitrile is further converted to an amino acid in the presence of acid and water.

[0259] Any of the processes disclosed herein using engineered polypeptides for the preparation of compounds of formula II, including but not limited to compound 2, can be carried out under a range of suitable reaction conditions, including but not limited to a range of substrates, a range of FAD cofactors, catalase, temperature, pH, solvent system, polypeptide loading, and a range of reaction times. In one example, in some embodiments, the preparation of compounds of formula II, including but not limited to compound 2, can be carried out where suitable reaction conditions include: (a) substrate loading of about 0.0001M to 1.0M of substrate compound; (b) FAD cofactor loading of about 0.005mM to 0.3mM; (c) engineered polypeptide loading of about 0.001g / L to 100g / L; (d) 0.01M to 1M sodium phosphate buffer; (e) 0.0005 to 1g / L of catalase (or equivalent as described herein); (f) pH of 6 to 9; (g) non-rate limiting O2; and (g) temperature of about 20°C to 60°C. In some embodiments, suitable reaction conditions include: (a) about 20 mM compound 1 substrate; (b) about 0.01 mM FAD cofactor; (c) about .004 g / L of engineered polypeptide; (d) 50 mM sodium phosphate buffer; (e) 0.02 g / L of bovine catalase; (f) a pH of 7.5; (g) non-rate-limiting O2; and (g) a temperature of about 25°C.

[0260] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting compound 1 to compound 2 comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO:6 or 414, wherein the polypeptide comprises an amino acid sequence where the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with respect to the amino acid sequence of SEQ ID NO:6 or 414, and the engineered monoamine oxidase polypeptide has 2-fold, 5-fold or 10-fold or greater activity of the engineered monoamine oxidase polypeptide of SEQ ID NO:6 or 414.

[0261] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting compound 1 to compound 2 comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO:6 or 414, wherein the polypeptide comprises an amino acid sequence where the amino acid residue corresponding to residue position 347 is a cysteine ​​or an asparagine and the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with respect to the amino acid sequence of SEQ ID NO:6 or 414, and the engineered monoamine oxidase polypeptide has 2-fold, 5-fold or 10-fold or more activity of the engineered monoamine oxidase polypeptide of SEQ ID NO:6 or 414.

[0262] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting compound 1 to compound 2 comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO:6 or 414, wherein the polypeptide comprises an amino acid sequence where the amino acid residue corresponding to residue position 347 is a cysteine ​​or asparagine, the amino acid residue corresponding to residue position 421 is a cysteine ​​and the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with respect to the amino acid sequence of SEQ ID NO:6 or 414, and the engineered monoamine oxidase polypeptide has 2-fold, 5-fold or 10-fold or greater activity of the engineered monoamine oxidase polypeptide of SEQ ID NO:6 or 414.

[0263] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting compound 1 to compound 2 comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO: 6 or 414, wherein the amino acid residue corresponding to residue position 145 is leucine, the amino acid residue corresponding to residue position 149 is methionine, the amino acid residue corresponding to residue position 198 is aspartic acid and the amino acid residue corresponding to residue position 260 is methionine. the amino acid residue corresponding to residue position 266 is a serine, the amino acid residue corresponding to residue position 267 is a leucine, the amino acid residue corresponding to residue position 338 is a glycine, the amino acid residue corresponding to residue position 347 is an asparagine, and the amino acid residue corresponding to residue position 429 is a glycine, wherein the residues are numbered with reference to the amino acid sequence of SEQ ID NO:6 or 414, and the engineered monoamine oxidase polypeptide has two-fold, five-fold, or ten-fold or more activity of the engineered monoamine oxidase polypeptide of SEQ ID NO:6 or 414.

[0264] In some embodiments, an engineered monoamine oxidase polypeptide capable of converting compound 1 to compound 2 comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence of SEQ ID NO:6 or 414, wherein the polypeptide comprises an amino acid sequence in which the amino acid residue corresponding to residue position 87 is leucine, the amino acid residue corresponding to residue position 113 is glycine, the amino acid residue corresponding to residue position 117 is lysine, the amino acid residue corresponding to residue position 260 is serine, the amino acid residue corresponding to residue position 338 is serine, and the amino acid residue corresponding to residue position 421 is serine, wherein the residues are numbered with respect to the amino acid sequence of SEQ ID NO:6 or 414, and the engineered monoamine oxidase polypeptide has two-fold, five-fold or ten-fold or more activity of the engineered monoamine oxidase polypeptide of SEQ ID NO:6 or 414.

[0265] In some embodiments, additional reaction components or techniques are performed to supplement the reaction conditions. These may include taking measures to stabilize enzymes or prevent enzyme inactivation, to reduce product inhibition, or to shift the equilibrium of the reaction toward the formation of the desired product.

[0266] In further embodiments, any of the above processes for converting one or more substrate compounds to product compounds may further comprise one or more steps selected from extraction, isolation, purification and crystallization of the product compounds. Methods, techniques and protocols for extracting, isolating, purifying and / or crystallizing products from the biocatalytic reaction mixtures produced by the above disclosed processes are known to those skilled in the art and / or obtained by routine experimentation. In addition, exemplary methods are provided in the examples below.

[0267] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting. EXAMPLES

[0268] experiment The following examples, including the experiments performed and the results, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0269] In the examples 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 (minutes) molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); CAM and cam (chloramphenicol); DMSO (dimethyl sulfoxide); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (Luria-Bertani Broth); TB (Terrific Broth; 12 g / L Bacto-tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4); PLP (pyridoxal 5'-phosphate), TEoA (triethanolamine buffer), HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory E. coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FIOPC (fold improvement over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Corning (Corning, Inc., Palo Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA).

[0270] Example 1 Preparation of HTP MAO-containing wet cell pellets E. coli cells containing the recombinant MAO-encoding gene from a monoclonal colony were inoculated into 180 μl of LB containing 1% glucose and 30 μg / mL chloramphenicol in the wells of a 96-well shallow-well microtiter plate. The plate was sealed with an O2-permeable seal and the cultures were grown overnight at 30°C, 200 rpm and 85% humidity. 10 μl of each of the cell cultures was then transferred to wells of a 96-well deep-well plate containing 390 mL of TB and 30 μg / mL CAM. The deep-well plate was sealed with an O2-permeable seal and incubated at an OD of 0.6-0.8. 600 The cells were then incubated at 30°C, 250 rpm and 85% humidity until a final concentration of 1 mM was reached. The cell culture was then induced with IPTG to a final concentration of 1 mM and incubated overnight at 30°C. The cells were then pelleted using centrifugation at 4,000 rpm for 10 minutes. The supernatant was discarded and the pellet was frozen at -80°C prior to lysis.

[0271] Example 2 Preparation of HTP MAO-containing wet cell lysates Frozen pellets prepared as described in Example 2 were lysed with 400 μl of lysis buffer containing 100 mM sodium phosphate buffer, pH 7.0. 1 g / L lysozyme and 0.5 g / L polymyxin b sulfate (PMBS) were added to the cell paste in each well. The cells were lysed at room temperature for 2 hours with shaking on a tabletop shaker. The plates were then centrifuged at 4,000 rpm for 10 minutes at 4° C. The clear supernatants were then used in biocatalysis to determine their activity levels.

[0272] Example 3 Preparation of freeze-dried monoamine oxidase lysate from shake flask (SF) cultures Single colonies containing the desired genes were picked from LB agar plates with 1% glucose and 30 μg / ml CAM and grown overnight at 37° C. A single colony from each culture was transferred to 6 ml of LB with 1% glucose and 30 μg / ml CAM. Cultures were grown for 18 hours at 30° C. and 250 rpm and subcultured approximately 1:50 into 250 ml of TB containing 30 μg / ml CAM to a final OD of 0.05. 600 The culture was grown for approximately 195 minutes at 30°C and 250 rpm to an OD between 0.6 and 0.8. 600 and induced with 1 mM IPTG. The culture was then grown for 20 hours at 30° C. and 250 rpm. The culture was centrifuged at 4,000 rpm for 20 minutes. The supernatant was discarded and the pellet was resuspended in 30 ml of 50 mM sodium phosphate, pH 7.0, and lysed at 18,000 psi using a Microfluidizer® processor system (Microfluidics). The lysate was pelleted (10,000 rpm, 60 minutes) and the supernatant was frozen and lyophilized to produce shake flake (SF) enzyme powder.

[0273] Example 4 Improved activity of MAO compared to SEQ ID NO:6 in high throughput screening SEQ ID NO:6, which has already been evolved for improved amine monooxygenase activity and stability and which was disclosed in WO2010 / 008828A2, was selected as the parent enzyme. SEQ ID NO:6 contains the following mutations compared to the wild type enzyme of SEQ ID NO:2: A289V / T315S / I316V / Q317H / A321P / T324P / E325Q / I327M / S328A / M330A / Q331N / A332I / M337Q / T339V / D346S / N347C / K348P / T354S / A357S / C364A / L379I / F382L / T384G / D385A / A386H / D392E / R396E / E397A / L399K / K400 M / G403E / Q404N / L405M / A406S / T409N / F410M / G411D / V412I / V421C / S433A / R434P / G436Q / M437L / V438L / E440K / C441S / Q443D / G444E / E447C / K448R / R450G / G451N / V453L / S465G / R479V / V480T / L482I / G486R / T487P / K488A / R489P / E490A / K492R / A493S / R494H. Libraries of engineered genes were generated using well-established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced by HTP, cell pellets were obtained and soluble lysates were produced as described in Example 2.

[0274] HTP reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. HTP reactions contained 3 g / L (18 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 4,000-fold diluted MAO-N lysate. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added, and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, and (ii) diluted MAO-N lysate (10 μL of 400-fold dilution) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30 °C with shaking at 600 rpm for 16-18 h. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 4]

[0275] Activity relative to SEQ ID NO:6 was calculated as the fold improvement over positive control (FIOPC), which was determined by dividing the integrated area under the curve of the imine signal in each sample by the area under the curve of the imine signal for the parent variant (positive control) present on the sample plate under the specified reaction conditions, and is shown in Table 4.2. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0276] Example 5 Improved activity of MAO compared to SEQ ID NO:6 in shake flask studies Reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. Reactions contained 3 g / L (18 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 0.01 g / L MAO-N SFP. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, (ii) diluted MAO-N SFP (10 μL of 0.1 g / L) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30 °C with shaking at 600 rpm for 16-18 hours. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 5]

[0277] Example 6 Improved activity of MAO compared to SEQ ID NO:6 in high throughput screening HTP reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. HTP reactions contained 3 g / L (18 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 4,000-fold diluted MAO-N lysate. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added, and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, and (ii) diluted MAO-N lysate (10 μL of 400-fold dilution) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30 °C with shaking at 600 rpm for 16-18 h. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

[0278] Example 7 Improved activity of MAO compared to SEQ ID NO:6 in shake flask studies Reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. Reactions contained 3 g / L (18 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 0.004 g / L MAO-N SFP. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, (ii) diluted MAO-N SFP (10 μL of 0.04 g / L) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30 °C with shaking at 600 rpm for 16-18 hours. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 7-1] [Table 7-2]

[0279] Example 8 Improved activity of MAO compared to SEQ ID NO:414 in high throughput Reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. Reactions included 12.7 g / L (75 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 4,000-fold diluted MAO-N lysate. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added, and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, and (ii) the diluted MAO-N lysate (10 μL of 400-fold dilution) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30°C with shaking at 600 rpm for 16-18 hours. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 8-1] [Table 8-2] [Table 8-3]

[0280] Example 9 Improved activity of MAO compared to SEQ ID NO: 414 in shake flask studies Reactions were performed in a 96-well format in 2 mL deep-well plates in a total volume of 100 μL. Reactions contained 12.7 g / L (75 mM) 6,6-dimethyl-3-azabicyclo[3.1.0]hexane acetate, 4 U catalase, 50 mM sodium phosphate, pH 7.4, and 0.01 g / L MAO-N SFP. Reactions were set up as follows: (i) all reaction components, except for catalase and MAO-N, were mixed in a single solution and the pH was adjusted to 7.4 using H3PO4. Catalase was added and the resulting solution (90 μL) was then aliquoted into each well of a 96-well plate, (ii) diluted MAO-N SFP (10 μL of 0.1 g / L) was then added to the well to initiate the reaction. The reaction plate was heat sealed with a foil seal and incubated at 30 °C with shaking at 600 rpm for 16-18 hours. The reaction was quenched with 100 μL of a 1:1 mixture of acetonitrile in 20 mM ammonium acetate. The quenched reaction was shaken on a benchtop shaker for 3 minutes, followed by centrifugation at 4,000 rpm for 10 minutes at 4° C. to pellet any precipitate. The supernatant (90 μL) was then transferred to a 96-well round-bottom plate. The samples were analyzed according to the RP-HPLC analytical method in Table 4.1. [Table 9-1] [Table 9-2]

[0281] Example 10 Validation of MAO activity in a scale-down process test of fermented powders To a 250 mL 3-neck flask equipped with an overhead stirrer at 300 rpm, 600 mL of Milli-Q water at about 25°C and 0.6 mL (approximately 0.5 g; approximately 4.5 mmol) of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane were added to obtain a homogenous solution with a pH of 11.8. Na2S2O5 (approximately 0.5 g) was added in small portions until a pH of 7.5 was reached. To the colorless solution, 150 μL of Antifoam-204 (Sigma catalog number A-6226) and 300 μL of bovine catalase solution (20 g / L) were added to obtain a colorless solution with a pH of 7.5. To this solution was added monoamine oxidase powder (produced by fermentation in BL21), resulting in a yellow solution. An air sparging probe was inserted (air flow rate was controlled at 60 mL / min using a mass flow controller). The pH of the reaction mixture immediately began to drop. The pH was maintained by feedback controlled dosing of 2N NaOH. After approximately 30 min, the pH remained stable and no further base dosing was performed. After an additional 10 min (40 min total), 75 mL of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane NaHSO3 solution (prepared by adding 13.05 g of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane to 82.5 mL of dH2O, and approximately 15 g of Na2S2O5 sufficient to reach pH 7.2) was added to the reaction at a rate of 3.5 mL / h. The pH of the reaction mixture immediately began to drop, so dosing of 2N NaOH was resumed. Addition of the 6,6-dimethyl-3-azabicyclo[3.1.0]hexane and NaHSO3 solutions was completed after 22 h (total reaction time of approximately 23 h). A 150 μL aliquot was removed, quenched with 150 μL of 8N NaOH (to cleave the aminosulfonate to the free imine) and extracted with 750 μL of CDCl3. 1 H-NMR analysis was used to monitor the conversion of the substrate to azabicyclo[3.1.0]hex-2-ene. [Table 10]

[0282] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0283] While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. 1. An engineered monoamine oxidase comprising a polypeptide sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6 and / or 414, or a functional fragment thereof, wherein said engineered monoamine oxidase polypeptide 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:6 and / or 414.

2. 2. The engineered monoamine oxidase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution at one or more positions selected from 429, 149, 198, 246, 260, 266, and 421, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

3. 2. The engineered monoamine oxidase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution selected from 429G, 149M, 198D, 246M, 260S, 266L, and 421S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

4. 2. The engineered monoamine oxidase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide comprises at least one substitution selected from A429G, L149M, E198D, I246M, R260S, A266L, and C421S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

5. the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide is / 284, 79, 80, 82, 85, 87, 87 / 185, 93, 100, 100 / 144 / 421 / 447, 100 / 144 / 447, 104, 107, 113, 114, 115, 117, 127, 135, 136, 144, 144 / 447, 145, 146, 147, 149, 149 / 157, 153, 155, 161, 162, 166, 167, 169, 170, 174, 177, 181, 184, 185, 187 / 242, 193, 196, 198, 199 , 208, 211, 218, 219, 219 / 332, 221, 229, 232, 234, 238, 239, 240, 242, 246, 250, 252, 254, 260, 262 / 266 / 421, 262 / 421 / 447, 266 / 447, 277, 277 / 347, 284, 290, 292, 294, 298, 300, 306, 310, 323, 332, 338, 347, 349, 359, 361, 366, 381, 385, 386, 387, 392, 393 2. The engineered monoamine oxidase polypeptide of claim 1, comprising at least one substitution or set of substitutions in the polypeptide sequence at one or more positions selected from SEQ ID NO:6, 400, 407, 411 / 445, 413, 418, 421, 425, 429, 433, 434, 441, 444, 445, 447, 449, 452, 475, and 483, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

6. 2. The engineered monoamine oxidase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and wherein the engineered monoamine oxidase polypeptide comprises at least one substitution or set of substitutions in the polypeptide sequence at one or more positions selected from 79, 80, 87, 146, 149, 219 / 332, 238, 260, 262 / 266 / 421, 338, and 429, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

7. the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide is 2 / 381 / 386 / 429, 80 / 188 / 190 / 332 / 386, 80 / 237 / 332, 80 / 238 / 332, 80 / 238 / 386 / 429, 80 / 284, 80 / 284 / 386, 80 / 332, 80 / 332 / 381 / 386, 80 / 332 / 386, 80 / 381, 80 / 386, 80 / 386 / 429, 80 / 429, 82, 82 / 87, 82 / 87 / 113, 82 / 87 / 113 / 117 / 218, 82 / 87 / 113 / 117 / 219, 82 / 87 / 113 / 117 / 219 / 418, 82 / 87 / 113 / 117 / 260 / 338, 82 / 87 / 113 / 117 / 338 / 418, 82 / 87 / 113 / 421, 82 / 87 / 117, 82 / 87 / 117 / 218 / 421, 82 / 87 / 117 / 260 / 338 / 421, 82 / 87 / 117 / 338 / 418, 82 / 87 / 218, 82 / 8 7 / 218 / 219 / 338, 82 / 87 / 218 / 219 / 421, 82 / 87 / 218 / 338, 82 / 87 / 219, 82 / 87 / 219 / 421, 82 / 87 / 338, 82 / 87 / 338 / 418, 82 / 87 / 338 / 421, 82 / 113 / 218 / 338, 82 / 113 / 218 / 338 / 418, 82 / 113 / 219 / 338, 82 / 113 / 338, 82 / 218 / 219 / 421, 82 / 260 / 338 / 421, 82 / 338 / 418 / 421, 82 / 418, 87, 87 / 113, 87 / 113 / 117 / 219 / 3 38 / 418 / 421, 87 / 113 / 117 / 260 / 338 / 421, 87 / 113 / 117 / 418 / 421, 87 / 113 / 218 / 219 / 260 / 338, 87 / 113 / 219 / 421, 87 / 113 / 260, 87 / 117, 87 / 117 / 218 / 219,８７／１１７／２１９／３１０／３３８、８７／２１８、８７／２１８／２１９、８７／２１８／２１９／３３８、８７／２１８／４１８、８７／２１９、８７／２１９／３３８、８７／２１９／４１８、８７／２６０、８７／３１０、８７／３１０／３３８、８７／３３８、８７／４１８、８７／４２１、９３／１８４／４２９、９３／２３８、９３／２３８／２８４、９３／２３８／３８６、９３／２３８／３８６／４２９、１００、１００／１４３／１４５／１４６／１４９／１９８／２４２／３４７／３４８／４４５／４４７、１００／１４３／１４５／１４９／３４７／４４７、１００／１４５／１４６／１４９、１００／１４５／１４６／１４９／１５４／３４８／４４５、１００／１４５／１４６／１４９／１９８、１００／１４５／１４６／１４９／１９８／２４２／２５０／２５２／３４７／３４８／４４７、１００／１４５／１４６／１４９／２０５／３４７／４４５／４４７、１００／１４５／１４６／１４９／２４２／４４５、１００／１４５／１４６／１４９／２５０／２７７／３４７／３４８／４４７、１００／１４５／１４９／１５４／２４６／２７７、１００／１４５／１４９／１９８／２４２／２４６／２７７／３４８／４４７、１００／１４５／１４９／１９８／２４２／２４６／４４５／４４７、１００／１４５／１４９／１９８／２４２／２７７／３４７／３４８／４４７、１００／１４５／１４９／１９８／２５２／３４８／４４５、１００／１４５／１４９／２０５、１００／１４５／１４９／２４２／２４６／２７７／３４７／４４５、１００／１４５／１４９／２４２／２４６／３４７／３４８／４４５／４４７、１００／１４５／１４９／２４２／４４５／４４７、１００／１４５／１４９／２４６／２５２／２７７／３４７／４４５／４４７、１００／１４５／１４９／２４６／３４７／４４５、１００／１４５／１４９／２７７／３４８／４４５、１００／１４５／１４９／３４７／４４５／４４７、１００／１４５／１９８／２４２／２４６／３４８、１００／１４５／１９８／２７７／３４８／４４５、１００／１４５／１９８／３００、１００／１４５／２０５／２４６／２７７／３４８／４４５、１００／１４５／２０５／２７７／３４８、１００／１４５／２４２／２４６／２７７／４４５／４４７、１００／１４５／２４６／３４７／３４８／４４５、１００／１４５／２７７／３４７、１００／１４５／３４８、１００／１４６／１４９／１９８／２４２／２４６／２７７／３４７／３４８／４４５／４４７、１００／１４６／１９８／３４７／３４８／４４７、１００／１４６／１９８／４４５、１００／１４６／２４２／２７７／４４７、１００／１４６／２５０／４４７、１００／１４６／３４７／３４８／４４５／４４７、１００／１４９／１９８、１００／１４９／２４２／２４６／２７７／３４７／３４８／４４５、１００／１４９／２４２／２４６／２７７／３４７／３４８／４４７、１００／１４９／２４２／３４７／３４８／４４７、１００／１４９／２４６／２７７／３４８／４４５、１００／１４９／２４６／４４７、１００／１４９／２５０／３４７／４４７、１００／１９８／２０５／３４８、１００／１９８／２７７／３４７／３４８、１００／１９８／３４７／３４８、１００／１９８／３４７／３４８／４４５、１００／１９８／３４７／４４５、１００／１９８／４４７、１００／２４２／２４６／２７７、１００／２４２／２４６／３４７、１００／２４２／２７７／３４８、１００／２４２／３４７／３４８／４４５、１００／２４２／３４７／３４８／４４５／４４７、１００／２４２／３４８／４４５、１００／２４６／２７７／３４８／４４５、１００／２７７／３４７／４４５／４４７、１００／２７７／３４８、１００／２７７／３４８／４４７、１００／３４７／３４８／４４５、１００／３４７／４４７、１００／４４５／４４７、１１０／３３８／４１８、１１３／２１８／３３８／４２１、１１３／３３８、１１３／３３８／４２１、１１７／２１９、１１７／２６０、１２１／１９８／３４７／４４５／４４７、１４５／１４６／１４９／１５４／２４６／２７７／３４７／３４８、１４５／１４６／１４９／１９８／２４２／２５２／２７７／３４７、１４５／１４６／１４９／１９８／２７７／４４５／４４７、１４５／１４６／１４９／２０５／３４８／４４７、１４５／１４６／１４９／２５０／２７７／３４７／３４８／４４７、１４５／１４６／１４９／２７７／３４７、１４５／１４６／１４９／３４７／３４８、１４５／１４６／１９８／２０５／２４２／２７７／３４８／４４７、１４５／１４６／１９８／４４５／４４７、１４５／１４６／２４６／２７７／４４７、１４５／１４６／２４６／３４７／３４８／４４７、１４５／１４６／２７７／３４７、１４５／１４６／３４７／４４７、１４５／１４９／１９８／２０５／３４８、１４５／１４９／１９８／２４２／２４６／２７７／３４７／４４７、１４５／１４９／１９８／２４２／２４６／３４８、１４５／１４９／１９８／２４２／２７７／３４７、１４５／１４９／１９８／２４６／４４７、１４５／１４９／１９８／３４７／４４７、１４５／１４９／２０５／２４２／３４７／３４８／４４５、１４５／１４９／２０５／２４６／２５０／２５２、１４５／１４９／２０５／２７７／４４５、１４５／１４９／２４２、１４５／１４９／２４２／３４８、１４５／１４９／２４６、１４５／１４９／２４６／３４７／４４５、１４５／１４９／２４６／４４７、１４５／１４９／３４７／３４８／４４５、１４５／１４９／３４７／４４７、１４５／１４９／４４７、１４５／１５５／４４５／４４７、１４５／１９８／２０５／３４８、１４５／２４６、１４５／２４６／２５２／３４７／４４７、１４５／２５０／２５２／３４８、１４５／２５２／３４８／４４７、１４５／２５５、１４５／２７７／３４７／４４５、１４５／２７７／３４８／４４７、１４５／３４７／３４８／４４５／４４７／４９１、１４５／４４５／４４７、１４６／１４９／１５１／２４６／３４８、１４６／１４９／２４２／２４６／４４５、１４６／１４９／２４６／２５２／４４７、１４６／１４９／２７７／３４８／４４７、１４６／１９８／２４６／２７７、１４６／１９８／２７７／３４８／４４７、１４６／２４２、１４６／２４２／２４６／２７７／３４７／３４８／４４５／４４７、１４６／２４６／２７７／３４８／４４５／４４７、１４６／２７７、１４６／２７７／３４７／３４８、１４６／２７７／３４７／４４５／４４７、１４６／２７７／４４５／４４７、１４６／３４７／３４８／４４７、１４６／３４８、１４６／４４５／４４７、１４９、１４９／１９８／２７７／３４７／３４８、１４９／１９８／３４８、１４９／１９８／４４５、１４９／２４２／２４６、１４９／２４２／２４６／３４８／４４５、１４９／３４７／３４８／４４５／４４７、１４９／４４５、１８４、１８４／２８４、１８４／２８４／４２９、１８４／３８６、１９８／２４２／３４８、１９８／２４６、198 / 250, 198 / 277 / 348 / 447, 198 / 445, 205, 205 / 246 / 347, 205 / 252 / 347 / 445 / 447, 205 / 347 / 348 / 445 / 447, 218, 218 / 260 / 338, 219, 219 / 338 / 418, 219 / 421, 238, 238 / 242 / 284, 238 / 284 / 386 / 429 , 238 / 332 / 429, 238 / 381 / 429, 238 / 386, 238 / 429, 242, 242 / 277 / 347 / 348, 242 / 284 / 381 / 429, 242 / 348, 242 / 445 / 447, 246, 246 / 277 / 348 / 447, 246 / 445 / 447, 260, 260 / 338, 260 / 338 / 418 / 421, 27 7 / 347 / 348 / 445, 277 / 347 / 447, 284, 284 / 332, 284 / 386, 284 / 386 / 429, 310 / 338 / 418, 310 / 418 / 421, 332, 332 / 386, 332 / 429, 338, 338 / 418, 347 / 348 / 445, 347 / 348 / 447, 348, 348 / 445 / 447, 348 / 447, 386, 386 / 429, 418, 429, and 447, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

8. the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, and the engineered monoamine oxidase polypeptide is selected from the group consisting of 80 / 429, 87 / 113 / 117 / 260 / 338 / 421, 87 / 421, 100 / 145 / 146 / 149 / 198 / 242 / 250 / 252 / 347 / 348 / 447, 100 / 145 / 149 / 198 / 242 / 277 / 347 / 348 2. The engineered monoamine oxidase polypeptide of claim 1, comprising at least one substitution or set of substitutions in the polypeptide sequence at one or more positions selected from: 145 / 149 / 205 / 246 / 250 / 252, 145 / 246 / 252 / 347 / 447, 110 / 338 / 418, 145 / 149 / 205 / 246 / 250 / 252, 145 / 246 / 252 / 347 / 447, 145 / 252 / 348 / 447, 238 / 386 / 429, and 242 / 284 / 381 / 429, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

6.

9. the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:414, and the engineered monoamine oxidase polypeptide is / 262 / 266 / 381, 87 / 198, 87 / 242 / 246 / 260 / 262 / 266 / 338, 87 / 284 / 338 / 347, 113 / 117 / 146 / 149 / 260 / 262, 113 / 117 / 266 / 381, 113 / 117 / 447, 113 / 145 / 146 / 149 / 242 / 447, 113 / 242 / 347, 113 / 242 / 381, 117 / 145 / 146 / 262 / 284, 117 / 145 / 149 / 284 / 447, 117 / 149, 117 / 198, 117 / 198 / 262, 117 / 242 / 24 6 / 260 / 266 / 284 / 381, 117 / 242 / 260 / 262, 117 / 246 / 260 / 262 / 381, 117 / 246 / 284, 117 / 266, 145 / 146 / 149 / 260 / 262 / 338 / 347, 145 / 146 / 242 / 260 / 262 / 338 / 347 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 145 / 149 / 260 / 266 / 338 / 347 / 381 / 447, 146 / 149 / 260 / 262 / 347, 149 / 198 / 246 / 260 / 266 / 421, 19 2. The engineered monoamine oxidase polypeptide of claim 1, comprising at least one substitution or set of substitutions in the polypeptide sequence at one or more positions selected from SEQ ID NO:414, ...

10. the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:414, and the engineered monoamine oxidase polypeptide is selected from the group consisting of 87 / 145 / 149 / 198 / 447, 87 / 149 / 260 / 262 / 266 / 381, 113 / 117 / 146 / 149 / 260 / 262, 117 / 145 / 149 / 284 / 447, 145 / 149 / 198 / 260 / 266 / 338 / 347, 146 / 149 / 260 / 262 / 347, and 149 / 198 / 246 / 260 / 266 / 421, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

414.

11. 2. The engineered monoamine oxidase polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to any of the even-numbered sequences set forth in SEQ ID NOs: 6-1084.

12. 2. The engineered monoamine oxidase polypeptide of claim 1, comprising a polypeptide sequence set forth in an even-numbered SEQ ID NO:6-1084.

13. 13. An engineered monoamine oxidase polypeptide according to any one of claims 1 to 12, comprising a polypeptide sequence that exhibits at least one improved property compared to the engineered monoamine oxidase polypeptides of SEQ ID NOs: 6 and / or 414.

14. The improved properties are 【Chemistry 31】 14. The engineered monoamine oxidase polypeptide of claim 13, comprising improved production of

15. The improved properties are obtained by comparing compound 1 with the reference sequence of SEQ ID NO: 6 or 414. 【Chemistry 32】 Compound 2 【Chemical 33】 14. The engineered monoamine oxidase polypeptide of claim 13, comprising a reduced enzyme load for conversion to.

16. The improved properties are 【Chemical 34】 Compound 2 【Chemistry 35】 14. The engineered monoamine oxidase polypeptide of claim 13, comprising 2-fold, 5-fold, 10-fold or more activity for conversion of

17. 17. The engineered monoamine oxidase polypeptide of any one of claims 1 to 16, which is purified.

18. 18. The engineered monoamine oxidase polypeptide of any one of claims 1 to 17, which is immobilized on a solid support.

19. 20. A composition comprising at least one engineered monoamine oxidase polypeptide according to any one of claims 1 to 18.

20. 20. An engineered polynucleotide encoding at least one engineered monoamine oxidase polypeptide according to any one of claims 1 to 18.

21. An engineered polynucleotide sequence encoding at least one engineered polypeptide, comprising 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:5 and / or 413, wherein said polynucleotide sequence of said polypeptide comprises at least one substitution at one or more positions.

22. 22. The engineered polynucleotide sequence of claim 20, comprising 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:5 and / or 413.

23. 23. The engineered polynucleotide sequence of any of claims 20 to 22, comprising SEQ ID NO: 5 and / or 413.

24. An engineered polynucleotide comprising any of the odd-numbered sequences set forth in SEQ ID NOs: 5 to 1083.

25. 25. A vector comprising an engineered polynucleotide according to any one of claims 20 to 24.

26. 26. The vector of claim 25, further comprising at least one regulatory sequence.

27. A host cell comprising the vector of claim 25 and / or 26.

28. 28. The host cell of claim 27, which produces at least one engineered monoamine oxidase polypeptide.

29. 29. A method for producing an engineered monoamine oxidase polypeptide in a host cell, comprising culturing a host cell according to claim 27 and / or 28 under suitable conditions such that at least one engineered monoamine oxidase polypeptide is produced.

30. 28. The method of claim 27, further comprising recovering the engineered monoamine oxidase polypeptide.

31. 31. The method of claims 29 and / or 30, further comprising the step of purifying the at least one engineered monoamine oxidase polypeptide.

32. A substantially stereoisomerically pure compound according to structural formula II: 【Chemical 36】 (Wherein, A is O, CR １ R ２ , -C=C-, or -CH ２ -CH ２ -, where R １ and R ２ are each independently -H, -COOH, -X, or -NH ２ , -CH ２ NHC (NH) NH ２ , -CX ３ , -CH ３ , -CH ２ CH ３ where X is selected from F, Cl and Br.

5. A process for preparing a compound according to claim 1, including salts and hydrates thereof, comprising the steps of: Amine Compounds According to Structural Formula I 【Chemical 37】 wherein A is as defined for structural formula II. with oxygen in the presence of an engineered monoamine oxidase enzyme with a cofactor under conditions whereby the engineered monoamine oxidase enzyme oxidizes the amine compound of structural formula I to the corresponding imine compound of structural formula II.

33. 33. The method of claim 32, wherein the cofactor is non-covalently associated with the engineered monoamine oxidase enzyme.

34. 33. The method of claim 32, wherein the cofactor is selected from the group consisting of FAD, FMN, NAD, and NADP.

35. Hydrogen peroxide (H ２ O ２ 33. The method of claim 32, further comprising a component that catalyzes the disproportionation of .

36. 36. The method of claim 35, wherein the component is selected from the group consisting of Pd, Fe, and catalase enzyme.

37. 37. The method of any of claims 32 to 36, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6 and / or 414, or a functional fragment thereof, and the engineered monoamine oxidase polypeptide comprises at least one substitution or set of substitutions in said polypeptide sequence, and the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6 and / or 414.

38. 37. The method of any of claims 32-36, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the even numbered sequences of SEQ ID NOs: 8-1084.

39. A mixture comprising a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b): 【Chemical 38】 wherein A is O, CRR, -C=C-, or -CH-CH-, where R and R are each independently selected from -H, -COOH, -X, -NH, -CHNHC(NH)NH, -CX, -CH, -CHCH, where X is selected from F, Cl, and Br.

5. A process for preparing a compound according to claim 1, including salts and hydrates thereof, comprising the steps of: Amine Compounds According to Structural Formula I 【Chemical 39】 wherein A is as defined for Structural Formula III. with oxygen in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite under conditions to produce said mixture comprising a substantially enantiomerically aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically aminosulfonic acid compound according to structural formula III(b). A method comprising:

40. 40. The method of claim 39, wherein the bisulfite is added to the reaction prior to or simultaneously with the engineered monoamine oxidase.

41. 40. The method of claim 39, wherein the hydrogen sulfate is added after the addition of the engineered monoamine oxidase.

42. 40. The method of claim 39, wherein the amine compound according to structural formula I is contacted with oxygen and the engineered monoamine oxidase enzyme in association with a cofactor prior to the addition of the bisulfite.

43. 43. The method of any of claims 39 to 42, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6 and / or 414, or a functional fragment thereof, and the engineered monoamine oxidase polypeptide comprises at least one substitution or set of substitutions in said polypeptide sequence, and the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6 and / or 414.

44. 43. The method of any of claims 39 to 42, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the even numbered sequences of SEQ ID NOs: 8-1084.

45. A substantially enantiomerically pure aminonitrile compound according to structural formula IV(a): 【Chemistry 40】 (Wherein, A is O, CR １ R ２ , -C=C-, or -CH ２ -CH ２ -, where R １ and R ２ are each independently -H, -COOH, -X, or -NH ２ , -CH ２ NHC (NH) NH ２ , -CX ３ , -CH ３ , -CH ２ CH ３ where X is selected from F, Cl and Br.

5. A process for preparing a compound according to claim 1, including salts and hydrates thereof, comprising the steps of: Amine Compounds According to Structural Formula I 【Chemistry 41】 wherein A is as defined for Structural Formula IV. in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite to produce a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b). 【Chemistry 42】 with oxygen under conditions to produce a mixture comprising: contacting said compound according to structural formula III(a) and III(b) with cyanide under conditions to yield said substantially stereoisomerically pure aminonitrile compound according to structural formula IV(a); A method comprising:

46. 46. ​​The method of claim 45, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6 and / or 414, or a functional fragment thereof, and the engineered monoamine oxidase polypeptide comprises at least one substitution or set of substitutions in said polypeptide sequence, and the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6 and / or 414.

47. 46. ​​The method of claim 45, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the even numbered sequences of SEQ ID NOs: 8-1084.

48. 46. ​​The method of claim 45, wherein the substantially enantiomerically pure amino nitrile compound according to structural formula IV(a) is contacted with an acid and water under conditions whereby the amino nitrile compound is converted to a substantially stereoisomerically pure amino acid compound.

49. A substantially stereoisomerically pure amino amide compound according to structural formula VII: 【Chemistry 43】 (Wherein, A is O, CR １ R ２ , -C=C-, or -CH ２ -CH ２ -, where R １ and R ２ are each independently -H, -COOH, -X, or -NH ２ , -CH ２ NHC (NH) NH ２ , -CX ３ , -CH ３ , -CH ２ CH ３ where X is selected from F, Cl and Br.

5. A process for preparing a compound according to claim 1, including a salt thereof, comprising the steps of: Amine Compounds According to Structural Formula I 【Chemistry 44】 wherein A is as defined for Structural Formula VII. in the presence of an engineered monoamine oxidase enzyme in association with a cofactor and bisulfite to produce a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(a) and a substantially enantiomerically pure aminosulfonic acid compound according to structural formula III(b). 【Chemistry 45】 with oxygen under conditions to produce a mixture comprising: A stereoisomerically pure amine bisulfite adduct according to structural formula III(a) or a substantially stereoisomerically pure amine bisulfite adduct according to structural formula III(b) or a mixture thereof is reacted with a substantially stereoisomerically pure aminonitrile compound according to structural formula IV(a) 【Chemistry 46】 where A is as defined for the amino compounds of formula VII. with cyanide under conditions to produce contacting said aminonitrile compound of structural formula IV(a) with an acid under conditions whereby said aminonitrile compound is converted to said substantially stereoisomerically pure aminoamide compound according to structural formula VII; A method comprising:

50. 50. The method of claim 49, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6 and / or 414, or a functional fragment thereof, and wherein the engineered monoamine oxidase polypeptide 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: 6 and / or 414.

51. 50. The method of claim 49, wherein the engineered monoamine oxidase comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the even numbered sequences of SEQ ID NOs: 8-1084.

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