Transaminase and variant thereof, and use of transaminase and variant thereof in synthesis of amine having high optical purity

Abstract

Provided are a transaminase and a variant thereof, and the use of the transaminase and the variant thereof in amine synthesis.

Description

Transaminases and their variants, and their applications in the synthesis of amines with high optical purity.

[0001] This application claims priority to Chinese Patent Application No. 202410907678.1, filed on July 8, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to transaminases and their variants, and the application of said transaminases and their variants in the synthesis of high optical purity amines in the pharmaceutical and fine chemical industries. Background Technology

[0003] Transaminases (EC 2.6.1.x) are a broad class of enzymes capable of transferring amino (NH2) functional groups from one part (donor) to another (acceptor), typically resulting in an optically pure NH2 functionalized product.

[0004] This is particularly interesting because many pharmaceutical intermediates or final drug substances are chiral amines, and there are strict requirements for the optical purity of chiral amines (usually expressed as enantiomeric excess value "ee").

[0005] Generally, chiral amines are required to have an optical purity greater than 99.0-99.5% ee, meaning that the ratio of the desired enantiomer to the unwanted enantiomer in two possible enantiomers, R or S, typically needs to be greater than 200-400:1. Sitagliptin is such a chiral amine and is a widely used active pharmaceutical ingredient for treating type II diabetes. Despite its wide application, more efficient, economical, and environmentally friendly production processes are still needed. Current processes for synthesizing sitagliptin involve the combined use of harmful metal hydrides or high-pressure hydrogen with toxic and expensive transition metal / ligand combinations, resulting in heavy waste streams that are unfriendly to the environment. Therefore, a mild and highly selective catalyst is needed to achieve the synthesis of sitagliptin. Enzymes are precisely such mild and selective catalysts. However, it is well known that enzymes and enzyme-catalyzed processes are generally incompatible with the harsh conditions of traditional chemical synthesis, which often requires organic solvents, high temperatures, and high substrate loadings. Existing biocatalytic methods for the synthesis of sitagliptin require the use of difficult-to-handle organic solvents such as DMSO, or methods that can only act on small / truncated substrates (Structural Formula 3; where A represents esters or amines). The truncated product (Structural Formula 4) requires additional chemical transformation steps to finally generate sitagliptin (Structural Formula 2), and these additional chemical steps negate the benefits of using biocatalytic synthesis of sitagliptin.

[0006] Therefore, a highly active and selective transaminase is still needed to synthesize sitagliptin from its precursor ketone, while avoiding the use of large amounts of difficult-to-handle organic solvents such as DMSO. Although a large number of wild-type transaminases are known to exist, these naturally occurring transaminases have not yet evolved to accept hydrophobic substrates with large molecular structures, such as the precursor ketone of sitagliptin (Structural Formula 1). Therefore, it is necessary to evolve wild-type transaminases through enzyme engineering to not only accept the precursor ketone of sitagliptin as a substrate, but also to produce the desired amine with sufficiently high optical purity at an economically feasible enzyme loading in a reaction environment that is naturally unfavorable to the enzyme. In addition, in this bio-enzymatic synthesis of sitagliptin, inexpensive, readily available, and easily removable isopropylamine (IPM) is selected as the amine donor, and the process produces a mild and easily removable byproduct, acetone. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present disclosure aims to provide an environmentally safe, mild reaction method for transaminases and their variants with high activity, high selectivity, and stability through enzyme engineering. The transaminases and their variants are used to convert sitagliptin precursor ketones into the corresponding (R)-amine, sitagliptin, with an optical purity of at least 90%ee, 95%ee, 98%ee, 99%ee, 99.5%ee, 99.8%ee, or 99.9%ee. Although wild-type transaminase sequences (e.g., SEQ ID NO:2, 4, 6, 8, 10, and 12, whose coding sequences are SEQ ID NO:1, 3, 5, 7, 9, and 11, respectively) do not exhibit activity against sitagliptin proketone, this invention utilizes enzyme engineering technology to express the amino acid aminotransferase gene (Uniprot: UPI00143F05B3) from *Thermomyces stellatus* in *E. coli*. By constructing transaminase libraries including site-saturated mutagenesis libraries and combinatorial libraries, mutant evolution of wild-type transaminase (Uniprot: UPI00143F05B3) was carried out to obtain variants (e.g., SEQ ID NO.:14, 16, 18, and 20) active against sitagliptin proketone (Structure 1), converting sitagliptin proketone (Structure 1) into the corresponding (R)-amine, sitagliptin (Structure 2). The mutant bacteria were cultured in 96-well plates, and multiple rounds of high-throughput screening were completed to finally obtain transaminases and their variants with high activity, high optical purity and stability. Sitagliptin was synthesized using this bio-enzymatic catalysis method.

[0008] Accordingly, in a first aspect, the present invention provides a method for preparing an amine, the method comprising reacting an amino receptor of Formula I or a pharmaceutically acceptable salt thereof with an amino donor in the presence of a transaminase:

[0009] in,

[0010] The transaminase comprises an amino acid sequence formed by mutating the sequence shown in SEQ ID NO:12, or a fragment or variant thereof having transaminase activity;

[0011] R1 and R2 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-10 Cycloalkyl, -L-3-10 membered heterocyclic, -LC 6-10 aryl and -L-5-10 heteroaryl groups, optionally bound by 1, 2 or 3 R groups s Substitution; and R1 and R2 are not both H at the same time;

[0012] Or R1, R2, and the carbon atoms they are connected to form C. 3-7 Cycloalkyl or 3-7 membered heterocyclic group, optionally surrounded by 1, 2 or 3 R groups. s replace;

[0013] R s Independently selected from H, halogen, CN, -L-OR a C 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 cycloalkyl groups and -L-3-7-membered heterocyclic groups;

[0014] L is independently selected from chemical bonds, -C 1-6 alkylene-C(O)- and C 1-6 Alkylene;

[0015] R a Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 cycloalkyl groups and -L-3-7-membered heterocyclic groups;

[0016] The aforementioned groups are optionally deuterated, up to and including complete deuteration;

[0017] The amino donor is a compound containing an amino group.

[0018] In a more specific implementation, R1 is -C 1-3 alkylene-phenyl, wherein the phenyl group is optionally surrounded by 1, 2 or 3 R groups. s Replace; Preferred

[0019] R2 is -C 1-3 Alkylene-C(O)-9-10-membered heterocyclic group, wherein the 9-10-membered heterocyclic group is optionally surrounded by 1, 2 or 3 R groups. sReplace; Preferred

[0020] R s Independently selected from H, halogens, CN, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0021] In a more specific embodiment, the present invention provides the above-described method, wherein,

[0022] The transaminase comprises an amino acid sequence formed by mutating the sequence shown in SEQ ID NO:12, or a fragment or variant thereof having transaminase activity;

[0023] R1 and R2 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 Cycloalkyl and -L-3-7-membered heterocyclic groups, optionally surrounded by 1, 2 or 3 R groups. s Substitution; and R1 and R2 are not both H at the same time;

[0024] Or R1, R2, and the carbon atoms they are connected to form C. 3-7 Cycloalkyl or 3-7 membered heterocyclic group, optionally surrounded by 1, 2 or 3 R groups. s replace;

[0025] R s Independently selected from H, halogen, CN, -L-OR a C 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 cycloalkyl groups and -L-3-7-membered heterocyclic groups;

[0026] L is independently selected from chemical bonds and C. 1-6 Alkylene;

[0027] R a Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 cycloalkyl groups and -L-3-7-membered heterocyclic groups;

[0028] The aforementioned groups are optionally deuterated, up to and including complete deuteration;

[0029] The amino donor is a compound containing an amino group.

[0030] In a more specific embodiment, the present invention provides the above-described method, wherein the amino donor is a compound of formula II, or a pharmaceutically acceptable salt thereof:

[0031] in,

[0032] R3 and R4 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 Cycloalkyl and -L-3-7-membered heterocyclic groups, which are optionally substituted with 1, 2 or 3 R*; and R3 and R4 are not both H;

[0033] Or R3, R4, and the carbon atoms they are attached to form C. 3-7 Cycloalkyl or 3-7-membered heterocyclic groups, which are optionally substituted with 1, 2 or 3 R* groups;

[0034] R* is independently selected from H, halogen, CN, -L-OR a C 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 cycloalkyl groups and -L-3-7-membered heterocyclic groups;

[0035] The aforementioned groups are optionally deuterated, up to and including complete deuteration.

[0036] R1 and R2

[0037] In one implementation, R1 is H; in another implementation, R1 is C. 1-6 Alkyl, such as C 1-3 Alkyl, such as methyl; in another embodiment, R1 is C 1-6 Halogenated alkyl groups, such as C 1-3 Halogenated alkyl; in another embodiment, R1 is -LC 3-10 cycloalkyl, for example -LC 3-7 cycloalkyl, such as C 3- 7-Cycloalkyl; in another embodiment, R1 is a -L-3-10-membered heterocyclic group, such as a -L-3-7-membered heterocyclic group, such as a 3-7-membered heterocyclic group; in another embodiment, R1 is a -LC 6-10 Aryl groups, such as -C 1-3 Alkylene-phenyl; in another embodiment, R1 is an L-5-10 heteroaryl group; in another embodiment, R1 is optionally surrounded by 1, 2, or 3 R groups. s In one embodiment, R1 is replaced; in another embodiment, R1 is not replaced; in yet another embodiment, R1 is...

[0038] In one implementation, R1 is optionally deuterated; in another implementation, R1 is not deuterated.

[0039] In one implementation, R2 is H; in another implementation, R2 is C. 1-6Alkyl, such as C 1-3 Alkyl; in another embodiment, R2 is C 1-6 Halogenated alkyl groups, such as C 1-3 Halogenated alkyl; in another embodiment, R2 is -LC 3-10 cycloalkyl, for example -LC 3-7 cycloalkyl, such as C 3-7 cycloalkyl; in another embodiment, R2 is an -L-3-10 membered heterocyclic group, such as -C 1-3 An alkylene-C(O)-9-10-membered heterocyclic group, such as an -L-3-7-membered heterocyclic group, such as a 3-7-membered heterocyclic group; in another embodiment, R2 is -LC. 6-10 aryl; in another embodiment, R2 is a -L-5-10 heteroaryl group; in another embodiment, R2 is optionally surrounded by 1, 2 or 3 R groups. s Replacement, for example, R2 is -C 1-6 Alkylene-OR a For example, R2 is -(CH2)2OH, or R2 is -C 1-6 Halogenated alkylene-OR a In another embodiment, R2 is not replaced; in another embodiment, R2 is...

[0040] In one implementation, R2 is optionally deuterated; in another implementation, R2 is not deuterated.

[0041] In one implementation, R1 and R2 are not both H.

[0042] In one implementation, R1, R2, and the carbon atoms they are attached to together form C. 3-7 Cycloalkyl; in another embodiment, R1, R2 together with the carbon atom to which they are attached form a 3-7 membered heterocyclic group; in another embodiment, the ring formed by R1, R2 and the carbon atom to which they are attached is optionally surrounded by 1, 2 or 3 R... s In one embodiment, the ring formed by R1, R2 and the carbon atoms connected to them is not substituted; in another embodiment, R1, R2 and the carbon atoms connected to them do not form a ring.

[0043] In one embodiment, the ring formed by R1, R2 and the carbon atoms connected to them is optionally deuterated; in another embodiment, the ring formed by R1, R2 and the carbon atoms connected to them is not deuterated.

[0044] In a more specific implementation, R1 and R2 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-10Cycloalkyl, -L-3-10 membered heterocyclic, -LC 6-10 aryl and -L-5-10 heteroaryl groups, optionally bound by 1, 2 or 3 R groups s The substitutions are made such that R1 and R2 are not both H; R1 and R2 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 Cycloalkyl and -L-3-7-membered heterocyclic groups, optionally surrounded by 1, 2 or 3 R groups. s Instead, R1 and R2 are not both H; in another more specific embodiment, R1 is selected from C. 1-6 Alkyl or C 1-6 Halogenated alkyl; in another more specific embodiment, R2 is selected from -C 1-6 Alkylene-OR a or -C 1-6 Halogenated alkylene-OR a In another, more specific implementation, R2 is -C 1-6 Alkylene-OR a In another more specific embodiment, R2 is -(CH2)2OH.

[0045] R s

[0046] In one implementation, R s For H; in another implementation, R s For halogen; in another embodiment, R s For CN; in another implementation, R s For -L-OR a For example, -OR a In another implementation, R s C 1-6 Alkyl; in another embodiment, R s C 1-6 Halogenated alkyl; in another embodiment, R s For -LC 3-7 cycloalkyl, such as C 3-7 cycloalkyl; in another embodiment, R s It is an -L-3-7 membered heterocyclic group, for example, a 3-7 membered heterocyclic group.

[0047] In one implementation, R s Optionally replaced by deuterium; in another implementation, R s It was not replaced by deuterium.

[0048] In a more specific implementation scheme, R s Independently selected from H, halogen, OR a C1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0049] L

[0050] In one embodiment, L is a chemical bond; in another embodiment, L is -C. 1-6 Alkylene-C(O)-, preferably -C 1-3 alkylene-C(O)-; in another embodiment, L is C 1-6 Alkylene, preferably C 1- 3-alkylene compounds.

[0051] In one implementation, L is optionally deuterated; in another implementation, L is not deuterated.

[0052] R a

[0053] In one implementation, R a For H; in another implementation, R a C 1-6 Alkyl; in another embodiment, R a C 1-6 Halogenated alkyl; in another embodiment, R a For -LC 3-7 cycloalkyl; in another embodiment, R a It is an -L-3-7 membered heterocyclic group.

[0054] In one implementation, R a Optionally replaced by deuterium; in another implementation, R a It was not replaced by deuterium.

[0055] In a more specific implementation scheme, R a Selected from H, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0056] R3 and R4

[0057] In one implementation, R3 is H; in another implementation, R3 is C. 1-6 Alkyl, such as C 1-3 Alkyl; in another embodiment, R3 is C 1-6 Halogenated alkyl groups, such as C 1-3 Halogenated alkyl group, such as methyl; in another embodiment, R3 is -LC. 3-7 cycloalkyl, such as C 3-7Cycloalkyl; in another embodiment, R3 is an -L-3-7-membered heterocyclic group, such as a 3-7-membered heterocyclic group; in another embodiment, R3 is optionally substituted with 1, 2 or 3 R*; in another embodiment, R3 is not substituted.

[0058] In one implementation, R3 is optionally deuterated; in another implementation, R3 is not deuterated.

[0059] In one implementation, R4 is H; in another implementation, R4 is C. 1-6 Alkyl, such as C 1-3 Alkyl, such as methyl; in another embodiment, R4 is C 1-6 Halogenated alkyl groups, such as C 1-3 Halogenated alkyl; in another embodiment, R4 is -LC 3-7 cycloalkyl, such as C 3-7 Cycloalkyl; in another embodiment, R4 is an -L-3-7-membered heterocyclic group, such as a 3-7-membered heterocyclic group; in another embodiment, R4 is optionally substituted with 1, 2 or 3 R*; in another embodiment, R4 is not substituted.

[0060] In one implementation, R4 is optionally deuterated; in another implementation, R4 is not deuterated.

[0061] In one implementation, R3 and R4 are not both H.

[0062] In one implementation, R3, R4, and the carbon atoms they are bonded to together form C. 3-7 Cycloalkyl; in another embodiment, R3, R4 together with the carbon atom to which they are attached form a 3-7 membered heterocyclic group; in another embodiment, the ring formed by R3, R4 together with the carbon atom to which they are attached is optionally substituted by 1, 2 or 3 R*; in another embodiment, the ring formed by R3, R4 together with the carbon atom to which they are attached is not substituted; in another embodiment, R3, R4 together with the carbon atom to which they are attached do not form a ring.

[0063] In one embodiment, the ring formed by R3, R4 and the carbon atoms connected to them is optionally deuterated; in another embodiment, the ring formed by R3, R4 and the carbon atoms connected to them is not deuterated.

[0064] In a more specific implementation, R3 and R4 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7Cycloalkyl and -L-3-7-membered heterocyclic groups, optionally substituted with 1, 2 or 3 R*, wherein R3 and R4 are not both H; in another more specific embodiment, R3 is selected from C 1-6 Alkyl and C 1-6 Halogenated alkyl; in another more specific embodiment, R4 is selected from C10. 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0065] R*

[0066] In one embodiment, R* is H; in another embodiment, R* is halogen; in yet another embodiment, R* is CN; in still another embodiment, R* is -L-OR a In another implementation, R* is C 1-6 Alkyl; in another embodiment, R* is C 1-6 Halogenated alkyl; in another embodiment, R* is -LC 3-7 Cycloalkyl; in another embodiment, R* is a -L-3-7-membered heterocyclic group.

[0067] In one implementation, R* is optionally deuterated; in another implementation, R* is not deuterated.

[0068] In a more specific implementation, R* is independently selected from H, halogen, C. 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0069] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof in R1 can be combined with R2, R3, R4, R s R a This invention relates to any technical solution such as L and R*, or any combination thereof. The present invention aims to include combinations of all these technical solutions; however, due to space limitations, they are not listed individually.

[0070] In a more specific implementation, R1 and R2 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 Cycloalkyl and -L-3-7-membered heterocyclic groups, optionally surrounded by 1, 2 or 3 R groups. s Substitution; and R1 and R2 are not both H at the same time;

[0071] R s Independently selected from H, halogen, OR a C 1-6 Alkyl and C 1-6Halogenated alkyl groups;

[0072] L is independently selected from chemical bonds and C. 1-6 Alkylene;

[0073] R a Selected from H, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0074] In a more specific implementation, R1 is selected from C. 1-6 Alkyl or C 1-6 Halogenated alkyl; preferably C 1-6 Alkyl; preferably C 1-3 Alkyl; preferably methyl;

[0075] R2 is selected from -C 1-6 Alkylene-OR a or -C 1-6 Halogenated alkylene-OR a Preferred -C 1-6 Alkylene-OR a Preferably -(CH2)2OH;

[0076] R a Selected from H, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0077] In a more specific implementation, R3 and R4 are independently selected from H and C. 1-6 Alkyl, C 1-6 Halogenated alkyl, -LC 3-7 Cycloalkyl and -L-3-7-membered heterocyclic groups, which are optionally substituted with 1, 2 or 3 R*; and R3 and R4 are not both H;

[0078] R* is independently selected from H, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups;

[0079] L is independently selected from chemical bonds and C. 1-6 Alkylene;

[0080] R a Selected from H, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups.

[0081] In a more specific implementation, R3 is selected from C. 1-6 Alkyl and C 1-6 Halogenated alkyl; preferably C 1-6 Alkyl; preferably C 1-3 Alkyl; preferably methyl;

[0082] R4 is selected from C 1-6 Alkyl and C 1-6 Halogenated alkyl; preferably C 1-6 Alkyl; preferably C 1-3 Alkyl; preferably methyl.

[0083] In a preferred embodiment, the amino receptor represented by Formula I is structural formula 1 (sitagliptin precursor ketone), and the compound represented by Formula III is structural formula 2 (sitagliptin).

[0084] In the method for preparing amines of the present invention, the transaminase variant has improved transaminase activity compared to the wild-type transaminase having the amino acid sequence shown in SEQ ID NO:12.

[0085] In some embodiments, the improved transaminase activity of the transaminase variant refers to increased activity in the reaction catalyzing the conversion of sitagliptin precursor ketone to sitagliptin.

[0086] In some embodiments, the transaminase variant is a transaminase variant with a mutated and / or truncated amino acid sequence compared to the wild-type transaminase, which contains the amino acid sequence shown in SEQ ID NO:12.

[0087] In some embodiments, the transaminase variant comprises an amino acid sequence having at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12, or ... The amino acid sequence shown in NO:12, when truncated at any one of the positions selected from 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, and 357, has an N-terminal sequence that is at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence.

[0088] In some respects, the differences between transaminase variants and wild-type transaminases include differences in one or more amino acid residues corresponding to amino acid sequence positions D8, Q24, M45, P47, E58, M61, R62, L65, V69, P70, S71, D81, L94, R102, A114, F122, E124, R135, S137, L149, Q151, V154, V156, P163, G180, L189, G214, A216, S222, V227, Y235, D238, Q243, K248, N255, K257, A270, H273, C280, T281, A283, I289, R290, K301, or A321 in SEQ ID NO:12. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at any one of the positions Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, and E357 corresponding to SEQ ID NO:12.

[0089] In some preferred embodiments, the differences between the transaminase variant and the wild-type transaminase include positions corresponding to SEQ ID NO:12: D8, Q24, M45, P47, E58, M61, R62, L65, V69, P70, S71, D81, L94, R102, A114, F122, E124, R135, S137, L149, Q151, V154, V156, P163, G180, L189, G214, A216. The amino acid residue differences at S222, V227, Y235, D238, Q243, K248, N255, K257, A270, H273, C280, T281, A283, I289, R290, K301 or A321, optionally truncated starting at position 329, 330, 331, 332, 333, 334 or 335.

[0090] In a more preferred embodiment, the difference between the transaminase variant and the wild-type transaminase includes differences in amino acid residues corresponding to positions 8, 45, 47, 58, 61, 62, 65, 69, 70, 71, 81, 122, 124, 135, 149, 151, 154, 156, 214, 222, 243, 281, or 283 of SEQ ID NO:12, optionally truncated starting at position 332, 333, 334, or 335.

[0091] In a further preferred embodiment, the difference between the transaminase variant and the wild-type transaminase includes differences in amino acid residues at positions 8, 47, 58, 61, 62, 65, 69, 70, 71, 122, 124, 135, 149, 151, 154, 214, 222, 243, 281, or 283 corresponding to SEQ ID NO:12, optionally truncated starting at position 332, 333, 334, or 335.

[0092] In some respects, the differences between the transaminase variants and wild-type transaminases include the amino acid sequence positions corresponding to SEQ ID NO:12: D8, M13, A21, Q24, E36, M45, P47, A51, E58, M61, R62, L65, T66, V69, P70, S71, D81, S85, L94, K97, L100, R102, E103, E104, K106, A114, K115, A121, F122, V 123, E124, I126, V127, K132, R135, S137, I142, R145, L149, V150, Q151, V154, V156, P163, D167, V169, G180, T185, L189, V195, R196, L198, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K257, G258, F259 , E260, V261, V263, A270, A271, H273, D275, M279, C280, T281, A283, M287, I289, R290 Differences in one or more amino acid residues of L292, G294, P296, N298, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355, or L356. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at any one of the positions Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, and E357 corresponding to SEQ ID NO:12.

[0093] In some preferred embodiments, the transaminase variant contains a variant corresponding to SEQ ID. NO:12 may be substituted with one or more of the following: D8A, D8T, D8S, D8K, D8N, M13I, A21S, Q24R, Q24L, Q24K, Q24F, E36Q, M45I, M45L, M45F, P47A, P47L, P47R, A51S, E58M, E58S, E58Q, M61L, R62T, R62K, R62A, R62G, R62I, R62Y, L65A, T66C, V69G, V69A, P70F, P70A, P70V, P70I, S71H, S71G, D81N, D81G, D81H, S85H, S85N, L94V, K97S. L100H, L100K, R102V, E103K, E104M, K106R, A114V, A114T, A114E, A114I, A114K, K115G, K115M, A121V, F122M, F122S, F122A, F122G, F122C, F122H, F 122N, F122T, V123C, E124L, E124M, I126M, V127I, K132V, K132M, K132I, K 132Q, R135A, R135Q, S137V, I142G, I142D, R145K, L149F, L149S, V150T, Q1 51C, V154Q, V154I, V156L, P163Y, P163K, P163R, D167S, V169L, V169I, G1 80C, G180L, T185K, L189K, L189F, V195N, R196N, L198I, S202K, T207R, G21 4C, G214Y, D215V, D215W, D215G, D215E, A216F, A216C, A216T, A216G, A21 6Q, N217H, S222P, I226V, V227F, V227L, L228T, D231G, Y235H, D238V, D238 S, D238I, D238H, Q243E, Q243P, Q243R, K248E, K248D, N255C, N255R, N255 L, K257A, G258K, F259H, F259I, E260K, V261A, V263L, A270T, A270C, A271C , H273N, H273T, D275E, D275N, M279C, C280S, T281S, A283G, M287C, I289V , R290T, R290S, L292M, G294F, P296T, P296L, N298G, K301A, K301N, P304L,E315D, H317V, A321K, Y322V, E325A, Y328P, A331K, L336A, Y339L, Y339I, F341I, Q342F, K344F, K344V, K344D, A346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S, or L356S.

[0094] In some respects, the differences between the transaminase variants and wild-type transaminases include the amino acid sequence positions corresponding to SEQ ID NO:12: D8, M10, A11, T12, M13, A21, Q24, E36, M45, P47, E50, A51, E58, M61, R62, L65, T66, V69, P70, S71, D81, S85, L94, K97, L100, R102, E103, E104, K106, K107, A114, K115, A121, F 122, V123, E124, I126, V127, L131, K132, R135, S137, E141, I142, V143, R145, L149, V150, Q15 1. V154, V156, E160, P163, D167, V169, G180, T185, L189, V195, R196, L198, F199, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K257, G2 58. F259, E260, V261, V263, P267, A270, A271, H273, D275, F278, M279, C280, T281, A283, M287 Differences in one or more amino acid residues of I289, R290, L292, G294, P296, N298, D299, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355, or L356. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at any one of the positions Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, and E357 corresponding to SEQ ID NO:12.

[0095] In some preferred embodiments, the transaminase variant contains a variant corresponding to SEQ ID. One or more of the following substitutions for NO:12: D8A, D8T, D8S, D8K, M10R, A11S, A11T, T12S, M13I, A21S, A21Y, Q24R, Q24L, Q24K, Q24F, E36Q, M45I, M45L, M45F, P47A, P47L, P47R, E50L, A51S, E58M, E58S, E58Q, M61L, R62T, R62K, R62A, R62G, R62I, R62Y, L65A, T66C, V69G, V69A, P70F, P70A, P70V, P70I, S71H, S71G, D81N, D81G. D81H, S85H, S85N, L94V, L94I, K97S, L100H, L100K, R102V, E103K, E104M, K106R, K107Q, A114V, A114T, A114E, A114I, A114K, K115G, K115M, A121V, F 122M, F122S, F122A, F122G, F122C, F122H, F122N, F122T, V123C, E124L, E 124M, I126M, V127I, L131Q, K132V, K132M, K132I, K132Q, K132S, R135A, R1 35Q, S137V, E141T, I142G, I142D, V143N, V143F, R145K, L149F, L149S, V1 50T, Q151C, V154Q, V154I, V156L, E160H, P163Y, P163K, P163R, D167S, V16 9L, V169I, G180C, G180L, T185K, L189K, L189F, V195N, R196N, L198I, F19 9H, S202K, T207R, G214C, G214Y, D215V, D215W, D215G, D215E, A216F, A216 C. A216T, A216G, A216Q, A216N, N217H, S222P, I226V, V227F, V227L, L228 T, D231G, Y235H, D238V, D238S, D238I, D238H, Q243E, Q243P, Q243R, K248E , K248D, N255C, N255R, N255L, N255V, N255R, K257A, G258K, F259H, F259I , F259Y, F259L, E260K, E260D, V261A, V263L, V263M, P267S, A270T, A270C,A271C, H273N, H273T, D275E, D275N, F278L, M279C, C280S, T281S, A283G, M287C, I289V, R29 0T, R290S, L292M, G294F, P296T, P296L, P296S, N298G, D299L, K301A, K301N, P304L, E315D, H317V, A321K, Y322V, E325A, Y328P, A331K, L336A, Y339L, Y339I, F341I, Q342F, K344F, K344V, K344D, A346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S, or L356S.

[0096] In some specific embodiments, the transaminase variant has substitutions selected from the group consisting of SEQ ID NO:12:

[0097] P47A+R62T+L65A+V69G+R135A+Q151C+V154Q+S222P+T281S+A283G;

[0098] P47A+R62T+L65A+V69G+R135A+V154Q+S222P+T281S+A283G;

[0099] P47A+R62T+V69G+R135A+V154Q+S222P+T281S+A283G.

[0100] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0101] A47K; D8A; A47L; A47R; T62K; D8T; M45I; M45L; L149F; D8S; D8K; T207R.

[0102] In some preferred embodiments, the transaminase variant also has substitutions selected from the following sites:

[0103] T62K; D8T; M45I; M45L; L149F.

[0104] In some preferred embodiments, the transaminase variant further has substitutions selected from the following sites:

[0105] A47K; D8A; A47L; A47R.

[0106] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0107] D8S+K47L+P70F+S71H+F122M+E124L+L149F+G214C+Q243E;

[0108] M45I+K47R+T62K+P70A+F122M+E124L+L149F+T207R+Q243E;

[0109] D8T+M45L+K47R+T62K+P70F+F122M+E124L+L149F+T207R;

[0110] D8A+M45L+K47R+T62K+P70F+F122M+E124L+L149F+Q243E;

[0111] D8T+K47R+T62K+P70F+F122M+E124L+L149F+T207R+Q243E;

[0112] D8K+K47R+T62K+P70F+S71H+F122M+E124L+L149F;

[0113] D8A+M45I+K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E;

[0114] K47L+P70F+F122M+E124L+V195N+G214C+Q243E;

[0115] K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E;

[0116] D8K+M45I+K47R+T62K+P70F+F122M+E124L+T207R+Q243E;

[0117] D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+T207R+Q243E;

[0118] D8A+M45I+K47R+T62K+F122M+E124L+L149F+T207R;

[0119] D8K+M45I+T62K+P70F+F122M+E124L+T207R+Q243E;

[0120] D8A+M45L+K47R+T62K+P70F+F122M+E124L+T207R;

[0121] D8K+M45L+K47R+P70F+F122M+E124L+L149F+T207R;

[0122] D8T+K47R+T62K+P70F+S71H+F122M+E124L+T207R;

[0123] D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+Q243E;

[0124] D8T+K47L+P70F+S71H+F122M+E124L+L149F+T207R;

[0125] D8A+K47R+P70F+S71H+F122M+E124L+T207R+Q243E;

[0126] D8A+P70F+S71H+F122M+E124L+T207R+Q243E;

[0127] D8A+P70F+F122M+E124L;

[0128] D8S+M45L+T62K+L149F+T207R;

[0129] D8A+M45L+K47L+T62K+L149F+T207R+E315D;

[0130] M45I+K47L+T62K+L149F+T207R;

[0131] M45I+K47R+T62K+L149F+T207R;

[0132] D8T+M45I+K47L+T62K+L149F+T207R;

[0133] D8S+M45L+K47L+T62K+D81N+L149F;

[0134] D8K+M45L+K47R+T62K+P70F+F122M+L149F+Q243E;

[0135] D8A+M45I+T62K+L149F+T207R+Q243E;

[0136] D8S+M45I+T62K+L149F+T207R;

[0137] D8A+M45I+K47L+T62K+L149F;

[0138] D8A+M45I+K47R+P70F+F122M+E124L+V195N+T207R+Q243E;

[0139] D8A+K47L+T62K+L149F;

[0140] D8T+K47L+T62K+L149F+T207R;

[0141] D8S+K47R+P70F+F122M+E124L+V195N+T207R;

[0142] D8A+M45L+K47L+T62K+L149F+T207R;

[0143] D8A+M45L+K47L+T62K+L149F+T207R+Q243E;

[0144] M45L+K47R+T62K+L149F+T207R;

[0145] D8T+M45L+T62K+L149F+T207R.

[0146] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0147] S8A+M45L+T62K+F70A+D81N+T207R;

[0148] S8K+M45L+L47R+D81G+L124M;

[0149] S8T+A21S+Q24R+M45I+F70A+D81G+E315D+Q354P;

[0150] S8T+M45L+T62K+F70A+D81N;

[0151] S8T+Q24R+T62K+F70A+D81G;

[0152] S8A+Q24R+D81G+L124M+Q354P;

[0153] S8T+A21S+F70A+D81G+Q354P.

[0154] In some specific embodiments, the transaminase variant also has any of the following substitutions:

[0155] M122S; M122A; M122G; M122C; K62A; M122H; R290T; V156L; R290S; C214Y; K62G; K62I; M122N; K62Y; K248E; A65L; E243P; V150T; K248D; V261A.

[0156] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0157] K62A+A65L+L124M+V150T+C214Y+K248E;

[0158] Q24L+K62A+P163Y+C214Y+A216F+K248D+K301A;

[0159] Q24L+K62A+A121V+L124M+V156L+P163Y+C214Y+A216F+K248D+R290T+K301A;

[0160] A65L+L124M+V156L+C214Y+E243R+K248D+K301A;

[0161] K62A+A65L+V150T+C214Y+A216F+K248E;

[0162] Q24L+K62A+A65L+L124M+C214Y+A216F+K248D+K301A;

[0163] A65L+L124M+V150T+V156L+C214Y+A216F+K248D+R290T;

[0164] Q24L+K62A+V156L+P163Y+C214Y+E243R+L292M;

[0165] L124M+V150T+V156L+C214Y+A216F+K248E+R290T;

[0166] V156L+P163Y+C214Y+K248E+R290T+K301A;

[0167] K62I+V150T+V156L+C214Y+A216F+K248E+R290T;

[0168] Q24L+A65L+L124M+V150T+C214Y+A216F+K248E+R290T+K301A;

[0169] Q24L+K62I+L124M+C214Y+K248D+K301A;

[0170] Q24L+K62A+L124M+K248E+R290T;

[0171] A65L+V156L+K248E;

[0172] A65L+L124M+V156L;

[0173] K62A+L124M+V156L+K248E+R290T;

[0174] Q24L+L124M+V156L+K248E;

[0175] Q24L+A65L+A121V+L124M+V150T+V156L+C214Y+A216F+E243R;

[0176] Q24L+K62A+S122A+K248E+R290T;

[0177] A65L+L124M+K248E;

[0178] V156L+K248E;

[0179] Q24L+K62A+A65L+S122T+V156L+K248E;

[0180] K62A+A65L+K248E+R290T;

[0181] K62I+V150T+K248E+K301A;

[0182] Q24L+S122G+L124M+V156L+K248E;

[0183] A121V+V150T+V156L+C214Y+K248D+K301A;

[0184] K62A+A65L+S122G+V156L+K248E+R290T;

[0185] K62I+S122G+L124M+V156L+K248E;

[0186] K62I+K248E;

[0187] Q24L+K62I+V150T+P163Y+A216F+E243R+K248E+K301A;

[0188] A65L+S122G+L124M+V156L+K248E;

[0189] A65L+V150T+A216F+K248E;

[0190] K62A+S122T+V156L+K248E;

[0191] Q24L+A121V+V150T+V156L+C214Y+A216F+E243R+K248D+R290T+K301A;

[0192] K62A+K248E.

[0193] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0194] A65L+L124M+V150T+E248D;

[0195] K62A+L124M+A216F;

[0196] V150T;

[0197] K62A+L124M+E248D;

[0198] Q24L+K62A+L124M;

[0199] L124M;

[0200] V150T+E248D;

[0201] A65L+L124M;

[0202] L124M+A216F+E243R;

[0203] K62I+L124M;

[0204] Q24L+A216F;

[0205] A65L+L156V+Y163P+A216F;

[0206] K62I+S122T+L124M+V150T+A216F+A301K;

[0207] Q24L+K62I+S122T+L124M+V150T;

[0208] Q24L+K62I+S122T+A216F+E248D;

[0209] Q24L+K62I+S122T+V150T+A216F;

[0210] K62A+A65L+A216F+E248D;

[0211] K62I+S122T+L124M+A301K;

[0212] A65L+A216F+E248D;

[0213] S122G+L124M+A216F+E243R;

[0214] K62I+S122T+L124M+A216F;

[0215] Q24L+K62I+S122T+L124M+A301K;

[0216] Q24L+K62I+S122T+L124M.

[0217] In some specific embodiments, the transaminase variant also has a substitution selected from any of the following:

[0218] A348T; K344F; G355K; E349P; L356S; K344V; L336A; Q342F; E349I; E58M; A348S; Y339L; L189K; F3 41I; Y339I; L351P; A346T; K344D; H273N; E349T; K115G; A70V; G355S; A331K;

[0219] In some specific embodiments, the transaminase variant also has substitutions selected from the group consisting of:

[0220] A70V+A114V+K115G+G180C+H273T;

[0221] A70V+K115G+G180C+H273T;

[0222] A70V+A114V+Q154V+G180C+D275E;

[0223] A70V+A114V+Q154V+G180C;

[0224] A114V+Q154V+G180C;

[0225] A70V+G180C+H273T;

[0226] A114V+G180C+H273T+D275E;

[0227] A70V+K115G+Q154V+G180C+H273T;

[0228] K115G+G180C+H273T+D275E;

[0229] K115G+G180C+H273T;

[0230] K115G+G180C+L189K+H273N+C280S;

[0231] K115G+G180C;

[0232] A114V+K115G+G180C+L189K;

[0233] A70V+K115G+H273T;

[0234] A70V+A114V+L189K+H273T+C280S;

[0235] Q154V+G180C+L189K+H273T+D275E;

[0236] A70I+A114T+Q154V+G180C+L189K+H273T;

[0237] A70V+A114V+K115G+Q154V+G180C+H273T+D275E;

[0238] A114V+K115G+G180C;

[0239] A70V+K115G;

[0240] G180C+D275E+C280S;

[0241] A70V+K115G+F149S+Q154V+L189K+H273T;

[0242] A70V+A114V+G180C+L189K+H273T+D275N+C280S。

[0243] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0244] A331*;

[0245] E332*;

[0246] G333*;

[0247] K334*;

[0248] P335*;

[0249] L94V;

[0250] L228T;

[0251] D275E+P335*;

[0252] G180C+D275E+E332*;

[0253] G180C+D275E+P335*;

[0254] F149S+A331*;

[0255] F149S+E332*;

[0256] D275E+A331*;

[0257] D275E+E332*;

[0258] D275E+P335*;

[0259] D238V;

[0260] D238S;

[0261] E104M;

[0262] V263L;

[0263] A321K.

[0264] In some specific embodiments, the transaminase variant also has substitutions selected from the following:

[0265] G69A; N255C; A216C; P296T; D215V; K132V; T66C; F259H; A216T; D215W.

[0266] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0267] L24K+L47P+R102V+K132M+A135Q+D167S+K189F;

[0268] L94V+D238V+L292M;

[0269] L94V+D238S+V261A+A321K;

[0270] L94V+D238V+A321K;

[0271] L94V+L292M;

[0272] L94V+D238S+V261A+E315D;

[0273] L94V+D238V;

[0274] L47P+K132M+A135Q+Y163K+K189F+V227F;

[0275] K189F+S280C;

[0276] L94V+D238S+V263L;

[0277] L94V+V261A+V263L+L292M;

[0278] L94V+D275N+L292M;

[0279] L47P+K132M+A135Q+Y163R+D167S+K189F+V227F;

[0280] L94V+D238V+V263L+L292M;

[0281] L47P+R102V+Y163K+K189F;

[0282] L94V+D238V+V261A;

[0283] L94V+D238S+V261A+V263L+L292M;

[0284] L94V+V263L;

[0285] L94V+D238S+D275N+L292M+A321K;

[0286] L94V+D238S+A321K;

[0287] L94V+D238S+L292M+A321K;

[0288] D238V+V261A+V263L+L292M;

[0289] L47P+K189F+A216G;

[0290] L94V+D238S+V263L+D275N;

[0291] L94V+V263L+A321K;

[0292] L94V+V261A;

[0293] L94V+D238V+V261A+V263L+L292M;

[0294] L47P+Y163K+K189F+A216G+F259I+H317V;

[0295] L94V+D238S+D275N;

[0296] V114E+K189L+V227F+N298G;

[0297] L47P+R196N;

[0298] R102V+A135Q+M279C+N298G;

[0299] M13I+E36Q+L45F+S85H+T122M+A135Q+D167S+S202K+A271C+M287C;

[0300] R102V+D167S+M279C+N298G;

[0301] E36Q+S85H+R102V+K106R+A135Q+D167S+N217H+A270T+M279C;

[0302] L47P+N255R;

[0303] L47P+R196N+A216G+N298G.

[0304] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0305] M58S+L94V+A135Q+A270T;

[0306] M58S+L94V+R102V+K132I+L198I+A270T+M279C;

[0307] L94V+K132M+A135Q+L198I+I226V;

[0308] L94V+K132M+I226V+A270T;

[0309] L94V+R102V+A135Q+I226V+A270T;

[0310] M58S+L94V+R102V+K132M+I226V+A270T;

[0311] L94V+K132M+A135Q+M279C;

[0312] L94V+R102V+K132M+A135Q+M279C;

[0313] L94V+R102V+A270T;

[0314] M58S+L94V+R102V+A270T;

[0315] L94V+K132I+L198I+A270T;

[0316] L94V+R102V+A135Q+L198I+I226V;

[0317] M58S+L94V+K132M+A135Q+L198I+I226V+M279C;

[0318] L94V+I126M+K132M+A135Q+N298G;

[0319] L94V+R102V+L198I+A270T;

[0320] M58S+L94V+K132I+A135Q+I226V;

[0321] M58S+L94V+R102V+K132I+A135Q+L198I+I226V+A270T;

[0322] L94V+I226V;

[0323] M58S+L94V+A135Q+L198I;

[0324] L94V+V123C+V127I+V261A+G294F;

[0325] L94V+V169L+I226V+N255C+V261A+P296T;

[0326] L94V+V123C+V127I+I226V+N255C+G294F+P296T+N298G;

[0327] L94V+V169L+T185K+V227L+N255C+N298G;

[0328] L94V+V123C+V127I+V150T+V169L+N255C;

[0329] L94V+V123C+V127I+V150T+V169I+N255C+V261A+G294F+P296T+N298G;

[0330] L94V+V123C+V127I+I226V+G294F+P296T+N298G;

[0331] L94V+V123C+V127I+V169L+V261A+G294F;

[0332] L94V+V123C+V127I+V261A+P296T;

[0333] L94V+V150T+I226V+N255C+V261A+G294F;

[0334] L94V+V123C+V127I+V169I+V227L+V261A+G294F.

[0335] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0336] L24K+R102V+E315D;

[0337] V263L;

[0338] K132M+E315D;

[0339] L24K+R102V+I226V+D238S+V261A+V263L+P296T+N298G+E315D;

[0340] R102V;

[0341] L24K+K132I;

[0342] L24K+R102V+K132M+E315D;

[0343] L24K+D238V+N298G;

[0344] L24K+I226V+D238V+V261A+V263L+M279C+L292M+N298G;

[0345] K132V;

[0346] R102V+K132V+L292M+E315D;

[0347] I226V+V227L+V261A+V263L+M279C+N298G+E315D;

[0348] K132M;

[0349] E103K;

[0350] L24K+R102V+K132V;

[0351] R102V+V227L+N255C+A321K;

[0352] R102V+V227L+E260K+V261A+V263L;

[0353] L24K+V169L+D238V+V261A+V263L+M279C+E315D;

[0354] R102V+K132V+V227L+A321K;

[0355] K132V+V169L;

[0356] V227L+D238V+V263L+M279C+N298G+E315D;

[0357] V227L+D238S+V263L+M279C+P296T+E315D;

[0358] R102V+K132V+A321K.

[0359] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0360] K132V+G180C+D238V+V263L;

[0361] K132V+V169I+D215G+D238V;

[0362] K132V+V263L+P296L;

[0363] M61L;

[0364] K132V+V263L+L292M;

[0365] D215V+D238V;

[0366] V123C+K132V+L292M;

[0367] S137V; D215E; L24F;

[0368] E104M+K132V+V169I+V263L+L292M+P296T;

[0369] D231G; K97S; K115M; I142G;

[0370] K132V+D238V+V263L+L292M+P296T;

[0371] I142D; C255L; H71G; A301N; E325A; V114I; N81H; S85N; K106R; Y328P.

[0372] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0373] K132Q+K257A;

[0374] L24K+K132Q+S137V+D231G+D238V;

[0375] L24F+S137V+C255L+P304L;

[0376] L24F+K132Q+S137V+D231G+C255L+K257A;

[0377] K132Q+S137V+D238V+C255L+K257A+V261A+V263L+L292M+N298G;

[0378] L24F+K132Q+C255L+K257A+T270C+P304L;

[0379] L24F+S137V+K257A;

[0380] L24K+K132Q+S137V+D238V+C255L+V263L+T270C+N298G;

[0381] L24F+S137V+D231G+K257A+T270C+298G;

[0382] N81H+S137V+C255L+T270C;

[0383] L24F+K132Q+S137V+I226V+D231G+D238V+C255L+V263L+N298G;

[0384] L24K+S137V+D238V+C255L+K257A+V263L+T270C+N298G;

[0385] S137V+C255L;

[0386] L24F+C255L+K257A;

[0387] N81H+S137V+K257A+Y322V;

[0388] N81H+K132Q+S137V+D231G+T270C;

[0389] N81H+K132Q+S137V+I226V+C255L+K257A+V261A+L292M+N298G;

[0390] K132Q+S137V+T270C+P304L;

[0391] N81H+D238V+V261A+V263L+N298G;

[0392] N81H+S137V+C255L+T270C;

[0393] L24K+N81H+K132Q+D231G+D238V+C255L+V261A+V263L;

[0394] L24F+N81H+K132Q+S137V+C255L+K257A+T270C;

[0395] D231G+D238V+L292M+N298G;

[0396] S137V+C255L+Y322V;

[0397] L24F+N81H+T270C;

[0398] L24F+N81H+S137V+T270C;

[0399] N81H+K132Q+I226V+D238V+C255L+V261A+V263L+N298G;

[0400] L24F+S137V+D231G+C255L+V261A+V263L+T270C;

[0401] L24K+S137V+D231G+D238V+C255L+L292M+N298G;

[0402] N81H+S137V+K257A;

[0403] L24F+S137V+C255L+K257A;

[0404] L24F+S137V+D231G+C255L+K257A+T270C;

[0405] L24K+K132Q+S137V+D231G+D238V+C255L+V263L+N298G;

[0406] D231G+K257A;

[0407] S137V+C255L+T270C+P304L;

[0408] N81H+C255L+K257A+T270C;

[0409] S137V+C255L+K257A;

[0410] L24F+T270C+P304L.

[0411] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0412] V114K; N81D; A51S; D238I; G216Q; Y235H; L100H; G180L; G258K; I289V; D238H; R145K.

[0413] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0414] L100K+I289V;

[0415] N81D+L100K+G180L+G216Q+D238H+I289V;

[0416] R145K+G216Q;

[0417] R145K+G180L+I289V;

[0418] N81D+L100K+R145K+G216Q;

[0419] N81D+L100K+D238H+G258K+I289V;

[0420] N81D+V114K+R145K+D238H+I289V;

[0421] N81D+G216Q+D238H+I289V;

[0422] N81D+L100K+D238H+G258K;

[0423] L100K+V114K+D238H+G258K;

[0424] N81D;

[0425] V114K+D238H+G258K+I289V;

[0426] G180L+D238H+I289V;

[0427] F24K+V150T+N298G;

[0428] F24K+V261A+N298G;

[0429] F24K+D167S+D238S+G294F+E315D;

[0430] V114K+R145K+D167S+G294F+N298G;

[0431] F24K+G180L+V261A+N298G;

[0432] V102R+V114A+Q135R+Q154I;

[0433] V102R+Q135R+Q154I;

[0434] Q135R+Q154V;

[0435] T8D+F24Q+V102R+V114A+Q154V;

[0436] S58Q+V102R+Q154V;

[0437] T8N+S58Q+Q135R+Q154V.

[0438] In some specific embodiments, the transaminase variant also has a selection of substitutions or combinations thereof:

[0439] A257K; L255V; K132S; L131Q; F24L; T8D+F24L+V102R+V114A+Q135R+Q154V.

[0440] In some specific embodiments, the transaminase variant also has a selection of substitutions or combinations thereof:

[0441] M10R+A257K+F259Y;

[0442] F259L;

[0443] M10R+L255V+F259L+P267S;

[0444] M10R+A257K;

[0445] M10R+F259L;

[0446] M10R.

[0447] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0448] K107Q+F199H+E260D+A301K;

[0449] F199H+E260D;

[0450] K107Q+V143N+F199H+E260D+A301K;

[0451] V143N+L255R.

[0452] In some specific embodiments, the transaminase variant also has substitutions selected from the following: A21Y; F278L; D299L.

[0453] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0454] A11S+E50L+E160H+V263M;

[0455] A11S+A21Y+N143F;

[0456] A11S+A21Y+E141T+V263M+F278L;

[0457] A11S+E50L+E141T+E160H;

[0458] A11S+E141T+N143F+V263M;

[0459] T122M+L198I.

[0460] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0461] V137S+E141T+N143F+H235Y+R255C+A257K+F259L+V263M;

[0462] A11S+V137S+E141T+A257K;

[0463] A11S+V137S+N143F+H235Y+R255C+A257K+V263M;

[0464] A11S+R255C+A257K;

[0465] E141T+R255C+F259L+V263M;

[0466] V70A+H235Y+R255C+F259L;

[0467] V137S+E141T+A257K;

[0468] A11S+V137S+E141T+R255C+F259L+V263M;

[0469] A11S+N81G+E141T+H235Y+A257K+V263M;

[0470] A11S+N81G+V94I+V137S+E141T+A257K+V263M;

[0471] E141T+N143F+G216N+H235Y+V263M;

[0472] A11S+E141T+A257K+V263M+P296S;

[0473] V94I+G216N+H235Y+A257K+V263M;

[0474] A11S+V137S+N143F+H235Y+A257K;

[0475] A11S+V137S;

[0476] A11S+V137S+N143F+H235Y+A257K+V263M+P296S;

[0477] A11S+E141T+G216N+V263M+P296S;

[0478] V94I+V137S+E141T+N143F+G216N+H235Y+V263M;

[0479] A11S+E141T+G216N+H235Y+V263M;

[0480] A11S+G216N+V263M;

[0481] N143F+G216N+A257K;

[0482] A11S+N81G+E141T+V263M;

[0483] A11S+N81G+V137S+N143F+V263M;

[0484] V137S+E141T+H235Y+A257K+V263M;

[0485] A11S+N81G+V137S+E141T+G216N+H235Y+A257K+V263M+P296S;

[0486] A11S+N81G+V94I+V137S+G216N+H235Y+A257K;

[0487] A11S+N81G+V94I+V137S+N143F+G216N+H235Y+V263M+P296S;

[0488] A11T+N81G+V94I+A257K+V263M;

[0489] G216N+H235Y+A257K+V263M;

[0490] T12S+D167S+N217H.

[0491] In some specific embodiments of the present invention, the reaction between the amino acceptor and the amino donor is as follows:

[0492] In some specific embodiments of the present invention, the reaction of Formula 2 is carried out under certain substrate (Formula 1) concentration, amino donor (Formula 5) concentration, reaction temperature, pH value of the reaction system and / or reaction time.

[0493] In some embodiments, a transaminase variant is obtained that is resistant to at least 1, 10, 20, 30, 40, or 50% of an aqueous miscible organic solvent (e.g., DMSO, PEG200, or PEG400), at pH 6, 7, 8, 9, or 10, in the presence of at least 0.5 M, 1 M, or 2 M isopropylamine, and at temperatures of at least 25°C, 30°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0494] In some specific embodiments of the present invention, the method is used for the synthesis of 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolone[4,3-a]pyrazine (sitagliptin) or for the synthesis of sitagliptin phosphate (CAS 654671-77-9).

[0495] In some specific embodiments of the present invention, transaminase activity can be characterized by the amount of target product generated under certain reaction conditions. For example, sitagliptin can be prepared using the transaminase or a variant thereof described in this invention under the conditions of 50 g / L sitagliptin precursor ketone, 50°C, pH 9, and 1 M isopropylamine hydrochloride for 24 hours. The transaminase activity or the activity of its variant can be expressed by the amount of sitagliptin generated after 24 hours of reaction under these conditions.

[0496] In a second aspect, the present invention provides a transaminase variant that has improved transaminase activity compared to a wild-type transaminase having the amino acid sequence shown in SEQ ID NO:12.

[0497] In some embodiments, the improved transaminase activity of the transaminase variant refers to increased activity in the reaction catalyzing the conversion of sitagliptin precursor ketone to sitagliptin.

[0498] In some embodiments, the transaminase variant comprises an amino acid sequence having at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the transaminase variant comprises an N-terminal sequence having at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12 at any one of positions selected from 329, 330, 331, 332, 333, 334, 335, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, and 357.

[0499] In some respects, the differences between transaminase variants and wild-type transaminases include one or more amino acid differences corresponding to amino acid sequence positions D8, Q24, M45, P47, E58, M61, R62, L65, V69, P70, S71, D81, L94, R102, A114, F122, E124, R135, S137, L149, Q151, V154, V156, P163, G180, L189, G214, A216, S222, V227, Y235, D238, Q243, K248, N255, K257, A270, H273, C280, T281, A283, I289, R290, K301, or A321 in SEQ ID NO:12. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at position Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, or E357 corresponding to SEQ ID NO:12.

[0500] In some preferred embodiments, the differences between the transaminase variant and the wild-type transaminase include differences in amino acid residues at positions 8, 45, 47, 58, 62, 65, 69, 70, 71, 81, 122, 124, 135, 149, 151, 154, 214, 222, 243, 281, or 283 corresponding to SEQ ID NO:12, optionally truncated starting at positions 329, 330, 331, 332, 333, 334, or 335.

[0501] In a more preferred embodiment, the difference between the transaminase variant and the wild-type transaminase includes differences in amino acid residues at positions 8, 47, 58, 62, 65, 69, 70, 71, 122, 124, 135, 149, 151, 154, 214, 222, 243, 281, or 283 corresponding to SEQ ID NO:12, optionally truncated starting at position 332, 333, 334, or 335.

[0502] In a further preferred embodiment, the difference between the transaminase variant and the wild-type transaminase includes differences in amino acid residues at positions 47, 62, 65, 69, 135, 151, 154, 222, 281, or 283 corresponding to SEQ ID NO:12, optionally truncated starting at position 332, 333, 334, or 335.

[0503] In some respects, the differences between the transaminase variants and wild-type transaminases include the amino acid sequence positions corresponding to SEQ ID NO:12: D8, M13, A21, Q24, E36, M45, P47, A51, E58, M61, R62, L65, T66, V69, P70, S71, D81, S85, L94, K97, L100, R102, E103, E104, K106, A114, K115, A121, F122, V 123, E124, I126, V127, K132, R135, S137, I142, R145, L149, V150, Q151, V154, V156, P163, D167, V169, G180, T185, L189, V195, R196, L198, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K257, G258, F259 , E260, V261, V263, A270, A271, H273, D275, M279, C280, T281, A283, M287, I289, R290 Differences in one or more amino acid residues of L292, G294, P296, N298, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355, or L356. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at any one of the positions Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, and E357 corresponding to SEQ ID NO:12.

[0504] In some preferred embodiments, the transaminase variant contains a variant corresponding to SEQ ID. One or more of the following substitutions for NO:12: D8A, D8T, D8S, D8K, M13I, A21S, Q24R, Q24L, Q24K, Q24F, E36Q, M45I, M45L, M45F, P47A, P47L, P47R, A51S, E58M, E58S, E58Q, M61L, R62T, R62K, R62A, R62G, R62I, R62Y, L65A, T66C, V69G, V69A, P70F, P70A, P70V, P70I, S71H, S71G, D81N, D81G, D81H, S85H, S85N, L94V, K97S, L100H , L100K, R102V, E103K, E104M, K106R, A114V, A114T, A114E, A114I, A114K , K115G, K115M, A121V, F122M, F122S, F122A, F122G, F122C, F122H, F122N, F122T, V123C, E124L, E124M, I126M, V127I, K132V, K132M, K132I, K132Q, R135A, R135Q, S137V, I142G, I142D, R145K, L149F, L149S, V150T, Q151C, V 154Q, V154I, V156L, P163Y, P163K, P163R, D167S, V169L, V169I, G180C, G 180L, T185K, L189K, L189F, V195N, R196N, L198I, S202K, T207R, G214C, G2 14Y, D215V, D215W, D215G, D215E, A216F, A216C, A216T, A216G, A216Q, N2 17H, S222P, I226V, V227F, V227L, L228T, D231G, Y235H, D238V, D238S, D23 8I, D238H, Q243E, Q243P, Q243R, K248E, K248D, N255C, N255R, N255L, K25 7A, G258K, F259H, F259I, E260K, V261A, V263L, A270T, A270C, A271C, H273 N, H273T, D275E, D275N, M279C, C280S, T281S, A283G, M287C, I289V, R290T , R290S, L292M, G294F, P296T, P296L, N298G, K301A, K301N, P304L, E315D,H317V, A321K, Y322V, E325A, Y328P, A331K, L336A, Y339L, Y339I, F341I, Q342F, K344F, K344V, K344D, A346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S, or L356S.

[0505] In some respects, the differences between the transaminase variants and wild-type transaminases include the amino acid sequence positions corresponding to SEQ ID NO:12: D8, M10, A11, T12, M13, A21, Q24, E36, M45, P47, E50, A51, E58, M61, R62, L65, T66, V69, P70, S71, D81, S85, L94, K97, L100, R102, E103, E104, K106, K107, A114, K115, A121, F 122, V123, E124, I126, V127, L131, K132, R135, S137, E141, I142, V143, R145, L149, V150, Q15 1. V154, V156, E160, P163, D167, V169, G180, T185, L189, V195, R196, L198, F199, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K257, G2 58. F259, E260, V261, V263, P267, A270, A271, H273, D275, F278, M279, C280, T281, A283, M287 Differences in one or more amino acid residues of I289, R290, L292, G294, P296, N298, D299, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355, or L356. Optionally, the transaminase variant comprises a polypeptide that is truncated starting at any one of the positions Q329, V330, A331, E332, G333, K334, P335, L336, A337, G338, Y339, R340, F341, Q342, E343, K344, L345, A346, A347, A348, E349, N350, L351, Y352, F353, Q354, G355, L356, and E357 corresponding to SEQ ID NO:12.

[0506] In some preferred embodiments, the transaminase variant contains a variant corresponding to SEQ ID. One or more of the following substitutions for NO:12: D8A, D8T, D8S, D8K, M10R, A11S, A11T, T12S, M13I, A21S, A21Y, Q24R, Q24L, Q24K, Q24F, E36Q, M45I, M45L, M45F, P47A, P47L, P47R, E50L, A51S, E58M, E58S, E58Q, M61L, R62T, R62K, R62A, R62G, R62I, R62Y, L65A, T66C, V69G, V69A, P70F, P70A, P70V, P70I, S71H, S71G, D81N, D81G. D81H, S85H, S85N, L94V, L94I, K97S, L100H, L100K, R102V, E103K, E104M, K106R, K107Q, A114V, A114T, A114E, A114I, A114K, K115G, K115M, A121V, F 122M, F122S, F122A, F122G, F122C, F122H, F122N, F122T, V123C, E124L, E 124M, I126M, V127I, L131Q, K132V, K132M, K132I, K132Q, K132S, R135A, R1 35Q, S137V, E141T, I142G, I142D, V143N, V143F, R145K, L149F, L149S, V1 50T, Q151C, V154Q, V154I, V156L, E160H, P163Y, P163K, P163R, D167S, V16 9L, V169I, G180C, G180L, T185K, L189K, L189F, V195N, R196N, L198I, F19 9H, S202K, T207R, G214C, G214Y, D215V, D215W, D215G, D215E, A216F, A216 C. A216T, A216G, A216Q, A216N, N217H, S222P, I226V, V227F, V227L, L228 T, D231G, Y235H, D238V, D238S, D238I, D238H, Q243E, Q243P, Q243R, K248E , K248D, N255C, N255R, N255L, N255V, N255R, K257A, G258K, F259H, F259I , F259Y, F259L, E260K, E260D, V261A, V263L, V263M, P267S, A270T, A270C,A271C, H273N, H273T, D275E, D275N, F278L, M279C, C280S, T281S, A283G, M287C, I289V, R29 0T, R290S, L292M, G294F, P296T, P296L, P296S, N298G, D299L, K301A, K301N, P304L, E315D, H317V, A321K, Y322V, E325A, Y328P, A331K, L336A, Y339L, Y339I, F341I, Q342F, K344F, K344V, K344D, A346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S, or L356S.

[0507] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0508] P47A+R62T+L65A+V69G+R135A+Q151C+V154Q+S222P+T281S+A283G;

[0509] P47A+R62T+L65A+V69G+R135A+V154Q+S222P+T281S+A283G;

[0510] P47A+R62T+V69G+R135A+V154Q+S222P+T281S+A283G.

[0511] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0512] A47K; D8A; A47L; A47R; T62K; D8T; M45I; M45L; L149F; D8S; D8K; T207R;

[0513] In some preferred embodiments, the transaminase variant also has substitutions selected from the following sites:

[0514] T62K; D8T; M45I; M45L; L149F;

[0515] In some preferred embodiments, the transaminase variant further has substitutions selected from the following sites:

[0516] A47K; D8A; A47L; A47R.

[0517] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0518] D8S+K47L+P70F+S71H+F122M+E124L+L149F+G214C+Q243E;

[0519] M45I+K47R+T62K+P70A+F122M+E124L+L149F+T207R+Q243E;

[0520] D8T+M45L+K47R+T62K+P70F+F122M+E124L+L149F+T207R;

[0521] D8A+M45L+K47R+T62K+P70F+F122M+E124L+L149F+Q243E;

[0522] D8T+K47R+T62K+P70F+F122M+E124L+L149F+T207R+Q243E;

[0523] D8K+K47R+T62K+P70F+S71H+F122M+E124L+L149F;

[0524] D8A+M45I+K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E;

[0525] K47L+P70F+F122M+E124L+V195N+G214C+Q243E;

[0526] K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E;

[0527] D8K+M45I+K47R+T62K+P70F+F122M+E124L+T207R+Q243E;

[0528] D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+T207R+Q243E;

[0529] D8A+M45I+K47R+T62K+F122M+E124L+L149F+T207R;

[0530] D8K+M45I+T62K+P70F+F122M+E124L+T207R+Q243E;

[0531] D8A+M45L+K47R+T62K+P70F+F122M+E124L+T207R;

[0532] D8K+M45L+K47R+P70F+F122M+E124L+L149F+T207R;

[0533] D8T+K47R+T62K+P70F+S71H+F122M+E124L+T207R;

[0534] D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+Q243E;

[0535] D8T+K47L+P70F+S71H+F122M+E124L+L149F+T207R;

[0536] D8A+K47R+P70F+S71H+F122M+E124L+T207R+Q243E;

[0537] D8A+P70F+S71H+F122M+E124L+T207R+Q243E;

[0538] D8A+P70F+F122M+E124L;

[0539] D8S+M45L+T62K+L149F+T207R;

[0540] D8A+M45L+K47L+T62K+L149F+T207R+E315D;

[0541] M45I+K47L+T62K+L149F+T207R;

[0542] M45I+K47R+T62K+L149F+T207R;

[0543] D8T+M45I+K47L+T62K+L149F+T207R;

[0544] D8S+M45L+K47L+T62K+D81N+L149F;

[0545] D8K+M45L+K47R+T62K+P70F+F122M+L149F+Q243E;

[0546] D8A+M45I+T62K+L149F+T207R+Q243E;

[0547] D8S+M45I+T62K+L149F+T207R;

[0548] D8A+M45I+K47L+T62K+L149F;

[0549] D8A+M45I+K47R+P70F+F122M+E124L+V195N+T207R+Q243E;

[0550] D8A+K47L+T62K+L149F;

[0551] D8T+K47L+T62K+L149F+T207R;

[0552] D8S+K47R+P70F+F122M+E124L+V195N+T207R;

[0553] D8A+M45L+K47L+T62K+L149F+T207R;

[0554] D8A+M45L+K47L+T62K+L149F+T207R+Q243E;

[0555] M45L+K47R+T62K+L149F+T207R;

[0556] D8T+M45L+T62K+L149F+T207R.

[0557] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0558] S8A+M45L+T62K+F70A+D81N+T207R;

[0559] S8K+M45L+L47R+D81G+L124M;

[0560] S8T+A21S+Q24R+M45I+F70A+D81G+E315D+Q354P;

[0561] S8T+M45L+T62K+F70A+D81N;

[0562] S8T+Q24R+T62K+F70A+D81G;

[0563] S8A+Q24R+D81G+L124M+Q354P;

[0564] S8T+A21S+F70A+D81G+Q354P.

[0565] In some specific embodiments, the transaminase variant also has substitutions selected from any of the following sites:

[0566] M122S; M122A; M122G; M122C; K62A; M122H; R290T; V156L; R290S; C214Y; K62G; K62I; M122N; K62Y; K248E; A65L; E243P; V150T; K248D; V261A.

[0567] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0568] K62A+A65L+L124M+V150T+C214Y+K248E;

[0569] Q24L+K62A+P163Y+C214Y+A216F+K248D+K301A;

[0570] Q24L+K62A+A121V+L124M+V156L+P163Y+C214Y+A216F+K248D+R290T+K301A;

[0571] A65L+L124M+V156L+C214Y+E243R+K248D+K301A;

[0572] K62A+A65L+V150T+C214Y+A216F+K248E;

[0573] Q24L+K62A+A65L+L124M+C214Y+A216F+K248D+K301A;

[0574] A65L+L124M+V150T+V156L+C214Y+A216F+K248D+R290T;

[0575] Q24L+K62A+V156L+P163Y+C214Y+E243R+L292M;

[0576] L124M+V150T+V156L+C214Y+A216F+K248E+R290T;

[0577] V156L+P163Y+C214Y+K248E+R290T+K301A;

[0578] K62I+V150T+V156L+C214Y+A216F+K248E+R290T;

[0579] Q24L+A65L+L124M+V150T+C214Y+A216F+K248E+R290T+K301A;

[0580] Q24L+K62I+L124M+C214Y+K248D+K301A;

[0581] Q24L+K62A+L124M+K248E+R290T;

[0582] A65L+V156L+K248E;

[0583] A65L+L124M+V156L;

[0584] K62A+L124M+V156L+K248E+R290T;

[0585] Q24L+L124M+V156L+K248E;

[0586] Q24L+A65L+A121V+L124M+V150T+V156L+C214Y+A216F+E243R;

[0587] Q24L+K62A+S122A+K248E+R290T;

[0588] A65L+L124M+K248E;

[0589] V156L+K248E;

[0590] Q24L+K62A+A65L+S122T+V156L+K248E;

[0591] K62A+A65L+K248E+R290T;

[0592] K62I+V150T+K248E+K301A;

[0593] Q24L+S122G+L124M+V156L+K248E;

[0594] A121V+V150T+V156L+C214Y+K248D+K301A;

[0595] K62A+A65L+S122G+V156L+K248E+R290T;

[0596] K62I+S122G+L124M+V156L+K248E;

[0597] K62I+K248E;

[0598] Q24L+K62I+V150T+P163Y+A216F+E243R+K248E+K301A;

[0599] A65L+S122G+L124M+V156L+K248E;

[0600] A65L+V150T+A216F+K248E;

[0601] K62A+S122T+V156L+K248E;

[0602] Q24L+A121V+V150T+V156L+C214Y+A216F+E243R+K248D+R290T+K301A;

[0603] K62A+K248E.

[0604] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0605] A65L+L124M+V150T+E248D;

[0606] K62A+L124M+A216F;

[0607] V150T;

[0608] K62A+L124M+E248D;

[0609] Q24L+K62A+L124M;

[0610] L124M;

[0611] V150T+E248D;

[0612] A65L+L124M;

[0613] L124M+A216F+E243R;

[0614] K62I+L124M;

[0615] Q24L+A216F;

[0616] A65L+L156V+Y163P+A216F;

[0617] K62I+S122T+L124M+V150T+A216F+A301K;

[0618] Q24L+K62I+S122T+L124M+V150T;

[0619] Q24L+K62I+S122T+A216F+E248D;

[0620] Q24L+K62I+S122T+V150T+A216F;

[0621] K62A+A65L+A216F+E248D;

[0622] K62I+S122T+L124M+A301K;

[0623] A65L+A216F+E248D;

[0624] S122G+L124M+A216F+E243R;

[0625] K62I+S122T+L124M+A216F;

[0626] Q24L+K62I+S122T+L124M+A301K;

[0627] Q24L+K62I+S122T+L124M.

[0628] In some specific embodiments, the transaminase variant also has a substitution selected from any of the following:

[0629] A348T; K344F; G355K; E349P; L356S; K344V; L336A; Q342F; E349I; E58M; A348S; Y339L; L189K; F3 41I; Y339I; L351P; A346T; K344D; H273N; E349T; K115G; A70V; G355S; A331K;

[0630] In some specific embodiments, the transaminase variant also has substitutions selected from the group consisting of:

[0631] A70V+A114V+K115G+G180C+H273T;

[0632] A70V+K115G+G180C+H273T;

[0633] A70V+A114V+Q154V+G180C+D275E;

[0634] A70V+A114V+Q154V+G180C;

[0635] A114V+Q154V+G180C;

[0636] A70V+G180C+H273T;

[0637] A114V+G180C+H273T+D275E;

[0638] A70V+K115G+Q154V+G180C+H273T;

[0639] K115G+G180C+H273T+D275E;

[0640] K115G+G180C+H273T;

[0641] K115G+G180C+L189K+H273N+C280S;

[0642] K115G+G180C;

[0643] A114V+K115G+G180C+L189K;

[0644] A70V+K115G+H273T;

[0645] A70V+A114V+L189K+H273T+C280S;

[0646] Q154V+G180C+L189K+H273T+D275E;

[0647] A70I+A114T+Q154V+G180C+L189K+H273T;

[0648] A70V+A114V+K115G+Q154V+G180C+H273T+D275E;

[0649] A114V+K115G+G180C;

[0650] A70V+K115G;

[0651] G180C+D275E+C280S;

[0652] A70V+K115G+F149S+Q154V+L189K+H273T;

[0653] A70V+A114V+G180C+L189K+H273T+D275N+C280S。

[0654] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0655] A331*;

[0656] E332*;

[0657] G333*;

[0658] K334*;

[0659] P335*;

[0660] L94V;

[0661] L228T;

[0662] D275E+P335*;

[0663] G180C+D275E+E332*;

[0664] G180C+D275E+P335*;

[0665] F149S+A331*;

[0666] F149S+E332*;

[0667] D275E+A331*;

[0668] D275E+E332*;

[0669] D275E+P335*; D238V;

[0670] D238S;

[0671] E104M;

[0672] V263L;

[0673] A321K.

[0674] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0675] G69A; N255C; A216C; P296T; D215V; K132V; T66C; F259H; A216T; D215W.

[0676] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0677] L24K+L47P+R102V+K132M+A135Q+D167S+K189F;

[0678] L94V+D238V+L292M;

[0679] L94V+D238S+V261A+A321K;

[0680] L94V+D238V+A321K;

[0681] L94V+L292M;

[0682] L94V+D238S+V261A+E315D;

[0683] L94V+D238V;

[0684] L47P+K132M+A135Q+Y163K+K189F+V227F;

[0685] K189F+S280C;

[0686] L94V+D238S+V263L;

[0687] L94V+V261A+V263L+L292M;

[0688] L94V+D275N+L292M;

[0689] L47P+K132M+A135Q+Y163R+D167S+K189F+V227F;

[0690] L94V+D238V+V263L+L292M;

[0691] L47P+R102V+Y163K+K189F;

[0692] L94V+D238V+V261A;

[0693] L94V+D238S+V261A+V263L+L292M;

[0694] L94V+V263L;

[0695] L94V+D238S+D275N+L292M+A321K;

[0696] L94V+D238S+A321K;

[0697] L94V+D238S+L292M+A321K;

[0698] D238V+V261A+V263L+L292M;

[0699] L47P+K189F+A216G;

[0700] L94V+D238S+V263L+D275N;

[0701] L94V+V263L+A321K;

[0702] L94V+V261A;

[0703] L94V+D238V+V261A+V263L+L292M;

[0704] L47P+Y163K+K189F+A216G+F259I+H317V;

[0705] L94V+D238S+D275N;

[0706] V114E+K189L+V227F+N298G;

[0707] L47P+R196N;

[0708] R102V+A135Q+M279C+N298G;

[0709] M13I+E36Q+L45F+S85H+T122M+A135Q+D167S+S202K+A271C+M287C;

[0710] R102V+D167S+M279C+N298G;

[0711] E36Q+S85H+R102V+K106R+A135Q+D167S+N217H+A270T+M279C;

[0712] L47P+N255R;

[0713] L47P+R196N+A216G+N298G.

[0714] In some specific embodiments, the transaminase variant also has substitutions selected from the following group of substitutions:

[0715] M58S+L94V+A135Q+A270T;

[0716] M58S+L94V+R102V+K132I+L198I+A270T+M279C;

[0717] L94V+K132M+A135Q+L198I+I226V;

[0718] L94V+K132M+I226V+A270T;

[0719] L94V+R102V+A135Q+I226V+A270T;

[0720] M58S+L94V+R102V+K132M+I226V+A270T;

[0721] L94V+K132M+A135Q+M279C;

[0722] L94V+R102V+K132M+A135Q+M279C;

[0723] L94V+R102V+A270T;

[0724] M58S+L94V+R102V+A270T;

[0725] L94V+K132I+L198I+A270T;

[0726] L94V+R102V+A135Q+L198I+I226V;

[0727] M58S+L94V+K132M+A135Q+L198I+I226V+M279C;

[0728] L94V+I126M+K132M+A135Q+N298G;

[0729] L94V+R102V+L198I+A270T;

[0730] M58S+L94V+K132I+A135Q+I226V;

[0731] M58S+L94V+R102V+K132I+A135Q+L198I+I226V+A270T;

[0732] L94V+I226V;

[0733] M58S+L94V+A135Q+L198I;

[0734] L94V+V123C+V127I+V261A+G294F;

[0735] L94V+V169L+I226V+N255C+V261A+P296T;

[0736] L94V+V123C+V127I+I226V+N255C+G294F+P296T+N298G;

[0737] L94V+V169L+T185K+V227L+N255C+N298G;

[0738] L94V+V123C+V127I+V150T+V169L+N255C;

[0739] L94V+V123C+V127I+V150T+V169I+N255C+V261A+G294F+P296T+N298G;

[0740] L94V+V123C+V127I+I226V+G294F+P296T+N298G;

[0741] L94V+V123C+V127I+V169L+V261A+G294F;

[0742] L94V+V123C+V127I+V261A+P296T;

[0743] L94V+V150T+I226V+N255C+V261A+G294F;

[0744] L94V+V123C+V127I+V169I+V227L+V261A+G294F.

[0745] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0746] L24K+R102V+E315D;

[0747] V263L;

[0748] K132M+E315D;

[0749] L24K+R102V+I226V+D238S+V261A+V263L+P296T+N298G+E315D;

[0750] R102V;

[0751] L24K+K132I;

[0752] L24K+R102V+K132M+E315D;

[0753] L24K+D238V+N298G;

[0754] L24K+I226V+D238V+V261A+V263L+M279C+L292M+N298G;

[0755] K132V;

[0756] R102V+K132V+L292M+E315D;

[0757] I226V+V227L+V261A+V263L+M279C+N298G+E315D;

[0758] K132M;

[0759] E103K;

[0760] L24K+R102V+K132V;

[0761] R102V+V227L+N255C+A321K;

[0762] R102V+V227L+E260K+V261A+V263L;

[0763] L24K+V169L+D238V+V261A+V263L+M279C+E315D;

[0764] R102V+K132V+V227L+A321K;

[0765] K132V+V169L;

[0766] V227L+D238V+V263L+M279C+N298G+E315D;

[0767] V227L+D238S+V263L+M279C+P296T+E315D;

[0768] R102V+K132V+A321K.

[0769] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0770] K132V+G180C+D238V+V263L;

[0771] K132V+V169I+D215G+D238V;

[0772] K132V+V263L+P296L;

[0773] M61L;

[0774] K132V+V263L+L292M;

[0775] D215V+D238V;

[0776] V123C+K132V+L292M;

[0777] S137V; D215E; L24F;

[0778] E104M+K132V+V169I+V263L+L292M+P296T;

[0779] D231G; K97S; K115M; I142G;

[0780] K132V+D238V+V263L+L292M+P296T;

[0781] I142D; C255L; H71G; A301N; E325A; V114I; N81H; S85N; K106R; Y328P.

[0782] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0783] K132Q+K257A;

[0784] L24K+K132Q+S137V+D231G+D238V;

[0785] L24F+S137V+C255L+P304L;

[0786] L24F+K132Q+S137V+D231G+C255L+K257A;

[0787] K132Q+S137V+D238V+C255L+K257A+V261A+V263L+L292M+N298G;

[0788] L24F+K132Q+C255L+K257A+T270C+P304L;

[0789] L24F+S137V+K257A;

[0790] L24K+K132Q+S137V+D238V+C255L+V263L+T270C+N298G;

[0791] L24F+S137V+D231G+K257A+T270C+298G;

[0792] N81H+S137V+C255L+T270C;

[0793] L24F+K132Q+S137V+I226V+D231G+D238V+C255L+V263L+N298G;

[0794] L24K+S137V+D238V+C255L+K257A+V263L+T270C+N298G;

[0795] S137V+C255L;

[0796] L24F+C255L+K257A;

[0797] N81H+S137V+K257A+Y322V;

[0798] N81H+K132Q+S137V+D231G+T270C;

[0799] N81H+K132Q+S137V+I226V+C255L+K257A+V261A+L292M+N298G;

[0800] K132Q+S137V+T270C+P304L;

[0801] N81H+D238V+V261A+V263L+N298G;

[0802] N81H+S137V+C255L+T270C;

[0803] L24K+N81H+K132Q+D231G+D238V+C255L+V261A+V263L;

[0804] L24F+N81H+K132Q+S137V+C255L+K257A+T270C;

[0805] D231G+D238V+L292M+N298G;

[0806] S137V+C255L+Y322V;

[0807] L24F+N81H+T270C;

[0808] L24F+N81H+S137V+T270C;

[0809] N81H+K132Q+I226V+D238V+C255L+V261A+V263L+N298G;

[0810] L24F+S137V+D231G+C255L+V261A+V263L+T270C;

[0811] L24F+S137V+D231G+C255L+K257A+T270C;

[0812] L24K+S137V+D231G+D238V+C255L+L292M+N298G;

[0813] N81H+S137V+K257A;

[0814] L24F+S137V+C255L+K257A;

[0815] L24K+K132Q+S137V+D231G+D238V+C255L+V263L+N298G;

[0816] D231G+K257A;

[0817] S137V+C255L+T270C+P304L;

[0818] N81H+C255L+K257A+T270C;

[0819] S137V+C255L+K257A;

[0820] L24F+T270C+P304L.

[0821] In some specific embodiments, the transaminase variant also has substitutions selected from the following sites:

[0822] V114K; N81D; A51S; D238I; G216Q; Y235H; L100H; G180L; G258K; I289V; D238H; R145K.

[0823] In some specific embodiments, the transaminase variant also has substitutions selected from the following substitution groups or sites:

[0824] L100K+I289V;

[0825] N81D+L100K+G180L+G216Q+D238H+I289V;

[0826] R145K+G216Q;

[0827] R145K+G180L+I289V;

[0828] N81D+L100K+R145K+G216Q;

[0829] N81D+L100K+D238H+G258K+I289V;

[0830] N81D+V114K+R145K+D238H+I289V;

[0831] N81D+G216Q+D238H+I289V;

[0832] N81D+L100K+D238H+G258K;

[0833] L100K+V114K+D238H+G258K;

[0834] N81D;

[0835] V114K+D238H+G258K+I289V;

[0836] G180L+D238H+I289V;

[0837] F24K+V150T+N298G;

[0838] F24K+V261A+N298G;

[0839] F24K+D167S+D238S+G294F+E315D;

[0840] V114K+R145K+D167S+G294F+N298G;

[0841] F24K+G180L+V261A+N298G;

[0842] V102R+V114A+Q135R+Q154I;

[0843] V102R+Q135R+Q154I;

[0844] Q135R+Q154V;

[0845] T8D+F24Q+V102R+V114A+Q154V;

[0846] S58Q+V102R+Q154V;

[0847] T8N+S58Q+Q135R+Q154V.

[0848] In some specific embodiments, the transaminase variant also has a selection of substitutions or combinations thereof:

[0849] A257K; L255V; K132S; L131Q; F24L; T8D+F24L+V102R+V114A+Q135R+Q154V.

[0850] In some specific embodiments, the transaminase variant also has a selection of substitutions or combinations thereof:

[0851] M10R+A257K+F259Y;

[0852] F259L;

[0853] M10R+L255V+F259L+P267S;

[0854] M10R+A257K;

[0855] M10R+F259L;

[0856] M10R.

[0857] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0858] K107Q+F199H+E260D+A301K;

[0859] F199H+E260D;

[0860] K107Q+V143N+F199H+E260D+A301K;

[0861] V143N+L255R.

[0862] In some specific embodiments, the transaminase variant also has substitutions selected from the following: A21Y; F278L; D299L.

[0863] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0864] A11S+E50L+E160H+V263M;

[0865] A11S+A21Y+N143F;

[0866] A11S+A21Y+E141T+V263M+F278L;

[0867] A11S+E50L+E141T+E160H;

[0868] A11S+E141T+N143F+V263M;

[0869] T122M+L198I.

[0870] In some specific embodiments, the transaminase variant also has a combination of substitutions selected from the following:

[0871] V137S+E141T+N143F+H235Y+R255C+A257K+F259L+V263M;

[0872] A11S+V137S+E141T+A257K;

[0873] A11S+V137S+N143F+H235Y+R255C+A257K+V263M;

[0874] A11S+R255C+A257K;

[0875] E141T+R255C+F259L+V263M;

[0876] V70A+H235Y+R255C+F259L;

[0877] V137S+E141T+A257K;

[0878] A11S+V137S+E141T+R255C+F259L+V263M;

[0879] A11S+N81G+E141T+H235Y+A257K+V263M;

[0880] A11S+N81G+V94I+V137S+E141T+A257K+V263M;

[0881] E141T+N143F+G216N+H235Y+V263M;

[0882] A11S+E141T+A257K+V263M+P296S;

[0883] V94I+G216N+H235Y+A257K+V263M;

[0884] A11S+V137S+N143F+H235Y+A257K;

[0885] A11S+V137S;

[0886] A11S+V137S+N143F+H235Y+A257K+V263M+P296S;

[0887] A11S+E141T+G216N+V263M+P296S;

[0888] V94I+V137S+E141T+N143F+G216N+H235Y+V263M;

[0889] A11S+E141T+G216N+H235Y+V263M;

[0890] A11S+G216N+V263M;

[0891] N143F+G216N+A257K;

[0892] A11S+N81G+E141T+V263M;

[0893] A11S+N81G+V137S+N143F+V263M;

[0894] V137S+E141T+H235Y+A257K+V263M;

[0895] A11S+N81G+V137S+E141T+G216N+H235Y+A257K+V263M+P296S;

[0896] A11S+N81G+V94I+V137S+G216N+H235Y+A257K;

[0897] A11S+N81G+V94I+V137S+N143F+G216N+H235Y+V263M+P296S;

[0898] A11T+N81G+V94I+A257K+V263M;

[0899] G216N+H235Y+A257K+V263M;

[0900] T12S+D167S+N217H.

[0901] The transaminase variants of the present invention exhibit improved enzymatic properties compared to wild-type transaminases, such as improved high activity, high stability, and high stereoselectivity. Compared to wild-type transaminases, the transaminase variants of the present invention can directly synthesize sitagliptin with high optical purity using the precursor ketone of sitagliptin as a substrate.

[0902] The transaminase variants of the present invention comprise an amino acid sequence selected from the following: SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 9 2, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 12 4, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 27 8, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 400, 4 02, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 6 28, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 666, 668, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 73 4, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938. In some embodiments, the transaminase variant of the present invention further comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the aforementioned amino acid sequence.

[0903] In a preferred embodiment, the transaminase variant of the present invention is used to catalyze the following reaction:

[0904] In a third aspect, the present invention provides a fusion protein comprising a transaminase variant of the second aspect of the present invention.

[0905] In a fourth aspect, the present invention provides a polynucleotide that encodes a transaminase variant of the second aspect of the present invention or a fusion protein of the third aspect of the present invention.

[0906] In some preferred embodiments, the polynucleotide comprises the same as SEQ ID. NO:13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 9 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 12 3. 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 27 7, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 399, 4 01, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 74 1, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 8 Nucleotide sequences of the following lengths: 51, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, or 937, having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some preferred embodiments, the polynucleotide comprises or consists of nucleotide sequences selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47.49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、223、225、227、229、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、261、263、265、267、269、271、273、275、277、279、281、283、285、287、289、291、293、295、297、299、301、303、305、307、309、311、313、315、317、319、321、323、325、327、329、331、333、335、337、339、341、343、345、347、349、351、353、355、357、359、361、363、365、367、369、371、373、375、377、379、381、383、385、387、389、391、393、395、399、401、403、405、407、409、411、413、415、417、419、421、423、425、427、429、431、433、435、437、439、441、443、445、447、449、451、453、455、457、459、461、463、465、467、469、471、473、475、477、479、481、483、485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 75 1, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 8 45, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, or 937.

[0907] In a fifth aspect, the present invention provides a nucleic acid construct comprising the polynucleotides of the fourth aspect of the present invention.

[0908] In a sixth aspect, the present invention provides a vector comprising the polynucleotide of the fourth aspect of the present invention or the nucleic acid construct of the fifth aspect of the present invention.

[0909] In a seventh aspect, the present invention provides a host cell comprising the polynucleotide of the fourth aspect of the present invention, the nucleic acid construct of the fifth aspect of the present invention, or the vector of the sixth aspect of the present invention.

[0910] In an eighth aspect, the present invention provides a composition comprising a transaminase variant of the second aspect of the present invention, a fusion protein of the third aspect of the present invention, a polynucleotide of the fourth aspect of the present invention, a nucleic acid construct of the fifth aspect of the present invention, a vector of the sixth aspect of the present invention, and / or a host cell of the seventh aspect of the present invention.

[0911] In a ninth aspect, the present invention provides the use of the transaminase variant of the second aspect, the fusion protein of the third aspect, the polynucleotide of the fourth aspect, the nucleic acid construct of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, or the composition of the eighth aspect in the preparation of amines.

[0912] In some preferred embodiments, the present invention provides the use of the transaminase variant of the second aspect of the invention, the fusion protein of the third aspect, the polynucleotide of the fourth aspect, the nucleic acid construct of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, or the composition of the eighth aspect in the preparation of 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolone[4,3-a]pyrazine (sitagliptin, CAS 486460-32-6) or in the synthesis of sitagliptin phosphate (CAS 654671-77-9). Attached Figure Description

[0913] Figure 1 shows the chromatogram-mass spectrum (black) of the racemic standard solutions containing (S)-amine and (R)-amine, and the chromatogram-mass spectrum (red) of the reaction solution of the first-generation mutant "Gen 1". The y-axis represents the signal intensity of (S)-amine and (R)-amine, and the x-axis represents the chromatographic elution time of (S)-amine and (R)-amine. Reaction conditions: 1 g / L substrate, room temperature, 2 M pH 8 isopropylamine*hydrochloric acid, reaction time 24 h. As shown in Figure 1, the first-generation mutant "Gen 1" has an optical purity >99.99%, and (S)-amine was not detected. Through multiple rounds of enzyme engineering evolution, a transaminase variant with high optical purity and high activity was obtained.

[0914] Figure 2 shows the relative activities of the 10th to 14th generation mutants "Gen 10–Gen 14" relative to the first-generation mutant "Gen 1". The y-axis represents the relative amount of sitagliptin produced compared to the first-generation mutant "Gen 1", normalized by setting the amount of sitagliptin produced by the first-generation mutant "Gen 1" transaminase to 1. Reaction conditions: 50 g / L substrate, 50 °C, 1 M pH 9 isopropylamine*hydrochloric acid, reaction for 24 hours. As shown in Figure 2, Gen 10, Gen 11, Gen 12, Gen 13, and Gen 14 underwent successive rounds of evolution, with each round screening for transaminase variants showing a significant increase in transaminase activity compared to the previous round.

[0915] Figure 3 shows the relative activities of the 19th to 24th generation mutants "Gen 19-Gen 24" relative to the 18th generation mutant "Gen 18". The y-axis represents the relative amount of sitagliptin produced compared to the 18th generation mutant "Gen 18", normalized by setting the amount of sitagliptin produced by the 18th generation mutant "Gen 18" to 1. Reaction conditions: 50 g / L substrate, 50 °C, 1 M pH 9 isopropylamine*hydrochloric acid, reaction for 24 hours. As shown in Figure 3, Gen 19, Gen 20, Gen 21, Gen 22, Gen 23, and Gen 24 underwent successive rounds of evolution, with each round screening for transaminase variants showing a significant increase in transaminase activity compared to the previous round. Detailed Implementation

[0916] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0917] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly specifies otherwise.

[0918] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0919] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0920] All numerical names, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values, appropriately varying in increments of 1.0 or 0.1, or optionally changing (+) or (-) by variations of + / - 15%, 10%, 5%, or 2%. It should be understood that all numerical names are preceded by the term "about." It should also be understood that the reagents described herein are exemplary only, and their equivalents are known in the art. When referring to measurable values ​​such as amount or concentration, the term "about" as used herein means a variation within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0921] In this document, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or mixed-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or substantially consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0922] The terms “protein,” “peptide,” “polypeptide,” and “amino acid sequence” are used interchangeably and, in their broadest sense, refer to a polymer of two or more amino acid subunits, amino acid analogs, or peptide mimics. “Protein,” “peptide,” “polypeptide,” and “amino acid sequence” contain at least two amino acids, and there is no limit to the maximum number of amino acids. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0923] Equivalents having one or more amino acid modifications compared to the protein or amino acid sequence described herein are also covered within the scope of this invention, provided that such modification does not affect or substantially does not affect the activity of the protein or amino acid sequence. In this document, amino acid modification can be amino acid substitution, amino acid deletion, or amino acid insertion. Amino acid substitution can be conserved or non-conserved. A conserved substitution (also called a conserved mutation, conserved replacement, or conserved variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, or size). In this document, "conserved substitution" means that an amino acid residue is replaced by another biologically similar residue. Examples of conserved substitution include one hydrophobic residue such as isoleucine, valine, leucine, or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. Other exemplary examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine, or glutamic acid; phenylalanine to tyrosine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and so on.

[0924] When referring to specific molecules, biological materials, or cellular substances, the terms "equivalent" and "functional variant" may be used interchangeably and refer to those that have minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polypeptide (e.g., the nucleotide sequence encoding the transaminase described in this invention); or polypeptides encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polynucleotide (e.g., the nucleotide sequence encoding the transaminase described in this invention).

[0925] In this article, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into peptides, polypeptides, amino acid sequences, or proteins. If the nucleic acid sequence originates from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0926] When the term "encoding" is applied to a nucleic acid sequence, it refers to a nucleic acid sequence that, if in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide, is called "encoding" a polypeptide. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0927] "Homology" or "identity" refers to the sequence similarity between two polypeptides or two nucleic acid sequences. The percentage of identity can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. "Irrelevant" or "non-homologous" sequences share less than 40% identity and less than 25% identity with one of the sequences of this invention. Tools for comparing sequence similarity are well known to those skilled in the art; for example, the alignment and percentage of sequence identity of the nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools bin / clustalw / ) and using ClustalW.

[0928] The term "nucleic acid construct" refers to a nucleotide sequence containing a complete expression cassette capable of expressing a target gene or protein. In addition to the coding sequence encoding the target gene or protein, a nucleic acid construct may also contain genetic elements such as promoters, enhancers, terminators, and / or polyadenylation signals for regulating gene expression. Those skilled in the art will understand that the genetic elements contained in a nucleic acid construct are combined in a "functionally linked" or "operably linked" manner to enable the target gene or protein to be expressed in a desired manner.

[0929] As used herein, the term "promoter" refers to an expression control sequence that controls the initiation and rate of transcription of a gene or transgene. Promoters can be, for example, constitutive, inducible, repressive, or tissue-specific. Promoters may contain genetic elements that regulate proteins and molecules such as RNA polymerases and transcription factors to which they can bind.

[0930] In this invention, "effective ligation" or "operationally ligation" refers to the connection of nucleic acid sequences, such that one sequence provides the function required for the linked sequences. In this invention, "operationally ligation" can refer to linking a promoter to a sequence of interest, such that the transcription of the sequence of interest is controlled and regulated by the promoter. When the sequence of interest encodes a protein and its expression is desired, "operationally ligation" means that a promoter is linked to the sequence in a manner that enables efficient transcription and translation of the sequence.

[0931] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host or capable of inserting a target gene fragment into the host genome, thereby transferring the carried target gene fragment into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0932] The term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," encompassing primary transformed cells and their derived progeny (regardless of passage number). The nucleic acid content of the progeny may not be entirely identical to that of the parent cells, and may contain mutations. This document includes mutant progeny that possess the same function or biological activity as those screened or selected in the original transformed cells.

[0933] "Amine" refers to a compound formed when the hydrogen atoms in an ammonia molecule (NH3) are replaced by hydrocarbon groups, in which the hydrocarbon groups are optionally replaced.

[0934] In this article, "aminotransferase" and "transaminase" are used interchangeably, referring to polypeptides that have the enzymatic ability to transfer amino (NH2) and hydrogen atoms from primary amines to acceptor carbonyl compounds, convert amino donors to their corresponding carbonyl compounds, and convert acceptors to their corresponding primary amines.

[0935] The term "substrate" as used in this article refers to an amino acceptor, such as a ketone, that accepts an amino group from an amino donor in a transaminase-mediated reaction.

[0936] The reaction formula for synthesizing sitagliptin from its precursor ketone (structural formula 1) is as follows:

[0937] An "amino donor" is an amino compound that can supply an amino group to an acceptor carbonyl compound (i.e., an amino acceptor), thereby becoming a carbonyl byproduct.

[0938] Sitagliptin, in its pharmaceutical form as sitagliptin phosphate monohydrate, is a dipeptidyl peptidase-4 (DPP-4) inhibitor. It can be used alone or in combination with other oral hypoglycemic agents (such as metformin) to treat type 2 diabetes. It improves glycemic control by increasing the level of active incretins. The structure of sitagliptin is shown in structural formula 2.

[0939] "Improved enzyme properties" refers to any enzyme property that is better or more desirable for a specific purpose compared to the properties exhibited by a reference enzyme. For the engineered transaminase peptides described herein, this comparison is generally made against a wild-type transaminase; however, in some embodiments, the reference transaminase may be another improved engineered transaminase. Improveable enzyme properties include, but are not limited to, enzyme activity (which can be expressed as the amount of target product generated or the percentage of substrate conversion over a period of time), thermal stability, solvent stability, pH activity profile, coenzyme requirement, reluctance to inhibitors (e.g., product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity). Therefore, "improved enzyme activity" can refer to an increase in the amount of target product generated or the percentage of substrate conversion over a period of time compared to a reference transaminase (e.g., a wild-type transaminase, or another improved engineered transaminase).

[0940] "Breatibility" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where one stereoisomer is formed more preferentially than the other, or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantiomer selectivity, which is the fraction (usually reported as a percentage) of one enantiomer in the sum of the two enantiomers. It is typically reported in the art, optionally, as (usually as a percentage) an enantiomer excess (ee) calculated from the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is called diastereomer selectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, typically reported, optionally, as a diastereomer excess (de). Enantiomer excess and diastereomer excess are types of stereoisomer excess.

[0941] "High stereoselectivity" refers to a chemical or enzymatic reaction that can convert a substrate into its corresponding product with an excess of at least about 85% stereoisomers.

[0942] In some embodiments of this application, "transaminase activity" refers to the ability of the transaminase to transfer an amino group from an amino donor to a substrate (amino acceptor, such as a ketone substrate), an ability that can be expressed by the amount of target product generated. Accordingly, a transaminase variant having "improved transaminase activity" refers to an increase in the amount of target product generated by the transaminase variant compared to a parental transaminase (e.g., a wild-type transaminase, or another improved engineered transaminase).

[0943] Chemical definition

[0944] The definitions of specific functional groups and chemical terms are described in more detail below.

[0945] When listing a range of values, it is assumed that each value and a subrange within that range will be included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2- 4. C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0946] “C 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl groups are preferred. In some embodiments, C 1-3 Alkyl groups are preferred. In some embodiments, C 1-2 Alkyl groups are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen (O), sulfur (S), nitrogen (N), boron (B), silicon (Si), phosphorus (P)). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0947] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0948] Therefore, "C" 1-6 "Halogenated alkyl" refers to the above "C 1-6 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-3 Halogenated alkyl groups are particularly preferred, and C4 groups are more preferred. 1-2 Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halide group can be substituted at any available junction, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0949] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-8 cycloalkyl, C 3-7 cycloalkyl, C4-6 Cycloalkyl groups are preferred, C 3-6 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9), etc. 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C9) 10 ), spiro[4.5]decyl(C 10 ), borneol, adamantyl, etc. Regardless of whether the cycloalkyl group is modified with "substituted", each cycloalkyl group is independently and optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, with suitable substituents defined as follows.

[0950] "3-10 membered heterocyclic groups" refer to groups with a 3- to 10-membered non-aromatic ring system having a ring carbon atom and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided that the valence allows. In some embodiments, 4-10 membered heterocyclic groups are preferred, which are 4-10 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; in some embodiments, 5-10 membered heterocyclic groups are preferred, which are 5-10 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; in some embodiments, 3-8 membered heterocyclic groups are preferred, which are 3-8 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; in some embodiments, 3-7 membered heterocyclic groups are preferred, which are 3-7 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; in some embodiments, 3-6 membered heterocyclic groups are particularly preferred, which are 3-6 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; more preferably, 5-6 membered heterocyclic groups are 5-6 membered non-aromatic ring systems having a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include cyclic systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl, aryl, or heteroaryl groups, wherein the connection point is on the heterocyclic ring; and in such cases, the number of ring members continues to indicate the number of ring members in the heterocyclic ring system. Regardless of whether the heterocyclic group is preceded by the word "substituted," each of the heterocyclic groups may be optionally substituted independently, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, with suitable substituents defined below.

[0951] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azircyclopropane, oxacyclopropane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azircyclobutane, oxacyclobutane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, and dioxane. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxeheptanyl, and thionylheptanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxeheptanyl, and thionylheptanyl. Exemplary 5-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinoneyl, etc. Exemplary 6-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0952] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C... 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, C 6-10Aryl groups are particularly preferred, and more preferably C6 aryl groups. Aryl groups also include ring systems in which the aforementioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Regardless of whether the aryl group is preceded by the word "substituted," each aryl group may be optionally substituted independently, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, with suitable substituents defined below.

[0953] "5-10-membered heteroaryl" refers to a group comprising a 4n+2 aromatic ring system of a 5-10-membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms (e.g., having 6 or 10 shared π electrons arranged in a ring), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6-membered heteroaryl is particularly preferred, which is a 4n+2 aromatic ring system of a 5-6-membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms. In some embodiments, a 5-membered heteroaryl group is particularly preferred, which is a 4n+2 aromatic ring system of a 5-membered monocyclic or bicyclic ring having a cyclic carbon atom and 1-4 cyclic heteroatoms. Regardless of whether the heteroaryl group is preceded by "substituted", each of the heteroaryl groups is optionally substituted independently, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, with suitable substituents defined as follows.

[0954] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to: triazinyl and tetraazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to: azirheptatrienyl, oxaheptatrienyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to: indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to: naphridinyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0955] In this application, the terms “optional” or “optionally” generally refer to an event or environment described subsequently that may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0956] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(Rbb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0957] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0958] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0959] R bb Each is independently selected from: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR)cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0960] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0961] R dd Each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(Rff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee -S(=O)R ee 、-Si(R ee 3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. Alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 Rgg groups, or two geminal Rdd substituents may combine to form =O or =S;

[0962] R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0963] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, or two R ff The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0964] R gg Each of these is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1- 6-alkyl, -ON(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - -NH(C 1-6 Alkyl)2 + X - -NH2(C 1-6 alkyl) + X - -NH3 + X - -N(OC) 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6 Alkyl group, -CO2H, -CO2(C 1- 6-alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6 Alkyl)2、-OC(=O)NH(C 1-6 Alkyl), -NHC(=O)(C 1-6 alkyl), -N(C) 1-6 Alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 Alkyl), -NHC(=O)N(C 1-6 Alkyl)2、-NHC(=O)NH(C 1-6 Alkyl groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl), -OC (=NH)(C 1-6Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1- 6-alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2、-SO2NH(C 1-6 Alkyl groups, -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C) 1- 6-alkyl)3、-OSi(C 1-6 Alkyl)3, -C(=S)N(C 1-6 Alkyl)2、C(=S)NH(C 1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC (=S)SC 1-6 Alkyl group, -P(=O)2(C 1-6 Alkyl), -P(=O)(C 1-6 Alkyl)2、-OP(=O)(C 1-6 Alkyl)2、-OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C7 carbocyclic, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two ethryl groups gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0965] Exemplary substituents on the nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2Raa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. cc The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0966] "Deuterated" or "deuterium" refers to the substitution of one or more hydrogen atoms in a compound or group by deuterium; deuteration can be monosubstituted, disubstituted, polysubstituted, or fully substituted. The terms "one or more deuterated" and "one or more deuterated" are used interchangeably. The deuterium isotope content at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, and more preferably greater than 99%.

[0967] As used herein, the terms “amino receptor” or “amino donor” refer to the amino receptor compound and amino donor compound of this application. The term also includes various pharmaceutically acceptable salts, stereoisomers, enantiomers, diastereomers, meso compounds, racemic compounds, or tautomers of the amino receptor or amino donor compound.

[0968] As used herein, the term "pharmaceutically acceptable salt" refers to acid or base addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0969] Pharmaceutically acceptable salts can be sulfates, pyrosulfates, bisulfates, sulfites, nitrates, borates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, etc., prepared from inorganic acids. Representative salts include hydrobromide, hydrochloride, sulfates, bisulfates, nitrates, borates, and phosphates. Salts can also be prepared from organic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, alkyl diacids, aromatic acids, and aliphatic and aromatic sulfonic acids. Representative salts include acetates, propionates, valerates, oleates, palmitates, stearates, laurates, caprylates, isobutyrates, oxalates, malonates, succinates, caprylates, sebacic acid salts, fumarates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, naphthates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, methanesulfonates, glucono-p-ethyl, lactobionates, laurylsulfonates, and hydroxyethanesulfonates. Pharmaceutically acceptable salts may include alkali metal and alkaline earth metal-based cations, such as sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. It also covers salts of amino acids, such as arginine salts, gluconates, galacturons, etc. (see, for example, Berge S. et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0970] This invention also includes isotopically labeled compounds (isotope variants) that are equivalent to the general formulas or specific compounds described in this application, but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、31 P, 32 P, 35 S, 18 F and 36 Cl. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Additionally, heavier isotopes such as deuterium (i.e.,...) are preferred. 2 H) substitution is preferred in some cases because its higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements. Isotope-labeled compounds of the present invention and their prodrugs can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0971] Example

[0972] Example 1. Culture and production of cells overexpressing transaminases

[0973] 1. Construction and expression of transaminase libraries

[0974] The amino acid aminotransferase gene (Uniprot: UPI00143F05B3) from *Thermomyces stellatus* was codon-optimized for expression in *E. coli*. The optimized coding sequence is shown in SEQ ID NO:11, and its encoded amino acid sequence is shown in SEQ ID NO:12. This gene was cloned into the expression vector pET-28a(+) driven by the T7 promoter. This pET-28a(+) expression vector contains the colE1 origin of replication and a kanamycin resistance gene. The vector was transformed into *E. coli* C43(DE3), and plasmid DNA was prepared from *E. coli* C43(DE3) using standard methods.

[0975] Transaminase libraries, including site-saturation mutagenesis libraries and combinatorial libraries, were constructed using the MegaWHOP and SOE methods. These libraries were then cloned into the pET-28a(+) vector, transformed into *E. coli* C43(DE3), and expressed. For the construction of the site-saturation mutagenesis library, degenerate primers were designed using NNK to introduce saturation mutations at each desired location. Each degenerate primer was used to obtain a megaprimer via conventional PCR, and a complete plasmid with single-site saturation mutagenesis was generated using MegaPrimer PCR (MegaWHOP). All MegaWHOP products were then mixed and transformed to generate a pooled site-saturation library. Sequence-defined primers were designed to introduce programmed mutations into the combinatorial mutagenesis library. Each primer contained one or more mutations or no mutations in each region where mutations were desired. Multiple primers for each mutation region were mixed in equimolar amounts for initial PCR. Further PCR was performed to fuse all initial PCR products to generate a combinatorial library with all desired mutations. The SOE product was then assembled into the pET-28a(+) vector using the Gibson method, and then transformed into E. coli C43(DE3) to obtain E. coli containing the plasmid of interest, thus generating a merged combinatorial library.

[0976] 2. Obtaining cryoprecipitates containing transaminase genes

[0977] Recombinant *E. coli* clones containing the gene of interest were selected using a colony selector and placed into 96-well plates containing 200 μL of TB or 1 g / L kanamycin (Kan) antibiotic. Cells were grown overnight in a humidity-controlled incubator at 85% humidity, 30°C, and 250 rpm. 10 μL aliquots were transferred to new deep-well plates containing 380 μL of TB with 1 g / L Kan, and incubated for 2–3 hours in a humidity-controlled incubator at 85% humidity, 30°C, and 250 rpm. Induction with 10 μL of 40 mM IPTG was then performed overnight. The cell pellet was collected by centrifugation at 4000 rpm / 4°C / 20 min. The supernatant was discarded, and the remaining cell pellet was stored at -80°C until assay.

[0978] Example 2. Determination of enzyme activity in 96-well plates

[0979] The frozen cell pellet obtained in Example 1 was thawed to room temperature in a 96-well deep-plate and treated with 200 μL / well of lysis buffer at room temperature for 90 min. This lysis buffer consisted of 2 g / L pyridoxal-phosphate (PLP), 0.5 g / L polymixin B sulfate (PMBS), 1 g / L lysozyme, and 0.1% v / v DNAase I in 100 mM triethanolamine*H2SO4 buffer at pH 8 or pH 9. The lysis mixture was centrifuged at 4000 rpm / 4 °C for 20 min. A certain volume of the lysate supernatant was added to another deep-plate, followed by 1 M–4 M isopropylamine*hydrochloric acid solution at pH 8 or pH 9 and a high concentration of precursor ketone substrate solution dissolved in an organic co-solvent. The volumes or concentrations of lysate, co-solvent, substrate, and IPM were adjusted as needed in each round of experiments. Seal the plate and place it in a shaking incubator at rt, 30, 40, 45, 50, 55 or 60°C for 24 hours.

[0980] The reaction was quenched with 200 μL of acetonitrile (ACN) and centrifuged at 4000 rpm for 10 minutes at room temperature. To determine optical purity, 100 μL of the supernatant was transferred to 100 μL of 10% NaHCO3 and treated at 40 °C with 500 μL of 2 g / L Marfey's reagent dissolved in ACN for 1 hour. The resulting solution could be directly analyzed by LC-MS / MS or further diluted before LC-MS / MS analysis.

[0981] The analytical method employed an Agilent Eclipse Plus C18 RRHD column (2.1 mm * 50 mm, 1.8 μm). 0.1% (v / v) formic acid was used as mobile phase A, and 0.1% (v / v) formic acid acetonitrile was used as mobile phase B. The column temperature was 40 °C, the flow rate was 0.4 mL / min, and isocratic elution was performed with a mobile phase A:mobile phase B ratio of 58:4. The analysis time was 1.8 min. The mass spectrometer was an Agilent 6460. Detailed mass spectrometry parameters were as follows: ESI positive ionization mode, MRM detection mode, precursor ion m / z 660.0, daughter ions m / z 570.2 and m / z 379.0, collision energy 25 eV, gas temperature 300 °C, dry gas nitrogen 5 L / min, nebulizer pressure 45 psi, sheath gas temperature 250 °C, sheath gas flow rate nitrogen 11 L / min, and capillary voltage 3500 V. Data analysis was performed using Agilent MassHunter workstation software. The retention times of (S)-sitalliptin-Marfey derivative and (R)-sitalliptin-Marfey derivative were 1.1 min and 1.6 min, respectively.

[0982] Example 3. Production of Shake Flask Powder (SFP)

[0983] A single colony of *E. coli* containing a plasmid of the gene for the enzyme of interest was inoculated into a 15 mL culture tube containing 7 mL of TB medium, 10 g / L glucose, and 1 g / L Kan. The tube was placed in a shaker at 250 rpm / 30 °C. After 16–24 hours, 6 mL of this culture was inoculated into 250 mL of TB w / 1 g / L Kan in a 1000 mL shaker-equipped flask. The flask was placed in a shaker at 250 rpm / 30 °C until the OD reached ~0.6–0.8 (usually after 2–4 hours), at which point 250 μL of 1 M IPTG was added. Shaking was continued overnight (16–24 hours). Cells were collected by centrifugation at 4000 rpm / 4 °C / 20 min. The supernatant was discarded, and the remaining cell pellet was resuspended in 35 mL of 50 mM pH 8 or pH 9 triethanolamine*H₂SO₄ and centrifuged again at 4000 rpm / 4 °C / 20 min. Discard the supernatant and resuspend the remaining cell pellet in 4–5 mL of 50 mM pH 8 or pH 9 triethanolamine*H2SO4 buffer at 4 °C. Lyse the cells using a Constant System “One-Shot” homogenizer at 35–40 kpsi. Centrifuge the lysed cell suspension at 7830 rpm / 4 °C / 1 h. Transfer the supernatant to a vial, freeze at -80 °C for 1 hour, and then freeze overnight to obtain a dry powder.

[0984] Example 4. Determination of SFP activity

[0985] Add a certain volume of 1M–4M pH 8 or pH 4.5 isopropylamine*HCl solution and a high-concentration precursor ketone substrate solution dissolved in an organic co-solvent to the wells of a 96-well deep-well plate. The volumes or concentrations of enzyme powder solution, co-solvent, substrate, and IPM are adjusted as needed in each round of experiments. Seal the plate and incubate it in a shaking incubator at rt, 30, 40, 45, 50, 55, or 60°C for 24 hours. Quench the reaction with 200 μL of acetonitrile (ACN) and centrifuge at 4000 rpm / 10 min at room temperature. To determine optical purity, transfer 100 μL of the supernatant to 100 μL of 10% NaHCO3 and treat it at 40°C with 500 μL of 2 g / L Marfey's reagent dissolved in ACN for 1 hour. The resulting solution can be directly analyzed by LC-MS / MS or further diluted before LC-MS / MS analysis.

[0986] Example 5. Post-reaction activity determination

[0987] The reaction was quenched with 200 μL of acetonitrile (ACN) and centrifuged at 4000 rpm for 10 minutes at room temperature. To determine optical purity, 100 μL of the supernatant was transferred to 100 μL of 10% NaHCO3 and treated at 40 °C for 1 hour with 500 μL of 2 g / L Marfey's reagent dissolved in ACN. The resulting solution could be directly analyzed by LC-MS / MS or further diluted before LC-MS / MS analysis.

[0988] The analytical method employed an Agilent Eclipse Plus C18 RRHD column (2.1 mm * 50 mm, 1.8 μm). 0.1% (v / v) formic acid was used as mobile phase A, and 0.1% (v / v) formic acid acetonitrile was used as mobile phase B. The column temperature was 40 °C, the flow rate was 0.4 mL / min, and isocratic elution was performed with a mobile phase A:mobile phase B ratio of 58:4. The analysis time was 1.8 min. The mass spectrometer was an Agilent 6460. Detailed mass spectrometry parameters were as follows: ESI positive ionization mode, MRM detection mode, precursor ion m / z 660.0, daughter ions m / z 570.2 and m / z 379.0, collision energy 25 eV, gas temperature 300 °C, dry gas nitrogen 5 L / min, nebulizer pressure 45 psi, sheath gas temperature 250 °C, sheath gas flow rate nitrogen 11 L / min, and capillary voltage 3500 V. Data analysis was performed using Agilent MassHunter workstation software. The retention times of (S)-sitalliptin-Marfey derivative and (R)-sitalliptin-Marfey derivative were 1.1 min and 1.6 min, respectively.

[0989] Example 6. Identification of Gen 1 transaminase (SEQ ID NO:13 / 14)

[0990] Following the procedures of Examples 1 and 2, *E. coli* expressing six wild-type transaminases (SEQ ID NO: 2, 4, 6, 8, 10, and 12) were lysed. The lysate was reacted with 1 g / L of precursor ketone substrate at room temperature and pH 8 using 1 M isopropylamine hydrochloric acid and 10% DMSO solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LC-MS-MS analysis was performed. No sitagliptin signal was detected, indicating no activity. Activity tests were conducted on variants under the same experimental conditions using site-saturated mutagenesis libraries and combinatorial libraries. LC-MS-MS results showed that some mutants of the wild-type transaminase (SEQ ID NO: 12) were active, and sitagliptin signals were detected. The results are shown in Table 1. Following the procedures of Examples 3 and 4, shake-flask powders (SFPs) of four active mutants (SEQ ID NO: 14, 16, 18, and 20) were prepared for activity testing. The best-performing variant (SEQ ID NO: 14) was selected as the parent for the next round of testing. In Tables 1-24, “nt” represents the coding sequence of the corresponding variant, and “aa” represents the amino acid sequence of the corresponding variant.

[0991] Table 1

[0992] +: (R)-amine, sitagliptin, was detected on LC-MS / MS.

[0993] The transaminase variant shown in SEQ ID NO:14 (which encodes the nucleic acid sequence SEQ ID NO:13) is designated as transaminase “Gen 1” and used as the parent for the next round of testing.

[0994] Figure 1 shows the chromatograms and mass spectra of racemic standard solutions containing (S)-amine and (R)-amine, and the reaction solution of the first-generation mutant "Gen 1". The chromatograms and mass spectra of the standard solutions containing (S)-amine and (R)-amine show peaks at retention times of approximately 1.1 min and 1.6 min, respectively, while the chromatogram and mass spectra of the "Gen 1" reaction solution show a peak only at a retention time of approximately 1.6 min, indicating that only (R)-amine was detected in this reaction solution, and (S)-amine was not detected. As can be seen from Figure 1, the optical purity of the (R)-amine synthesized by the first-generation mutant "Gen 1" is >99.99%. Through multiple rounds of enzyme engineering evolution, transaminase variants with high optical purity and high activity can be obtained.

[0995] Example 7. Identification of Gen 2 transaminase (SEQ ID NO:21 / 22)

[0996] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 1 g / L of the precursor ketone substrate at 45°C and pH 9 using 1 M isopropylamine hydrochloric acid and 10% DMSO solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LC-MS / MS were measured. The results are shown in Table 2.

[0997] Table 2 +++: Relative activity greater than 2.5 compared to Gen 1 transaminase activity; ++: Relative activity between 2.0 and 2.5 compared to Gen 1 transaminase activity; +: Relative activity between 1.5 and 2.0 compared to Gen 1 transaminase activity.

[0998] The transaminase variant shown in SEQ ID NO:22 (which encodes the nucleic acid sequence SEQ ID NO:21) is designated as transaminase “Gen 2” and used as the parent for the next round of testing.

[0999] Example 8. Identification of Gen 3 transaminase (SEQ ID NO:45 / 46)

[1000] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 1 g / L of the precursor ketone substrate at 60°C and pH 9 using 1 M isopropylamine hydrochloric acid and 10% DMSO solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 3.

[1001] Table 3 +++++: Relative activity greater than 20 compared to Gen 2 transaminase activity; ++++: Relative activity between 15 and 20 compared to Gen 2 transaminase activity; +++: Relative activity between 10 and 15 compared to Gen 2 transaminase activity; ++: Relative activity between 5 and 10 compared to Gen 2 transaminase activity; +: Relative activity between 2 and 5 compared to Gen 2 transaminase activity.

[1002] The transaminase variant shown in SEQ ID NO:46 (which encodes the nucleic acid sequence SEQ ID NO:45) was designated as transaminase “Gen 3” and used as the parent for the next round of testing.

[1003] Example 9. Identification of Gen 4 transaminase (SEQ ID NO:129 / 130)

[1004] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 10 g / L of the precursor ketone substrate at 50°C and pH 9 using 2 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 4.

[1005] Table 4 +++: Relative activity greater than 3 compared to Gen 3 transaminase activity; ++: Relative activity between 2 and 3 compared to Gen 3 transaminase activity; +: Relative activity between 1.5 and 2 compared to Gen 3 transaminase activity.

[1006] The transaminase variant shown in SEQ ID NO:130 (which encodes the nucleic acid sequence SEQ ID NO:129) was designated as transaminase “Gen 4” and used as the parent for the next round of testing.

[1007] Example 10. Identification of the 5th generation transaminase “Gen 5” (SEQ ID NO:137 / 138)

[1008] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 10 g / L of the precursor ketone substrate at 50°C and pH 9 using 2M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 5.

[1009] Table 5 ++++: Relative activity greater than 15 compared to Gen 4 transaminase activity; +++: Relative activity between 10 and 15 compared to Gen 4 transaminase activity; ++: Relative activity between 5 and 10 compared to Gen 4 transaminase activity; +: Relative activity between 2 and 5 compared to Gen 4 transaminase activity.

[1010] The transaminase variant shown in SEQ ID NO:138 (which encodes the nucleic acid sequence SEQ ID NO:137) was designated as transaminase “Gen 5” and used as the parent for the next round of testing.

[1011] Example 11. Identification of Gen 6 transaminase (SEQ ID NO: 195 / 196)

[1012] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 10 g / L of the precursor ketone substrate at 50°C and pH 9 using 2 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 6.

[1013] Table 6 +++++: Relative activity greater than 20 compared to Gen 5 transaminase activity; ++++: Relative activity between 15 and 20 compared to Gen 5 transaminase activity; +++: Relative activity between 10 and 15 compared to Gen 5 transaminase activity; ++: Relative activity between 5 and 10 compared to Gen 5 transaminase activity; +: Relative activity between 2 and 5 compared to Gen 5 transaminase activity.

[1014] The transaminase variant shown in SEQ ID NO:196 (which encodes the nucleic acid sequence SEQ ID NO:195) was designated as transaminase “Gen 6” and used as the parent for the next round of testing.

[1015] Example 12. Identification of the 7th generation transaminase “Gen 7” (SEQ ID NO:295 / 296)

[1016] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 10 g / L of the precursor ketone substrate at 50°C and pH 9 using 2 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMSMS were measured. The results are shown in Table 7.

[1017] Table 7 ++: Relative activity greater than 1.5 compared to Gen 6 transaminase activity; +: Relative activity less than 1.5 compared to Gen 6 transaminase activity.

[1018] The transaminase variant shown in SEQ ID NO:296 (which encodes the nucleic acid sequence SEQ ID NO:295) is designated as transaminase “Gen 7” and used as the parent for the next round of testing.

[1019] Example 13. Identification of the 8th generation transaminase “Gen 8” (SEQ ID NO:315 / 316)

[1020] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 50°C and pH 9 using 2M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 8.

[1021] Table 8 ++: Relative activity greater than 2 compared to Gen 7 transaminase activity; +: Relative activity between 1.5 and 2 compared to Gen 7 transaminase activity.

[1022] The transaminase variant shown in SEQ ID NO:316 (which encodes the nucleic acid sequence SEQ ID NO:315) is designated as transaminase “Gen 8” and used as the parent for the next round of testing.

[1023] Example 14. Identification of the 9th generation transaminase “Gen 9” (SEQ ID NO:379 / 380)

[1024] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 50 °C and pH 9 using 2 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 9.

[1025] Table 9 ++: Relative activity greater than 2 compared to Gen 8 transaminase activity; +: Relative activity between 1.5 and 2 compared to Gen 8 transaminase activity.

[1026] Note: "L228L" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 228.

[1027] The transaminase variant shown in SEQ ID NO:380 (which encodes the nucleic acid sequence SEQ ID NO:379) is designated as transaminase “Gen 9” and used as the parent for the next round of testing.

[1028] Example 15. Identification of the 10th generation transaminase “Gen 10” (SEQ ID NO:399 / 400)

[1029] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 50°C and pH 9 using 2 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 10.

[1030] Table 10 *: A stop codon is present; the amino acid sequence is truncated when the stop codon first appears. +: The relative activity is greater than 1 compared to the activity of transaminase Gen 9.

[1031] The transaminase variant shown in SEQ ID NO:400 (which encodes the nucleic acid sequence SEQ ID NO:399) is designated as transaminase “Gen 10” and used as the parent for the next round of testing.

[1032] Example 16. Identification of the 11th generation transaminase “Gen 11” (SEQ ID NO:423 / 424)

[1033] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 60°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 11.

[1034] Table 11 ++: Relative activity greater than 1.5 compared to the activity of transaminase Gen 10; +: Relative activity between 1.1 and 1.5 compared to the activity of transaminase Gen 10.

[1035] The transaminase variant shown in SEQ ID NO:424 (which encodes the nucleic acid sequence SEQ ID NO:423) is designated as transaminase “Gen 11” and used as the parent for the next round of testing.

[1036] Example 17. Identification of the 12th generation transaminase “Gen 12” (SEQ ID NO:489 / 490)

[1037] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 50 °C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 12.

[1038] Table 12 ++: Relative activity greater than 1.2 compared to the activity of transaminase Gen 11; +: Relative activity between 1.0 and 1.2 compared to the activity of transaminase Gen 11.

[1039] Note: "R129R" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 129.

[1040] The transaminase variant shown in SEQ ID NO:490 (which encodes the nucleic acid sequence SEQ ID NO:489) is designated as transaminase “Gen 12” and used as the parent for the next round of testing.

[1041] Example 18. Identification of the 13th generation transaminase “Gen 13” (SEQ ID NO:519 / 520)

[1042] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 60°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 13.

[1043] Table 13 ++: Relative activity greater than 1.5 compared to the activity of transaminase Gen 12; +: Relative activity between 1.0 and 1.5 compared to the activity of transaminase Gen 12.

[1044] The transaminase variant shown in SEQ ID NO:520 (which encodes the nucleic acid sequence SEQ ID NO:519) is designated as transaminase “Gen 13” and used as the parent for the next round of testing.

[1045] Example 19. Identification of the 14th generation transaminase “Gen 14” (SEQ ID NO:609 / 610)

[1046] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 45°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 14.

[1047] Table 14 +: The relative activity is greater than 1.0 compared to the activity of transaminase Gen 13.

[1048] Note: "G31G" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 31.

[1049] The transaminase variant shown in SEQ ID NO:610 (which encodes the nucleic acid sequence SEQ ID NO:609) is designated as transaminase “Gen 14” and used as the parent for the next round of testing.

[1050] Figure 2 shows the relative activities of the 10th to 14th generation mutants "Gen 10-Gen 14" relative to the first generation mutant "Gen 1". As can be seen from Figure 2, Gen 10, Gen 11, Gen 12, Gen 13 and Gen 14 underwent successive rounds of evolution, with each round screening out transaminase variants with significantly increased transaminase activity compared to the previous round.

[1051] Example 20. Identification of the 15th generation transaminase “Gen 15” (SEQ ID NO:631 / 632)

[1052] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 60°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 15.

[1053] Table 15 ++: Relative activity greater than 1.2 compared to the activity of transaminase Gen 14; +: Relative activity between 1.0 and 1.2 compared to the activity of transaminase Gen 14.

[1054] Note: "E220E" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 220; "V229V" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 229.

[1055] The transaminase variant shown in SEQ ID NO:632 (which encodes the nucleic acid sequence SEQ ID NO:631) is designated as transaminase “Gen 15” (generation 15) and used as the parent for the next round of testing.

[1056] Example 21. Identification of the 16th generation transaminase “Gen 16” (SEQ ID NO:743 / 744)

[1057] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 50 g / L of the precursor ketone substrate at 60°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 16.

[1058] Table 16 ++: Relative activity greater than 2 compared to the activity of transaminase Gen 15; +: Relative activity between 1 and 2 compared to the activity of transaminase Gen 15.

[1059] The transaminase variant shown in SEQ ID NO:744 (which encodes the nucleic acid sequence SEQ ID NO:743) is designated as transaminase “Gen 16” and used as the parent for the next round of testing.

[1060] Example 22. Identification of the 17th generation transaminase “Gen 17” (SEQ ID NO:767 / 768)

[1061] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45 °C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 17.

[1062] Table 17 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 16.

[1063] The transaminase variant shown in SEQ ID NO:768 (which encodes the nucleic acid sequence SEQ ID NO:767) is designated as transaminase “Gen 17” and used as the parent for the next round of testing.

[1064] Example 23. Identification of the 18th generation transaminase “Gen 18” (SEQ ID NO:805 / 806)

[1065] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 60 °C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 18.

[1066] Table 18 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 17.

[1067] The transaminase variant shown in SEQ ID NO:806 (which encodes the nucleic acid sequence SEQ ID NO:805) is designated as transaminase “Gen 18” and used as the parent for the next round of testing.

[1068] Example 24. Identification of the 19th generation transaminase “Gen 19” (SEQ ID NO:839 / 840)

[1069] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45 °C and pH 8.5 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 19.

[1070] Table 19 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 18.

[1071] The transaminase variant shown in SEQ ID NO:840 (which encodes the nucleic acid sequence SEQ ID NO:839) is designated as transaminase “Gen 19” and used as the parent for the next round of testing.

[1072] Example 25. Identification of the 20th generation transaminase “Gen 20” (SEQ ID NO:851 / 852)

[1073] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45 °C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 20.

[1074] Table 20 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 19.

[1075] The transaminase variant shown in SEQ ID NO:852 (which encodes the nucleic acid sequence SEQ ID NO:851) is designated as transaminase “Gen 20” and used as the parent for the next round of testing.

[1076] Example 26. Identification of the 21st generation transaminase “Gen 21” (SEQ ID NO:859 / 860)

[1077] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 21.

[1078] Table 21 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 20.

[1079] The transaminase variant shown in SEQ ID NO:860 (which encodes the nucleic acid sequence SEQ ID NO:859) is designated as transaminase “Gen 21” and used as the parent for the next round of testing.

[1080] Example 27. Identification of the 22nd generation transaminase “Gen 22” (SEQ ID NO:865 / 866)

[1081] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 60 °C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 22.

[1082] Table 22 +: The relative activity is between 1 and 2 compared to the activity of transaminase Gen 21.

[1083] The transaminase variant shown in SEQ ID NO:866 (which encodes the nucleic acid sequence SEQ ID NO:865) is designated as transaminase “Gen 22” and used as the parent for the next round of testing.

[1084] Example 28. Identification of the 23rd generation transaminase “Gen 23” (SEQ ID NO:877 / 878)

[1085] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 23.

[1086] Table 23 +: The relative activity is between 1 and 1.5 compared to the activity of transaminase Gen 22.

[1087] The transaminase variant shown in SEQ ID NO:878 (which encodes the nucleic acid sequence SEQ ID NO:877) is designated as transaminase “Gen 23” and used as the parent for the next round of testing.

[1088] Example 29. Further optimization of the 23rd generation transaminase "Gen 23" (SEQ ID NO:877 / 878)

[1089] Following the procedures of Examples 1 and 2, *E. coli* expressing the mutant enzyme were lysed. The lysate was reacted with 100 g / L of the precursor ketone substrate at 45°C and pH 9 using 1 M isopropylamine hydrochloric acid and 50% PEG200 solvent for 24 hours. The reaction mixture was quenched as described in Example 5, and LCMS / MS were measured. The results are shown in Table 24.

[1090] Table 24 +: The relative activity is between 1 and 1.5 compared to the activity of transaminase Gen 23.

[1091] Note: "I251I" indicates that the coding sequence of the corresponding transaminase variant has a synonymous mutation at the position encoding amino acid 251.

[1092] Figure 3 shows the relative activities of the 19th to 24th generation mutants "Gen 19-Gen 24" relative to the 18th generation mutant "Gen 18". As can be seen from Figure 3, Gen 19, Gen 20, Gen 21, Gen 22, Gen 23, and Gen 24 underwent successive rounds of evolution, with each round screening for transaminase variants with significantly increased transaminase activity compared to the previous round.

[1093] This application involved multiple rounds of screening, resulting in several transaminase variants with improved transaminase activity. These transaminase variants show great promise for the synthesis of drugs containing chiral amines.

Claims

1. A transaminase variant having improved transaminase activity compared to a wild-type transaminase having the amino acid sequence shown in SEQ ID NO:12, and the transaminase variant comprising an amino acid sequence having at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12, or ...0%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12, or comprising an amino acid sequence having at least about 80%, 82%, 85%, 87%, 90%, 90%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12, or comprising an amino acid sequence having at least about 80%, The amino acid sequence shown in NO:12, when truncated at any one of positions 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, and 357, has an N-terminal sequence that is at least about 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence.

2. The transaminase variant of claim 1, wherein the transaminase variant comprises one or more substitutions at the following positions corresponding to SEQ ID NO:12: D8, M13, A21, Q24, E36, M45, P47, A51, E58, M61, R62, L65, T66, V69, P70, S71, D81 , S85, L94, K97, L100, R102, E103, E104, K106, A114, K115, A121, F122, V123, E12 4. I126, V127, K132, R135, S137, I142, R145, L149, V150, Q151, V154, V156, P163 , D167, V169, G180, T185, L189, V195, R196, L198, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K257, G258, F 259, E260, V261, V263, A270, A271, H273, D275, M279, C280, T281, A283, M287, I2 89, R290, L292, G294, P296, N298, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355 or L356; Preferably, the transaminase variant comprises one or more of the following substitutions corresponding to SEQ ID NO:12: D8A、D8T、D8S、D8K、M13I、A21S、Q24R、Q24L、Q24K、Q24F、E36Q、M45I、M45L、M45F、P47A、P47L、P47R、A51S、E58M、E58S、E58Q、M61L、R62T、R62K、R62A、R62G、R62I、R62Y、L65A、T66C、V69G、V69A、P70F、P70A、P70V、P70I、S71H、S71G、D81N、D81G、D81H、S85H、S85N、L94V、K97S、L100H、L100K、R102V、E103K、E104M、K106R、A114V、A114T、A114E、A114I、A114K、K115G、K115M、A121V、F122M、F122S、F122A、F122G、F122C、F122H、F122N、F122T、V123C、E124L、E124M、I126M、V127I、K132V、K132M、K132I、K132Q、R135A、R135Q、S137V、I142G、I142D、R145K、L149F、L149S、V150T、Q151C、V154Q、V154I、V156L、P163Y、P163K、P163R、D167S、V169L、V169I、G180C、G180L、T185K、L189K、L189F、V195N、R196N、L198I、S202K、T207R、G214C、G214Y、D215V、D215W、D215G、D215E、A216F、A216C、A216T、A216G、A216Q、N217H、S222P、I226V、V227F、V227L、L228T、D231G、Y235H、D238V、D238S、D238I、D238H、Q243E、Q243P、Q243R、K248E、K248D、N255C、N255R、N255L、K257A、G258K、F259H、F259I、E260K、V261A、V263L、A270T、A270C、A271C、H273N、H273T、D275E、D275N、M279C、C280S、T281S、A283G、M287C、I289V、R290T、R290S、L292M、G294F、P296T、P296L、N298G、K301A、K301N、P304L、E315D、H317V、A321K、Y322V、E325A、Y328P、A331K、L336A、Y339L、Y339I, F341I, Q342F, K344F, K344V, K344D, A346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S, or L356S.

3. The transaminase variant of claim 1 or 2, wherein the transaminase variant comprises one or more substitutions at the following positions corresponding to SEQ ID NO:12: D8, M10, A11, T12, M13, A21, Q24, E36, M45, P47, E50, A51, E58, M61, R62, L65, T66, V69, P7 0. S71, D81, S85, L94, K97, L100, R102, E103, E104, K106, K107, A114, K115, A121, F122, V1 23. E124, I126, V127, L131, K132, R135, S137, E141, I142, V143, R145, L149, V150, Q151, V 154, V156, E160, P163, D167, V169, G180, T185, L189, V195, R196, L198, F199, S202, T207, G214, D215, A216, N217, S222, I226, V227, L228, D231, Y235, D238, Q243, K248, N255, K25 7. G258, F259, E260, V261, V263, P267, A270, A271, H273, D275, F278, M279, C280, T281, A2 83, M287, I289, R290, L292, G294, P296, N298, D299, K301, P304, E315, H317, A321, Y322, E325, Y328, A331, L336, Y339, F341, Q342, K344, A346, A348, E349, L351, Q354, G355 or L356; Preferably, the transaminase variant comprises one or more of the following substitutions corresponding to SEQ ID NO:12: D8A、D8T、D8S、D8K、D8N、M10R、A11S、A11T、T12S、M13I、A21S、A21Y、Q24R、Q24L、Q24K、Q24F、E36Q、M45I、M45L、M45F、P47A、P47L、P47R、E50L、A51S、E58M、E58S、E58Q、M61L、R62T、R62K、R62A、R62G、R62I、R62Y、L65A、T66C、V69G、V69A、P70F、P70A、P70V、P70I、S71H、S71G、D81N、D81G、D81H、S85H、S85N、L94V、L94I、K97S、L100H、L100K、R102V、E103K、E104M、K106R、K107Q、A114V、A114T、A114E、A114I、A114K、K115G、K115M、A121V、F122M、F122S、F122A、F122G、F122C、F122H、F122N、F122T、V123C、E124L、E124M、I126M、V127I、L131Q、K132V、K132M、K132I、K132Q、K132S、R135A、R135Q、S137V、E141T、I142G、I142D、V143N、V143F、R145K、L149F、L149S、V150T、Q151C、V154Q、V154I、V156L、E160H、P163Y、P163K、P163R、D167S、V169L、V169I、G180C、G180L、T185K、L189K、L189F、V195N、R196N、L198I、F199H、S202K、T207R、G214C、G214Y、D215V、D215W、D215G、D215E、A216F、A216C、A216T、A216G、A216Q、A216N、N217H、S222P、I226V、V227F、V227L、L228T、D231G、Y235H、D238V、D238S、D238I、D238H、Q243E、Q243P、Q243R、K248E、K248D、N255C、N255R、N255L、N255V、N255R、K257A、G258K、F259H、F259I、F259Y、F259L、E260K、E260D、V261A、V263L、V263M、P267S、A270T、A270C、A271C、H273N、H273T、D275E、D275N、F278L、M279C、C280S, T281S, A283G, M287C, I289V, R290T, R290S, L292M, G294F, P296T, P296 L, P296S, N298G, D299L, K301A, K301N, P304L, E315D, H317V, A321K, Y322V, E32 5A, Y328P, A331K, L336A, Y339L, Y339I, F341I, Q342F, K344F, K344V, K344D, A 346T, A348T, A348S, E349P, E349I, E349T, L351P, Q354P, G355K, G355S or L356S. , 4. The transaminase variant of any one of claims 1-3, wherein the transaminase variant comprises, compared to SEQ ID NO:12, a combination of substitutions selected from: P47A+R62T+L65A+V69G+R135A+Q151C+V154Q+S222P+T281S+A283G; P47A+R62T+L65A+V69G+R135A+V154Q+S222P+T281S+A283G; P47A+R62T+V69G+R135A+V154Q+S222P+T281S+A283G; Preferably, the transaminase variant comprises the following substitution combinations compared to SEQ ID NO:12: P47A+R62T+L65A+V69G+R135A+Q151C+V154Q+S222P+T281S+A283G.

5. The transaminase variant of claim 4, wherein the transaminase variant further comprises one or more substitutions selected from: A47K; D8A; A47L; A47R; T62K; D8T; M45I; M45L; L149F; D8S; D8K; T207R; Preferably, the transaminase variant further comprises an A47K substitution.

6. The transaminase variant of claim 5, wherein the transaminase variant further comprises a combination of substitutions selected from: D8S+K47L+P70F+S71H+F122M+E124L+L149F+G214C+Q243E; M45I+K47R+T62K+P70A+F122M+E124L+L149F+T207R+Q243E; D8T+M45L+K47R+T62K+P70F+F122M+E124L+L149F+T207R; D8A+M45L+K47R+T62K+P70F+F122M+E124L+L149F+Q243E; D8T+K47R+T62K+P70F+F122M+E124L+L149F+T207R+Q243E; D8K+K47R+T62K+P70F+S71H+F122M+E124L+L149F; D8A+M45I+K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E; K47L+P70F+F122M+E124L+V195N+G214C+Q243E; K47R+T62K+P70F+S71H+F122M+E124L+L149F+T207R+Q243E; D8K+M45I+K47R+T62K+P70F+F122M+E124L+T207R+Q243E; D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+T207R+Q243E; D8A+M45I+K47R+T62K+F122M+E124L+L149F+T207R; D8K+M45I+T62K+P70F+F122M+E124L+T207R+Q243E; D8A+M45L+K47R+T62K+P70F+F122M+E124L+T207R; D8K+M45L+K47R+P70F+F122M+E124L+L149F+T207R; D8T+K47R+T62K+P70F+S71H+F122M+E124L+T207R; D8K+M45L+K47R+T62K+P70F+S71H+F122M+E124L+Q243E; D8T+K47L+P70F+S71H+F122M+E124L+L149F+T207R; D8A+K47R+P70F+S71H+F122M+E124L+T207R+Q243E; D8A+P70F+S71H+F122M+E124L+T207R+Q243E; D8A+P70F+F122M+E124L; D8S+M45L+T62K+L149F+T207R; D8A+M45L+K47L+T62K+L149F+T207R+E315D; M45I+K47L+T62K+L149F+T207R; M45I+K47R+T62K+L149F+T207R; D8T+M45I+K47L+T62K+L149F+T207R; D8S+M45L+K47L+T62K+D81N+L149F; D8K+M45L+K47R+T62K+P70F+F122M+L149F+Q243E; D8A+M45I+T62K+L149F+T207R+Q243E; D8S+M45I+T62K+L149F+T207R; D8A+M45I+K47L+T62K+L149F; D8A+M45I+K47R+P70F+F122M+E124L+V195N+T207R+Q243E; D8A+K47L+T62K+L149F; D8T+K47L+T62K+L149F+T207R; D8S+K47R+P70F+F122M+E124L+V195N+T207R; D8A+M45L+K47L+T62K+L149F+T207R; D8A+M45L+K47L+T62K+L149F+T207R+Q243E; M45L+K47R+T62K+L149F+T207R; D8T+M45L+T62K+L149F+T207R; Preferably, the transaminase variant further comprises the following substitution combinations: D8S+K47L+P70F+S71H+F122M+E124L+L149F+G214C+Q243E.

7. The transaminase variant of claim 6, wherein the transaminase variant further comprises a combination of substitutions selected from: S8A+M45L+T62K+F70A+D81N+T207R; S8K+M45L+L47R+D81G+L124M; S8T+A21S+Q24R+M45I+F70A+D81G+E315D+Q354P; S8T+M45L+T62K+F70A+D81N; S8T+Q24R+T62K+F70A+D81G; S8A+Q24R+D81G+L124M+Q354P; S8T+A21S+F70A+D81G+Q354P; Preferably, the transaminase variant further comprises the following substitution combinations: S8T+M45L+T62K+F70A+D81N.

8. The transaminase variant of claim 7, wherein the transaminase variant further comprises a substitution selected from any one of the following sites: M122S; M122A; M122G; M122C; K62A; M122H; R290T; V156L; R290S; C214Y; K62G; K62I; M122N; K62Y; K248E; A65L; E243P; V150T; K248D; V261A; Preferably, the transaminase variant further comprises an M122S substitution.

9. The transaminase variant of claim 8, wherein the transaminase variant further comprises a combination of substitutions selected from: K62A+A65L+L124M+V150T+C214Y+K248E; Q24L+K62A+P163Y+C214Y+A216F+K248D+K301A; Q24L+K62A+A121V+L124M+V156L+P163Y+C214Y+A216F+K248D+R290T+K301A; A65L+L124M+V156L+C214Y+E243R+K248D+K301A; K62A+A65L+V150T+C214Y+A216F+K248E; Q24L+K62A+A65L+L124M+C214Y+A216F+K248D+K301A; A65L+L124M+V150T+V156L+C214Y+A216F+K248D+R290T; Q24L+K62A+V156L+P163Y+C214Y+E243R+L292M; L124M+V150T+V156L+C214Y+A216F+K248E+R290T; V156L+P163Y+C214Y+K248E+R290T+K301A; K62I+V150T+V156L+C214Y+A216F+K248E+R290T; Q24L+A65L+L124M+V150T+C214Y+A216F+K248E+R290T+K301A; Q24L+K62I+L124M+C214Y+K248D+K301A; Q24L+K62A+L124M+K248E+R290T; A65L+V156L+K248E; A65L+L124M+V156L; K62A+L124M+V156L+K248E+R290T; Q24L+L124M+V156L+K248E; Q24L+A65L+A121V+L124M+V150T+V156L+C214Y+A216F+E243R; Q24L+K62A+S122A+K248E+R290T; A65L+L124M+K248E; V156L+K248E; Q24L+K62A+A65L+S122T+V156L+K248E; K62A+A65L+K248E+R290T; K62I+V150T+K248E+K301A; Q24L+S122G+L124M+V156L+K248E; A121V+V150T+V156L+C214Y+K248D+K301A; K62A+A65L+S122G+V156L+K248E+R290T; K62I+S122G+L124M+V156L+K248E; K62I+K248E; Q24L+K62I+V150T+P163Y+A216F+E243R+K248E+K301A; A65L+S122G+L124M+V156L+K248E; A65L+V150T+A216F+K248E; K62A+S122T+V156L+K248E; Q24L+A121V+V150T+V156L+C214Y+A216F+E243R+K248D+R290T+K301A; K62A+K248E; Preferably, the transaminase variant comprises the following substitution combinations: V156L+P163Y+C214Y+K248E+R290T+K301A.

10. The transaminase variant of claim 9, wherein the transaminase variant further comprises a selection of substitutions or combinations thereof: A65L+L124M+V150T+E248D; K62A+L124M+A216F; V150T; K62A+L124M+E248D; Q24L+K62A+L124M; L124M; V150T+E248D; A65L+L124M; L124M+A216F+E243R; K62I+L124M; Q24L+A216F; A65L+L156V+Y163P+A216F; K62I+S122T+L124M+V150T+A216F+A301K; Q24L+K62I+S122T+L124M+V150T; Q24L+K62I+S122T+A216F+E248D; Q24L+K62I+S122T+V150T+A216F; K62A+A65L+A216F+E248D; K62I+S122T+L124M+A301K; A65L+A216F+E248D; S122G+L124M+A216F+E243R; K62I+S122T+L124M+A216F; Q24L+K62I+S122T+L124M+A301K; Q24L+K62I+S122T+L124M; Preferably, the transaminase variant further comprises the following substitution combinations: Q24L+K62I+S122T+L124M.

11. The transaminase variant of claim 10, wherein the transaminase variant further comprises substitutions selected from any one or more of the following: A348T; K344F; G355K; E349P; L356S; K344V; L336A; Q342F; E349I; E58M; A348S; Y339L; L189K; F3 41I; Y339I; L351P; A346T; K344D; H273N; E349T; K115G; A70V; G355S; A331K; Preferably, the transaminase variant further comprises an E58M substitution.

12. The transaminase variant of claim 11, wherein the transaminase variant further comprises a combination of substitutions selected from: A70V+A114V+K115G+G180C+H273T; A70V+K115G+G180C+H273T; A70V+A114V+Q154V+G180C+D275E; A70V+A114V+Q154V+G180C; A114V+Q154V+G180C; A70V+G180C+H273T; A114V+G180C+H273T+D275E; A70V+K115G+Q154V+G180C+H273T; K115G+G180C+H273T+D275E; K115G+G180C+H273T; K115G+G180C+L189K+H273N+C280S; K115G+G180C; A114V+K115G+G180C+L189K; A70V+K115G+H273T; A70V+A114V+L189K+H273T+C280S; Q154V+G180C+L189K+H273T+D275E; A70I+A114T+Q154V+G180C+L189K+H273T; A70V+A114V+K115G+Q154V+G180C+H273T+D275E; A114V+K115G+G180C; A70V+K115G; G180C+D275E+C280S; A70V+K115G+F149S+Q154V+L189K+H273T; A70V+A114V+G180C+L189K+H273T+D275N+C280S; Preferably, the transaminase variant further comprises the following substitution combinations: A70V+A114V+L189K+H273T+C280S.

13. The transaminase variant of claim 12, wherein the transaminase variant further comprises a mutation or combination of mutations selected from: A331*； E332*； G333*； K334*； P335*； L94V; L228T; D275E+P335*; G180C+D275E+E332*; G180C+D275E+P335*; F149S+A331*; F149S+E332*; D275E+A331*; D275E+E332*; D275E+P335*; D238V; D238S; E104M; V263L; A321K; Preferably, the transaminase variant further comprises the following mutations: E332*。 14. The transaminase variant of claim 13, wherein the transaminase variant further comprises one or more substitutions selected from: G69A; N255C; A216C; P296T; D215V; K132V; T66C; F259H; A216T; D215W; Preferably, the transaminase variant further comprises a G69A substitution.

15. The transaminase variant of claim 14, wherein the transaminase variant further comprises a combination of substitutions selected from: L24K+L47P+R102V+K132M+A135Q+D167S+K189F; L94V+D238V+L292M; L94V+D238S+V261A+A321K; L94V+D238V+A321K; L94V+L292M; L94V+D238S+V261A+E315D; L94V+D238V; L47P+K132M+A135Q+Y163K+K189F+V227F; K189F+S280C; L94V+D238S+V263L; L94V+V261A+V263L+L292M; L94V+D275N+L292M; L47P+K132M+A135Q+Y163R+D167S+K189F+V227F; L94V+D238V+V263L+L292M; L47P+R102V+Y163K+K189F; L94V+D238V+V261A; L94V+D238S+V261A+V263L+L292M; L94V+V263L; L94V+D238S+D275N+L292M+A321K; L94V+D238S+A321K; L94V+D238S+L292M+A321K; D238V+V261A+V263L+L292M; L47P+K189F+A216G; L94V+D238S+V263L+D275N; L94V+V263L+A321K; L94V+V261A; L94V+D238V+V261A+V263L+L292M; L47P+Y163K+K189F+A216G+F259I+H317V; L94V+D238S+D275N; V114E+K189L+V227F+N298G; L47P+R196N; R102V+A135Q+M279C+N298G; M13I+E36Q+L45F+S85H+T122M+A135Q+D167S+S202K+A271C+M287C; R102V+D167S+M279C+N298G; E36Q+S85H+R102V+K106R+A135Q+D167S+N217H+A270T+M279C; L47P+N255R; L47P+R196N+A216G+N298G; Preferably, the transaminase variant further comprises the following substitution combinations: L47P+K189F+A216G.

16. The transaminase variant of claim 15, wherein the transaminase variant further comprises a combination of substitutions selected from: M58S+L94V+A135Q+A270T; M58S+L94V+R102V+K132I+L198I+A270T+M279C; L94V+K132M+A135Q+L198I+I226V; L94V+K132M+I226V+A270T; L94V+R102V+A135Q+I226V+A270T; M58S+L94V+R102V+K132M+I226V+A270T; L94V+K132M+A135Q+M279C; L94V+R102V+K132M+A135Q+M279C; L94V+R102V+A270T; M58S+L94V+R102V+A270T; L94V+K132I+L198I+A270T; L94V+R102V+A135Q+L198I+I226V; M58S+L94V+K132M+A135Q+L198I+I226V+M279C; L94V+I126M+K132M+A135Q+N298G; L94V+R102V+L198I+A270T; M58S+L94V+K132I+A135Q+I226V; M58S+L94V+R102V+K132I+A135Q+L198I+I226V+A270T; L94V+I226V; M58S+L94V+A135Q+L198I; L94V+V123C+V127I+V261A+G294F; L94V+V169L+I226V+N255C+V261A+P296T; L94V+V123C+V127I+I226V+N255C+G294F+P296T+N298G; L94V+V169L+T185K+V227L+N255C+N298G; L94V+V123C+V127I+V150T+V169L+N255C; L94V+V123C+V127I+V150T+V169I+N255C+V261A+G294F+P296T+N298G; L94V+V123C+V127I+I226V+G294F+P296T+N298G; L94V+V123C+V127I+V169L+V261A+G294F; L94V+V123C+V127I+V261A+P296T; L94V+V150T+I226V+N255C+V261A+G294F; L94V+V123C+V127I+V169I+V227L+V261A+G294F; Preferably, the transaminase variant further comprises the following substitution combinations: M58S+L94V+A135Q+A270T.

17. The transaminase variant of claim 16, further comprising a selection of substitutions or combinations thereof: L24K+R102V+E315D; V263L; K132M+E315D; L24K+R102V+I226V+D238S+V261A+V263L+P296T+N298G+E315D; R102V; L24K+K132I; L24K+R102V+K132M+E315D; L24K+D238V+N298G; L24K+I226V+D238V+V261A+V263L+M279C+L292M+N298G; K132V; R102V+K132V+L292M+E315D; I226V+V227L+V261A+V263L+M279C+N298G+E315D; K132M; E103K; L24K+R102V+K132V; R102V+V227L+N255C+A321K; R102V+V227L+E260K+V261A+V263L; L24K+V169L+D238V+V261A+V263L+M279C+E315D; R102V+K132V+V227L+A321K; K132V+V169L; V227L+D238V+V263L+M279C+N298G+E315D; V227L+D238S+V263L+M279C+P296T+E315D; R102V+K132V+A321K; Preferably, the transaminase variant further comprises the following substitution combinations: R102V+V227L+N255C+A321K.

18. The transaminase variant of claim 17, further comprising a selection of substitutions or combinations thereof: K132V+G180C+D238V+V263L; K132V+V169I+D215G+D238V; K132V+V263L+P296L; M61L; K132V+V263L+L292M; D215V+D238V; V123C+K132V+L292M; S137V; D215E; L24F; E104M+K132V+V169I+V263L+L292M+P296T; D231G; K97S; K115M; I142G; K132V+D238V+V263L+L292M+P296T; I142D; C255L; H71G; A301N; E325A; V114I; N81H; S85N; K106R; Y328P; Preferably, the transaminase variant contains an M61L substitution.

19. The transaminase variant of claim 18, further comprising a combination of substitutions selected from: K132Q+K257A; L24K+K132Q+S137V+D231G+D238V; L24F+S137V+C255L+P304L; L24F+K132Q+S137V+D231G+C255L+K257A; K132Q+S137V+D238V+C255L+K257A+V261A+V263L+L292M+N298G; L24F+K132Q+C255L+K257A+T270C+P304L; L24F+S137V+K257A; L24K+K132Q+S137V+D238V+C255L+V263L+T270C+N298G; L24F+S137V+D231G+K257A+T270C+298G; N81H+S137V+C255L+T270C; L24F+K132Q+S137V+I226V+D231G+D238V+C255L+V263L+N298G; L24K+S137V+D238V+C255L+K257A+V263L+T270C+N298G; S137V+C255L; L24F+C255L+K257A; N81H+S137V+K257A+Y322V; N81H+K132Q+S137V+D231G+T270C; N81H+K132Q+S137V+I226V+C255L+K257A+V261A+L292M+N298G; K132Q+S137V+T270C+P304L; N81H+D238V+V261A+V263L+N298G; N81H+S137V+C255L+T270C; L24K+N81H+K132Q+D231G+D238V+C255L+V261A+V263L; L24F+N81H+K132Q+S137V+C255L+K257A+T270C; D231G+D238V+L292M+N298G; S137V+C255L+Y322V; L24F+N81H+T270C; L24F+N81H+S137V+T270C; N81H+K132Q+I226V+D238V+C255L+V261A+V263L+N298G; L24F+S137V+D231G+C255L+V261A+V263L+T270C; L24F+S137V+D231G+C255L+K257A+T270C; L24K+S137V+D231G+D238V+C255L+L292M+N298G; N81H+S137V+K257A; L24F+S137V+C255L+K257A; L24K+K132Q+S137V+D231G+D238V+C255L+V263L+N298G; D231G+K257A; S137V+C255L+T270C+P304L; N81H+C255L+K257A+T270C; S137V+C255L+K257A; L24F+T270C+P304L; Preferably, the transaminase variant further comprises the following substitution combinations: L24F+S137V+C255L+K257A.

20. The transaminase variant of claim 19, further comprising one or more substitutions selected from: V114K; N81D; A51S; D238I; G216Q; Y235H; L100H; G180L; G258K; I289V; D238H; R145K; Preferably, the transaminase variant further comprises a Y235H substitution.

21. The transaminase variant of claim 20, further comprising a selection of substitutions or combinations thereof: L100K+I289V; N81D+L100K+G180L+G216Q+D238H+I289V; R145K+G216Q; R145K+G180L+I289V; N81D+L100K+R145K+G216Q; N81D+L100K+D238H+G258K+I289V; N81D+V114K+R145K+D238H+I289V; N81D+G216Q+D238H+I289V; N81D+L100K+D238H+G258K; L100K+V114K+D238H+G258K; N81D; V114K+D238H+G258K+I289V; G180L+D238H+I289V; F24K+V150T+N298G; F24K+V261A+N298G; F24K+D167S+D238S+G294F+E315D; V114K+R145K+D167S+G294F+N298G; F24K+G180L+V261A+N298G; V102R+V114A+Q135R+Q154I; V102R+Q135R+Q154I; Q135R+Q154V; T8D+F24Q+V102R+V114A+Q154V; S58Q+V102R+Q154V; T8N+S58Q+Q135R+Q154V; Preferably, the transaminase variant further comprises the following substitution combinations: G180L+D238H+I289V.

22. The transaminase variant of claim 21, wherein the transaminase variant further comprises a substitution or combination of substitutions selected from: A257K; L255V; K132S; L131Q; F24L; T8D+F24L+V102R+V114A+Q135R+Q154V; Preferably, the transaminase variant further comprises the following substitution combination: T8D+F24L+V102R+V114A+Q135R+Q154V.

23. The transaminase variant of claim 22, wherein the transaminase variant further comprises a substitution or combination of substitutions selected from: M10R+A257K+F259Y; F259L; M10R+L255V+F259L+P267S; M10R+A257K; M10R+F259L; M10R; Preferably, the transaminase variant further comprises the following substitution: M10R.

24. The transaminase variant of claim 23, wherein the transaminase variant further comprises a combination of substitutions selected from: K107Q+F199H+E260D+A301K; F199H+E260D; K107Q+V143N+F199H+E260D+A301K; V143N+L255R; Preferably, the transaminase variant further comprises the following substitution combination: V143N+L255R.

25. The transaminase variant of claim 24, wherein the transaminase variant further comprises a substitution selected from: A21Y; F278L; D299L; Preferably, the transaminase variant further comprises the following substitution: D299L.

26. The transaminase variant of claim 25, wherein the transaminase variant further comprises a combination of substitutions selected from: A11S+E50L+E160H+V263M; A11S+A21Y+N143F; A11S+A21Y+E141T+V263M+F278L; A11S+E50L+E141T+E160H; A11S+E141T+N143F+V263M; T122M+L198I; Preferably, the transaminase variant further comprises the following substitution combination: T122M+L198I.

27. The transaminase variant of claim 26, wherein the transaminase variant further comprises a combination of substitutions selected from: V137S+E141T+N143F+H235Y+R255C+A257K+F259L+V263M; A11S+V137S+E141T+A257K; A11S+V137S+N143F+H235Y+R255C+A257K+V263M; A11S+R255C+A257K; E141T+R255C+F259L+V263M; V70A+H235Y+R255C+F259L; V137S+E141T+A257K; A11S+V137S+E141T+R255C+F259L+V263M; A11S+N81G+E141T+H235Y+A257K+V263M; A11S+N81G+V94I+V137S+E141T+A257K+V263M; E141T+N143F+G216N+H235Y+V263M; A11S+E141T+A257K+V263M+P296S; V94I+G216N+H235Y+A257K+V263M; A11S+V137S+N143F+H235Y+A257K; A11S+V137S; A11S+V137S+N143F+H235Y+A257K+V263M+P296S; A11S+E141T+G216N+V263M+P296S; V94I+V137S+E141T+N143F+G216N+H235Y+V263M; A11S+E141T+G216N+H235Y+V263M; A11S+G216N+V263M; N143F+G216N+A257K; A11S+N81G+E141T+V263M; A11S+N81G+V137S+N143F+V263M; V137S+E141T+H235Y+A257K+V263M; A11S+N81G+V137S+E141T+G216N+H235Y+A257K+V263M+P296S; A11S+N81G+V94I+V137S+G216N+H235Y+A257K; A11S+N81G+V94I+V137S+N143F+G216N+H235Y+V263M+P296S; A11T+N81G+V94I+A257K+V263M; G216N+H235Y+A257K+V263M; T12S+D167S+N217H.

28. The transaminase variant of any one of claims 1-27, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 ,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90 ,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122 ,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 27 4, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 3 94, 396, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、666、668、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938。、 29. A fusion protein comprising any one of the transaminase variants of claims 1-28.

30. A polynucleotide encoding a transaminase variant of any one of claims 1-28 or a fusion protein of claim 29; Preferably, the polynucleotide comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、223、225、227、229、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、261、263、265、267、269、271、273、275、277、279、281、283、285、287、289、291、293、295、297、299、301、303、305、307、309、311、313、315、317、319、321、323、325、327、329、331、333、335、337、339、341、343、345、347、349、351、353、355、357、359、361、363、365、367、369、371、373、375、377、379、381、383、385、387、389、391、393、395、399、401、403、405、407、409、411、413、415、417、419、421、423、425、427、429、431、433、435、437、439、441、443、445、447、449、451、453、455、457、459、461、463、465、467、469、471、473、475、477、479、481、483、485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、925、927、929、931、933、935、937。、 31. A nucleic acid construct comprising the polynucleotide of claim 30.

32. A vector comprising the polynucleotide of claim 30 or the nucleic acid construct of claim 31.

33. A host cell comprising the polynucleotide of claim 30, the nucleic acid construct of claim 31, or the vector of claim 32.

34. A composition comprising any one of the transaminase variants of claims 1-28, the fusion protein of claim 29, the polynucleotide of claim 30, the nucleic acid construct of claim 31, the vector of claim 32, and / or the host cell of claim 33.

35. Use of any transaminase variant of claims 1-28, the fusion protein of claim 29, the polynucleotide of claim 30, the nucleic acid construct of claim 31, the vector of claim 32, the host cell of claim 33, or the composition of claim 34 in the preparation of an amine; preferably, the amine is 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolone[4,3-a]pyrazine (sitagliptin, CAS 486460-32-6) or sitagliptin phosphate (CAS 654671-77-9).

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