Novel transaminase mutant and method for producing chiral amines

Abstract

The present application discloses a novel transaminase mutant and a method for producing chiral amines. The transaminase mutant is obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, and the transaminase mutant comprises mutations at one or more positions selected from the group consisting of positions 25, 78, 87, 94, 140, 145, 146, 147, 160, 178, 197, 225, 243, 246, 271, 296, 303, 307, 319, 327, 367, 381 and 444 corresponding to SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and specifically relates to enzymes, and more specifically to novel aminotransferase mutants and methods for producing chiral amines. Background Art

[0002] Chiral amines are a class of compounds with amino functional groups attached to the chiral center. They are an important class of pesticide and pharmaceutical intermediates. For example, the synthesis of neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. all use chiral amines as intermediates. However, the traditional chemical method for producing chiral amine compounds has a complex synthesis route, high cost, harsh reaction conditions, low yield and optical purity of the product, and is also prone to cause serious pollution to the environment. In recent years, transaminase enzymatic reactions have gradually begun to be used to produce chiral amines.

[0003] Transaminase, also known as aminotransferase, belongs to the transferase class. It is widely present in nature and plays an important role in amino transfer in the nitrogen metabolism of cells. It can catalyze the transfer of the amino group on the amino donor to the prochiral acceptor ketone to obtain chiral amine and byproduct ketone or α-keto acid. However, most wild-type transaminases have low activity and poor stability. They are easily inactivated by various factors during the catalytic reaction, which affects their industrial application. For example, due to the low enzyme activity, in order to achieve the catalytic effect, it is often necessary to increase the amount of enzyme added, which will increase the difficulty of post-processing; in addition, the wild-type enzyme has poor thermal stability, pH stability, and organic solvent tolerance. In the reaction, especially in the amplification process, once uneven heating, uneven stirring, and too fast addition when adjusting the pH, the local enzyme may be inactivated, thereby affecting the overall reaction effect.

[0004] There is still an urgent need in the art for a transaminase having high enzyme activity, high thermal stability, high pH stability and high organic solvent tolerance and a method for producing chiral amines therefrom. Summary of the invention

[0005] In order to solve the above technical problems, in one aspect, the present application provides a transaminase mutant, which is obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, and the transaminase mutant includes mutations at one or more positions selected from the following group: corresponding to position 25, position 78, position 87, position 94, position 140, position 145, position 146, position 147, position 160, position 178, position 197, position 225, position 243, position 246, position 271, position 296, position 303, position 307, position 319, position 327, position 367, position 381 and position 444 of SEQ ID NO: 1.

[0006] In another aspect, the present application provides a nucleic acid molecule encoding a transaminase mutant, wherein the transaminase mutant is obtained by mutation of the transaminase having an amino acid sequence as shown in SEQ ID NO: 1, and the transaminase mutant comprises a mutation at one or more positions selected from the group consisting of: position 25, position 78, position 87, position 94, position 140, position 145, position 146, position 147, position 160, position 178, position 197, position 225, position 243, position 246, position 271, position 296, position 303, position 307, position 319, position 327, position 367, position 381 and position 444 corresponding to SEQ ID NO: 1.

[0007] In another aspect, the present application provides an expression vector comprising a nucleic acid molecule encoding a transaminase mutant, wherein the transaminase mutant is obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, and the transaminase mutant comprises a mutation at one or more positions selected from the group consisting of: position 25, position 78, position 87, position 94, position 140, position 145, position 146, position 147, position 160, position 178, position 197, position 225, position 243, position 246, position 271, position 296, position 303, position 307, position 319, position 327, position 367, position 381 and position 444 corresponding to SEQ ID NO: 1.

[0008] In another aspect, the present application provides a host cell, comprising a nucleic acid molecule or an expression vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes a transaminase mutant, wherein the transaminase mutant is obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, and the transaminase mutant comprises a mutation at one or more positions selected from the group consisting of: position 25, position 78, position 87, position 94, position 140, position 145, position 146, position 147, position 160, position 178, position 197, position 225, position 243, position 246, position 271, position 296, position 303, position 307, position 319, position 327, position 367, position 381 and position 444 corresponding to SEQ ID NO: 1.

[0009] In another aspect, the present application provides a method for producing a chiral amine, the method comprising:

[0010] (a) obtaining a catalyst comprising a transaminase mutant, the transaminase mutant being obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, the transaminase mutant comprising a mutation at one or more positions selected from the group consisting of: position 25, position 78, position 87, position 94, position 140, position 145, position 146, position 147, position 160, position 178, position 197, position 225, position 243, position 246, position 271, position 296, position 303, position 307, position 319, position 327, position 367, position 381 and position 444 corresponding to SEQ ID NO: 1; and

[0011] (b) reacting a ketone compound with an amino donor in the presence of the catalyst to produce the chiral amine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present application is described in more detail below with reference to the accompanying drawings, in which:

[0013] Figure 1 A comparison of the temperature stability of the wild type and the S25W mutant;

[0014] Figure 2 is a comparison of the stability of the wild type and the S25W mutant at different pH levels;

[0015] Figure 3 A comparison of the tolerance of the wild type and the S25W mutant in different organic solvents;

[0016] Figure 4 Comparison of the tolerance of the wild type and S25W mutant in different DMSO volumes. DETAILED DESCRIPTION

[0017] The present application relates to a transaminase mutant, which is obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, wherein the transaminase mutant comprises mutations at one or more positions selected from the group consisting of: positions 25, 78, 87, 94, 140, 145, 146, 147, 160, 178, 197, 225, 243, 246, 271, 296, 303, 307, 319, 327, 367, 381 and 444 corresponding to SEQ ID NO: 1. In the present application, the thermotolerant Bacillus ( Bacillus thermotolerans) The amino acid sequence of the aminotransferase from the source is shown in SEQ ID NO: 1. In one embodiment, the aminotransferase mutant comprises a mutation at one or more positions selected from the group consisting of: corresponding to position 25, position 160, position 178, position 225, position 271, position 303, position 307, position 319, position 327, position 367 and position 444 of SEQ ID NO: 1. In one embodiment, the aminotransferase mutant comprises a mutation at one or more positions selected from the group consisting of: corresponding to position 25, position 178, position 225, position 303, position 307, position 319, position 367 and position 444 of SEQ ID NO: 1. In a preferred embodiment, the amino acid mutant comprises a mutation at one or more positions selected from the group consisting of: corresponding to position 25, position 178 and position 444 of SEQ ID NO: 1. In a preferred embodiment, the amino acid mutant comprises a mutation at position 25 of SEQ ID NO: 1. In a preferred embodiment, the amino acid mutant comprises a mutation at at least one of position 178 and position 25 and position 444. In one embodiment, the mutation at position 25 is a mutation of serine to any amino acid other than serine. In one embodiment, the mutation at position 25 is a mutation of serine to tryptophan. In one embodiment, the mutation at position 78 is a mutation of alanine to any amino acid other than alanine. In one embodiment, the mutation at position 78 is a mutation of alanine to glutamine. In one embodiment, the mutation at position 87 is a mutation of asparagine to any amino acid other than asparagine. In one embodiment, the mutation at position 87 is a mutation of asparagine to proline. In one embodiment, the mutation at position 94 is a mutation of lysine to any amino acid other than lysine. In one embodiment, the mutation at position 94 is a mutation of lysine to arginine. In one embodiment, the mutation at position 140 is a mutation of leucine to any amino acid other than leucine. In one embodiment, the mutation at position 140 is a mutation of leucine to lysine. In one embodiment, the mutation at position 145 is glutamic acid to any amino acid except glutamic acid. In one embodiment, the mutation at position 145 is glutamic acid to tyrosine. In one embodiment, the mutation at position 146 is histidine to any amino acid except histidine. In one embodiment, the mutation at position 146 is histidine to arginine. In one embodiment, the mutation at position 147 is glycine to any amino acid except glycine. In one embodiment, the mutation at position 147 is glycine to serine. In one embodiment, the mutation at position 160 is arginine to any amino acid except arginine. In one embodiment, the mutation at position 160 is arginine to glycine.In one embodiment, the mutation at position 178 is that leucine is mutated to any amino acid except leucine. In one embodiment, the mutation at position 178 is that leucine is mutated to alanine, cysteine, glutamine, histidine, asparagine, tyrosine, serine, methionine, lysine, valine, isoleucine, phenylalanine, threonine or arginine. In a preferred embodiment, the mutation at position 178 is that leucine is mutated to glutamine, tyrosine, methionine, lysine, valine, isoleucine, phenylalanine, threonine, arginine. In a preferred embodiment, the mutation at position 178 is that leucine is mutated to arginine or glutamine. In one embodiment, the mutation at position 197 is that phenylalanine is mutated to any amino acid except phenylalanine. In one embodiment, the mutation at position 197 is that phenylalanine is mutated to tyrosine. In one embodiment, the mutation at position 225 is that tyrosine is mutated to any amino acid except tyrosine. In one embodiment, the mutation at position 225 is that tyrosine is mutated to glutamic acid. In one embodiment, the mutation at position 243 is a mutation from serine to any amino acid except serine. In one embodiment, the mutation at position 243 is a mutation from serine to glycine. In one embodiment, the mutation at position 246 is a mutation from histidine to any amino acid except histidine. In one embodiment, the mutation at position 246 is a mutation from histidine to proline. In one embodiment, the mutation at position 271 is a mutation from leucine to any amino acid except leucine. In one embodiment, the mutation at position 271 is a mutation from leucine to methionine. In one embodiment, the mutation at position 296 is a mutation from glycine to any amino acid except glycine. In one embodiment, the mutation at position 296 is a mutation from glycine to alanine. In one embodiment, the mutation at position 303 is a mutation from serine to any amino acid except serine. In one embodiment, the mutation at position 303 is a mutation from serine to tyrosine. In one embodiment, the mutation at position 307 is a mutation from alanine to any amino acid except alanine. In one embodiment, the mutation at position 307 is a mutation from alanine to glycine. In one embodiment, the mutation at position 319 is methionine to any amino acid except methionine. In one embodiment, the mutation at position 319 is methionine to phenylalanine. In one embodiment, the mutation at position 327 is valine to any amino acid except valine. In one embodiment, the mutation at position 327 is valine to glycine. In one embodiment, the mutation at position 367 is phenylalanine to any amino acid except phenylalanine. In one embodiment, the mutation at position 367 is phenylalanine to leucine. In one embodiment, the mutation at position 381 is aspartic acid to any amino acid except aspartic acid.In one embodiment, the mutation at position 381 is aspartic acid to arginine. In one embodiment, the mutation at position 444 is alanine to any amino acid except alanine. In one embodiment, the mutation at position 444 is alanine to proline. In one embodiment, the amino acid sequence of the transaminase mutant comprises an amino acid sequence having more than 80% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57. SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83. SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 and SEQ ID NO: 93.In one embodiment, the transaminase mutant comprises an amino acid sequence having more than 85% homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 and SEQ ID NO: 93.In one embodiment, the transaminase mutant comprises an amino acid sequence having more than 90% homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 and SEQ ID NO: 93.In one embodiment, the transaminase mutant comprises an amino acid sequence having more than 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 and SEQ ID NO: 93.In one embodiment, the amino acid sequence of the present invention is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 and SEQ ID NO: 93.In one embodiment, the transaminase mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 31, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 33, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 35, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 NO: 93, SEQ ID NO: 37 and SEQ ID NO: 39. In a preferred embodiment, the aminotransferase mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29. In a more preferred embodiment, the aminotransferase mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 29. In a most preferred embodiment, the aminotransferase mutant comprises the amino acid sequence SEQ ID NO: 3. In one embodiment, the mutant has enhanced enzyme activity. In one embodiment, the mutant has an improved substrate conversion rate. In one embodiment, the mutant is tolerant to high temperatures. In one embodiment, the mutant is tolerant to temperatures of at least 50°C. In one embodiment, the mutant is tolerant to temperatures of at least 55°C. In one embodiment, the mutant has high pH stability. In one embodiment, the mutant is tolerant to at least pH 10.In one embodiment, the mutant tolerates at least pH 10.5. In one embodiment, the mutant has enhanced organic solvent tolerance or compatibility. In one embodiment, the organic solvent comprises one or more selected from the group consisting of n-heptane, toluene, 1,4-dioxane, acetonitrile, methanol, ethanol, isopropanol, dimethyl sulfoxide and N,N-dimethylformamide.

[0018] The present application also relates to a nucleic acid molecule encoding a transaminase mutant, which may be the transaminase mutant described in any of the aforementioned embodiments. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having more than 80% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60. SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86. SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 94.In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having more than 85% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 94.In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having more than 90% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60. SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92 and SEQ ID NO:94.In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having more than 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 94.In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having more than 99% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60. SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86. SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 94.In one embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62. SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92 and SEQ ID NO:94. In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30. In a more preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 30.In a most preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 4.

[0019] The present application also relates to an expression vector, wherein the vector comprises a nucleic acid molecule, which may be a nucleic acid molecule as described in any of the foregoing embodiments. In one embodiment, the expression vector comprises a recombinant expression vector. In one embodiment, the recombinant expression vector comprises a plasmid. In one embodiment, the plasmid vector comprises pET21a(+), pET28a(+), pET19b(+), pET30a(+), pRSFDuet -1 , pTrc99A, pPICZA or pPICZαA. In a preferred embodiment, the plasmid vector comprises pET21a(+).

[0020] The present application also relates to a host cell, the cell comprising a nucleic acid molecule or an expression vector, the nucleic acid molecule may be the nucleic acid molecule described in any of the foregoing embodiments, and the expression vector may be the expression vector described in any of the foregoing embodiments. In one embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell comprises a prokaryotic cell. In one embodiment, the prokaryotic cell comprises an Escherichia coli cell. In one embodiment, the Escherichia coli cell comprises a BL21 (DE3) cell or a Rosetta (DE3) cell. In a preferred embodiment, the Escherichia coli cell comprises a BL21 (DE3) cell. In one embodiment, the host cell comprises a eukaryotic cell. In one embodiment, the eukaryotic cell comprises yeast.

[0021] The present application also relates to a method for producing chiral amines, the method comprising: (a) obtaining a catalyst, the catalyst comprising a transaminase mutant, and the transaminase mutant may be the transaminase mutant described in any of the foregoing embodiments. In one embodiment, the catalyst comprises wet cells obtained by centrifugation after fermentation and culture of host cells, host cells immobilized with wet cells, transaminase mutants extracted after ultrasonic disruption of wet cells, or immobilized transaminase mutants, and the host cells may be the host cells described in any of the foregoing embodiments. In one embodiment, the catalyst comprises transaminase mutant powder. In one embodiment, the catalyst comprises freeze-dried crude enzyme powder.

[0022] The method for producing a chiral amine further comprises: (b) reacting a ketone compound with an amino donor in the presence of a catalyst to produce the chiral amine. In one embodiment, the ketone compound comprises , , or , wherein R is independently selected from Ph, F, Cl, Br, CH3, OCH3, NO2, CF3 and COOR'. In one embodiment, R' is selected from alkyl and aromatic groups. In a preferred embodiment, R' is selected from methyl, ethyl and benzyl. In a preferred embodiment, the ketone compound includes or . In one embodiment, the amino donor comprises alanine or isopropylamine (i-PrNH2). In a preferred embodiment, the amino donor comprises isopropylamine. In one embodiment, the reaction system of the reaction has a pH of 7-11. In a preferred embodiment, the pH is 8.5-10. In a preferred embodiment, the pH is about 10. In one embodiment, the reaction system of the reaction has a temperature of 30-60°C. In a preferred embodiment, the temperature is 45-55°C. In a preferred embodiment, the temperature is about 55°C. In one embodiment, the reaction system of the reaction comprises an organic solvent. In one embodiment, the organic solvent comprises one or more selected from the group consisting of n-heptane, toluene, 1,4-dioxane, acetonitrile, methanol, ethanol, isopropanol, dimethyl sulfoxide and N,N-dimethylformamide. In a preferred embodiment, the organic solvent comprises one or more selected from the group consisting of n-heptane, toluene, methanol and dimethyl sulfoxide. In a preferred embodiment, the organic solvent comprises toluene. In a preferred embodiment, the organic solvent comprises dimethyl sulfoxide. In one embodiment, the volume content of the organic solvent is 5-50%. In one embodiment, the volume content of the organic solvent is 10-30%. In one embodiment, in the reaction system of the reaction, the concentration of the ketone compound is 2-30 g / L. In one embodiment, in the reaction system of the reaction, the concentration of the ketone compound is 1-150 mM. In one embodiment, in the reaction system of the reaction, the concentration of the ketone compound is 5-100 mM. In one embodiment, in the reaction system of the reaction, the concentration of the ketone compound is about 10 mM. In one embodiment, in the reaction system of the reaction, the concentration of the amino donor is 5-50 g / L. In one embodiment, in the reaction system of the reaction, the concentration of the amino donor is 0.1-2 M. In one embodiment, in the reaction system of the reaction, the concentration of the amino donor is 0.2-1 M. In one embodiment, in the reaction system of the reaction, the concentration of the amino donor is about 0.5 M. In one embodiment, the reaction system of the reaction further includes a coenzyme. In one embodiment, the coenzyme includes pyridoxal phosphate (PLP). In one embodiment, in the reaction system of the reaction, the concentration of the coenzyme is 0.2-0.5 g / L. In one embodiment, in the reaction system of the reaction, the concentration of the coenzyme is 0.5-2 mM. In one embodiment, in the reaction system of the reaction, the concentration of the coenzyme is about 1 mM. In one embodiment, in the reaction system of the reaction, the amount of the catalyst is 1-5 g / L by weight of the lyophilized crude enzyme powder. In one embodiment, the catalyst is a lyophilized crude enzyme powder comprising protein and salt. In one embodiment, the protein in the lyophilized crude enzyme powder comprises about 70%-90% of transaminase. In one embodiment, the reaction time is 2-100 hours.In a preferred embodiment, the reaction time is 12-36 hours. In a preferred embodiment, the reaction time is about 24 hours. Example

[0023] The present application will be described in detail by the following exemplary specific examples. The following examples are only used to help those skilled in the art better understand the inventions of the present application. It should be noted that the spirit of the present application and the scope of protection of the claims are not limited by the following specific examples.

[0024] List of reagents and equipment used

[0025]

[0026] Example 1: Preparation of mutant library

[0027] 1. Establishment of wild-type transaminase genetic engineering bacteria

[0028] According to NCBI Bacillus thermotolerans The wild-type transaminase gene sequence (SequenceID: WP_046128564.1) was optimized and the whole gene fragment was artificially synthesized. The gene was inserted into the pET-21a(+) plasmid by a gene synthesis company. The vector carrying the target gene was transferred into Escherichia coli BL21(DE3) (Shanghai Biotechnology Co., Ltd.) to establish the transaminase gene engineering bacteria.

[0029] 2. Construction of transaminase mutant library

[0030] Using the plasmid carrying the wild-type transaminase gene as a template, the 25th, 78th, 87th, 94th, 140th, 145 / 146 / 147th, 160th, 178th, 197th, 225th, 243 / 246th, 271st, 296th, 303th, 307th, 319th, 327th, 367th, 381st, 444th positions in the wild-type amino acid sequence were subjected to site-directed mutagenesis by polymerase chain reaction (PCR), and the corresponding primers were designed and synthesized, as shown in the following table:

[0031]

[0032] At the same time, saturation mutation was performed on position 178 by designing saturation mutation primers using the degenerate codon NNK; positions 25, 178, and 444 were subjected to combination mutations in pairs or in triplicate, that is, the constructed single-point mutation plasmid was used as a template and the corresponding primers were used to complete the construction of the mutation plasmid of the combination site through PCR.

[0033] After the mutant plasmid was sequenced and verified to be correct, it was transformed into Escherichia coli BL21 (DE3) to obtain the transaminase mutant engineered bacteria.

[0034] 3. Preparation of crude enzyme (induced expression of each mutant)

[0035] The plasmids carrying the wild-type aminotransferase sequence and the mutant plasmids were transformed into E. coli BL21 (DE3) cells and overexpressed in E. coli. The induction conditions were: 0.1 mM IPTG, 25°C overnight induction (≥16 hours). The cells induced overnight were collected, resuspended in 100 mM PBS (pH7.5) buffer at a ratio of cells: buffer = 1:5, and then ultrasonically disrupted, the supernatant was collected, and the supernatant was vacuum freeze-dried to obtain freeze-dried crude enzyme powder, which was stored at -20°C.

[0036] Example 2: The mutant acquired the catalytic ability towards sterically hindered substrates.

[0037] Using 1-([1,1-biphenyl]-2-yl)ethanone (CAS: 2142-66-7) as a substrate, the mutant library obtained in Example 1 was screened for activity. Reaction conditions A: 10mM ketone substrate, 2.5 times the weight (wt) of the substrate enzyme (lyophilized crude enzyme powder in Example 1), 1mM PLP, 0.5M i-PrNH2, 10% DMSO, 0.1M PBS 8.5, 40°C, 24 hours. The peak areas of the product and the raw material were monitored by LCMS, and the enzyme activity was calculated according to the formula: enzyme activity is the product conversion rate = product peak area / (product + raw material peak area). The results show that the wild-type enzyme has poor catalytic activity for the substrate, while the mutant enzyme has good catalytic activity, and the mutant S25W can increase the product conversion rate from 2% to more than 40%. At the same time, the chiral selectivity of the product that the wild type can achieve is guaranteed (verified by the SFC method, 99% ee). This indicates that after the modification of the present invention, the aminotransferase mutant has gained catalytic activity for substrates with large steric hindrance, and has expanded the substrate spectrum. The reaction formula is as follows, and the results are shown in Table 1.

[0038]

[0039] Table 1

[0040]

[0041] Among them, - indicates a conversion rate of 0~2%, + indicates a conversion rate of 2~10%, ++ indicates a conversion rate of 10%~30%, +++ indicates a conversion rate of 30%~40%, and ++++ indicates a conversion rate >40%.

[0042] Enzyme activity is product conversion rate = product peak area / (product + raw material peak area).

[0043] The peak areas of the product and the raw material were measured by LCMS.

[0044] Example 3: The activity of the S25W mutant on various substrates was significantly improved.

[0045] The wild-type transaminase and the S25W mutant catalyze different types of ketone compounds to generate chiral amines, and the activity is tested under the combination conditions of B~G. The results show that the catalytic activity of the S25W mutant on various substrates is higher than that of the wild-type transaminase. After the modification of the present invention, the transaminase mutant S25W has improved catalytic activity. The reaction substrates and results are shown in Table 2.

[0046] Experimental condition B: 5 mM ketone substrate, 8 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 40°C, 24 h.

[0047] Experimental condition C: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 45°C, 24 h.

[0048] Experimental condition D: 20 mM ketone substrate, 1.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 40°C, 24 h.

[0049] Experimental condition E: 25 mM ketone substrate, 1.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 40°C, 24 h.

[0050] Experimental condition F: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 55°C, 24 h.

[0051] Experimental condition G: 150 mM ketone substrate, 0.25 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 40°C, 24 h.

[0052] Table 2

[0053]

[0054] Example 4: The high temperature stability of the mutant was significantly improved, and the activity did not change significantly at 55°C.

[0055] The wild-type transaminase and its S25W mutant were tested for activity under experimental condition H using 2,5-dimethoxyacetophenone (CAS: 1201-38-3) as substrate. The results showed that the activity of the wild-type enzyme was only about 15% at 55°C, while the activity of the S25W mutant enzyme did not change significantly at 55°C. After the modification of the present invention, the high temperature stability of the transaminase mutant was improved and it could remain stable at 55°C. The reaction formula is as follows, and the results are as follows: Figure 1 shown.

[0056] Experimental conditions H: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5, 40-60 °C, 24 h.

[0057]

[0058] Example 5: The mutant has an improved ability to tolerate alkaline pH.

[0059] The wild-type transaminase and its S25W mutant were tested under experimental condition I using 2,5-dimethoxyacetophenone (CAS: 1201-38-3) as substrate. The results showed that the activity of the wild-type enzyme began to decrease at pH 10, and the activity was less than 10% at pH 10.5; while the activity of the S25W mutant did not change significantly at pH 10, and still retained about 60% of its activity at pH 10.5. After the modification of the present invention, the pH stability of the transaminase mutant is improved, and a good catalytic effect can be maintained under the condition of pH 10-10.5. The reaction formula is as follows, and the results are as follows Figure 2 shown.

[0060]

[0061] Experimental condition I: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% DMSO, 0.1 M PBS pH 8.5~11, 45°C, 24 h.

[0062] Example 6: The mutant has improved ability to tolerate organic solvents.

[0063] The wild-type transaminase and its S25W mutant were tested for activity using 2,5-dimethoxyacetophenone (CAS: 1201-38-3) as a substrate under experimental conditions J and K. The results showed that when 1,4-dioxane was used as a cosolvent, the wild-type transaminase lost 80% of its activity, while the S25W mutant only lost about 10% of its activity. In the presence of 30% DMSO, the enzymatic activity of the wild-type transaminase was lost by more than 60%, while the enzymatic activity of the S25W mutant did not change significantly. After the modification of the present invention, the transaminase mutant has better tolerance to the type and concentration of organic solvents than the wild type. The reaction formula is as follows, and the results are as follows. Figure 3 and Figure 4 shown.

[0064]

[0065] Experimental conditions J: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10% different organic solvents, 0.1 M PBS pH 8.5, 45 °C, 24 h.

[0066] Experimental conditions K: 10 mM ketone substrate, 2.5 wt enzyme (lyophilized crude enzyme powder), 1 mM PLP, 0.5 M i-PrNH2, 10%~50% DMSO, 0.1 M PBS pH 8.5, 45°C, 24 h.

[0067] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the mutation of the present invention expands the substrate spectrum of the enzyme for large steric hindrance substrates and improves the catalytic ability of the enzyme; enhances the stability of the enzyme in temperature, pH and organic solvent, and can catalyze reactions under high temperature, high alkalinity and high concentration solvent conditions, thereby increasing the reaction rate, reducing the amount of enzyme used, and reducing the difficulty of post-processing in industrial applications. At the same time, its high stability ensures the stability of the mutant enzyme in the process and its industrial production application.

[0068] In summary, compared with the prior art, the present invention has the following advantages:

[0069] 1. The enzyme activity of the mutant of the present invention can be increased by up to 70% compared with the wild type, and the enzyme activity is enhanced for 7 test substrates;

[0070] 2. The present invention broadens the catalytic effect of the enzyme on large steric hindrance substrates. For example, the wild type previously had only a 2% conversion rate, but after mutation, the conversion rate is more than 50%;

[0071] 3. The mutant of the present invention can tolerate 55°C without a significant decrease in activity, while the wild type can only tolerate 45°C;

[0072] 4. The mutant of the present invention has almost no activity loss under alkaline conditions of pH 10, while the wild type can only reach pH 9.5;

[0073] 5. The mutant of the present invention has better tolerance and compatibility with organic solvents, and can tolerate 30% DMSO cosolvent without significant change in activity, while the activity of the wild type begins to decline at 20% DMSO;

[0074] 6. The mutant of the present invention is compatible with a variety of organic solvents such as n-heptane, toluene, 1,4-dioxane, methanol, etc. as reaction co-solvents, while the wild type cannot use 1,4-dioxane as a reaction co-solvent;

[0075] 7. The properties of the mutant enzyme of the present invention are better than those of the wild type, and still retain a high degree (>99% ee) of configuration selectivity, which is very beneficial for industrial production and application.

Claims

1. A transaminase mutant, the transaminase mutant being obtained by mutation of a transaminase having an amino acid sequence as shown in SEQ ID NO: 1, the transaminase mutant comprising mutations at one or more positions selected from the group consisting of: positions 25, 178 and 444 corresponding to SEQ ID NO: 1, and the amino acid sequence of the transaminase mutant being selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO:

89.

2. The transaminase mutant according to claim 1, wherein the amino acid sequence of the transaminase mutant is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO:

29.

3. The transaminase mutant according to claim 1, wherein the amino acid sequence of the transaminase mutant is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 25 and SEQ ID NO:

29.

4. The transaminase mutant according to claim 1, wherein the amino acid sequence of the transaminase mutant is SEQ ID NO:

3.

5. A nucleic acid molecule encoding the transaminase mutant according to any one of claims 1 to 4. An expression vector comprising the nucleic acid molecule according to claim 5 .

7. A host cell, comprising the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. A method for producing a chiral amine, the method comprising: (a) obtaining a catalyst, wherein the catalyst comprises the aminotransferase mutant according to any one of claims 1 to 4; and (b) reacting a ketone compound with an amino donor in the presence of the catalyst to produce the chiral amine.

9. The method of claim 8, wherein the ketone compound is selected from , , or , wherein R is independently selected from Ph, F, Cl, Br, CH3, OCH3, NO2, CF3 and COOR', and R' is selected from alkyl and aromatic groups.

10. The method of claim 9, wherein R' is selected from the group consisting of methyl, ethyl and benzyl.

11. The method of claim 9, wherein the ketone compound is selected from or .

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