Aminotransferase mutant and method for producing chiral amine compound

Abstract

The present invention provides an aminotransferase mutant and a method for producing a chiral amine compound. Here, the aminotransferase mutant includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, or (c) a protein in which an amino acid mutation occurs at at least one site such as Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452 in the amino acid sequence in (b) and has an aminotransferase function, (d) a protein having 80% or more homology with the amino acid sequence defined in any one of (a), (b), or (c) and having an aminotransferase function. It can solve the problem of low activity of aminotransferase in the prior art and is suitable for the field of enzyme catalysis.

Description

Cross - reference to related applications

[0001] This application is based on and claims priority to a Chinese application with a CN application number of 202210707289.5 and a filing date of June 21, 2022. The disclosure content of the CN application is hereby incorporated into this application in its entirety again.

Technical Field

[0002] The present invention relates to the field of enzyme catalysis, and specifically to an aminotransferase mutant and a method for producing a chiral amine compound.

Background Art

[0003] A chiral amine refers to a type of compound containing an amino group at the chiral center of a small - molecule compound, and is a structural unit of many important biologically active molecules and an important intermediate for synthesizing many chiral drugs. Currently, the production of chiral amines is mainly achieved by chemical methods, biological resolution methods, and biological asymmetric synthesis methods. Chemical methods have drawbacks such as long reaction routes, harsh conditions, the use of toxic transition - metal catalysts, and low stereoselectivity of products. Since the theoretical maximum yield of the biological resolution method is only 50%, both have certain limitations in large - scale production.

[0004] The asymmetric synthesis method catalyzed by aminotransferase has a high theoretical yield and advantages such as high selectivity, high conversion rate, and mild reaction conditions, so it has become the first choice for synthesizing chiral amines. However, the drawbacks of most wild - type enzymes are that their substrate ranges are limited, and they generally have difficulty accepting a group larger than a methyl substituent on one side adjacent to the carbonyl group (referred to as the synthesis of highly sterically hindered chiral amine compounds in this application). As a result, there are not many aminotransferases that are actually industrially applicable. Catalytically synthesizing highly sterically hindered chiral amine compounds with aminotransferase has been a long - standing problem.

[0005] By modifying wild-type enzymes as a means of directed evolution, the substrate acceptance range can be improved, and in particular, it is an excellent method for solving this problem in the synthesis of highly sterically hindered chiral amine compounds. There are two preferred examples as follows. Codexis in the United States directed the evolution of wild-type aminotransferases from different origins and used the obtained mutants for efficient catalysis of precursor ketones to synthesize Sitagliptin [Science, 329 (Jul. 16 TN. 5989), 305-309] and Sacubitril [ACS Catal. 2021, 11 (6), 3762-3770], the corresponding highly sterically hindered chiral amine compounds. The team of the Biosynthesis Technology Research and Development Center of Asymchem Life Sciences & Technology (Tianjin) Co., Ltd. screened one strain of aminotransferase mutants derived from Chromobaterium violaceum in previous studies and could use it for the catalytic synthesis of highly sterically hindered chiral amines. Subsequently, in order to further adapt to the high substrate concentration, low enzyme amount, and extreme environmental requirements in industrial production, Asymchem has been continuously developing aminotransferases that can synthesize highly sterically hindered chiral amine compounds to improve production efficiency, reduce the cost of industrial production, and reduce the emissions of industrial waste gas, waste water, and waste.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The main object of the present invention is to provide a method for producing aminotransferase mutants and chiral amine compounds in order to solve the problem of low activity of aminotransferases in the prior art.

Means for Solving the Problems

[0007] To achieve the above object, according to a first aspect of the present invention, there is provided an aminotransferase mutant comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, or (c) a protein having an amino acid mutation at at least one site of Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F409, T402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77 or N317 in the amino acid sequence of (b) and having an aminotransferase function, (d) a protein having a homology of 80% or more with the amino acid sequence defined in any one of (a), (b) or (c) and having an aminotransferase function.

[0008] Furthermore, the amino acid mutations in (c) are each independently selected from S424A or S424F or S424Q or S424P or S424R or S424N or S424V or S424Y or S424E or S424I, L380A, S417A or S417Q or S417F or S417I, Y89A or Y89D or Y89S or Y89H or Y89M or Y89G or Y89F, F409A or F409S or F409Q or F409L or F409H or F409P or F409K or F409G or F409V or F409N, N86M or N86P or N86D or N86A or N86V or N86H, Y85M or Y85F or Y85R, P83C or P83A or P83S or P83G or P83R, T91M or T91A or T91V or T91N or T91I, K90Y or K90G or K90A or K90V or K90S, F301S, G164S, T452S, M180V, F449L, F320H, Y322T, D315V or D315C or D315R or D315M, A31V, L295Q or L295M or L295F or L295Q, V64M or V64A or V64S, H154S, T402K or T402S, T126C, F364L, A433T, L379A or L379S, D416A, N151A, H274Y, I311F, V327S, R77Q, N317Y, where the alphabet before the number represents the original amino acid and the alphabet after the number represents the mutated amino acid. Preferably, in (d), the protein has at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99% homology with the amino acid sequence defined in (a), (b) or (c) and has aminotransferase function.

[0009] Furthermore, the mutations of the aminotransferase mutant include any of the following amino acid mutations. W60A; Y89A; N151A; F166A; E168A; V234A; I262A; L379A; L380A; R405A; D416A; S417A; C418A; S424A; V234A + S424A; V234A + L380A; V234A + L379A; V234A + D416A; V234A + S417A; V234A + Y89A; V234A + N151A; V234A + L379A + L380A; V234A + D416A + S417A; V234A + D416A + S424A; V234A + S417A + S424A; V234A + L380A + Y89D; V234A + L380A + Y89S; V234A + L380A + Y89H; V234A + L380A + Y89M; V234A + L380A + F409A; V234A + L380A + F409S; V234A + L380A + F409Q; V234A + L380A + F409L; V234A + L380A + S424F; V234A + L380A + S424Q; V234A + L380A + S424P; V234A + L380A + S424R; V234A + L380A + S424N; V234A + L380A + N86M; V234A + L380A + N86P; V234A + L380A + N86D; V234A + L380A + N86A; V234A + L380A + N86V; V234A + L380A + N86H; V234A + L380A + N86M + Y89D; V234A + L380A + N86M + Y89S; V234A + L380A + N86M + Y89G; V234A + L380A + N86M + Y89H; V234A + L380A + N86M + Y89F; V234A + L380A + N86M + Y89A; V234A + L380A + Y89D + F409H; V234A + L380A + Y89D + F409A; V234A + L380A + Y89D + F409P; V234A + L380A + Y89D + F409K; V234A + L380A + Y89D + F409G; V234A + L380A + Y89D + S424A; V234A + L380A + Y89D + S424V; V234A + L380A + Y89D + S424Y; V234A + L380A + Y89D + S424E; V234A + L380A + Y89D + S424Q; V234A + L380A + Y89D + N86H; V234A + L380A + Y89D + N86A;V234A+L380A+Y89D+N86H+Y85M; V234A+L380A+Y89D+N86H+Y85F; V234A+L380A+Y89D+N86H+Y85R; V234A+L380A+Y89D+N86H+Y85M+S417Q; V234A+L380A+Y89D+N86H+Y85M+S417F; V234A+L380A+Y89D+N86H+Y85M+S417I; V234A+L380A+Y89D+N86H+Y85M+S424I; V234A+L380A+Y89D+N86H+Y85M+P83C; V234A+L380A+Y89D+N86H+Y85M+P83A; V234A+L380A+Y89D+N86H+Y85M+P83S; V234A+L380A+Y89D+N86H+Y85M+P83G; V234A+L380A+Y89D+N86H+Y85M+T91M; V234A+L380A+Y89D+N86H+Y85M+T91A; V234A+L380A+Y89D+N86H+Y85M+T91V; V234A+L380A+Y89D+N86H+Y85M+T91N; V234A+L380A+Y89D+N86H+Y85M+T91I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83C; V234A+L380A+Y89D+N86H+Y85M+T91M+P83A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83G; V234A+L380A+Y89D+N86H+Y85M+T91M+P83R; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I;V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417A; V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A; V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+A239S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y+F409L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S;V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+I311F; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315C+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+V327S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295M;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295F;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T; 452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64A;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409Q;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N+T402K;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+R77Q;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+N317Y;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+A433T;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y。;

[0010] In order to achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above aminotransferase mutant.

[0011] In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid to which the above DNA molecule is ligated.

[0012] To achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell in which the above recombinant plasmid is transformed inside.

[0013] Furthermore, the host cell includes a prokaryotic cell, and preferably, the prokaryotic cell includes Escherichia coli.

[0014] To achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing a chiral amine compound, and the production method includes performing an amino group transfer reaction on a ketone substrate represented by Formula I under the action of an amino group donor by using the above aminotransferase mutant to produce a chiral amine compound

Chemical formula

[0015] Ar1 is selected from a first substituted aryl group, a first unsubstituted aryl group, a substituted arylene group, an unsubstituted arylene group, a substituted heteroarylene group, or an unsubstituted heteroarylene group; Ar2 is selected from a second substituted aryl group, a second unsubstituted aryl group, a substituted cycloalkyl group, an unsubstituted cycloalkyl group, an alkyl group, or an alkylene group; R is selected from H, an alkyl group, an alkylene group, or an alkylidene group, the number of C atoms of the alkylene group or alkylidene group is selected from 1 to 5, and the alkylene group or alkylidene group includes a substituted alkylene group or alkylidene group or an unsubstituted alkylene group or alkylidene group. When R is selected from an alkylene group or alkylidene group, the alkylene group or alkylidene group is linked to Ar1 and / or Ar2 to form a ring. The substituents in the first substituted aryl group, the substituted arylene group, the substituted heteroarylene group, the second substituted aryl group, or the substituted alkylene group or alkylidene group are each independently selected from halogen, a hydroxy group, an amino group, a methyl group, an ethyl group, or -CH2CH2OH, and the heteroatom in the substituted heteroarylene group is selected from N, O, or S.

[0016] Furthermore, the substituents in the first substituted aryl group, the second substituted aryl group or the substituted arylene group are each independently selected from halogen or -CH2CH2OH, and preferably, the substituents are each independently located at any one or more positions of the ortho-position, meta-position or para-position of the first substituted aryl group, the second substituted aryl group or the substituted arylene group, and preferably, the halogen is selected from F, Cl or Br.

[0017] Furthermore, Ar1 is selected from a first substituted aryl group, a substituted arylene group, a first unsubstituted aryl group or an unsubstituted arylene group, Ar2 is selected from a second unsubstituted aryl group, R is selected from an unsubstituted alkylene group, the number of C atoms of the unsubstituted alkylene group is selected from 1 to 5, and the unsubstituted alkylene group is linked to Ar1 to form a ring.

[0018] Furthermore, Ar1 is selected from an unsubstituted heteroarylene group, Ar2 is selected from a second substituted aryl group, the substituent of the second substituted aryl group is selected from halogen, R is selected from a substituted alkylene group, the substituent of the substituted alkylene group is selected from a hydroxy group, the number of C atoms of the substituted alkylene group is selected from 1 to 5, and the substituted alkylene group is linked to Ar1 to form a ring.

[0019] Furthermore, Ar1 is selected from a substituted arylene group, the substituent of the substituted arylene group is halogen, Ar2 is selected from a substituted cycloalkyl group or an unsubstituted cycloalkyl group, the number of C atoms of the substituted cycloalkyl group or the unsubstituted cycloalkyl group is selected from 3 to 8, and R is selected from H.

[0020] Furthermore, Ar1 is selected from a first unsubstituted aryl group, Ar2 is selected from a second substituted aryl group, a second unsubstituted aryl group, a substituted cycloalkyl group or an unsubstituted cycloalkyl group, and R is selected from H, a methyl group, a methylene group or a methine group.

[0021] Furthermore, Ar1 is selected from a cycloalkyl group or a first substituted aryl group, the substituent is selected from a hydroxy group, a methyl group, an ethyl group, or -CH2CH2OH, Ar2 is selected from a second unsubstituted aryl group or an alkyl group, and R is selected from H, a methyl group, a methylene group, or a methine group.

[0022] Furthermore, the ketone substrate is

Chemical formula

[0023] Furthermore, the amino group donor includes isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine, or aniline.

Advantages of the Invention

[0024] Applying the technical solution of the present invention, using the aminotransferase mutant (SEQ ID NO: 1) derived from Chromobaterium violaceum as the parent, protein engineering modifications such as single-site site-directed mutagenesis, saturation mutagenesis, combinatorial mutagenesis, and error-prone PCR are carried out to obtain an aminotransferase mutant with a broad substrate spectrum, capable of catalytic synthesis of highly sterically hindered chiral amine compounds, high enzyme activity, and high ability to withstand extreme environments.

Modes for Carrying Out the Invention

[0025] In addition, the examples and features of the examples in this application can be combined with each other as long as they do not conflict. Hereinafter, the present invention will be described in detail with reference to the examples.

[0026] As mentioned in the background art, the asymmetric synthesis method catalyzed by aminotransferase has high theoretical yields and advantages such as high selectivity, high conversion rates, and mild reaction conditions, making it the first choice for synthesizing chiral amines. However, the drawback of most wild-type enzymes is that their substrate ranges are limited, and it is generally difficult to synthesize highly sterically hindered chiral amine compounds using highly sterically hindered ketone substrates as substrates.

[0027] Therefore, in this application, the inventors attempted to mutate aminotransferase, discovered multiple important active amino acid sites, thereby obtained the above-mentioned multiple types of aminotransferase mutants, and were able to catalytically synthesize highly sterically hindered chiral amine compounds. Thus, a series of protection proposals of this application are proposed.

[0028] In the first exemplary embodiment of this application, (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, or (c) at least one site of Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F409, T402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77 or N317 in the amino acid sequence in (b) has an amino acid mutation and has an aminotransferase function, (d) including a protein having 80% or more homology with the amino acid sequence limited by any one of (a), (b) or (c) and having an aminotransferase function, to provide an aminotransferase mutant.

[0029] The amino acid sequence shown in SEQ ID NO: 1 is an amino acid mutant derived from Chromobaterium violaceum. By performing computer simulations such as homology modeling, active site simulation, site-directed mutagenesis, etc. and molecular biology experiments on the amino acid sequence, an important amino acid site V234 was discovered. The V was point-mutated to A to obtain the amino acid sequence shown in SEQ ID NO: 2, or mutated to C to obtain the amino acid sequence shown in SEQ ID NO: 3. Based on the amino acids with mutated V234, it was discovered that amino acid sites such as Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F409, T402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77 or N317 in the active site also have a great impact on the activity of the protein. By mutating the above amino acid sites, a protein having aminotransferase function and further enhanced aminotransferase function can be obtained. The obtained protein may change at unimportant mutation sites and active sites, and a protein having more than 80% homology with the above amino acid sequence and having aminotransferase function can be obtained.

[0030] SEQ ID NO: 1: JPEG2025520599000003.jpg42163 SEQ ID NO: 2: JPEG2025520599000004.jpg40163 SEQ ID NO: 3: JPEG2025520599000005.jpg42163 In a preferred embodiment, the amino acid mutations in (c) are each independently S424A or S424F or S424Q or S424P or S424R or S424N or S424V or S424Y or S424E or S424I, L380A, S417A or S417Q or S417F or S417I, Y89A or Y89D or Y89S or Y89H or Y89M or Y89G or Y89F, F409A or F409S or F409Q or F409L or F409H or F409P or F409K or F409G or F409V or F409N, N86M or N86P or N86D or N86A or N86V or N86H, Y85M or Y85F or Y85R, P83C or P83A or P83S or P83G or P83R, T91M or T91A or T91V or T91N or T91I, K90Y or K90G or K90A or K90V or K90S, F301S, G164S, T452S, M180V, F449L, F320H, Y322T, D315V or D315C or D315R or D315M, A31V, L295Q or L295M or L295F or L295Q, V64M or V64A or V64S, H154S, T402K or T402S, T126C, F364L, A433T, L379A or L379S, D416A, N151A, H274Y, I311F, V327S, R77Q, N317Y, where the alphabet before the number represents the original amino acid and the alphabet after the number represents the mutant amino acid. Preferably, in (d), it is a protein having a homology of 85% or more, preferably 90% or more, more preferably 95%, 96%, 97% or 98% or more, still more preferably 99%, 99.9% or more with the amino acid sequence defined in (a), (b) or (c) and having aminotransferase function.

[0031] In the present application, the applicant has continued to conduct further research on the above active site, and found that when the active site mutates to different amino acids, there are also differences in the activity of the corresponding protein, and the activity of aminotransferase is enhanced by specific mutations. Through experimental research, it was found that when the above specific mutation is performed on the active site, a protein with enhanced activity can be obtained. Flexible selection and combination can be performed among the above mutations with respect to the amino acid mutation site of the aminotransferase protein.

[0032] In a preferred embodiment, the mutation of the aminotransferase mutant includes any one of the following amino acid mutations. W60A; Y89A; N151A; F166A; E168A; V234A; I262A; L379A; L380A; R405A; D416A; S417A; C418A; S424A; V234A + S424A; V234A + L380A; V234A + L379A; V234A + D416A; V234A + S417A; V234A + Y89A; V234A + N151A; V234A + L379A + L380A; V234A + D416A + S417A; V234A + D416A + S424A; V234A + S417A + S424A; V234A + L380A + Y89D; V234A + L380A + Y89S; V234A + L380A + Y89H; V234A + L380A + Y89M; V234A + L380A + F409A; V234A + L380A + F409S; V234A + L380A + F409Q; V234A + L380A + F409L; V234A + L380A + S424F; V234A + L380A + S424Q; V234A + L380A + S424P; V234A + L380A + S424R; V234A + L380A + S424N; V234A + L380A + N86M; V234A + L380A + N86P; V234A + L380A + N86D; V234A + L380A + N86A; V234A + L380A + N86V; V234A + L380A + N86H; V234A + L380A + N86M + Y89D; V234A + L380A + N86M + Y89S; V234A + L380A + N86M + Y89G; V234A + L380A + N86M + Y89H; V234A + L380A + N86M + Y89F; V234A + L380A + N86M + Y89A; V234A + L380A + Y89D + F409H; V234A + L380A + Y89D + F409A; V234A + L380A + Y89D + F409P; V234A + L380A + Y89D + F409K; V234A + L380A + Y89D + F409G; V234A + L380A + Y89D + S424A; V234A + L380A + Y89D + S424V; V234A + L380A + Y89D + S424Y; V234A + L380A + Y89D + S424E; V234A + L380A + Y89D + S424Q; V234A + L380A + Y89D + N86H; V234A + L380A + Y89D + N86A;V234A + L380A + Y89D + N86H + Y85M; V234A + L380A + Y89D + N86H + Y85F; V234A + L380A + Y89D + N86H + Y85R; V234A + L380A + Y89D + N86H + Y85M + S417Q; V234A + L380A + Y89D + N86H + Y85M + S417F; V234A + L380A + Y89D + N86H + Y85M + S417I; V234A + L380A + Y89D + N86H + Y85M + S424I; V234A + L380A + Y89D + N86H + Y85M + P83C; V234A + L380A + Y89D + N86H + Y85M + P83A; V234A + L380A + Y89D + N86H + Y85M + P83S; V234A + L380A + Y89D + N86H + Y85M + P83G; V234A + L380A + Y89D + N86H + Y85M + T91M; V234A + L380A + Y89D + N86H + Y85M + T91A; V234A + L380A + Y89D + N86H + Y85M + T91V; V234A + L380A + Y89D + N86H + Y85M + T91N; V234A + L380A + Y89D + N86H + Y85M + T91I; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83C; V234A + L380A + Y89D + N86H + Y85M + T91M + P83A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83G; V234A + L380A + Y89D + N86H + Y85M + T91M + P83R; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417A; V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A; V234C+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+A239S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+L379S+H274Y+F409L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+T452S; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91M+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+I311F; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315C+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+V327S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295M; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295F; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64A; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409Q; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409N+T402K; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315R+A31V+L295Q+V64M+H154S+F409V+T402S+T126C; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315M+A31V+L295Q+V64M+H154S+F409V+T402S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+R77Q; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+N317Y; V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L+A433T; or V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y。

[0033] All of the above amino acid mutations have been tested and studied in the examples of this application. All of them have aminotransferase activity, and compared with the parent strain having the amino acid sequence shown in SEQ ID NO: 1, they have a broader substrate spectrum, can catalyze the synthesis of highly sterically hindered chiral amine compounds, have high enzyme activity, and / or can obtain aminotransferase mutants with strong ability to withstand extreme environments.

[0034] In a second exemplary embodiment of the present application, a DNA molecule encoding the above aminotransferase mutant is provided.

[0035] In a third exemplary embodiment of the present application, a recombinant plasmid linked with the above DNA molecule is provided.

[0036] The above DNA can encode the above aminotransferase mutant and can be ligated to a recombinant plasmid to form circular DNA. Both the above DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the above aminotransferase mutant.

[0037] In a fourth exemplary embodiment of the present application, a host cell transformed with the above recombinant plasmid inside is provided.

[0038] In a preferred example, the host cell includes prokaryotic cells, and preferably, the prokaryotic cells include Escherichia coli.

[0039] Using the above host cells, recombinant plasmids can be copied in the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large amount of aminotransferase mutants. Using existing techniques, the protein can be disrupted and purified from the host cells, and after disruption, crude enzyme catalysis or other methods can be used to obtain aminotransferase mutants, and subsequent catalysis of amine compounds can be carried out. The host cell is a host cell derived from a non-plant source.

[0040] In the fifth typical embodiment of the present application, a method for producing a chiral amine compound is provided. The production method includes performing an amino group transfer reaction on a ketone substrate represented by Formula I under the action of an amino group donor using the above aminotransferase mutant to obtain a chiral amine compound. Ar1 is selected from a first substituted aryl group, a first unsubstituted aryl group, a substituted arylene group, an unsubstituted arylene group, a substituted heteroarylene group, or an unsubstituted heteroarylene group. Ar2 is selected from a second substituted aryl group, a second unsubstituted aryl group, a substituted cycloalkyl group, an unsubstituted cycloalkyl group, an alkyl group, or an alkylene group. R is selected from H, an alkyl group, an alkylene group, or an alkylidene group. The number of C atoms in the alkylene group or alkylidene group is selected from 1 to 5. The alkylene group or alkylidene group includes a substituted alkylene group or alkylidene group or an unsubstituted alkylene group or alkylidene group. When R is selected from an alkylene group or alkylidene group, the alkylene group or alkylidene group is linked to Ar1 and / or Ar2 to form a ring. The substituents in the first substituted aryl group, substituted arylene group, substituted heteroarylene group, second substituted aryl group, or substituted alkylene group or alkylidene group are each independently selected from halogen, a hydroxy group, an amino group, a methyl group, an ethyl group, or -CH2CH2OH. The heteroatom in the substituted heteroarylene group is selected from N, O, or S.

[0041]

Chemical formula

[0042] In a preferred embodiment, the substituents in the first substituted aryl group, the second substituted aryl group, or the substituted arylene group are each independently selected from halogen or -CH2CH2OH, preferably, the substituents are each independently located at any one or more positions of the ortho position, meta position, or para position of the first substituted aryl group, the second substituted aryl group, or the substituted arylene group, and preferably, the halogen is selected from F, Cl, or Br.

[0043] In a preferred embodiment, Ar1 is selected from the first substituted aryl group, the substituted arylene group, the first unsubstituted aryl group, or the unsubstituted arylene group, Ar2 is selected from the second unsubstituted aryl group, R is selected from the unsubstituted alkylene group, the number of C atoms of the unsubstituted alkylene group is selected from 1 to 5, and the unsubstituted alkylene group is linked to Ar1 to form a ring.

[0044] In a preferred embodiment, Ar1 is selected from the unsubstituted heteroarylene group, Ar2 is selected from the second substituted aryl group, the substituent of the second substituted aryl group is selected from halogen, R is selected from the substituted alkylene group, the substituent of the substituted alkylene group is selected from the hydroxy group, the number of C atoms of the substituted alkylene group is selected from 1 to 5, and the substituted alkylene group is linked to Ar1 to form a ring.

[0045] In a preferred embodiment, Ar1 is selected from substituted arylene groups, the substituents of the substituted arylene groups are halogens, Ar2 is selected from substituted cycloalkyl groups or unsubstituted cycloalkyl groups, the number of C atoms of the substituted cycloalkyl groups or unsubstituted cycloalkyl groups is selected from 3 to 8, and R is selected from H.

[0046] In a preferred embodiment, Ar1 is selected from first unsubstituted aryl groups, Ar2 is selected from second substituted aryl groups, second unsubstituted aryl groups, substituted cycloalkyl groups or unsubstituted cycloalkyl groups, and R is selected from H, methyl groups, methylene groups or methine groups.

[0047] In a preferred embodiment, Ar1 is selected from cycloalkyl groups or first substituted aryl groups, the substituents are selected from hydroxy groups, methyl groups, ethyl groups or -CH2CH2OH, Ar2 is selected from second unsubstituted aryl groups or alkyl groups, and R is selected from H, methyl groups, methylene groups or methine groups.

[0048] In a preferred embodiment, the ketone substrate is

Chemical formula

[0049] The substrate range of the above ketone substrates is wide, and it has a group larger than the methyl substituent on one side adjacent to the carbonyl group. Due to the influence of steric hindrance, it is difficult to chirally catalyze the above substrates using aminotransferases in the prior art. Also, in the prior art, even if there is an aminotransferase that can perform chiral catalysis in industrial production such as high substrate concentration, low enzyme amount, and extreme environment, large-scale industrial production has been difficult. By using the above aminotransferase mutant, the above ketone substrates can be efficiently chirally catalyzed, the carbonyl group is catalyzed into a chiral amino group, and a chiral amine compound with stereoselectivity is obtained.

[0050] In a preferred embodiment, the amino group donor includes, but is not limited to, one or more of isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine, or aniline. The amino group donor can make a flexible selection or combination among the amino group donors commonly used in the prior art.

[0051] In this application, the inventor performed homology modeling on the cleaned mutants according to the three-dimensional structure (PDB: 4BA5) of the protein that has already been analyzed, and performed molecular docking on different highly sterically hindered ketone compounds according to the model structure. By analyzing the docking results, 18 residues near the active center that may affect the catalytic activity of the protein were selected, and these residues include L59, W60, F88, Y89, N151, Y153, F166, E168, V234, I262, L379, L380, F397, R405, D416, S417, C418, S424. Single-point mutations were made to these residues, all of which were mutated to alanine, and an "alanine scan" was performed to observe the effects of these changes on protein function.

[0052] Site-directed mutagenesis refers to introducing necessary changes including base addition, deletion, point mutation, etc. (changes characterizing the advantageous direction are common) into a target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR). Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of the target protein expressed by DNA, and is a very useful means in gene research. The method of introducing site-directed mutagenesis using full plasmid PCR is simple and effective, and is currently a widely used means. Its principle is that after annealing a pair of primers (forward and reverse) containing the mutation site and a template plasmid, polymerase is used for "circular extension". So-called circular extension means that the polymerase extends the primer according to the template, ends when it extends one round and returns to the 5' end of the primer, and then continuously repeats the cycle of heating, annealing, and extension. This reaction is different from rolling circle amplification and does not form multiple tandem copies. After annealing the extension products of the forward and reverse primers and then pairing them, a nicked open circular plasmid is formed. The product by DpnI enzyme cleavage and extension, since the original template plasmid is derived from common Escherichia coli and has undergone dam methylation modification, is sensitive to DpnI and is cleaved. The plasmid with the mutated sequence synthesized in vitro is not methylated and thus is not cleaved. Therefore, subsequent transformation is successful and clones of the mutated plasmid can be obtained.

[0053] After identifying the correct site-directed mutant bacteria by sequencing, the expression of ketoreductase was induced under the condition of inducing overnight at 25°C with 0.2 mM IPTG. Then, crude enzyme for detecting reaction characteristics was obtained by a method of disrupting cells with ultrasonic waves. After verifying the reaction characteristics, the sites that can clearly improve the catalytic characteristics of aminotransferase are those from Y89A, N151A, V234A, L379A, L380A, D416A, S417A, S424A.

[0054] Next, beneficial amino acid sites were combined to obtain mutants with better properties. The construction method of two-point mutations in combinatorial mutations is the same as that of single-point mutations, and it is constructed using the full plasmid PCR method. Multisite mutations where two or more sites mutate simultaneously are performed using overlap-extension-PCR amplification to obtain mutant genes containing multisite mutations. After both ends are enzymatically cleaved by restriction endonucleases, they are ligated into an expression vector, transformed into E. coli cells, spread on an LB culture dish containing 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain combinatorial mutants, which were sequenced and identified. The activities of the obtained correct mutants were verified to obtain mutants with improved catalytic properties, which were used as templates for the next round of saturation mutagenesis. Subsequently, according to the molecular docking model, 20 points were selected at the positions of the substrate channel and an important loop region of a section to perform saturation mutagenesis.

[0055] Saturation mutagenesis is a method of obtaining mutants in which the amino acids at the target site are each replaced by 19 other amino acids within a short period by modifying the coding gene of the target protein. This method is a powerful tool for directed modification of proteins and an important means for studying the structure-function relationship of proteins. Saturation mutagenesis can often obtain more ideal evolved products than single-point mutagenesis. These problems that cannot be solved by site-directed mutagenesis methods are the unique advantages of saturation mutagenesis methods. By sequencing and identification, the activities of the mutants obtained by saturation mutagenesis against different substrates and their resistance to high temperature were tested respectively.

[0056] After obtaining amino transferase mutants with significantly improved activity and resistance, random mutations were performed on them using the error-prone PCR method to construct a high-quality mutant library, and an appropriate high-throughput screening method was developed to screen the library to obtain mutants with further improved activity.

[0057] Error-prone PCR refers to PCR under error-prone conditions, that is, a PCR technique in which errors are likely to occur in the copied DNA sequence, and is also called mismatch PCR or biased error PCR. Specifically, by using a low-fidelity Taq DNA polymerase and changing the PCR reaction conditions, the fidelity of DNA copying is reduced, and base mismatches are increased during the synthesis process of the new DNA strand, so that many point mutations occur in the amplification product, which is a method for inducing DNA sequence mutations in vitro.

[0058] The following high-throughput screening method was developed to screen an error-prone mutant library.

[0059] 1. Cultivation of mutants: Add 300 μL of LB medium to each well of a 96-well plate, inoculate a single clone on an agar plate into a deep-well 96-well plate, culture overnight at 37 °C and 200 rpm. Transfer the overnight-cultured bacterial solution to another 96-well plate with 800 μL of LB medium added per well using Qpix, culture at 37 °C and 200 rpm for 5 h. When the OD600 of the bacterial solution in the 96-well plate reaches 0.6 - 0.9, add IPTG solution to the 96-well plate again using Qpix so that the final concentration of IPTG in the well plate is 0.1 mM, induce at 25 °C and 200 rpm overnight for about 16 h, centrifuge at 4000 rpm for 5 min, discard the supernatant, and react with the whole cells.

[0060] 2. 96-well plate high-throughput screening system: Add 8 μL of PLP stock solution (1 mg / mL), 3.5 μL of 6 M isopropylamine hydrochloride (20 eq), and add 0.1 M Tris-Cl 9.0 until the volume reaches 100 μL, mix well, dispense the mixed sample into a 96-well plate filled with activated sludge in each well, and finally add the substrate stock solution (dissolve 0.3 mg of substrate in 15 μL of DMSO to a total volume of 15%), mix well, and react at 50 °C on a constant temperature shaker at 700 rpm for 18 h.

[0061] After the error-prone PCR was completed, the inventors had already obtained mutants with significantly improved activity and resistance at the laboratory scale. However, when applied industrially, there was still a problem that the amount of enzyme was large and the resistance was insufficient. Subsequently, using the same mutagenesis method described above, saturation mutagenesis and combinatorial mutagenesis evolution were performed multiple times to obtain a series of aminotransferase mutants. As can be seen from the results, these aminotransferase mutants had a further greatly improved catalytic efficiency for multiple ketone substrates, could be used for the synthesis of multiple chiral amine compounds, especially the efficient synthesis of highly sterically hindered chiral amine compounds, and also had a certain improvement in resistance to extreme environments.

[0062] Hereinafter, with reference to specific examples, the beneficial effects of the present application will be described in more detail. (Example 1) Using the above method, site-directed mutagenesis was performed based on the parental sequence number 1. For specific mutagenesis sites, refer to Table 1, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0063] A 1 mL reaction system contained 2 mg of substrate 1 or substrate 2 or substrate 3 or substrate 4, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 400 μL of crude enzyme solution (prepared from 200 mg of wet bacterial sludge), and 100 mM phosphate buffer at pH 8.0, and was reacted at 50 °C for 42 h. The above wet bacterial sludge was obtained by centrifuging the Escherichia coli fermentation broth of the corresponding mutant, and the crude enzyme solution was obtained by adding 100 mM phosphate buffer at pH 8.0 to the obtained wet bacterial sludge, homogenizing the cell wall by ultrasonic wave or homogenate machine, and then concentrating.

[0064] The detection results are as shown in Table 1.

[0065]

Table 1

[0066]

Table 2

[0067] A 1 mL reaction system contained 4 mg of Substrate 1 or Substrate 2 or Substrate 3 or Substrate 4, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 400 μL of crude enzyme solution (prepared from 200 mg of wet bacterial sludge), and 100 mM phosphate buffer at pH 8.0, and was reacted at 50 °C for 18 h. The results are as shown in Table 3.

[0068]

Table 3

[0069] A 1 mL reaction system contained 4 mg of substrate 1 or substrate 2 or substrate 3 or substrate 4, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 400 μL of crude enzyme solution (prepared from 200 mg of wet bacterial sludge) after treatment at 70 °C for 1 h, and 100 mM phosphate buffer at pH 8.0, and was reacted at 50 °C for 18 h. The results are as shown in Table 4.

[0070]

Table 4

[0071] The relative residual activity refers to the ratio of the enzyme activity measured with the enzyme solution after treatment with an appropriate amount under extreme conditions such as high temperature, alkaline, and organic solvents, to the enzyme activity under the optimal conditions of the enzyme solution that has not undergone treatment in an extreme environment. Under the same treatment conditions, the higher the relative residual activity, the higher the stability of the enzyme under this condition. (Example 5) Based on Example 3, error-prone PCR and combinatorial mutagenesis were performed multiple times, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0072] The 1 mL reaction system contained 10 mg of Substrate 1 or Substrate 2 or Substrate 3 or Substrate 4, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 100 μL of crude enzyme solution (prepared from 50 mg of wet bacterial sludge), and 100 mM phosphate buffer at pH 8.0, and was reacted at 50 °C for 18 h. The results are as shown in Table 5.

[0073]

Table 5

[0074] The 1 mL reaction system contained 50 mg of Substrate 1 or Substrate 2 or Substrate 3 or Substrate 4, 1 mg of PLP, 10 mg of isopropylamine hydrochloride, 50 μL of crude enzyme solution (prepared from 25 mg of wet bacterial sludge), and 100 mM phosphate buffer at pH 8.0, and was reacted at 45 °C for 18 h. The results are as shown in Table 6.

[0075]

Table 6

[0076] A 1 mL reaction system contained 10 mg of Substrate 1 or Substrate 2 or Substrate 3 or Substrate 4, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 100 μL of a crude enzyme solution (prepared from 50 mg of wet bacterial sludge) after treatment at 70 °C for 1 h, and 100 mM phosphate buffer at pH 8.0, and was reacted at 50 °C for 18 h. The results are as shown in Table 7.

[0077] [Table 7] Note: In the above table, + indicates that the relative residual activity is 50% or more and less than 60%, ++ indicates that the relative residual activity is 60% or more and less than 70%, and +++ indicates that the relative residual activity is 70% or more. (Example 8) 50 mL of 100 mmol / L phosphate buffer (5 vol) and 20 mL of 5 mol / L isopropylamine hydrochloride solution (2 vol) were added into a 250 mL four-necked flask at room temperature and adjusted to pH = 8.5 - 9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%), 10 g of

Chem.

Chem.

[0078] By HPLC detection, the purity > 99%, de value > 99%, and yield 87%. (Example 9) At room temperature, 50 mL of 100 mmol / L phosphate buffer (5 vol) and 20 mL of 5 mol / L isopropylamine hydrochloride solution (2 vol) were added into a 250 mL four-necked flask and the pH was adjusted to 8.5 - 9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of

Chem.

[0079] By HPLC detection, the purity was > 98%, the de value was > 99%, and the yield was 84%. (Example 10) At room temperature, 50 mL of 100 mmol / L phosphate buffer (5 vol) and 20 mL of 5 mol / L isopropylamine hydrochloride solution (2 vol) were added into a 250 mL four-necked flask and the pH was adjusted to 8.5 - 9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of [Chemical formula] (Substrate 3) was added and stirred uniformly. Subsequently, 2 mL (0.1 wt, 0.5 g / mL) of the enzyme solution of the CvTA aminotransferase mutant (V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64M+H154S+F409V+T402S+T126C+F364L) mutated based on SEQ ID NO: 1 was added, and the pH was adjusted to 8.5 - 9.0. The temperature was raised to 45 °C and the reaction was carried out with stirring overnight. After the reaction was completed, the acidity of the system was adjusted to pH = 2 - 3 to denature the protein. The filtrate after filtration was extracted with 50 mL of methyl tert-butyl ether. The aqueous phase was adjusted to pH = 12 and then extracted twice with 50 mL of methyl tert-butyl ether. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated under the conditions of T < 40 °C and P ≤ -0.06 Mpa until there was no distillate. The target product

Chemical formula

[0080] By HPLC detection, the purity was > 98%, the ee value was > 99%, and the yield was 82%. (Example 11) At room temperature, 50 mL of 100 mmol / L phosphate buffer (5 vol) and 20 mL of 5 mol / L isopropylamine hydrochloride solution (2 vol) were added into a 250 mL four-necked flask and the pH was adjusted to 8.5 - 9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of

Chemical formula

Chemical formula

[0081] By HPLC detection, the purity > 99%, de value > 99%, and yield 91%. (Example 12) Using the enzyme solution of the aminotransferase mutant (V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C + F364) mutated based on SEQ ID NO: 1, substrates 5 - 16 were subjected to catalytic reaction with reference to the catalytic synthesis steps of Examples 6 - 9, and the results are as shown in 8.

[0082]

Table 8

[0083] The above are only preferred embodiments of the present invention and do not limit the present invention. Those skilled in the art can make various modifications and changes to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. (a)A protein having the amino acid sequence shown in SEQ ID NO: 1, or (b)A protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, or (c)A protein in which an amino acid mutation occurs at at least one site of Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F409, T402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77 or N317 of the amino acid sequence in (b), and having aminotransferase function (d)A protein having a homology of 80% or more with the amino acid sequence defined in any one of (a), (b) or (c) and having aminotransferase function Comprising, characterized in that it is an aminotransferase mutant

2. The amino acid mutation in (c) is, independently of each other, S424A or S424F or S424Q or S424P or S424R or S424N or S424V or S424Y or S424E or S424I, L380A, S417A or S417Q or S417F or S417I, Y89A or Y89D or Y89S or Y89H or Y89M or Y89G or Y89F, F409A or F409S or F409Q or F409L or F409H or F409P or F409K or F409G or F409V or F409N, N86M or N86P or N86D or N86A or N86V or N86H, Y85M or Y85F or Y85R, P83C or P83A or P83S or P83G or P83R, T91M or T91A or T91V or T91N or T91I, K90Y or K90G or K90A or K90V or K90S, F301S, G164S, T452S, M180V, F449L, F320H, Y322T, D315V or D315C or D315R or D315M, A31V, L295Q or L295M or L295F or L295Q, V64M or V64A or V64S, H154S, T402K or T402S, T126C, F364L, A433T, L379A or L379S, D416A, N151A, H274Y, I311F, V327S, R77Q, selected from N317Y, wherein the alphabet before the number represents the original amino acid and the alphabet after the number represents the mutant amino acid, the aminotransferase mutant according to claim 1, characterized in that. **Claim 3** In the above (d), a protein having a homology of 85% or more with the amino acid sequence defined in (a), (b) or (c) and having an aminotransferase function, the aminotransferase mutant according to claim 2, characterized in that. **Claim 4** The mutation of the aminotransferase mutant includes any of the following amino acid mutations, and the aminotransferase mutant according to claim 2 or 3, characterized in that. W60A; Y89A; N151A; F166A; E168A; V234A; I262A; L379A; L380A; R405A; D416A; S417A; C418A; S424A; V234A + S424A; V234A + L380A; V234A + L379A; V234A + D416A; V234A + S417A; V234A + Y89A; V234A + N151A; V234A + L379A + L380A; V234A + D416A + S417A; V234A + D416A + S424A; V234A + S417A + S424A; V234A + L380A + Y89D; V234A + L380A + Y89S; V234A + L380A + Y89H; V234A + L380A + Y89M; V234A + L380A + F409A; V234A + L380A + F409S; V234A + L380A + F409Q; V234A + L380A + F409L; V234A + L380A + S424F; V234A + L380A + S424Q; V234A + L380A + S424P; V234A + L380A + S424R; V234A + L380A + S424N; V234A + L380A + N86M; V234A + L380A + N86P; V234A + L380A + N86D; V234A + L380A + N86A; V234A + L380A + N86V; V234A + L380A + N86H; V234A + L380A + N86M + Y89D; V234A + L380A + N86M + Y89S; V234A + L380A + N86M + Y89G; V234A + L380A + N86M + Y89H; V234A + L380A + N86M + Y89F; V234A + L380A + N86M + Y89A; V234A + L380A + Y89D + F409H; V234A + L380A + Y89D + F409A; V234A + L380A + Y89D + F409P; V234A + L380A + Y89D + F409K; V234A + L380A + Y89D + F409G; V234A + L380A + Y89D + S424A; V234A + L380A + Y89D + S424V; V234A + L380A + Y89D + S424Y; V234A + L380A + Y89D + S424E; V234A + L380A + Y89D + S424Q; V234A + L380A + Y89D + N86H; V234A + L380A + Y89D + N86A; V234A + L380A + Y89D + N86H + Y85M; V234A + L380A + Y89D + N86H + Y85F; V234A + L380A + Y89D + N86H + Y85R; V234A + L380A + Y89D + N86H + Y85M + S417Q; V234A + L380A + Y89D + N86H + Y85M + S417F; V234A + L380A + Y89D + N86H + Y85M + S417I; V234A + L380A + Y89D + N86H + Y85M + S424I; V234A + L380A + Y89D + N86H + Y85M + P83C; V234A + L380A + Y89D + N86H + Y85M + P83A; V234A + L380A + Y89D + N86H + Y85M + P83S; V234A + L380A + Y89D + N86H + Y85M + P83G; V234A + L380A + Y89D + N86H + Y85M + T91M; V234A + L380A + Y89D + N86H + Y85M + T91A; V234A + L380A + Y89D + N86H + Y85M + T91V; V234A + L380A + Y89D + N86H + Y85M + T91N; V234A + L380A + Y89D + N86H + Y85M + T91I; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83C; V234A + L380A + Y89D + N86H + Y85M + T91M + P83A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83G; V234A + L380A + Y89D + N86H + Y85M + T91M + P83R; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90V; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417A; V234C + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A; V234C + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + L379S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + L379S + H274Y; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + L379S + A239S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + L379S + H274Y + F409L; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + G164S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + T452S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S + G164S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S + G164S + T452S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + T452S; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + Y322T; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T; V234A + L380A + Y89D + N86H + Y85M + T91M + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + I311F; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315C + A31V; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + V327S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295M; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295F; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64A; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409Q; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409V; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409N; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409N + T402K; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409V + T402S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409V + T402S + T126C; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315M + A31V + L295Q + V64M + H154S + F409V + T402S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90Y + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315R + A31V + L295Q + V64M + H154S + F409V + T402S + T126C; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315M + A31V + L295Q + V64M + H154S + F409V + T402S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C + F364L; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C + F364L + R77Q; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C + F364L + N317Y; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M + H154S + F409V + T402S + T126C + F364L + A433T; V234A + L380A + Y89D + N86H + Y85M + T91I + P83S + K90G + S417I + S424A + F301S + G164S + T452S + M180V + F449L + F320H + Y322T + D315V + A31V + L295Q + V64M H154S + F409V + T402S + T126C + F364L + N317Y。

5. Encoding the aminotransferase mutant according to any one of Claims 1 to 4, A DNA molecule characterized by this.

6. Wherein the DNA molecule according to Claim 5 is linked, A recombinant plasmid characterized by this.

7. Wherein the recombinant plasmid according to Claim 6 is transformed inside, A host cell characterized by this.

8. Including prokaryotic cells, The host cell according to Claim 7, characterized by this.

9. Including Escherichia coli, The host cell according to Claim 8, characterized by this.

10. A method for producing a chiral amine compound, comprising: Using the aminotransferase mutant according to any one of Claims 1 to 4, under the action of an amino group donor, performing an amino group transfer reaction on the ketone substrate represented by Formula I to produce the chiral amine compound. 【Chemical 1】 Ar1 is selected from a first substituted aryl group, a first unsubstituted aryl group, a substituted arylene group or an unsubstituted arylene group, a substituted heteroarylene group or an unsubstituted heteroarylene group, Ar2 is selected from a second substituted aryl group, a second unsubstituted aryl group, a substituted cycloalkyl group, an unsubstituted cycloalkyl group, an alkyl group or an alkylene group, R is selected from H, an alkyl group, an alkylene group or an alkylidene group, the number of C atoms of the alkylene group or the alkylidene group is selected from 1 to 5, and the alkylene group or the alkylidene group includes a substituted alkylene group or a substituted alkylidene group or an unsubstituted alkylene group or an unsubstituted alkylidene group, When R is selected from an alkylene group or an alkylidene group, the alkylene group or the alkylidene group is linked to the Ar1 and / or the Ar2 to form a ring, The substituents in the first substituted aryl group, the substituted arylene group, the substituted heteroarylene group, the second substituted aryl group, or the substituted alkylene group or alkylidene group are each independently halogen, a hydroxy group, an amino group, a methyl group, an ethyl group, or -CH ２ CH ２ OH, and are selected from The heteroatom in the substituted heteroarylene group is selected from N, O or S, A method for producing a chiral amine compound, characterized in that.

11. The substituents of the first substituted aryl group, the second substituted aryl group or the substituted arylene group are each independently the halogen or the -CH ２ CH ２ selected from OH, The production method according to claim 10, characterized in that.

12. The substituents are each independently located at any one or more positions of the ortho position, meta position, or para position of the first substituted aryl group, the second substituted aryl group or the substituted arylene group, The production method according to claim 11, characterized in that.

13. The halogen is selected from F, Cl or Br, The production method according to claim 12, characterized in that.

14. Ar1 is selected from the first substituted aryl group, the substituted arylene group, the first unsubstituted aryl group or the unsubstituted arylene group, Ar2 is selected from the second unsubstituted aryl group, R is selected from the unsubstituted alkylene group, the number of C atoms of the unsubstituted alkylene group is selected from 1 to 5, The unsubstituted alkylene group is linked to the Ar1 to form a ring, The production method according to claim 13, characterized in that.

15. Ar1 is selected from the unsubstituted heteroarylene group, Ar2 is selected from the second substituted aryl group, and the substituent of the second substituted aryl group is selected from the halogen, R is selected from the substituted alkylene group, the substituent of the substituted alkylene group is selected from the hydroxy group, and the number of C atoms of the substituted alkylene group is selected from 1 to 5, The substituted alkylene group is linked to the Ar1 to form a ring. The production method according to claim 13, characterized in that.

16. The Ar1 is selected from the substituted arylene groups, and the substituent of the substituted arylene group is the halogen. The Ar2 is selected from the substituted cycloalkyl group or the unsubstituted cycloalkyl group, and the number of C atoms of the substituted cycloalkyl group or the unsubstituted cycloalkyl group is selected from 3 to 8. The R is selected from the H. The production method according to claim 13, characterized in that.

17. The Ar1 is selected from the first unsubstituted aryl groups. The Ar2 is selected from the second substituted aryl group, the second unsubstituted aryl group, the substituted cycloalkyl group or the unsubstituted cycloalkyl group. The R is selected from the H, methyl group, methylene group or methine group. The production method according to claim 13, characterized in that.

18. Ar1 is selected from a cycloalkyl group or the first substituted aryl group, and the substituent is the hydroxy group, the methyl group, the ethyl group or the -CH ２ CH ２ OH, The Ar2 is selected from the second unsubstituted aryl group or the alkyl group. The R is selected from the H, methyl group, methylene group or methine group. The production method according to claim 13, characterized in that.

19. The ketone substrate is 【Chemical 2】 selected from The production method according to any one of claims 10 to 18, characterized in that.

20. The amino group donor includes isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine or aniline. The production method according to claim 19, characterized in that.

Images