Method for producing aminotransferase mutants and chiralamine compounds

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, and the disclosure content of the CN application is 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] Chiral amines refer to a type of compound containing an amino group at the chiral center of a small molecule compound, and are structural units of many important biologically active molecules and important intermediates 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 disadvantages 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 disadvantages of most wild-type enzymes are that their substrate ranges are limited and they generally do not accept groups larger than methyl substituents on one side adjacent to the carbonyl group (referred to as the synthesis of highly sterically hindered chiral amine compounds in this application), which results in few aminotransferases that are actually industrially applicable. Catalytic synthesis of highly sterically hindered chiral amine compounds with aminotransferase has been a long-standing problem.

[0005] Modifying wild-type enzymes through targeted evolution improves their substrate acceptance range, which is an excellent method for solving the problem, particularly in the synthesis of highly sterically hindered chiral amine compounds. Two preferred examples are: Codexis, a US company, has evolved wild-type aminotransferases from different sources using targeted evolution, and the resulting mutants can be used for efficient catalysis of precursor ketones such as sitagliptin [Science, 329 (Jul. 16 TN. 5989), 305-309] and sacubitril [ACS Catal. 2021, 11(6), 3762-3770], and the corresponding highly sterically hindered chiral amine compounds. In a previous study, a team at the Biosynthesis Technology Research and Development Center of Kai Lai Ying Life Science and Technology (Tianjin) Co., Ltd. (Asymchem) screened one aminotransferase mutant from Chromobaterium violaceum that can be used for catalytic synthesis of highly sterically hindered chiral amines. Subsequently, Asymchem has continued to develop aminotransferases capable of synthesizing highly sterically hindered chiral amine compounds to further adapt to the high substrate concentrations, low enzyme levels, and extreme environmental requirements of industrial production, thereby improving production efficiency, reducing the cost of industrial production, and decreasing the emissions of exhaust gases, wastewater, and waste from industrial production. [Overview of the project] [Problems that the invention aims to solve]

[0006] The main objective of the present invention is to provide a method for producing aminotransferase mutants and chiralamine compounds in order to solve the problem of low aminotransferase activity in conventional techniques. [Means for solving the problem]

[0007] To achieve the above objective, according to a first aspect of the present invention, (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) the amino acid sequence in (b) Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F4 The present invention provides aminotransferase mutants comprising a protein having an amino acid mutation at at least one site of 09, T402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77, or N317 and possessing aminotransferase function, and a protein having 80% or more homology to any one of the amino acid sequences of (d)(a), (b), or (c) and possessing aminotransferase function.

[0008] Furthermore, the amino acid mutations in (c) are, 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, F301 S, 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, V32 Selected from 7S, R77Q, and N317Y, where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid, preferably having 85% or more homology, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology in (d) to the amino acid sequence limited by (a), (b), or (c), and having aminotransferase function.

[0009] Furthermore, the mutations in the aminotransferase mutant include 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+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+T 126C+F364L+R77Q;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F301S+G164S+T452S+M180V+F4 49L+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+V64 M+H154S+F409V+T402S+T126C+F364L+A433T;V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90G+S417I+S424A+F30 1S+G164S+T452S+M180V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y. ;

[0010] To achieve the above objective, a second aspect of the present invention provides a DNA molecule encoding the above-mentioned aminotransferase mutant.

[0011] To achieve the above objective, a third aspect of the present invention provides a recombinant plasmid in which the above-mentioned DNA molecules are linked.

[0012] To achieve the above objective, according to a fourth aspect of the present invention, a host cell is provided which has been transformed with the above-mentioned recombinant plasmid inside.

[0013] Furthermore, the host cells include prokaryotic cells, preferably including E. coli.

[0014] To achieve the above objective, a fifth aspect of the present invention provides a method for producing a chiral amine compound, the method of which utilizes the above-mentioned aminotransferase mutant to carry out an amino group transfer reaction with a ketone substrate represented by formula I under the action of an amino group donor, thereby producing a chiral amine compound [ka] This includes manufacturing.

[0015] 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 in the alkylene group or alkylidene group is selected from 1 to 5, and the alkylene group or alkylidene group may be a substituted alkylene group or alkylidene group Alternatively, if an unsubstituted alkylene group or alkylidene group is included, and R is selected from the alkylene group or alkylidene group, the alkylene group or alkylidene group is linked to Ar1 and / or Ar2 to form a ring, and 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, hydroxyl group, amino group, methyl group, 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 halogens or -CH2CH2OH, and preferably, each substituent is independently located at one or more of the ortho, meta, or para positions 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 in 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 a halogen, R is selected from a substituted alkylene group, the substituent of the substituted alkylene group is selected from a hydroxyl group, the number of C atoms in 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 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 in the substituted cycloalkyl group or 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] By applying the technical solution of the present invention, using the aminotransferase mutant (SEQ ID NO: 1) derived from Chromobaterium violaceum as the parent, performing protein engineering modifications such as single-site site-specific mutation, saturation mutation, combinatorial mutation and error-prone PCR, 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 is obtained.

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 information, asymmetric synthesis using aminotransferase catalysts is the first choice for synthesizing chiral amines due to its advantages such as high theoretical yield, high selectivity, high conversion rate, and mild reaction conditions. However, a drawback of most wild-type enzymes is their limited substrate range, and the fact that synthesizing highly sterically hindered chiral amine compounds using highly sterically hindered ketone substrates is generally difficult.

[0027] Therefore, in this application, the inventors attempt to mutate aminotransferases, discover several important active amino acid sites, and thereby obtain the above-mentioned multiple types of aminotransferase mutants, which can be used to catalytically synthesize highly sterically hindered chiral amine compounds. Thus, the present invention proposes a series of protection measures.

[0028] In a first typical embodiment of the present 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) the amino acid sequence in (b) Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452, M180, F449, F320, Y322, D315, A31, L295, V64, H154, F409, T The present invention provides aminotransferase mutants comprising a protein having an amino acid mutation at at least one site of 402, T126, F364, A433, L379, D416, N151, H274, I311, V327, R77, or N317 and possessing aminotransferase function, and a protein having 80% or more homology to any one of the amino acid sequences of (d)(a), (b), or (c) and possessing aminotransferase function.

[0029] The amino acid sequence shown in Sequence ID No. 1 above is an amino acid mutant derived from Chromobaterium violaceum. By performing computer simulations such as homology modeling, active site simulation, and site-directed mutation, as well as molecular biological experiments, an important amino acid site called V234 was discovered. Point mutations were then performed at V, and the amino acid sequence shown in Sequence ID No. 2 was obtained by mutating V to A, or by mutating to C to obtain the amino acid sequence shown in Sequence ID No. 3. Based on the mutated amino acids of V234, we discovered that active 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 also have a significant impact on protein activity. By mutating these amino acid sites, it is possible to obtain proteins that possess aminotransferase function, and even proteins with enhanced aminotransferase function. The resulting protein may have alterations in non-essential mutation sites and active sites, and it is possible to obtain a protein that has 80% or more homology to the above amino acid sequence and possesses aminotransferase function.

[0030] Sequence ID 1: JPEG0007839313000003.jpg42163 Sequence ID 2: JPEG0007839313000004.jpg40163 Sequence ID 3: JPEG0007839313000005.jpg42163 In a preferred embodiment, the amino acid mutations in (c) are, 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, G16 4S, 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, N317 Selected from Y, where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid, preferably having 85% or more homology, preferably 90% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99%, 99.9% or more homology in (d) with the amino acid sequence limited by (a), (b), or (c), and having aminotransferase function.

[0031] In this application, the applicant continued to study the above-mentioned active site and discovered that when the active site is mutated to a different amino acid, there are differences in the activity of the corresponding protein, and that specific mutations enhance the activity of aminotransferase. Through experimental research, it was discovered that by performing the above-mentioned specific mutations on the active site, a protein with enhanced activity can be obtained. Flexible selection and combination of the above-mentioned mutations can be made for the amino acid mutation site of the aminotransferase protein.

[0032] In a preferred embodiment, the mutation in the aminotransferase mutant includes 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+M1 80V+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+M180 V+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+M180 V+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, and all of them possess aminotransferase activity. Compared to the parent plant having the amino acid sequence shown in SEQ ID NO: 1, it is possible to obtain aminotransferase mutants with a broader substrate spectrum, capable of catalytic synthesis of highly sterically hindered chiral amine compounds, high enzyme activity, and / or strong tolerance to extreme environments.

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

[0035] A third typical embodiment of the present application provides a recombinant plasmid in which the above-mentioned DNA molecules are linked.

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

[0037] A fourth typical embodiment of the present application provides a host cell transformed internally with the above-described recombinant plasmid.

[0038] In a preferred embodiment, the host cells include prokaryotic cells, preferably including Escherichia coli.

[0039] Using the above-mentioned host cells, recombinant plasmids can be copied within the host cells, and the DNA molecules carried by the recombinant plasmids can be transcribed and translated to obtain a large number of aminotransferase mutants. Using existing techniques, proteins can be disrupted and purified from host cells, and aminotransferase mutants can be obtained by crude enzyme catalysis or other methods after disruption, and then used for subsequent catalysis of amine compounds. The host cells in question are non-plant-derived host cells.

[0040] A fifth typical embodiment of the present application provides a method for producing a chiral amine compound, the method comprising using the above-mentioned aminotransferase mutant to perform an amino group transfer reaction with a ketone substrate represented by formula I under the action of an amino group donor to obtain a chiral amine compound, wherein 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, and R is selected from H, an alkyl group, an alkylene group or an alkylidene group. The number of carbon atoms in the alkylene or alkylidene group is selected from 1 to 5, and the alkylene or alkylidene group includes substituted alkylene or alkylidene groups or unsubstituted alkylene or alkylidene groups. When R is selected from the alkylene or alkylidene group, the alkylene 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 or alkylidene group are independently selected from halogen, hydroxyl group, amino group, methyl group, ethyl group, or -CH2CH2OH, and the heteroatom in the substituted heteroarylene group is selected from N, O, or S.

[0041] [ka] By utilizing the above manufacturing method, the above aminotransferase mutant can be used to catalyze ketone compounds shown in formula I under the action of the provided amino group donor, catalyzing the carbonyl group to a chiral amino group to obtain a chiral amine compound. The above aminotransferase mutant can chiral catalyze the above raw material ketone compounds, which include a ketone compound having a group larger than a methyl substituent on one side adjacent to the carbonyl group, to synthesize highly sterically hindered chiral amines. Furthermore, it can catalyze under industrial production conditions such as high substrate concentration, low enzyme amount, and extreme environments, thereby improving production efficiency, reducing industrial production costs, and decreasing the discharge of exhaust gases, wastewater, and waste from industrial production.

[0042] In a preferred embodiment, the substituents on the first substituted aryl group, the second substituted aryl group, or the substituted arylene group are each independently selected from halogens or -CH2CH2OH, preferably each substituent is independently located at one or more of the ortho, meta, or para positions 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 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 in 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 an unsubstituted heteroarylene group, Ar2 is selected from a second substituted aryl group, the substituent of the second substituted aryl group is selected from a halogen, R is selected from a substituted alkylene group, the substituent of the substituted alkylene group is selected from a hydroxyl group, the number of C atoms in 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 a substituted arylene group, the substituent of the substituted arylene group is a 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 unsubstituted cycloalkyl group is selected from 3 to 8, and R is selected from H.

[0046] In a preferred embodiment, 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.

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

[0048] In one preferred embodiment, the ketone substrate is: [ka] Selected from.

[0049] The above-mentioned ketone substrates have a broad substrate range, and one side of the carbonyl group has a group larger than a methyl substituent. Due to steric hindrance, it is difficult to chiralize these substrates using conventional aminotransferases. Furthermore, even if there are aminotransferases that can perform chiral catalysis in industrial production using high substrate concentrations, low enzyme levels, and extreme environments, industrial mass production is difficult. By using the above-mentioned aminotransferase mutant, the above-mentioned ketone substrates can be efficiently chiralized, and the carbonyl group can be converted to a chiral amino group to obtain stereoselective chiral amine compounds.

[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 be flexibly selected or combined from among amino group donors commonly used in the prior art.

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

[0052] Site-directed mutation (SMU) refers to the introduction of necessary changes (generally changes that characterize a favorable direction) into a target DNA fragment (which may be a genome or a plasmid) using methods such as polymerase chain reaction (PCR), including the addition, deletion, and point mutation of bases. SMU is a very useful tool in genetic research because it can rapidly and efficiently improve the properties and characterization of the target protein expressed by the DNA. The method of introducing SMU using full plasmid PCR is simple, effective, and currently widely used. The principle is to anneal a pair of primers (forward and reverse) containing the mutation site and a template plasmid, and then "circular extension" with polymerase. So-called circular extension refers to the polymerase extending the primer according to the template, ending when it extends one full turn and returns to the 5' end of the primer, and then repeating the heating, annealing, and extension cycle. Unlike rolling circle amplification, this reaction does not form multiple tandem copies. After annealing the extended products of the forward and reverse primers, they are paired to form a ring-open plasmid with a cut end. The product obtained by DpnI enzyme cleavage and extension is sensitive to DpnI because the original template plasmid originates from common E. coli and has undergone dam methylation modification. However, the plasmid containing the mutant sequence synthesized in vitro is not methylated and therefore not cleaved. As a result, subsequent transformation is successful, and a clone of the mutant plasmid is obtained.

[0053] After identifying site-directed mutants by sequencing, ketoreductase expression was induced overnight at 25°C with 0.2 mM IPTG. Crude enzymes were then obtained for detection of reaction characteristics by sonication of the cells. After verifying the reaction characteristics, the sites that clearly improve the catalytic properties of the aminotransferase were found to be Y89A, N151A, V234A, L379A, L380A, D416A, S417A, and S424A.

[0054] Next, we combined beneficial amino acid sites to obtain mutants with superior properties. The method for constructing two-point mutations in combination mutations is the same as that for single-point mutations, and is constructed using full plasmid PCR. Multipoint mutations, in which two or more sites mutate simultaneously, were performed using overlap-extension-PCR amplification to obtain mutant genes containing multipoint mutations. After enzymatic cleavage of both ends with restriction endonucleases, the genes were ligated into expression vectors, transformed into E. coli cells, spread on LB culture dishes containing 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain combination mutants, which were then sequenced and identified. The activity of the obtained correct mutants was verified to obtain mutants with improved catalytic properties, which were used as templates for subsequent saturation mutations. Subsequently, depending on the molecular docking model, saturation mutations were performed at substrate channels and key loop regions within a single section, and at 20 selected points.

[0055] Saturated mutation is a method for obtaining mutants in which the target amino acid is replaced by one of 19 other amino acids within a short period of time by modifying the coding gene of a target protein. This method is a powerful tool for targeted protein modification and an important means of studying protein structure-function relationships. Saturated mutation often yields more ideal evolutionary products than single-point mutation. These problems that cannot be solved by site-directed mutation methods are unique advantages of saturated mutation. Sequencing identification was used to test the activity of mutants obtained by saturated mutation to different substrates and their tolerance to high temperatures.

[0056] After obtaining aminotransferase mutants with significantly improved activity and resistance, we performed random mutations against them using error-prone PCR to construct a high-quality mutant library. We then developed an appropriate high-throughput screening method to screen the library and obtain mutants with even greater activity.

[0057] Error-prone PCR refers to PCR performed under error-prone conditions, i.e., a PCR technique where errors are likely to occur in the copied DNA sequence. It is also called mismatch PCR or tendency error PCR. Specifically, it is a method of inducing DNA sequence mutations in vitro by reducing the fidelity of DNA copying and increasing base mismatches in the new DNA strand synthesis process through the use of low-fidelity TaqDNA polymerase and modification of PCR reaction conditions, resulting in many point mutations in the amplified product.

[0058] We developed the following high-throughput screening method to screen the error-prone mutant library.

[0059] 1. Culture of mutants: Add 300 μL of LB medium to each well of a 96-well plate. Inoculate a single clone from an agar plate into a 96-well plate with deep wells and culture overnight at 37°C and 200 rpm. Transfer the bacterial suspension from the overnight culture to another 96-well plate with 800 μL of LB medium per well using Qpix, and culture at 37°C and 200 rpm for 5 hours. When the OD600 of the bacterial suspension in the 96-well plate reaches 0.6-0.9, add IPTG solution to the 96-well plate again using Qpix until the final IPTG concentration in the well plate is 0.1 mM. Induce overnight at 25°C and 200 rpm for approximately 16 hours, then centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and allow the whole cell to react.

[0060] 2. 96-well plate high-throughput screening system: 8 μL of PLP stock solution (1 mg / mL) and 3.5 μL of 6 M isopropylamine hydrochloride (20 eq) were mixed with 0.1 M Tris-Cl 9.0 until the total volume reached 100 μL. The mixture was then dispensed into a 96-well plate with activated sludge in each well. Finally, the substrate stock solution (0.3 mg of substrate dissolved in 15 μL of DMSO, representing 15% of the total volume) was added and mixed thoroughly. The mixture was then reacted at 700 rpm for 18 hours on a 50°C constant temperature shaking bed.

[0061] After error-prone PCR was completed, the inventors had already obtained mutants with significantly improved activity and resistance on a laboratory scale. However, when applied industrially, problems remained, such as high enzyme levels and insufficient resistance. Subsequently, using the same mutation method described above, multiple evolutions of saturated and combined mutations were performed to obtain a series of aminotransferase mutants. As can be seen from the results, these aminotransferase mutants showed even greater catalytic efficiency for multiple ketone substrates, could be used for the efficient synthesis of various chiral amine compounds, especially highly sterically hindered chiral amine compounds, and also showed some degree of improved resistance to extreme environments.

[0062] The beneficial effects of this application will be explained in more detail below with reference to specific examples. (Example 1) Using the method described above, site-directed mutations were performed based on the parental sequence number 1. Refer to Table 1 for the specific mutation sites, and the catalytic activity of the mutants was detected according to the reaction conditions described below.

[0063] A 1 mL reaction system contained 2 mg of substrate 1, substrate 2, 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 hours. The wet bacterial sludge was obtained by centrifugation of the fermentation broth of the corresponding mutant E. coli, 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 wall with ultrasound or a homogenizing machine, and then concentrating it.

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

[0065] [Table 1] Note: In the table above, 0 indicates a conversion rate < 1%, + indicates a conversion rate of 1% or more but less than 5%, and ++ indicates a conversion rate of 5% or more but 10% or less. (Example 2) Based on Example 1, combination mutations were performed, and activity screening was conducted for the combination mutations according to the same reaction conditions as in Example 1. The results are shown in Table 2.

[0066] [Table 2] Note: In the table above, 0 indicates a conversion rate < 1%, + indicates a conversion rate of 1% or more but less than 5%, ++ indicates a conversion rate of 5% or more but 10% or less, +++ indicates a conversion rate of 10% or more but less than 15%, and ++++ indicates a conversion rate of 15% or more but less than 20%. (Example 3) Based on Example 2, multiple saturation mutations were performed, and the catalytic activity of the mutants was detected according to the reaction conditions described below.

[0067] A 1 mL reaction system contained 4 mg of substrate 1, substrate 2, 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 hours. The results are shown in Table 3.

[0068] [Table 3] JPEG0007839313000011.jpg237167 Note: In the table above, 0 indicates a conversion rate < 1%, + indicates a conversion rate of 1% or more but less than 5%, ++ indicates a conversion rate of 5% or more but less than 10%, +++ indicates a conversion rate of 10% or more but less than 15%, ++++ indicates a conversion rate of 15% or more but less than 20%, +++++ indicates a conversion rate of 20% or more but less than 40%, and ++++++ indicates a conversion rate of 40% or more. (Example 4) Some of the mutants from Examples 1, 2, and 3 were selected, and their resistance was detected according to the reaction conditions described below.

[0069] The 1 mL reaction system contained 4 mg of substrate 1, substrate 2, 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 hour, and a 100 mM phosphate buffer at pH 8.0, and was reacted at 50°C for 18 hours. The results are shown in Table 4.

[0070] [Table 4] Note: In the table above, + indicates a relative remaining vitality of 10% or more and less than 30%, ++ indicates a relative remaining vitality of 30% or more and less than 40%, +++ indicates a relative remaining vitality of 40% or more and less than 50%, and ++++ indicates a relative remaining vitality of 50% or more and less than 60%.

[0071] Relative residual activity refers to the ratio of enzyme activity measured in an enzyme solution after treatment under extreme conditions such as high temperature, alkalinity, and organic solvents, to enzyme activity under optimal conditions in an enzyme solution that has not been treated under extreme conditions. Under the same treatment conditions, a higher relative residual activity indicates greater stability of the enzyme under those conditions. (Example 5) Based on Example 3, error-prone PCR and combined mutation were performed multiple times, and the catalytic activity of the mutants was detected according to the reaction conditions described below.

[0072] A 1 mL reaction system contained 10 mg of substrate 1, 2, 3, or 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 hours. The results are shown in Table 5.

[0073] [Table 5] JPEG0007839313000014.jpg239167JPEG0007839313000015.jpg98166 Note: In the table above, 0 indicates a conversion rate < 1%, + indicates a conversion rate of 1% or more but less than 5%, ++ indicates a conversion rate of 5% or more but less than 10%, +++ indicates a conversion rate of 10% or more but less than 15%, ++++ indicates a conversion rate of 15% or more but less than 20%, +++++ indicates a conversion rate of 20% or more but less than 40%, and ++++++ indicates a conversion rate of 40% or more. (Example 6) Based on Example 5, saturated mutations and combined mutations were performed multiple times, and the catalytic activity of the mutants was detected according to the reaction conditions described below.

[0074] A 1 mL reaction system contained 50 mg of substrate 1, 2, 3, or 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 hours. The results are shown in Table 6.

[0075] [Table 6] JPEG0007839313000017.jpg239166JPEG0007839313000018.jpg170166 Note: In the table above, 0 indicates a conversion rate < 1%, + indicates a conversion rate of 1% or more but less than 5%, ++ indicates a conversion rate of 5% or more but less than 10%, +++ indicates a conversion rate of 10% or more but less than 20%, ++++ indicates a conversion rate of 20% or more but less than 40%, +++++ indicates a conversion rate of 40% or more but less than 60%, and ++++++ indicates a conversion rate of 60% or more. (Example 7) Some of the mutants from Examples 5 and 6 were selected, and their resistance was detected according to the reaction conditions described below.

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

[0077] [Table 7] Note: In the table above, + indicates that the relative remaining vitality is 50% or more but less than 60%, ++ indicates that the relative remaining vitality is 60% or more but less than 70%, and +++ indicates that the relative remaining vitality is 70% or more. (Example 8) 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 to a 250 mL four-necked flask to adjust the pH to 8.5-9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of [ka] Substrate 1 was added and mixed uniformly. Subsequently, 2 mL (0.1 wt, 0.5 g / mL) of an enzyme solution of an 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 Sequence 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 allowed to proceed overnight with stirring. After the reaction was complete, 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 subsequently extracted twice with 50 mL of methyl tert-butyl ether. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated under conditions T<40°C and P≦-0.06 MPa until no fraction remained. Target product [ka] I obtained it.

[0078] HPLC detection revealed a purity of >99%, a DE value of >99%, and a yield of 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 to a 250 mL four-necked flask to adjust the pH to 8.5-9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of [ka] Substrate 2 was added and mixed uniformly. Subsequently, 2 mL (0.1 wt, 0.5 g / mL) of an enzyme solution of an 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 Sequence 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 allowed to proceed overnight with stirring. After the reaction was complete, 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 subsequently extracted twice with 50 mL of methyl tert-butyl ether. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated under conditions T<40°C and P≦-0.06 MPa until no fraction remained. Target product [ka] I obtained it.

[0079] HPLC detection revealed a purity of >98%, a DE value of >99%, and a yield of 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 to a 250 mL four-necked flask to adjust the pH to 8.5-9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of [ka] Substrate 3 was added and mixed uniformly. Subsequently, 2 mL (0.1 wt, 0.5 g / mL) of enzyme solution (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) of a CvTA aminotransferase mutant mutated based on Sequence 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 allowed to proceed overnight with stirring. After the reaction was complete, 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 subsequently extracted twice with 50 mL of methyl tert-butyl ether. The integrated organic phase was dried over anhydrous magnesium sulfate and then concentrated under conditions T < 40°C and P ≤ -0.06 MPa until no fraction remained. Target product [ka] I obtained it.

[0080] HPLC detection revealed a purity of >98%, an ee value of >99%, and a yield of 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 to a 250 mL four-necked flask to adjust the pH to 8.5-9.0. Subsequently, 0.1 g of pyridoxal phosphate (1 wt%) and 10 g of [ka] Substrate 4 was added and mixed uniformly. Subsequently, 2 mL (0.1 wt, 0.5 g / mL) of an enzyme solution of an 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 Sequence 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 allowed to proceed overnight with stirring. After the reaction was complete, 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 subsequently extracted twice with 50 mL of methyl tert-butyl ether. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated under conditions T<40°C and P≦-0.06 MPa until no fraction remained. Target product [ka] I obtained it.

[0081] HPLC detection revealed a purity of >99%, a DE value of >99%, and a yield of 91%. (Example 12) Using an enzyme solution of an 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 Sequence ID No. 1, substrates 5-16 were catalytically reacted according to the catalytic synthesis steps of Examples 6-9, and the results are shown in 8.

[0082] [Table 8] From the above description, it can be seen that the following technical effects have been achieved by the above embodiments of the present invention. In addition to obtaining an aminotransferase parent with excellent activity, the present application has discovered multiple sites that can improve aminotransferase activity and / or resistance through research on the active site of aminotransferase, and has obtained multiple aminotransferase mutants with excellent performance through research on combinations of one or more sites. These aminotransferase mutants have advantages such as a broad substrate spectrum, the ability to catalytically synthesize highly sterically hindered chiral amine compounds, high enzyme activity, and strong resistance to extreme environments, and can meet the requirements of process production.

[0083] The foregoing are merely preferred embodiments of the present invention and do not limit it; those skilled in the art will know that the present invention can be modified and altered in various ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be within the scope of protection of the present invention.

Claims

1. An aminotransferase mutant characterized in that it is obtained by causing one amino acid mutation selected from the following in Sequence ID No. 1: V234A+L380A+Y89D+N86H+Y85M+T91I+P83S+K90Y+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+K90 G+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+M180 V+F449L+F320H+Y322T+D315V+A31V+L295Q+V64MH154S+F409V+T402S+T126C+F364L+N317Y.

2. Encoding the aminotransferase mutant described in claim 1, A DNA molecule characterized by the following features.

3. The DNA molecule described in claim 2 is linked, A recombinant plasmid characterized by the following features.

4. The recombinant plasmid described in claim 3 is transformed inside. A host cell characterized by the following features.

5. Including prokaryotic cells, The host cell according to feature 4.

6. Including E. coli, The host cell according to feature 5.

7. A method for producing chiralamine compounds, The process involves using the aminotransferase mutant described in claim 1 to carry out an amino group transfer reaction with a ketone substrate under the action of an amino group donor to produce the chiral amine compound, The ketone substrates are 【Chemistry 2】 Selected from, A method for producing chiral amine compounds, characterized by the following features.

8. The amino group donor includes isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine, or aniline. The manufacturing method according to claim 7, characterized in that it

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