Imine reductase mutant and method for preparing chiral hydrazine compound

Abstract

Provided are an imine reductase mutant and a method for preparing a chiral hydrazine compound. The imine reductase mutant comprises: (a) a protein having a mutation based on the wild-type imine reductase as represented in SEQ ID NO: 1, wherein the mutation comprises a mutation at position A233; or (b) a protein having 70% or higher homology to the amino acid sequence defined in (a) and having imine reductase activity. The present invention can solve the problem in the prior art of low activity of wild-type imine reductase, and is suitable for the field of enzyme catalysis.

Description

Preparation method of imine reductase mutant and chiral hydrazine compound

[0001] This application is based on the Chinese application with CN application number 202410185492.X and application date February 19, 2024, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of enzyme catalysis, and in particular to a method for preparing an imine reductase mutant and a chiral hydrazine compound. Background Art

[0003] Chiral hydrazines are an important class of structural units that are widely present in various drugs, pesticides and natural products. Chiral hydrazines can also be used as chiral catalysts in organic synthesis and as important intermediates for the synthesis of heterocyclic compounds. Traditional methods for synthesizing chiral hydrazines mainly include racemic separation and asymmetric synthesis. Racemic separation is challenging due to the similarity between enantiomers and has the problem of low yield (usually less than 50%). Methods using metal catalysts or organic agents to catalyze the reduction of hydrazones to chiral hydrazines usually have the problems of using expensive metal catalysts or ligands, harsh reaction conditions, tedious protection and deprotection steps, and low optical purity of the products.

[0004] Compared to chemical synthesis, biocatalysis offers numerous advantages, such as mild reaction conditions, good enzyme stereoselectivity, and environmental friendliness. Consequently, it has been widely used in the industrial production of numerous pharmaceutical intermediates and fine chemicals. A large number of imine reductases or reductive aminases have been discovered and applied to the reduction of imines and the reductive amination of ketones or aldehydes to generate amines. However, due to the poor activity of wild-type imine reductases, product yields are low, making them difficult to meet the demands of industrial production.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for preparing an imine reductase mutant and a chiral hydrazine compound, so as to solve the problem of low activity of wild-type imine reductase in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, an imine reductase mutant is provided, comprising: (a) a protein mutated based on the wild-type imine reductase shown in SEQ ID NO: 1, wherein the mutation includes a mutation at position A233; or (b) a protein having greater than 70% homology to the amino acid sequence defined in (a) and having imine reductase activity.

[0008] Furthermore, the mutation occurring at the A233 site is A233C; preferably, the mutation also includes a mutation occurring at any one or more of the following sites: F215, F132, L92, A118, L134, I122, I119, M120, A218, T241, L181, V212, A251, G214, S68, G278, A194, V239, D243, S217, A220, M177, A211, V216 or Y219.

[0009] Furthermore, the mutation also includes mutations at any one or more of the following sites: A220S; L92Y or L92F; A118C or A118I; I122A; I119V; M120F or M120H; F132M, F132L, F132V, F132Q or F132L; L181M, L181I, L181V or L181R; F215M or F215L; A218S, A218I, A218R, A218L, A218V or A218M; V239S, V239M, V239G, V239C, V239I, V239F or V239R; T241C; G278 S; A194F, A194K, A194E, A194M, A194V, A194L, or A194I; V212L or V212I; A211S, A211R, or A211C; A251R, A251G, A251S, A251F, or A251C; G214R or G214C; S68A, S68C, S68K, or S68T; L134F or L134Y; D243R, D243L, D243V, D243C, D243M, or D243G; M177L; V216I or V216L; S217M, S217V, S217L, or S217W; Y219V.

[0010] Furthermore, the mutation includes any of the following amino acid mutations: A233C; A233C+L92Y; A233C+A118C; A233C+I122A; A233C+I119V; A233C+M120F; A233C+F132M; A233C+F132L; A233C+L181M; A233C+F215M; A233C+F215L; A233C+A218S; A233C+V239S; A233C+T241C; A233C+G278S; A233C+A194F; A233C+F215L+L92F; A233C+F215L+F132L; A233C+F 215L+F132M; A233C+F215L+F132V; A233C+F215L+F132Q; A233C+F215L+L18 1M; A233C+F215L+L181V; A233C+F215L+V212I; A233C+F215L+V239S; A233C +F215L+F132M+L181M; A233C+F215L+F132M+L181V; A233C+F215L+F132M+T 241C; A233C+F215L+F132M+A251R; A233C+F215L+F132M+A251G; A233C+F215 L+F132M+L92Y; A233C+F215L+F132M+L92F; A233C+F215L+F132M+L92F+A25 1R; A233C+F215L+F132M+L92F+A251S; A233C+F215L+F132M+L92F+A251F; A 233C+F215L+F132M+L92F+A251C; A233C+F215L+F132M+L92F+A251G; A233C +F215L+F132M+L92F+G214R; A233C+F215L+F132M+L92F+S68A; A233C+F215 L+F132M+L92F+S68C; A233C+F215L+F132M+L92F+S68K; A233C+F215L+F132 M+L92F+G278S; A233C+F215L+F132M+L92F+A118C; A233C+F215L+F132M+L9 2F+A118I; A233C+F215L+F132M+L92F+M120F; A233C+F215L+F132M+L92F+M 120H; A233C+F215L+F132M+L92F+L181V; A233C+F215L+F132M+L92F+L181M;A233C+F215L+F132M+L92F+L134Y;A233C+F215L+F132M+L92F+L134F;A233C+F215L+F132M+L92F+A194K;A233C+F215L+F132M+L92F+A194E;A233C+F215L+F132M+L92F+A118I+V239S;A233C+F215L+F132M+L92F+A118I+V239M;A233C+F215L+F132M+L92F+A118I+V239R;A233C+F215L+F132M+L92F+A118I+D243R;A233C+F215L+F132M+L92F+A118I+D243L;A233C+F215L+F132M+L92F+A118I+L134Y;A233C+F215L+F132M+L92F+A118I+L134F;A233C+F215L+F132M+L92F+A118I+L181M;A233C+F215L+F132M+L92F+A118I+G278S;A233C+F215L+F132M+L92F+A118I+A194M;A233C+F215L+F132M+L92F+A118I+A194V;A233C+F215L+F132M+L92F+A118I+A194L;A233C+F215L+F132M+L92F+A118I+G278S+D243V;A233C+F215L+F132M+L92F+A118I+G278S+D243L;A233C+F215L+F132M+L92F+A118I+G278S+D243R;A233C+F215L+F132M+L92F+A118I+G278S+D243C;A233C+F215L+F132M+L92F+A118I+G278S+L181M; A233C+F215L+F132M+L92F+A118I+G278S+L181R;A233C+F215L+F132M+L92F+A118I+G278S+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F;A233C+F215L+F132M+L92F+A118I+G278S+A194M;A233C+F215L+F132M+L92F+A118I+G278S+A194V;A233C+F215L+F132M+L92F+A118I+G278S+A194I;A233C+F215L+F132M+L92F+A118I+G278S+I119V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A220S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S68T;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134F;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134Y;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+M177L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+G214C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+G214C; A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217W;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S68T;A233C+F215L+F132M+L92F+A118I+G278S+A194I+Y219V; A233C+F215L +F132M+L92F+A118I+G278S+A194I+V239F; A233C+F215L+F132M+L92F+ A118I+G278S+A194I+V239I; A233C+F215L+F132M+L92F+A118I+G278S+ A194I+A211S; A233C+F215L+F132M+L92F+A118I+G278S+A194I+A211R. ;

[0011] Furthermore, the imine reductase mutants include proteins having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) and having imine reductase activity.

[0012] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which encodes any one of the above-mentioned imine reductase mutants.

[0013] In order to achieve the above object, according to the third aspect of the present invention, a recombinant vector is provided, wherein the recombinant vector is connected to the above DNA molecule.

[0014] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or the above recombinant vector.

[0015] Furthermore, the host cell includes a prokaryotic cell.

[0016] To achieve the above object, according to a fourth aspect of the present invention, a method for preparing a chiral hydrazine compound is provided, the method comprising: using any of the above-mentioned imine reductase mutants to catalyze a reduction reaction of a substrate ketone compound as shown in Formula I and a substrate hydrazine compound as shown in Formula II to obtain a chiral hydrazine compound as shown in Formula III;

[0017] The technical solution of the present invention is applied, and the above-mentioned imine reductase mutant is utilized. Compared with the wild-type enzyme, the above-mentioned imine reductase mutant has high enzyme activity, can significantly reduce the amount of enzyme used in the production process, thereby reducing production costs, and is suitable for industrial scale-up production. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0019] As mentioned in the background, wild-type imine reductases in the prior art have low activity, making them difficult to meet the requirements of industrial production. Therefore, in this application, the inventors attempted to develop an imine reductase mutant with higher enzymatic activity for use in industrial scale-up production. Based on this, a series of protection schemes are proposed in this application.

[0020] In a first typical embodiment of the present application, an imine reductase mutant is provided, comprising: (a) a protein mutated based on the wild-type imine reductase shown in SEQ ID NO: 1 (named SrIRED in this application), wherein the mutation includes a mutation at position A233; or (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having imine reductase activity.

[0021] SEQ ID NO: 1:

[0022] In this application, the wild-type imine reductase amino acid sequence shown in SEQ ID NO: 1 (source: Streptosporangium roseum, NCBI Gene Bank: WP_012887675.1) was modified through enzyme evolution to obtain an imine reductase mutant with enhanced activity by subjecting the enzyme to at least amino acid mutations at position A233. SEQ ID NO: 2 is the nucleotide sequence encoding the wild-type imine reductase.

[0023] SEQ IN NO: 2:

[0024] In a preferred embodiment, in the above (a), the mutation occurring at the A233 site is A233C; preferably, the mutation further includes a mutation occurring at any one or more of the following sites: F215, F132, L92, A118, L134, I122, I119, M120, A218, T241, L181, V212, A251, G214, S68, G278, A194, V239, D243, S217, A220, M177, A211, V216 or Y219.

[0025] In a preferred embodiment, the types of amino acids substituted at each position are independently selected from the following: A233C; L92Y or L92F; A118C or A118I; I122A; I119V; M120F or M120H; F132M, F132L, F132V, F132Q or F132L; L181M, L181I, L181V or L181R; F215M or F215L; A218S, A218I, A218R, A218L, A218V or A218M; V239S, V239M, V239G, V239C, V239I, V239F or V239R; T241C; G278S; A194F, A194 4K, A194E, A194M, A194V, A194L or A194I; V212L or V212I; A211S, A211R or A211C; A251R, A251G, A251S, A251F or A251C; G214R or G214C; S68A, S68C, S68K or S68T; L134F or L134Y; D243R, D243L, D243V, D243C, D243M or D243G; M177L; V216I or V216L; S217M, S217V, S217L or S217W; Y219V; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0026] In a preferred embodiment, the mutation further comprises mutations at any one or more of the following sites: A233C; A233C+L92Y; A233C+A118C; A233C+I122A; A233C+I119V; A233C+M120F; A233C+F132M; A233C+F132L; A233C+L181M; A233C+F215M; A233C+F215L; A233C+A218S; A233C+V239S; A233C+T241C; A233C+G278S; A233C+A194F; A233C+F215L+L92F; A233C+F215 L+F132L; A233C+F215L+F132M; A233C+F215L+F132V; A233C+F215L+F132Q ;A233C+F215L+L181M;A233C+F215L+L181V;A233C+F215L+V212I;A233C+ F215L+V239S; A233C+F215L+F132M+L181M; A233C+F215L+F132M+L181V; A 233C+F215L+F132M+T241C; A233C+F215L+F132M+A251R; A233C+F215L+F1 32M+A251G; A233C+F215L+F132M+L92Y; A233C+F215L+F132M+L92F; A233C +F215L+F132M+L92F+A251R; A233C+F215L+F132M+L92F+A251S; A233C+F2 15L+F132M+L92F+A251F; A233C+F215L+F132M+L92F+A251C; A233C+F215L +F132M+L92F+A251G; A233C+F215L+F132M+L92F+G214R; A233C+F215L+F1 32M+L92F+S68A; A233C+F215L+F132M+L92F+S68C; A233C+F215L+F132M+L 92F+S68K; A233C+F215L+F132M+L92F+G278S; A233C+F215L+F132M+L92F+ A118C; A233C+F215L+F132M+L92F+A118I; A233C+F215L+F132M+L92F+M12 0F; A233C+F215L+F132M+L92F+M120H; A233C+F215L+F132M+L92F+L181V;A233C+F215L+F132M+L92F+L181M; A233C+F215L+F132M+L92F+L134Y;A233C+F215L+F132M+L92F+L134F;A233C+F215L+F132M+L92F+A194K;A233C+F215L+F132M+L92F+A194E;A233C+F215L+F132M+L92F+A118I+V239S;A233C+F215L+F132M+L92F+A118I+V239M;A233C+F215L+F132M+L92F+A118I+V239R;A233C+F215L+F132M+L92F+A118I+D243R;A233C+F215L+F132M+L92F+A118I+D243L;A233C+F215L+F132M+L92F+A118I+L134Y;A233C+F215L+F132M+L92F+A118I+L134F;A233C+F215L+F132M+L92F+A118I+L181M;A233C+F215L+F132M+L92F+A118I+G278S;A233C+F215L+F132M+L92F+A118I+A194M;A233C+F215L+F132M+L92F+A118I+A194V;A233C+F215L+F132M+L92F+A118I+A194L;A233C+F215L+F132M+L92F+A118I+G278S+D243V;A233C+F215L+F132M+L92F+A118I+G278S+D243L;A233C+F215L+F132M+L92F+A118I+G278S+D243R;A233C+F215L+F132M+L92F+A118I+G278S+D243C;A233C+F215L+F132M+L92F+A118I+G278S+L181M;A233C+F215L+F132M+L92F+A118I+G278S+L181R;A233C+F215L+F132M+L92F+A118I+G278S+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F;A233C+F215L+F132M+L92F+A118I+G278S+A194M;A233C+F215L+F132M+L92F+A118I+G278S+A194V;A233C+F215L+F132M+L92F+A118I+G278S+A194I;A233C+F215L+F132M+L92F+A118I+G278S+I119V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A220S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S68T;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134F;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134Y;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181V; A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+M177L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+G214C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+G214C;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217W;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S68T; A233C+F215L+F132M+L92F+A 118I+G278S+A194I+Y219V; A233C+F215L+F132M+L92F+A118I+G278S+A194I+V239F; A233C+F215L+F132M+L92F+A118I+G278S+A194I+V239I; A233C+F215L+F132M+L92F+ A118I+G278S+A194I+A211S; A233C+F215L+F132M+L92F+A118I+G278S+A194I+A211R. ;

[0027] The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0028] or a protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more homology to the above amino acid sequence.

[0029] In a preferred embodiment, the imine reductase mutant comprises a protein having more than 75%, more than 80%, more than 85%, more preferably more than 95%, and further preferably more than 99% homology to the amino acid sequence defined in (a) and having imine reductase activity.

[0030] The above amino acid mutations were all experimentally explored in the examples of this application. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, they all have the activity of catalyzing the reaction of the substrate 3-cyclopentyl-3-oxopropionitrile with hydrazine hydrate to obtain chiral hydrazine compounds. The above mutation sites are all mutations made around the amino acid active site. Such mutations can improve the binding ability and / or catalytic ability of the mutant with the substrate. For mutations far away from the active site, the catalytic ability of the enzyme is less affected. Therefore, it is possible to obtain proteins with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more homology with the above amino acid sequence and the same catalytic activity.

[0031] The term "identity" used herein refers to the "homology" between amino acid sequences, that is, the total ratio of identical amino acid residues in an amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0032] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the protein provided by sequence (a), are homologous proteins obtained by amino acid mutations.

[0033] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0034] Substitution and replacement rules generally refer to the fact that amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0035] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0036] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0037] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0038] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0039] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0040] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model that can predict the conformation of protein complexes. Its predictions of protein 3D structures are very close to those observed in real experiments using instruments such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thus guiding the study of protein structure and activity.

[0041] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes any one of the above-mentioned imine reductase mutants.

[0042] In a third typical embodiment of the present application, a recombinant vector is provided, wherein the recombinant vector is connected to the above-mentioned DNA molecule.

[0043] The DNA can encode the imine reductase mutant and can be linked to a recombinant vector to form a circular DNA. Both the DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the imine reductase mutant.

[0044] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or recombinant vector.

[0045] In a preferred embodiment, the host cell comprises a prokaryotic cell; preferably, the prokaryotic cell comprises Escherichia coli; preferably, the Escherichia coli comprises BL21 (DE3). Preferably, the host cell is not a plant cell or an animal cell.

[0046] The host cells described above can replicate the recombinant vector within the host cells and transcribe and translate the DNA molecules carried by the recombinant vector, thereby obtaining a large number of imine reductase mutants. The imine reductase mutants can be obtained using whole cells or by fragmenting the host cells using existing technologies to purify the protein, obtain the crude enzyme after fragmentation, or other methods, and subsequently catalyze the substrate ketone. The host cells are not of plant or animal origin.

[0047] In a fifth typical embodiment of the present application, a method for preparing a chiral hydrazine compound is provided, the method comprising: providing a method for preparing a chiral hydrazine compound, the method comprising: using any of the above-mentioned imine reductase mutants to catalyze a reduction reaction of a substrate ketone compound as shown in Formula I and a substrate hydrazine compound as shown in Formula II to obtain a chiral hydrazine compound as shown in Formula III;

[0048] The substrate ketone compound includes 3-cyclopentyl-3-oxopropionitrile, and the substrate hydrazine compound includes hydrazine hydrate.

[0049] Preferably, the reaction temperature of the above preparation method is 10-40°C, including but not limited to 10, 15, 20, 25, 30, 35 or 40°C; preferably, the reaction time of the above preparation method is 0.1-24 hours, including but not limited to 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22 or 24 hours; preferably, the concentration of the substrate ketone compound in the above preparation method is 1 μM to 1000 mM, including but not limited to 1 μM, 2 μM, 3 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 2mM, 3mM, 5mM, 10mM, 20mM, 50mM, 100mM, 200mM, 500mM or 1000mM; preferably, the concentration of the substrate hydrazine compound in the above preparation method is 1μM to 1000mM, including but not limited to 1μM, 2μM, 3μM, 5μM, 10μM, 20μM, 50μM, 100μM, 200μM, 500μM, 1mM, 2mM, 3mM, 5mM, 10mM, 20mM, 50mM, 100mM, 200mM, 500mM or 1000mM. Preferably, the concentration of the imine reductase mutant (based on the wet cell crude enzyme solution) is 0.1 to 1000mg mL -1 , including but not limited to 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 70, 100, 150, 200, 300, 500, 700 or 1000 mg·mL -1 .

[0050] Ruxolitinib is a selective JAK1 / JAK2 tyrosine kinase inhibitor developed jointly by Incyte and Novartis. It was the first drug approved by the US FDA in 2011 for the treatment of myelofibrosis (trade name: Jakafi). Its indications are intermediate- and high-risk myelofibrosis, including primary myelofibrosis, secondary polycythemia vera myelofibrosis, and essential thrombocythemia myelofibrosis. Its trade name is Jakafi.

[0051] In the prior art, a racemate resolution method is used to synthesize this chiral hydrazine intermediate to further synthesize ruxolitinib. The substrate (E)-3-cyclopentyl acrylonitrile is subjected to an addition reaction with hydrazine hydrate to generate racemic 3-cyclopentyl-3-hydrazine propionitrile, which is then resolved using tartaric acid to obtain (R)-3-cyclopentyl-3-hydrazine propionitrile. However, as with the drawbacks of the racemate resolution method described above, this method has a low yield. In addition, (R)-3-cyclopentyl-3-hydrazine propionitrile can also be prepared by an enzymatic synthesis method, such as by using imine reductase SrIRED to catalyze the reaction of a substrate keto compound 3-cyclopentyl-3-oxopropionitrile with a substrate hydrazine compound hydrazine hydrate to prepare it. However, due to the poor activity of the imine reductase, the product yield is low, thereby limiting the commercial production of ruxolitinib intermediates synthesized by biological methods.

[0052] In the present application, any of the above-mentioned imine reductase mutants is used to react 3-cyclopentyl-3-oxopropionitrile with hydrazine hydrate in an aqueous or biphasic medium to generate a chiral hydrazine compound, ruxolitinib intermediate (R)-3-cyclopentyl-3-hydrazinylpropionitrile. When the imine reductase mutants in the present application are used to catalyze the reaction of 3-cyclopentyl-3-oxopropionitrile with hydrazine hydrate to prepare the ruxolitinib intermediate (R)-3-cyclopentyl-3-hydrazinylpropionitrile, compared with wild-type imine reductase and reductive amination enzyme, the imine reductase mutants in the present application have high enzyme activity and good stereoselectivity, and the product yield is significantly improved.

[0053] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0054] Example 1

[0055] 1. Using the wild-type imine reductase SrIRED as the parent, a mutant library was constructed by site-directed saturation mutagenesis. The constructed mutant library was then subjected to high-throughput activity screening using a 96-deep-well plate to obtain imine reductase mutants.

[0056] 1) Mutant Culture: 500 μL of LB medium was added to each well of a 96-well plate. Single colonies from the agar plate were inoculated into deep-well 96-well plates and cultured overnight at 37°C, 700 rpm. 100 μL of the overnight culture was transferred to another 96-well plate and 700 μL of LB medium was added to each well. The culture was cultured at 37°C, 700 rpm for 3 h. When the OD600 of the culture reached 0.6 to 0.8, 0.1 mM IPTG solution was added and the cells were induced overnight at 25°C, 200 rpm for approximately 16 h. The cells were centrifuged at 4000 rpm for 10 min, the supernatant discarded, and the whole cells were used for the reaction.

[0057] 2) 96-well plate high-throughput screening system: 475 μL of 200 mM hydrazine hydrate and 1 mM NADP were added to each well of the 96-well plate containing the bacterial sludge obtained above. + , 60 mM glucose monohydrate, 1 mg mL -1 Dissolve GDH enzyme (glutamate dehydrogenase) powder in 100mM Tris-HCl buffer, resuspend by vortexing, then add 25μL of 4M substrate 3-cyclopentyl-3-oxopropionitrile. Incubate on a shaker at 700 rpm at 20°C for 5 hours. Terminate the reaction by adding 500μL of acetonitrile, centrifuge, and remove the supernatant for analysis by LC-MS or HPLC.

[0058] 3) Mutant rescreening: Through the above mutant primary screening, mutants with improved activity were obtained. Induction culture was carried out in a 2L shake flask (optimal conditions for induction expression: 25°C, 0.1mM IPTG overnight induction), and after centrifugation to obtain bacterial sludge, the crude enzyme solution was obtained by ultrasonic cell disruption, and then the crude enzyme solution was used for rescreening. The screening conditions were: 2mL reaction system, 20mM substrate 3-cyclopentyl-3-oxopropionitrile, 200mM hydrazine hydrate, 1mg·mL -1 GDH, 1 mM NADP + , 60 mM glucose monohydrate, 100 mg mL -1 The crude enzyme solution from wet cells was added to a 100 mM Tris-HCl solution containing 5% DMSO and incubated at 20°C on a shaker at 200 rpm for 5 hours. After the reaction, 2 mL of acetonitrile was added to terminate the reaction. After centrifugation, the supernatant was collected and analyzed by LC-MS or HPLC.

[0059] 2. Construction of saturation mutant library The whole plasmid PCR amplification method was used. After obtaining the PCR product, it was digested with DpnI enzyme to remove the template in the PCR product and then transformed into Escherichia coli BL21 (DE3).

[0060] Example 2

[0061] Based on the parent SEQ ID NO: 1, saturation mutagenesis at specific sites was performed to construct a mutant library. The mutant library was then subjected to high-throughput activity screening using a 96-well plate. The mutants with improved activity were subjected to combinatorial mutagenesis. After induction culture of the mutants with improved activity in 2L shake flasks, the catalytic activity of the mutants was tested under the following reaction conditions: 2mL reaction system, 20mM substrate 3-cyclopentyl-3-oxopropionitrile, 200mM hydrazine hydrate, 1mg·mL -1GDH, 1mM NADP+, 60mM glucose monohydrate, 100mg mL -1 The crude enzyme solution of wet cells was added to a 100mM Tris-HCl solution containing 5% DMSO and the reaction was carried out in a constant temperature shaker at 200 rpm at 20°C for 5 hours. After the reaction was completed, 2mL of acetonitrile was added to the system to terminate the reaction. After centrifugation, the supernatant was collected and the yield was analyzed by LC-MS or HPLC. Take 100μL of the reaction system, add 100μL of 1M NaHCO3 solution, add 400μL of 5mg·mL -1 Marfey reagent (CAS: 95713-52-3) was placed at 50° C. for 2 hours, 100 μL of 1 M HCl solution was added, and the ee value of the reaction was detected by HPLC.

[0062] The ee value refers to the enantiomeric excess, which is used to describe the relative abundance of chiral isomers in the product. In this application, ee = ((R)-3-cyclopentyl-3-hydrazinepropionitrile - (S)-3-cyclopentyl-3-hydrazinepropionitrile) ÷ ((R)-3-cyclopentyl-3-hydrazinepropionitrile + (S)-3-cyclopentyl-3-hydrazinepropionitrile). The units in the formula can be the proportion of the enantiomeric mixture, or the concentration, or the peak area, or other parameters. The ee value can be calculated by unifying the units in the formula.

[0063] Yield = (actual amount of product produced / theoretical amount of product produced) × 100%.

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

[0065] Table 1

[0066] Example 3

[0067] The optimal mutant A233C+F215L obtained in Example 2 was used as the parent for the next round of saturation mutagenesis and combined mutagenesis. The mutants with improved activity were initially screened and cultured in 2 L shake flasks. Activity screening was performed according to the following reaction conditions: 2 mL reaction system, 20 mM substrate 3-cyclopentyl-3-oxopropionitrile, 200 mM hydrazine hydrate, 1 mg mL -1 GDH, 1 mM NADP + , 60 mM glucose monohydrate, 100 mg mL -1The crude enzyme solution from wet cells was added to a 100 mM Tris-HCl solution containing 5% DMSO and incubated at 200 rpm at 20°C for 5 hours. After completion of the reaction, 2 mL of acetonitrile was added to terminate the reaction. After centrifugation, the supernatant was collected and analyzed for yield by HPLC. The reaction system was derivatized with Marfey's reagent to determine the ee value. The results are shown in Table 2.

[0068] Table 2

[0069] Example 4

[0070] The optimal mutant A233C+F215L+F132M+L92F obtained in Example 3 was used as the parent, and the next round of saturation mutagenesis and combined mutagenesis were performed. After the mutants with improved activity were initially screened and cultured in 2L shake flasks, the activity screening was performed according to the following reaction conditions: 2mL reaction system, 20mM substrate 3-cyclopentyl-3-oxopropionitrile, 200mM hydrazine hydrate, 1mg·mL -1 GDH, 1 mM NADP + , 60 mM glucose monohydrate, 100 mg mL -1 The crude enzyme solution from wet cells was added to a 100 mM Tris-HCl solution containing 5% DMSO and incubated at 200 rpm at 20°C for 5 hours. After completion of the reaction, 2 mL of acetonitrile was added to terminate the reaction. After centrifugation, the supernatant was collected and analyzed for yield by HPLC. The reaction system was derivatized with Marfey's reagent to determine the ee value. The results are shown in Table 3.

[0071] Table 3

[0072] Example 5

[0073] The optimal mutant A233C+F215L+F132M+L92F+A118I+G278S obtained in Example 4 was used as the parent, and the next round of saturation mutagenesis and combined mutagenesis were performed. After the mutants with improved activity were initially screened and cultured in a 2L system, the activity screening was performed according to the following reaction conditions: 2mL reaction system, 20mM substrate 3-cyclopentyl-3-oxopropionitrile, 200mM hydrazine hydrate, 1mg·mL -1 GDH, 1 mM NADP + , 60 mM glucose monohydrate, 100 mg mL -1The crude enzyme solution from wet cells was added to a 100 mM Tris-HCl solution containing 5% DMSO and incubated at 200 rpm at 20°C for 5 hours. After completion of the reaction, 2 mL of acetonitrile was added to terminate the reaction. After centrifugation, the supernatant was collected and analyzed for yield by HPLC. The reaction system was derivatized with Marfey's reagent to determine the ee value. The results are shown in Table 4.

[0074] Table 4

[0075] From the above description, it can be seen that the above-mentioned embodiments of the present application have achieved the following technical effects: after multiple rounds of evolution, the present application has obtained a series of imine reductase mutants with high activity and good stereoselectivity compared to wild-type imine reductase, therefore when the imine reductase mutants obtained by the present application are used to catalyze the reaction of 3-cyclopentyl-3-oxopropionitrile and hydrazine hydrate to synthesize chiral hydrazine compounds, catalytic efficiency has been improved, yield is improved, and the stereoselectivity of the reaction is good, and the target product (R) -3-cyclopentyl-3-hydrazine-propionitrile with higher purity can be obtained, while the output of the corresponding isomer (S) -3-cyclopentyl-3-hydrazine-propionitrile is very little, which is convenient for subsequent purification. In the technical scheme of the present application, the reaction steps of enzyme synthesis are simple, the usage amount of enzyme can be greatly reduced, and the pollution of organic solvent to the environment is reduced, thereby reducing production costs and being environmentally friendly, and being suitable for industrialized amplification production.

[0076] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An imine reductase mutant, characterized in that The imine reductase mutants include: (a) a protein comprising a mutation based on the wild-type imine reductase shown in SEQ ID NO: 1, wherein the mutation comprises a mutation at position A233; or (b) A protein having 70% or more homology to the amino acid sequence defined in (a) and having imine reductase activity.

2. The imine reductase mutant according to claim 1, characterized in that The mutation at the A233 site is A233C; Preferably, the mutation further includes a mutation occurring at any one or more of the following sites: F215, F132, L92, A118, L134, I122, I119, M120, A218, T241, L181, V212, A251, G214, S68, G278, A194, V239, D243, S217, A220, M177, A211, V216 or Y219.

3. The imine reductase mutant according to claim 2, characterized in that The mutation also includes mutations at any one or more of the following sites: A220S; L92Y or L92F; A118C or A118I; I122A; I119V; M120F or M120H; F132M, F132L, F132V, F132Q or F132L; L181M, L181I, L181V, or L181R; F215M or F215L; A218S, A218I, A218R, A218L, A218V, or A218M; V239S, V239M, V239G, V239C, V239I, V239F, or V239R; T241C; G278S; A194F, A194K, A194E, A194M, A194V, A194L, or A194I; V212L or V212I; A211S, A211R, or A211C; A251R, A251G, A251S, A251F, or A251C; G214R or G214C; S68A, S68C, S68K or S68T; L134F or L134Y; D243R, D243L, D243V, D243C, D243M, or D243G; M177L; V216I or V216L; S217M, S217V, S217L, or S217W; Y219V.

4. The imine reductase mutant according to claim 1, characterized in that The mutation includes any of the following amino acid mutations: A233C;A233C+L92Y;A233C+A118C;A233C+I122A;A233C+I119V;A233C+M120F;A233C+F132M;A233C+F132L;A233C+L181M;A233C+F215M;A233C+F215L;A233C+A218S;A233C+V239S;A233C+T241C;A233C+G278S;A233C+A194F;A233C+F215L+L92F;A233C+F215L+F132L;A233C+F215L+F132M;A233C+F215L+F132V;A233C+F215L+F132Q;A233C+F215L+L181M;A233C+F215L+L181V;A233C+F215L+V212I;A233C+F215L+V239S;A233C+F215L+F132M+L181M;A233C+F215L+F132M+L181V;A233C+F215L+F132M+T241C;A233C+F215L+F132M+A251R;A233C+F215L+F132M+A251G;A233C+F215L+F132M+L92Y;A233C+F215L+F132M+L92F;A233C+F215L+F132M+L92F+A251R;A233C+F215L+F132M+L92F+A251S;A233C+F215L+F132M+L92F+A251F;A233C+F215L+F132M+L92F+A251C;A233C+F215L+F132M+L92F+A251G;A233C+F215L+F132M+L92F+G214R;A233C+F215L+F132M+L92F+S68A;A233C+F215L+F132M+L92F+S68C;A233C+F215L+F132M+L92F+S68K;A233C+F215L+F132M+L92F+G278S;A233C+F215L+F132M+L92F+A118C;A233C+F215L+F132M+L92F+A118I;A233C+F215L+F132M+L92F+M120F;A233C+F215L+F132M+L92F+M120H;A233C+F215L+F132M+L92F+L181V;A233C+F215L+F132M+L92F+L181M;<h2 style=";text-align:left;direction:ltr">A233C+F215L+F132M+L92F+L134Y;A233C+F215L+F132M+L92F+L134F;A233C+F215L+F132M+L92F+A194K;A233C+F215L+F132M+L92F+A194E;A233C+F215L+F132M+L92F+A118I+V239S;A233C+F215L+F132M+L92F+A118I+V239M;A233C+F215L+F132M+L92F+A118I+V239R; A233C+F215L+F132M+L92F+A118I+D243R;A233C+F215L+F132M+L92F+A118I+D243L;A233C+F215L+F132M+L92F+A118I+L134Y;A233C+F215L+F132M+L92F+A118I+L134F;A233C+F215L+F132M+L92F+A118I+L181M;A233C+F215L+F132M+L92F+A118I+G278S;A233C+F215L+F132M+L92F+A118I+A194M;A233C+F215L+F132M+L92F+A118I+A194V;A233C+F215L+F132M+L92F+A118I+A194L;A233C+F215L+F132M+L92F+A118I+G278S+D243V;A233C+F215L+F132M+L92F+A118I+G278S+D243L;A233C+F215L+F132M+L92F+A118I+G278S+D243R;A233C+F215L+F132M+L92F+A118I+G278S+D243C;A233C+F215L+F132M+L92F+A118I+G278S+L181M;A233C+F215L+F132M+L92F+A118I+G278S+L181R;A233C+F215L+F132M+L92F+A118I+G278S+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F;A233C+F215L+F132M+L92F+A118I+G278S+A194M;A233C+F215L+F132M+L92F+A118I+G278S+A194V;A233C+F215L+F132M+L92F+A118I+G278S+A194I;A233C+F215L+F132M+L92F+A118I+G278S+I119V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A220S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+D243R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+A211S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+S68T;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134F;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L134Y;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181I; A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181V;A233C+F215L+F132M+L92F+A118I+G278S+A194F+L181M;A233C+F215L+F132M+L92F+A118I+G278S+A194F+M177L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+G214C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239R;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239G;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239C;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239S;A233C+F215L+F132M+L92F+A118I+G278S+A194F+V239I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V216L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+G214C;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217V;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S217W;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V212I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218R;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218M;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A218L;A233C+F215L+F132M+L92F+A118I+G278S+A194I+S68T;A233C+F215L+F132M+L92F+A118I+G278S+A194I+Y219V;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V239F;A233C+F215L+F132M+L92F+A118I+G278S+A194I+V239I;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A211S;A233C+F215L+F132M+L92F+A118I+G278S+A194I+A211R。; 5. The imine reductase mutant according to any one of claims 1 to 4, characterized in that The imine reductase mutants include proteins having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) and having imine reductase activity.

6. A DNA molecule, characterized in that The DNA molecule encodes the imine reductase mutant according to any one of claims 1 to 5.

7. A recombinant vector, characterized in that The recombinant vector is connected to the DNA molecule according to claim 6.

8. A host cell, characterized in that The host cell contains the DNA molecule according to claim 6 or the recombinant vector according to claim 7.

9. The host cell according to claim 8, characterized in that The host cells include prokaryotic cells.

10. A method for preparing a chiral hydrazine compound, characterized in that: The preparation method comprises: using the imine reductase mutant according to any one of claims 1 to 5 to catalyze a reduction reaction of a substrate ketone compound as shown in formula I and a substrate hydrazine compound as shown in formula II to obtain the chiral hydrazine compound as shown in formula III;

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