Transaminase mutants and their use

Abstract

The present invention provides a transaminase mutant and its use. The transaminase mutant includes: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; (b) a protein having transaminase activity function, in which at least one of the following sites in the amino acid sequence of (a) is mutated: V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107, or L449; and (c) a protein having 80% or more homology with the amino acid sequence defined by any of (a) and (b), and having transaminase function. The problem of the prior art that transaminases have poor activity or resistance to catalyze the industrial production of large sterically hindered chiral amine compounds can be solved.

Description

Technical Field

[0001] This application claims priority based on a Chinese application with Chinese Patent Application No. 202211538995.8 and a filing date of December 2, 2022, and the disclosure content of the Chinese patent application is incorporated into this application as a whole again.

[0002] The present invention relates to the field of enzyme catalysis, and specifically to a transaminase mutant and its use.

Background Art

[0003] Chiral amines are structural units of many important biologically active molecules and are key intermediates in the synthesis of many chiral drugs. Currently, mainly three methods are used for the synthesis of chiral amines, including chemical methods, biological resolution methods, and biological asymmetric synthesis methods. Among them, chemical methods have drawbacks such as a long reaction route, harsh conditions, the use of toxic transition metal catalysts for synthesis, low stereoselectivity of products, and low yields. The theoretically maximum yield of the biological resolution method is only 50%, and both methods have certain limitations for scale-up production.

[0004] The asymmetric synthesis of chiral amines catalyzed by transaminases has attracted increasing attention due to advantages such as high selectivity, high conversion rate, mild reaction conditions, and environmental friendliness, and is currently a widely used method for the production of chiral amines. However, most wild-type transaminases have a limited substrate range, and it is often difficult to synthesize large steric hindrance chiral amine compounds (which means that the substituent adjacent to the carbonyl of the catalytic substrate is larger than methyl). CN114875006A discloses transaminase mutants that can efficiently synthesize chiral amines, especially large steric hindrance chiral amines. However, when these mutants are applied in industrialization, their stability is relatively poor, and it is difficult to cope with extreme environments such as high temperature and high-concentration organic solvents in the scale-up process.

[0005] Transaminases can be improved through enzyme evolution. Cao, J. et al. modified the (R) type transaminases derived from Aspergillus tereus for thermal stability, constructing 13 mutants through site-directed mutation. Experiments confirmed that three mutants (E133Q, D224K, and E253A) were advantageous for improving the enzyme's thermal stability, with mutant D224K showing a 4.23-fold improvement in stability at 40°C (Front.Chem.2021,9:664156). Furthermore, Cai, B. et al. constructed an enzymatic process for the efficient production of (R)-α-phenylethylamine using (R) type transaminases derived from Aspergillus fumigatus Af293. The mutants obtained through directed evolution showed more than a 3000-fold increase in activity and significantly improved resistance to isopropylamine (2M), reaching industrial-scale production of 168g L. -1 d -1 This has resulted in high yields of (R)-α-phenylethylamine (Org. Process Res. Dev. 2022, 26, 7, 2004-2012).

[0006] Therefore, to better adapt to the industrial production requirements for the synthesis of highly sterically hindered chiral amines, the stability of transaminases under extreme conditions can be improved through methods of enzyme evolution. This will further promote the immobilization and continuous use of transaminases, improve production efficiency, reduce industrial production costs, and decrease the discharge of industrial wastewater, exhaust, and solid waste. [Overview of the project] [Problems that the invention aims to solve]

[0007] The main objective of the present invention is to provide a transaminase mutant and its use that can solve the problem of poor transaminase activity or resistance during the process of catalyzing the industrial production of highly sterically hindered chiralamine compounds in the prior art. [Means for solving the problem]

[0008] To achieve the above objective, according to the first aspect of the present invention, (a) A protein having the amino acid sequence shown in Sequence ID No. 1, or (b) The amino acid sequence of (a) has undergone an amino acid mutation at least one of the following sites: V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107, or L449, and the protein has transaminase activity. (c) Provides a transaminase mutant comprising a protein having 80% or more homology to an amino acid sequence limited by either (a) or (b) and possessing transaminase function.

[0009] Furthermore, (b) the amino acid mutations are, independently of each other, V315T, V315R, V315D, V315A, V315K, or V315H; I91Q, I91M, I91N, I91G, I91F, or I91D; V124C, V124S, V124Y, V124P, V124M, V124A, or V124I; Y116F, Y116Y, Y116A, Y116R, Y116S, Y116 G, Y116H, or Y116L; A286I, A286R, A286Q, A286F, A286D, A286M, or A286I; C418L, C418W, C418R, C418D, C418F, or C418A; T87A, T87Y, T87N, T87V, T87F, or T87E; S301A, S301G, S301H, S301K, S301W, S301I, S301M, or S3 01N;S27A, S27V, S27N, S27M, S27E, S27R, or S27;S101A, S101R, S101Y, or S101H;R34H, R34K, R34N, R34T, R34F, or R34M;T117V, T117M, T117L, T117T, T117R, or T117S;T285M, T285Y, T285I, T285A, or T285G;T107S, T1 Selected from 07Q, T107A, or T107G, where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid, preferably (c) is a protein having 85% or more, preferably 90% or more, more preferably 95% or more, homology to the amino acid sequence limited in (a) or (b), and having transaminase function.

[0010] Furthermore, the transaminase mutant includes one of the following amino acid mutations.

[0011] V315T、V315R、V315D、V315A、V315K、V315H、V315T+I91Q、V315T+I91M、V315T+I91N、V315T+I91G、V315T+I91F、V315T+I91D、V315T+V124C、V315T+V124S、V315T+V124Y、V315T+V124P、V315T+V124M、V315T+V124A、V315T+V124C+I91N、V315T+V124C+I91Q、V315T+V124C+I91M、V315T+V124C+I91G、V315T+V124C+I91F、V315T+I91Q+V124S、V315T+I91Q+V124I、V315T+I91Q+V124P、V315T+I91Q+V124M、V315T+I91Q+V124A、V315T+I91Q+V124Y、V315T+I91Q+V124S+Y116F、V315T+I91Q+V124S+Y116Y、V315T+I91Q+V124S+Y116A、V315T+I91Q+V124S+Y116R、V315T+V124C+I91N+Y116F、V315T+V124C+I91N+Y116A、V315T+V124C+I91N+Y116S、V315T+V124C+I91N+Y116G、V315T+V124C+I91N+Y116H、V315T+V124C+I91N+Y116L、V315T+V124C+I91N+Y116F+A286I、V315T+V124C+I91N+Y116F+A286R、V315T+V124C+I91N+Y116F+A286Q、V315T+V124C+I91N+Y116F+A286F、V315T+V124C+I91N+Y116F+A286D、V315T+V124C+I91N+Y116F+A286M、V315T+V124C+I91N+Y116F+A286I+C418L、V315T+V124C+I91N+Y116F+A286I+C418W、V315T+V124C+I91N+Y116F+A286I+C418R、V315T+V124C+I91N+Y116F+A286I+C418D、V315T+V124C+I91N+Y116F+A286I+C418F、V315T+V124C+I91N+Y116F+A286I+C418A、V315T+V124C+I91N+Y116F+A286I+T87A、V315T+V124C+I91N+Y116F+A286I+T87Y、V315T+V124C+I91N+Y116F+A286I+T87N、V315T+V124C+I91N+Y116F+A286I+T87V、V315T+V124C+I91N+Y116F+A286I+T87F、V315T+V124C+I91N+Y116F+A286I+T87E、V315T+V124C+I91N+Y116F+A286I+T87A+S301A、V315T+V124C+I91N+Y116F+A286I+T87A+S301G、V315T+V124C+I91N+Y116F+A286I+T87A+S301H、V315T+V124C+I91N+Y116F+A286I+T87A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S301W、V315T+V124C+I91N+Y116F+A286I+T87A+S27A、V315T+V124C+I91N+Y116F+A286I+T87A+S27V、V315T+V124C+I91N+Y116F+A286I+T87A+S27N、V315T+V124C+I91N+Y116F+A286I+T87A+S27M、V315T+V124C+I91N+Y116F+A286I+T87A+S27E、V315T+V124C+I91N+Y116F+A286I+T87A+S27R、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R, V315T+V124C+I91N+Y116F+A286 I+T87A+S27A+S101A+R34H+T117S, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V +T285M, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y, V315T+V124C+I91 N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I, V315T+V124C+I91N+Y116F+A286I+T87A+S27 A+S101A+R34H+T117V+T285A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T2 85G, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S, V315T+V124C+I91N+Y 116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S 101A+R34H+T117V+T107A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G. ,

[0012] To achieve the above objective, a second aspect of the present invention provides a DNA molecule encoding the above-mentioned transaminase mutant.

[0013] 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.

[0014] To achieve the above objective, according to a fourth aspect of the present invention, host cells transformed with the above-mentioned recombinant plasmid are provided.

[0015] To achieve the above objective, according to a fifth aspect of the present invention, a method for producing a chiral amine compound is provided, comprising the step of using the above-mentioned transaminase mutant to carry out an amino group transfer reaction with a ketone substrate represented by formula I under the action of an amino donor to obtain a chiral amine compound.

[0016] [ka] (Ar1 is selected from primary substituted aryl, primary unsubstituted aryl, substituted arylene or unsubstituted arylene, substituted heteroarylene or unsubstituted heteroarylene; Ar2 is selected from secondary substituted aryl, secondary unsubstituted aryl, substituted cycloalkyl or unsubstituted cycloalkyl, alkyl or alkylene; R is selected from H, alkyl, alkylene or alkylidene; the number of carbon atoms in alkyl, alkylene or alkylidene is selected from 1 to 5; alkyl, alkylene or alkylidene is substituted alkyl, alkylene, alkylidene or unsubstituted) The compound comprises an alkyl, alkylene, or alkylidene, where R is selected from alkyl, alkylene, or alkylidene. In this case, the alkyl, alkylene, or alkylidene is linked to Ar1 and / or Ar2 to form a ring, and the substituents in the primary substituted aryl, substituted arylene, substituted heteroarylene, secondary substituted aryl, or substituted alkyl, alkylene, or alkylidene are independently selected from halogen, hydroxy, amino, methyl, ethyl, or -CH2CH2OH, and the heteroatom in the substituted heteroarylene is selected from N, O, or S.

[0017] Furthermore, the substituents of the first substituted aryl, second substituted aryl, or substituted arylene are each independently selected from halogens.

[0018] Furthermore, each substituent is independently located at one or more of the ortho, meta, or para positions of the primary substituted aryl, secondary substituted aryl, or substituted arylene.

[0019] Furthermore, the halogen is selected from F, Cl or Br.

[0020] Furthermore, Ar1 is selected from unsubstituted heteroarylene, Ar2 is selected from secondary-substituted aryl, the substituent of the secondary-substituted aryl is selected from halogen, R is selected from substituted alkylene, the number of carbon atoms of the substituted alkylene is selected from 1 to 5, the substituent is selected from hydroxy, and the substituted alkylene is linked to Ar1 to form a ring.

[0021] Furthermore, Ar1 is selected from primary-substituted aryl or primary-unsubstituted aryl, the substituent of the primary-substituted aryl is selected from methyl or Cl, Ar2 is selected from unsubstituted cycloalkyl, alkyl or secondary-unsubstituted aryl, the number of carbon atoms of the unsubstituted cycloalkyl is selected from 3 to 5, the alkyl is selected from isopropyl or ethyl, R is selected from alkyl, and the alkyl is selected from isopropyl, methyl or ethyl.

[0022] Furthermore, Ar1 is selected from primary-substituted aryl or primary-unsubstituted aryl, the substituent of the primary-substituted aryl is selected from methyl or -CH2CH2OH, Ar2 is selected from secondary-unsubstituted aryl or alkyl, the alkyl is selected from methyl or isopropyl, R is selected from substituted alkylene, the number of carbon atoms of the substituted alkylene is selected from 1 to 5, the substituent is selected from hydroxy, and the substituted alkylene is linked to Ar1 to form a ring.

[0023] Furthermore, the ketone substrate is selected from the following.

[0024]

Chemical formula

[0025] Using the technical solution of the present invention, a transaminase mutant derived from Chromobacterium violaceum (SEQ ID NO: 1) is used as the parent plant, and protein engineering modifications such as saturation mutations and combination mutations are performed to obtain a transaminase mutant in which both activity and resistance are greatly improved in reactions catalyzed by highly sterically hindered chiral amines. [Modes for carrying out the invention]

[0026] The embodiments and features of the embodiments described herein may be combined with each other without contradiction. The present invention will be described in detail below with reference to the embodiments.

[0027] Explanation of terms: Large sterically hindered chiral compounds: In this application, this refers to ketone compounds in which the group adjacent to the prochiral carbonyl group is larger than methyl. The group larger than methyl may be ethyl, propyl, tert-butyl, or phenyl tert-butyl, etc.

[0028] As mentioned in the background technology section, chemical synthesis and biological resolution methods for chiral amines have various drawbacks. However, synthesizing chiral amines using biological asymmetric methods can achieve higher efficiency while also resulting in milder reaction conditions. However, since most wild-type transaminases have a limited substrate range, when synthesizing highly sterically hindered chiral amine compounds, transaminases often suffer from low activity and poor tolerance, making scale-up production impossible.

[0029] Therefore, in this application, the inventors attempt to modify transaminases by enzyme evolution, thereby improving the properties of transaminases under extreme conditions and increasing production efficiency in industrial production, and thus propose the series of protective measures of this application.

[0030] In a first representative embodiment of the present application, a transaminase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having transaminase activity, in which at least one of the amino acid sequences V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107, or L449 of the amino acid sequence of (a) has undergone an amino acid mutation; and (c) a protein having 80% or more homology to an amino acid sequence limited by either (a) or (b) and having transaminase function.

[0031] The amino acid sequence shown in Sequence ID No. 1 above is a transaminase mutant derived from Chromobacterium violaceum. Computer simulations performing homology modeling of this amino acid sequence analyzed the model structure, and molecular docking with different sterically hindrance ketone compounds revealed 19 amino acid residues, including S27, V31, R34, M64, A74, R77, T87, I91, S101, Y116, T117, V124, N151, L213, T285, A286, V315, C418, and L449. These amino acid sites may affect the catalytic activity and stability of the protein. By mutating these amino acid sites, a protein with transaminase function, or a protein with further enhanced transaminase function, can be obtained. The obtained protein may also have non-essential mutation sites or active sites altered, thereby yielding a protein with over 80% homology to the above amino acid sequence and possessing transaminase function.

[0032] The sequence for sequence number 1 is as follows: MQKQRTCSQWRELDAAHHLHPFTDTASLNQVGARVMTRGEGVYLWDCEGNKIIDGMAGAWCVNMGYGRKDFAEAARRQMEELSFMHTADGITHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDCMIRMVRRYWDVQGKPEKKTLIGRWNGYSGSTIGGASLSGFKEMHEQGDLPIPGVAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGAIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYQPIGAVSVGKRVAEGLIAGGVFYHGHTTSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETLSRFEHVDDVRGVGMLAAFTLVKNKAKRELFPDFGEIGSLCRDIFVRNNLIMDICGDHIVAAPPLVMTRAEVDEMLAVAERCLEELEQSLKARGLA。

[0033] In one preferred embodiment, the amino acid mutations in (b) are, independently, V315T, V315R, V315D, V315A, V315K, or V315H; I91Q, I91M, I91N, I91G, I91F, or I91D; V124C, V124S, V124Y, V124P, V124M, V124A, or V124I; Y116F, Y116Y, Y116A, Y116R, Y11 6S, Y116G, Y116H, or Y116L; A286I, A286R, A286Q, A286F, A286D, A286M, or A286I; C418L, C418W, C418R, C418D, C418F, or C418A; T87A, T87Y, T87N, T87V, T87F, or T87E; S301A, S301G, S301H, S301K, S301W, S301I, S301M , or S301N; S27A, S27V, S27N, S27M, S27E, S27R, or S27; S101A, S101R, S101Y, or S101H; R34H, R34K, R34N, R34T, R34F, or R34M; T117V, T117M, T117L, T117T, T117R, or T117S; T285M, T285Y, T285I, T285A, or T285G; T107 Selected from S, T107Q, T107A, or T107G, where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid, preferably (c) is a protein having 85% or more, preferably 90% or more, more preferably 95% or more, homology to the amino acid sequence limited in (a) or (b), and having transaminase function.

[0034] As used herein, the abbreviations for amino acid residues are 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).

[0035] The general rule for substitutions and exchanges is that if the properties of amino acids are similar, the effect after the exchange will also be similar. For example, in the homologous proteins mentioned above, the exchange of conserved amino acids can occur. "Exchange of conserved amino acids" includes, but is not limited to, the following. Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are substituted with other hydrophobic amino acids. Hydrophobic amino acids with thick side chains (Phe, Tyr, Trp) can be substituted with other hydrophobic amino acids with thick side chains. Amino acids with positively charged side chains (Arg, His, Lys) can be substituted with other amino acids with positively charged side chains. Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) can be substituted with other amino acids with polar, uncharged side chains.

[0036] Those skilled in the art can also perform conservative amino acid replacements according to amino acid replacement rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0037] In this application, the applicant, through continued investigation of the active site, discovered that when the active site is mutated to a different amino acid, the activity of the corresponding protein also differs, and that specific mutations enhance transaminase activity. Tests revealed that by applying the specific mutations described above to the active site, a protein with enhanced activity can be obtained. The amino acid mutation sites of the transaminase protein can be flexibly selected and combined from the above mutations.

[0038] In one preferred embodiment, the mutation in the transaminase mutant includes one of the following amino acid mutations: V315T、V315R、V315D、V315A、V315K、V315H、V315T+I91Q、V315T+I91M、V315T+I91N、V315T+I91G、V315T+I91F、V315T+I91D、V315T+V124C、V315T+V124S、V315T+V124Y、V315T+V124P、V315T+V124M、V315T+V124A、V315T+V124C+I91N、V315T+V124C+I91Q、V315T+V124C+I91M、V315T+V124C+I91G、V315T+V124C+I91F、V315T+I91Q+V124S、V315T+I91Q+V124I、V315T+I91Q+V124P、V315T+I91Q+V124M、V315T+I91Q+V124A、V315T+I91Q+V124Y、V315T+I91Q+V124S+Y116F、V315T+I91Q+V124S+Y116Y、V315T+I91Q+V124S+Y116A、V315T+I91Q+V124S+Y116R、V315T+V124C+I91N+Y116F、V315T+V124C+I91N+Y116A、V315T+V124C+I91N+Y116S、V315T+V124C+I91N+Y116G、V315T+V124C+I91N+Y116H、V315T+V124C+I91N+Y116L、V315T+V124C+I91N+Y116F+A286I、V315T+V124C+I91N+Y116F+A286R、V315T+V124C+I91N+Y116F+A286Q、V315T+V124C+I91N+Y116F+A286F、V315T+V124C+I91N+Y116F+A286D、V315T+V124C+I91N+Y116F+A286M、V315T+V124C+I91N+Y116F+A286I+C418L、V315T+V124C+I91N+Y116F+A286I+C418W、V315T+V124C+I91N+Y116F+A286I+C418R、V315T+V124C+I91N+Y116F+A286I+C418D、V315T+V124C+I91N+Y116F+A286I+C418F、V315T+V124C+I91N+Y116F+A286I+C418A、V315T+V124C+I91N+Y116F+A286I+T87A、V315T+V124C+I91N+Y116F+A286I+T87Y、V315T+V124C+I91N+Y116F+A286I+T87N、V315T+V124C+I91N+Y116F+A286I+T87V、V315T+V124C+I91N+Y116F+A286I+T87F、V315T+V124C+I91N+Y116F+A286I+T87E、V315T+V124C+I91N+Y116F+A286I+T87A+S301A、V315T+V124C+I91N+Y116F+A286I+T87A+S301G、V315T+V124C+I91N+Y116F+A286I+T87A+S301H、V315T+V124C+I91N+Y116F+A286I+T87A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S301W、V315T+V124C+I91N+Y116F+A286I+T87A+S27A、V315T+V124C+I91N+Y116F+A286I+T87A+S27V、V315T+V124C+I91N+Y116F+A286I+T87A+S27N、V315T+V124C+I91N+Y116F+A286I+T87A+S27M、V315T+V124C+I91N+Y116F+A286I+T87A+S27E、V315T+V124C+I91N+Y116F+A286I+T87A+S27R、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T、V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R, V315T+V124C+I91N+Y116F+A286 I+T87A+S27A+S101A+R34H+T117S, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V +T285M, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y, V315T+V124C+I91 N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I, V315T+V124C+I91N+Y116F+A286I+T87A+S27 A+S101A+R34H+T117V+T285A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T2 85G, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S, V315T+V124C+I91N+Y 116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S 101A+R34H+T117V+T107A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G. ,

[0039] All of the above amino acid mutations were experimentally investigated in the examples of this application, and all of them possess transaminase activity. Compared to the parent plant having the amino acid sequence shown in SEQ ID NO: 1, transaminase mutants are obtained that are more stable, more resistant to extreme environments, have higher enzyme activity, and can be used for industrial scale-up production.

[0040] A second representative embodiment of the present application provides a DNA molecule encoding the above-mentioned transaminase mutant.

[0041] A third representative embodiment of the present application provides a recombinant plasmid in which the above-mentioned DNA molecules are linked.

[0042] The above DNA can encode the transaminase mutant described above, and can also be ligated to a recombinant plasmid to form circular DNA. Both the above DNA and the recombinant plasmid can be transcribed and translated by RNA polymerase, ribosomes, tRNA, etc., to obtain the above transaminase mutant.

[0043] A fourth representative embodiment of the present invention provides a host cell transformed with the above-described recombinant plasmid. The host cell may be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell may be Escherichia coli, and the eukaryotic cell may be yeast.

[0044] Using the host cells described above, it is possible to copy recombinant plasmids within the host cells, transcribe and translate the DNA molecules contained in the recombinant plasmids, and obtain a large number of transaminase mutants. Using conventional techniques, transaminase mutants can be obtained by lysing the host cells and purifying the proteins, or by catalyzing them with crude enzymes after lysing, or by other methods, after which amine compounds can be catalyzed. These host cells are non-plant derived host cells.

[0045] A fifth representative embodiment of the present application provides a method for producing a chiral amine compound, comprising the step of using the above-mentioned transaminase mutant to perform an amino group transfer reaction on a ketone substrate represented by formula I under the action of an amino donor to obtain a chiral amine compound. Ar1 is selected from a first-substituted aryl, a first-unsubstituted aryl, a substituted arylene or unsubstituted arylene, a substituted heteroarylene or unsubstituted heteroarylene; Ar2 is selected from a second-substituted aryl, a second-unsubstituted aryl, a substituted cycloalkyl or unsubstituted cycloalkyl, an alkyl or alkylene; R is selected from H, an alkyl, an alkylene or alkylidene; the number of carbon atoms of the alkyl, alkylene or alkylidene is selected from 1 to 5; and the alkyl, alkylene or alkylidene is a substituted alkyl, alkylene, alkylidene or unsubstituted The material comprises an alkyl, alkylene, or alkylidene, where R is selected from alkyl, alkylene, or alkylidene, the alkyl, alkylene, or alkylidene is linked to Ar1 and / or Ar2 to form a ring, and the substituents in the primary substituted aryl, substituted arylene, substituted heteroarylene, secondary substituted aryl, or substituted alkyl, alkylene, or alkylidene are each independently selected from halogen, hydroxy, amino, methyl, ethyl, or -CH2CH2OH, and the heteroatom in the substituted heteroarylene is selected from N, O, or S.

[0046] [ka]

[0047] By the above manufacturing method, the above transaminase mutant is used to perform an amino group transfer reaction on a ketone compound represented by formula I under the action of an amino donor to obtain a chiral amine compound. The above transaminase mutant can perform chiral catalysis on the above ketone compound and synthesize the required highly sterically hindered chiral amine. The activity and tolerance of the transaminase are greatly improved, enabling it to exert catalytic activity under industrial production conditions such as extreme environments, thereby improving production efficiency and reducing industrial production costs.

[0048] In one preferred embodiment, the substituents of the first substituted aryl, second substituted aryl, or substituted arylene are each independently selected from halogens.

[0049] In one preferred embodiment, the substituents are independently located at one or more of the ortho, meta, or para positions of the first substituted aryl, second substituted aryl, or substituted arylene.

[0050] In one preferred embodiment, the halogen is selected from F, Cl, or Br.

[0051] In one preferred embodiment, Ar1 is selected from an unsubstituted heteroarylene, Ar2 is selected from a secondary substituted aryl, the substituent of the secondary substituted aryl is selected from a halogen, R is selected from a substituted alkylene, the carbon number of the substituted alkylene is selected from 1 to 5, the substituent is selected from hydroxy, and the substituted alkylene is linked to Ar1 to form a ring.

[0052] In one preferred embodiment, Ar1 is selected from a first substituted aryl or a first unsubstituted aryl, the substituent of the first substituted aryl is selected from methyl or Cl, Ar2 is selected from an unsubstituted cycloalkyl, alkyl or second unsubstituted aryl, the number of carbon atoms of the unsubstituted cycloalkyl is selected from 3 to 5, the alkyl is selected from isopropyl or ethyl, R is selected from alkyl, the alkyl is selected from isopropyl, methyl or ethyl.

[0053] In one preferred embodiment, Ar1 is selected from a first substituted aryl or a first unsubstituted aryl, the substituent of the first substituted aryl is selected from methyl or -CH2CH2OH, Ar2 is selected from a second unsubstituted aryl or alkyl, the alkyl is selected from methyl or isopropyl, R is selected from a substituted alkylene, the number of carbon atoms of the substituted alkylene is selected from 1 to 5, the substituent is selected from hydroxy, and the substituted alkylene is linked to Ar1 to form a ring.

[0054] In one preferred embodiment, the ketone compound is selected from the following:

[0055] [ka]

[0056] In this application, the inventors perform homology modeling on screened mutants based on the analyzed three-dimensional structure of the protein (PDB: 4BA5), perform molecular docking with different sterically hindrance ketone compounds based on the model structure, and modify 19 residues that may affect the catalytic activity and stability of the protein, guided by the above strategies for improving protein stability. These residues include S27, V31, R34, M64, A74, R77, T87, I91, S101, Y116, T117, V124, N151, L213, T285, A286, V315, C418, and L449. The protein modification methods include saturated mutations and combined mutations.

[0057] Saturated mutation is a method of modifying the gene encoding a target protein to obtain a mutant in which the amino acid at the target site is replaced with one of 19 other amino acids in a short time. This method is not only a powerful tool for targeted protein modification but also an important means of studying the relationship between protein structure and function. Saturated mutations often yield more ideal evolutionary forms than single-point mutations. These problems that site-directed mutagenesis cannot solve are precisely what saturated mutagenesis excels at. Saturated mutations were constructed using whole-plasmid PCR, the PCR products were digested with DPNI enzyme to remove the template, and transformed into E. coli BL21(DE3). Since high-throughput screening methods are often required for mutant screening, the following method for screening mutant resistance was developed.

[0058] We developed the following high-throughput screening method for screening mutant libraries.

[0059] 1. Culture of mutants: 300 μL of LB medium was added to each well of a 96-well plate. A single clone from an agar plate was inoculated into a deep-well 96-well plate and cultured overnight at 37°C and 200 rpm. Using Qpix, the overnight cultured bacterial suspension was transferred to a 96-well plate containing 800 μL of LB medium per well. After culturing at 37°C and 200 rpm for 5 hours, when the OD600 of the bacterial suspension in the 96-well plate reached 0.6-0.9, IPTG solution was added to the 96-well plate again using Qpix to bring the final IPTG concentration in the well plate to 0.1 mM. Induction was carried out overnight at 25°C and 200 rpm for approximately 16 hours. The cells were centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the whole cells were used for the reaction.

[0060] High-throughput screening system for 2.96-well plates: 8 μL of PLP mother liquor (1 mg / mL), 3.5 μL of 6 M isopropylamine hydrochloride (20 eq), and 0.1 M Tris-Cl 9.0 (replenishment up to 100 μL) were homogeneously mixed. The mixed sample was dispensed into a 96-well plate with bacterial slime in each well. Finally, the substrate mother liquor (0.3 mg of substrate dissolved in methanol of different concentrations) was added and homogeneously mixed. The mixture was reacted at a constant temperature of 50°C for 18 hours using a shaker at 700 rpm. Next, three times the volume of methanol was added per well, the mixture was centrifuged, and the supernatant sample was sent to a UPLC for analysis.

[0061] Preliminary screening of the above mutants yielded mutants with improved properties. Next, the optimal mutants were induced in a 2L shaking flask (optimal conditions for induction expression: 25°C, 0.2 mM IPTG overnight), centrifuged to obtain bacterial sediment, and then the cells were sonicated to obtain a crude enzyme solution. Finally, a g-grade scale reaction was performed to confirm the properties of the optimal mutants. Since single-site saturation mutations often do not show a clear improvement, it is usually necessary to perform iterative saturation mutagenesis multiple times to obtain mutants with significantly improved properties.

[0062] Based on saturated mutations to obtain mutants with improved activity, beneficial amino acid sites obtained through screening can be combined to obtain mutants with superior properties. The method for constructing double-point mutations in combination mutations is the same as for single-point mutations, using whole-plasmid PCR. For multiple-point mutations, where two or more sites are mutated simultaneously, overlap extension PCR amplification is used to obtain mutant genes containing multiple point mutations. After digesting both ends with restriction enzymes, these genes are ligated into expression vectors, transformed into E. coli cells, spread in LB culture dishes containing 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain combination mutants. After the above combination mutants were accurately identified by sequencing, the above 2L shaking flask induction culture and reaction verification were performed in the same manner as above.

[0063] Through multiple evolutionary cycles, a series of transaminase mutants were obtained, and these mutants were found to exhibit significantly improved activity and tolerance during the catalysis of highly sterically hindered chiral amine reactions. These mutants proved to be highly suitable for the industrial production of highly sterically hindered chiral amines.

[0064] The present invention will be described in more detail below with reference to specific examples, but these examples should not be understood as limiting the scope of protection of this application.

[0065] In Examples 1-4 below, the change in enzyme stability is represented by the conversion rate at various methanol concentrations and temperatures.

[0066] Example 1 Based on the parental seed sequence number 1, two saturation mutations were performed. Specific mutation sites are shown in the table below, and the catalytic activity of the mutants was detected according to the reaction conditions described below.

[0067] Each 1 mL reaction system contained 30 mg of substrate 1 or substrate 2, 300 μL of ethanol, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 300 μL of crude enzyme solution (prepared from 30 mg of wet bacterial mud), and a pH 8.0 100 mM phosphate buffer. The reaction was carried out at 40°C for 18 hours.

[0068] The detection results are shown in the table below.

[0069] [Table 1] Note: In the table above, + indicates a conversion rate of less than 10%, ++ indicates a conversion rate of 10% or more and 20% or less, +++ indicates a conversion rate of 20% or more and 30% or less, ++++ indicates a conversion rate of 30% or more and 40% or less, +++++ indicates a conversion rate of 40% or more and 50% or less, and ++++++ indicates a conversion rate of 50% or more and 60% or less.

[0070] Example 2 Based on Example 1, saturated mutations and combined mutations were subsequently performed, and activity screening for the mutations was carried out according to the following reaction conditions.

[0071] Each 1 mL reaction system contained 30 mg of substrate 1 or substrate 2, 400 μL of ethanol, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 300 μL of crude enzyme solution (prepared from 30 mg of wet bacterial mud), and a pH 8.0 100 mM phosphate buffer. The reaction was carried out at 40°C for 18 hours.

[0072] The results are shown in the table below.

[0073] [Table 2] Note: In the table above, + indicates a conversion rate of less than 10%, ++ indicates a conversion rate of 10% or more and 20% or less, +++ indicates a conversion rate of 20% or more and 30% or less, ++++ indicates a conversion rate of 30% or more and 40% or less, +++++ indicates a conversion rate of 40% or more and 50% or less, and ++++++ indicates a conversion rate of 50% or more and 60% or less.

[0074] Example 3 Based on Example 2, multiple saturation mutations were performed, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0075] Each 1 mL reaction system contained 30 mg of substrate 1 or substrate 2, 500 μL of methanol, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 150 μL of crude enzyme solution (prepared from 15 mg of wet bacterial mud), and a pH 8.0 100 mM phosphate buffer. The reaction was carried out at 40°C for 18 hours.

[0076] The results are shown in the table below.

[0077] [Table 3] Note: In the table above, + indicates a conversion rate of less than 10%, ++ indicates a conversion rate of 10% or more and 20% or less, +++ indicates a conversion rate of 20% or more and 30% or less, ++++ indicates a conversion rate of 30% or more and 40% or less, +++++ indicates a conversion rate of 40% or more and 50% or less, and ++++++ indicates a conversion rate of 50% or more and 60% or less.

[0078] Example 4 Based on Example 3, multiple saturation mutations were subsequently performed, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0079] Each 1 mL reaction system contained 50 mg of substrate 1 or substrate 2, 600 μL of methanol, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 100 μL of crude enzyme solution (prepared from 10 mg of wet bacterial mud), and a pH 8.0 100 mM phosphate buffer. The reaction was carried out at 50°C for 18 hours.

[0080] [Table 4-1]

[0081] [Table 4-2] Note: In the table above, + indicates a conversion rate of less than 10%, ++ indicates a conversion rate of 10% or more and 20% or less, +++ indicates a conversion rate of 20% or more and 30% or less, ++++ indicates a conversion rate of 30% or more and 40% or less, +++++ indicates a conversion rate of 40% or more and 50% or less, and ++++++ indicates a conversion rate of 50% or more and 60% or less.

[0082] Example 5 Some of the mutants obtained in the above examples were selected, and the stability of the enzyme was tested after treatment with high temperature and different organic solvents. The reaction was then carried out according to the following conditions.

[0083] Each 1 mL reaction system contained 50 mg of substrate 1 or substrate 2, 1 mg of PLP, 2 mg of isopropylamine hydrochloride, 100 μL of crude enzyme solution (prepared from 10 mg of wet bacterial mud) treated at 70°C for 1 hour, or treated with methanol, ethanol, acetonitrile, ethyl acetate, dichloromethane, methyl t-butyl ether, and n-heptane for 1 hour each (the control was the same volume of untreated enzyme solution), and a pH 8.0 100 mM phosphate buffer. The reaction was carried out at 40°C for 18 hours.

[0084] The results are shown in the table below.

[0085] [Table 5]

[0086] [Table 6]

[0087] [Table 7]

[0088] [Table 8] Note: 1) In the table above, relative residual activity refers to the percentage difference between the enzyme activity measured in an enzyme solution appropriately treated under extreme conditions such as high temperature and organic solvents, and the 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 that the enzyme is more stable under those conditions. 2) In contrast, * This indicates that the activity is 0.1 to 0.5 times higher than that of the parent species. ** This indicates that the activity is 0.5 to 1.0 times higher than that of the parent species. *** This indicates that the activity is 1.0 to 1.5 times higher than that of the parent species. **** This indicates that the activity level is 1.5 to 2 times higher than that of the parent species. 3) + indicates that the relative residual activity is 10% or more but less than 30%, ++ indicates that the relative residual activity is 30% or more but less than 50%, +++ indicates that the relative residual activity is 50% or more but less than 70%, and ++++ indicates that the relative residual activity is 70% or more but less than 90%.

[0089] From the description of the above example, the following can be understood. 1) The mutant obtained in this invention exhibits significantly improved stability at high temperatures. 2) Regarding resistance to organic solvents, in addition to a clear improvement in resistance to methanol, resistance to the six commonly used organic solvents listed above has improved to varying degrees. 3) Furthermore, the above control experiments show that, in addition to improved stability of the mutants, enzyme activity also improved to varying degrees due to the evolution of the enzyme.

[0090] Example 6 In a 250 mL four-necked flask, 20 mL of 100 mmol / L phosphate buffer, 20 mL of 6 mol / L isopropylamine hydrochloride solution (2 vol), and 60 mL of methanol were added at room temperature to adjust the pH to 8.5-9.0. Next, 0.1 g of pyridoxal phosphate was added.

[0091] [ka] 5g of (substrate 1) was added and mixed uniformly. 2mL of enzyme solution (0.5g wet bacterial mud / mL enzyme solution) of a transaminase mutant (V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M) mutated based on SEQ ID NO: 1 was added, and the pH was adjusted to 8.5-9.0. The mixture was heated to 50°C and stirred to allow the reaction to proceed. After the reaction was complete, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50mL of methyl t-butyl ether. The aqueous phase was adjusted to pH 12 and then extracted twice with 50mL of methyl t-butyl ether. The organic phase was dried over anhydrous magnesium sulfate and concentrated under conditions of T<40°C and P≦-0.06Mpa until no fraction remained. Target product

[0092] [ka] I obtained it.

[0093] HPLC detection revealed a purity of >99%, a DE value of >99%, and a yield of 85%.

[0094] Example 7 In a 250 mL four-necked flask, 20 mL of 100 mmol / L phosphate buffer, 20 mL of 6 mol / L isopropylamine hydrochloride solution (2 vol), and 60 mL of methanol were added at room temperature to adjust the pH to 8.5-9.0. Next, 0.1 g of pyridoxal phosphate was added.

[0095] [ka] 5g of (substrate 2) was added and mixed uniformly. 2mL of enzyme solution (0.5g wet bacterial mud / mL enzyme solution) of a transaminase mutant (V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M) mutated based on SEQ ID NO: 1 was added, and the pH was adjusted to 8.5-9.0. The mixture was heated to 50°C and stirred to allow the reaction to proceed. After the reaction was complete, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50mL of methyl t-butyl ether. The aqueous phase was adjusted to pH 12 and then extracted twice with 50mL of methyl t-butyl ether. The organic phase was dried over anhydrous magnesium sulfate and concentrated under conditions of T<40°C and P≦-0.06Mpa until no fraction remained. Target product

[0096] [ka] I obtained it.

[0097] HPLC detection revealed a purity of >99%, a DE value of >99%, and a yield of 84%.

[0098] Example 8 In a 250 mL four-necked flask, 20 mL of 100 mmol / L phosphate buffer, 20 mL of 6 mol / L isopropylamine hydrochloride solution (2 vol), and 60 mL of methanol were added at room temperature to adjust the pH to 8.5-9.0. 0.1 g of pyridoxal phosphate was added.

[0099] [ka] 5g of (substrate 16) was added and mixed uniformly. 2mL of enzyme solution (0.5g wet bacterial mud / mL enzyme solution) of a transaminase mutant (V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M) mutated based on SEQ ID NO: 1 was added, and the pH was adjusted to 8.5-9.0. The mixture was heated to 50°C and stirred to allow the reaction to proceed. After the reaction was complete, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50mL of methyl t-butyl ether. The aqueous phase was adjusted to pH 12 and then extracted twice with 50mL of methyl t-butyl ether. The organic phase was dried over anhydrous magnesium sulfate and concentrated under conditions of T<40°C and P≦-0.06Mpa until no fraction remained. Target product

[0100] [ka] I obtained it.

[0101] HPLC detection revealed a purity of >99%, a DE value of >99%, and a yield of 75%.

[0102] Example 9 In a 250 mL four-necked flask, 20 mL of 100 mmol / L phosphate buffer, 20 mL of 6 mol / L isopropylamine hydrochloride solution (2 vol), and 60 mL of methanol were added at room temperature to adjust the pH to 8.5-9.0. 0.1 g of pyridoxal phosphate was added.

[0103] [ka] 5g of (substrate 17) was added and mixed uniformly. 2mL of enzyme solution (0.5g wet bacterial mud / mL enzyme solution) of a transaminase mutant (V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M) mutated based on SEQ ID NO: 1 was added, and the pH was adjusted to 8.5-9.0. The mixture was heated to 50°C and stirred to allow the reaction to proceed. After the reaction was complete, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50mL of methyl t-butyl ether. The aqueous phase was adjusted to pH 12 and then extracted twice with 50mL of methyl t-butyl ether. The organic phase was combined and dried over anhydrous magnesium sulfate, then concentrated under conditions of T<40°C and P≦-0.06Mpa until no fraction remained. Target product

[0104] [ka] I obtained it.

[0105] HPLC detection results show purity >99%, DE value >99%, and yield 80%.

[0106] Example 10 Using an enzyme solution of a transaminase mutant (V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M) mutated based on Sequence ID No. 1, catalytic reactions were carried out with substrates 3-15, referring to the catalytic synthesis steps of Examples 6-9, and the results are shown in the table below.

[0107] [Table 9] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects. 1) Through the evolution of transaminases, mutants with improved activity and stability can be obtained, enabling the synthesis of various highly sterically hindered chiral amines under high temperature and high organic solvent conditions without the need for heavy metal catalysts or toxic reagents, thus realizing green chemistry. 2) This transaminase mutant exhibits high biocatalytic activity, enzyme stability, high reaction substrate concentration, high product yield, significant reduction in wastewater, exhaust, and solid waste, and thus saves production costs.

[0108] The foregoing describes only preferred embodiments of the present invention and does not limit it, and various modifications and changes are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made in the spirit and principles of the present invention are all within the scope of protection of the present invention.

Claims

1. A transaminase mutant having a sequence in which the amino acid sequence shown in Sequence ID No. 1 contains one of the following amino acid mutations. V315T, V315R, V315D, V315A, V315K, V315H, V315T+I91Q, V315T+I91M, V315T+I91N, V315T+I91G, V315T+I91F, V315T+I91D, V315T+V124C, V315T+V124S, V315T+V124Y, V315T+V124P, V315T+V124M, V315T+V124A, V315T+V124C+I91N, V315T+V124C+I91Q, V315T+V124C+I91M, V315T+V124C+I91G, V315T+V124C+I91F, V315T+I91Q+V124S, V315T+I91Q+V124I, V315T+I91Q+V124P, V315T+I91Q+V124M, V315T+I91Q+V124A, V315T+I91Q+V124Y, V315T+I91Q+V124S+Y116F, V315T+I91Q+V124S+Y116Y, V315T+I91Q+V124S+Y116A, V315T+I91Q+V124S+Y116R, V315T+V124C+I91N+Y116F, V315T+V124C+I91N+Y116A, V315T+V124C+I91N+Y116S, V315T+V124C+I91N+Y116G, V315T+V124C+I91N+Y116H, V315T+V124C+I91N+Y116L, V315T+V124C+I91N+Y116F+A286I, V315T+V124C+I91N+Y116F+A286R, V315T+V124C+I91N+Y116F+A286Q, V315T+V124C+I91N+Y116F+A286F, V315T+V124C+I91N+Y116F+A286D, V315T+V124C+I91N+Y116F+A286M, V315T+V124C+I91N+Y116F+A286I+C418L, V315T+V124C+I91N+Y116F+A286I+C418W, V315T+V124C+I91N+Y116F+A286I+C418R, V315T+V124C+I91N+Y116F+A286I+C418D, V315T+V124C+I91N+Y116F+A286I+C418F、 V315T+V124C+I91N+Y116F+A286I+C418A、 V315T+V124C+I91N+Y116F+A286I+T87A、 V315T+V124C+I91N+Y116F+A286I+T87Y、 V315T+V124C+I91N+Y116F+A286I+T87N、 V315T+V124C+I91N+Y116F+A286I+T87V、 V315T+V124C+I91N+Y116F+A286I+T87F、 V315T+V124C+I91N+Y116F+A286I+T87E、 V315T+V124C+I91N+Y116F+A286I+T87A+S301A、 V315T+V124C+I91N+Y116F+A286I+T87A+S301G、 V315T+V124C+I91N+Y116F+A286I+T87A+S301H、 V315T+V124C+I91N+Y116F+A286I+T87A+S301K、 V315T+V124C+I91N+Y116F+A286I+T87A+S301W、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A、 V315T+V124C+I91N+Y116F+A286I+T87A+S27V、 V315T+V124C+I91N+Y116F+A286I+T87A+S27N、 V315T+V124C+I91N+Y116F+A286I+T87A+S27M、 V315T+V124C+I91N+Y116F+A286I+T87A+S27E、 V315T+V124C+I91N+Y116F+A286I+T87A+S27R、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V、 V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117S, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285G, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107A, V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G.

2. A DNA molecule characterized by encoding the transaminase mutant described in claim 1.

3. A recombinant plasmid characterized by having the DNA molecule described in claim 2 linked to it.

4. A host cell characterized by being transformed with the recombinant plasmid described in claim 3.

5. A method for producing a chiral amine compound, characterized by comprising the step of using the transaminase mutant described in claim 1 to perform an amino group transfer reaction with a ketone substrate represented by formula I under the action of an amino donor to obtain a chiral amine compound. 【Chemistry 1】 (Ar1 is selected from the first substituted aryl, the first unsubstituted aryl, substituted arylene or unsubstituted arylene, substituted heteroarylene or unsubstituted heteroarylene, Ar2 is selected from a secondary substituted aryl, a secondary unsubstituted aryl, a substituted cycloalkyl or unsubstituted cycloalkyl, an alkyl or alkylene. R is selected from H, alkyl, alkylene or alkylidene, the number of carbon atoms of the alkyl, alkylene or alkylidene is selected from 1 to 5, and the alkyl, alkylene or alkylidene includes substituted alkyl, substituted alkylene, substituted alkylidene or unsubstituted alkyl, unsubstituted alkylene or unsubstituted alkylidene. When R is selected from alkyl, alkylene or alkylidene, the alkyl, alkylene or alkylidene is linked to Ar1 and / or Ar2 to form a ring. The substituents in the first substituted aryl, the substituted arylene, the substituted heteroarylene, the second substituted aryl, or the substituted alkyl, alkylene, or alkylidene are each independently halogen, hydroxy, amino, methyl, ethyl, or -CH ２ CH ２ Selected from OH, The heteroatom in the substituted heteroarylene is selected from N, O, or S.

6. The manufacturing method according to claim 5, characterized in that the substituents of the first substituted aryl, the second substituted aryl, or the substituted arylene are each independently selected from the halogens.

7. The manufacturing method according to claim 6, characterized in that each substituent is independently located at one or more of the ortho, meta, or para positions of the first substituted aryl, the second substituted aryl, or the substituted arylene.

8. The manufacturing method according to claim 7, characterized in that the halogen is selected from F, Cl, or Br.

9. The Ar1 is selected from the unsubstituted heteroarylenes, The Ar2 is selected from the second substituted aryl, and the substituent of the second substituted aryl is selected from the halogen. The R is selected from the substituted alkylenes, the number of carbon atoms in the substituted alkylene is selected from 1 to 5, and the substituent is selected from hydroxyl. The manufacturing method according to claim 8, characterized in that the substituted alkylene is linked to the Ar1 to form a ring.

10. The Ar1 is selected from the first substituted aryl or the first unsubstituted aryl, and the substituent of the first substituted aryl is selected from methyl or Cl. The Ar2 is selected from the unsubstituted cycloalkyl, alkyl, or second unsubstituted aryl, the number of carbon atoms in the unsubstituted cycloalkyl is selected from 3 to 5, and the alkyl is selected from isopropyl or ethyl. The manufacturing method according to claim 8, characterized in that R is selected from the alkyl groups, and the alkyl group is selected from isopropyl, methyl, or ethyl.

11. The Ar1 is selected from the first substituted aryl or the first unsubstituted aryl, and the substituent of the first substituted aryl is methyl or -CH ２ CH ２ Selected from OH, The Ar2 is selected from the second unsubstituted aryl or alkyl, and the alkyl is Selected from methyl or isopropyl, The R is selected from substituted alkylenes, the number of carbon atoms in the substituted alkylene is selected from 1 to 5, and the substituent is selected from hydroxyl. The manufacturing method according to claim 8, characterized in that the substituted alkylene is linked to the Ar1 to form a ring.

12. The manufacturing method according to claim 8, characterized in that the ketone substrate is selected from the following. 【Chemistry 2】

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