Transaminase mutants and uses thereof

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 from Chinese patent application No. 202211538995.8, filed on December 2, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to the field of enzyme catalysis, and in particular to transaminase mutants and uses thereof. [Background technology]

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

[0004] The asymmetric synthesis of chiral amines catalyzed by transaminases has attracted increasing attention due to its advantages such as high selectivity, high conversion, mild reaction conditions, and environmental friendliness, and has now become a widely used method for producing chiral amines. However, most wild-type transaminases have a limited substrate range, and it is often difficult to synthesize large sterically hindered chiral amine compounds (meaning that the substituent next to the carbonyl of the catalytic substrate is larger than methyl). CN114875006A discloses transaminase mutants that can efficiently synthesize chiral amines, especially large sterically hindered chiral amines. However, these mutants have relatively poor stability when applied in industrialization, and are difficult to cope with extreme environments such as high temperatures and high concentrations of organic solvents in scale-up processes.

[0005] The resistance of transaminase can be improved by enzyme evolution. Cao, J. et al. modified the (R)-type transaminase from Aspergillus terreus in terms of thermostability, constructed 13 mutants by site-directed mutagenesis, and confirmed through experiments that three mutants (E133Q, D224K, and E253A) were favorable for improving the thermostability of the enzyme, among which, the stability of mutant D224K at 40 °C is improved by 4.23 times (Front. Chem. 2021, 9: 664156.). In addition, Cai, B. et al. used the (R)-type transaminase from Aspergillus fumigatus Af293 to construct an enzyme process for the efficient production of (R)-α-phenylethylamine. The mutant obtained by directed evolution showed an activity improvement of more than 3000 times and a significant improvement in the tolerance to isopropylamine (2M), with an industrial test scale of 168 g L -1 d -1 and achieved high production of (R)-α-phenylethylamine (Org. Process Res. Dev. 2022, 26, 7, 2004-2012).

[0006] Therefore, in order to better meet the industrial production requirements for the synthesis of highly sterically hindered chiral amines, the stability of transaminases under extreme conditions can be improved through the method of enzyme evolution, which further promotes the immobilization and continuous use of transaminases, improves production efficiency, reduces industrial production costs, and reduces the discharge of industrial wastewater, gas and solid waste. Summary of the Invention [Problem to be solved by the invention]

[0007] The main object of the present invention is to provide a transaminase mutant and its use, which can solve the prior art problem of poor activity or resistance of transaminase during the process of catalyzing the industrial production of large sterically hindered chiral amine compounds. [Means for solving the problem]

[0008] In order to achieve the above object, according to a first aspect of the present invention, (a) a protein having the amino acid sequence set forth in SEQ ID NO:1, or (b) a protein having transaminase activity, in which at least one of the sites V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107 or L449 in the amino acid sequence of (a) has been subjected to amino acid mutation; (c) a protein having 80% or more homology to the amino acid sequence defined by either (a) or (b) and having transaminase function.

[0009] Further, the targeted amino acid mutations in (b) are, each 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, 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 07Q, T107A, or T107G, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid, and preferably, (c) has a homology of 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the amino acid sequence defined in (a) or (b), and is a protein having transaminase function.

[0010] Further, the mutation of the transaminase mutant may include any 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 object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above transaminase mutant.

[0013] To achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid in which the above DNA molecule is ligated.

[0014] To achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell transformed with the above recombinant plasmid.

[0015] In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing a chiral amine compound, comprising the steps of: utilizing the above transaminase mutant to carry out a transamination reaction on 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 a first substituted aryl, a first unsubstituted aryl, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; Ar2 is selected from a second substituted aryl, a second unsubstituted aryl, a substituted or unsubstituted cycloalkyl, an alkyl, or an alkylene; R is selected from H, an alkyl, an alkylene, or an 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 selected from a substituted alkyl, alkylene, alkylidene, or an unsubstituted alkyl, alkylene, or alkylidene. R includes an alkyl, alkylene, or alkylidene, and when R is selected from an alkyl, alkylene, or alkylidene, the alkyl, alkylene, or alkylidene is linked to Ar1 and / or Ar2 to form a ring, and the substituents on the first substituted aryl, substituted arylene, substituted heteroarylene, second substituted aryl, or substituted alkyl, alkylene, or alkylidene are each independently selected from halogen, hydroxy, amino, methyl, ethyl, or -CHCHOH, and the heteroatom on the substituted heteroarylene is selected from N, O, or S.

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

[0018] Further, the substituents are each independently located at any one or more of the ortho, meta, or para positions of the first substituted aryl, the second substituted aryl, or the substituted arylene.

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

[0020] Further, Ar1 is selected from unsubstituted heteroarylene, Ar2 is selected from a second substituted aryl, a substituent of the second substituted aryl is selected from halogen, 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.

[0021] Further, Ar1 is selected from a first substituted aryl or a first unsubstituted aryl, and a substituent of the first substituted aryl is selected from methyl or Cl; Ar2 is selected from an unsubstituted cycloalkyl, an alkyl or a second unsubstituted aryl, and the carbon number of the unsubstituted cycloalkyl is selected from 3 to 5, and the alkyl is selected from isopropyl or ethyl; R is selected from an alkyl, and the alkyl is selected from isopropyl, methyl or ethyl.

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

[0023] Additionally, the ketone-based substrate is selected from:

[0024] [ka]

[0025] Using the technical solution of the present invention, a transaminase mutant (SEQ ID NO: 1) derived from Chromobacterium violaceum is used as the parent species, and protein engineering modifications such as saturation mutation and combinatorial mutation are carried out to obtain a transaminase mutant with greatly improved activity and tolerance in the reaction catalyzing a highly sterically hindered chiral amine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In addition, the embodiments and features of the embodiments of the present application may be combined with each other without causing any contradiction.

[0027] Explanation of terms: Largely hindered chiral compound: as used herein, refers to a ketone-based compound in which the group next to the prochiral carbonyl is larger than methyl, which may be ethyl, propyl, tert-butyl, or phenyl tert-butyl, etc.

[0028] As mentioned in the background art, the chemical synthesis and biological resolution methods of chiral amines have various drawbacks, but by synthesizing chiral amines using biological asymmetric methods, higher efficiency can be achieved and the reaction conditions are milder. However, most wild-type transaminases have a limited substrate range, so when synthesizing large sterically hindered chiral amine compounds, the transaminases often suffer from low activity and poor tolerance, making it impossible to scale up production.

[0029] Therefore, in this application, the inventors attempt to modify transaminase by the method of enzyme evolution, thereby improving the properties of transaminase under extreme conditions and improving the production efficiency in industrial production, and thus propose a series of protection schemes in this application.

[0030] In a first exemplary embodiment of the present application, there is provided a transaminase mutant comprising: (a) a protein having an amino acid sequence as set forth 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) has been subjected to an amino acid mutation: V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107, or L449; or (c) a protein having 80% or more homology to an amino acid sequence defined by any one of (a) and (b), and having transaminase function.

[0031] The amino acid sequence shown in SEQ ID NO: 1 above is a transaminase mutant derived from Chromobacterium violaceum. A computer simulation was performed to perform homology modeling of this amino acid sequence, and the model structure was analyzed and molecular docking was performed with different large sterically hindered ketone compounds, and 19 amino acid residues were found, 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 the above amino acid sites, a protein having transaminase function or a protein with further enhanced transaminase function can be obtained. Non-essential mutation sites or active sites of the above obtained protein may be changed, thereby obtaining a protein having 80% or more homology with the above amino acid sequence and having transaminase function.

[0032] The sequence of SEQ ID NO:1 is as follows: MQKQRTCSQWRELDAAHHLHPFTDTASLNQVGARVMTRGEGVYLWDCEGNKIIDGMAGAWCVNMGYGRKDFAEAARRQMEELSFMHTADGITHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDCMIRMVRRYWDVQGKPEKKTLIGRWNGYSGSTIGGASLSGFKEMHEQGDLPIPGVAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGAIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYQPIGAVSVGKRVAEGLIAGGVFYHGHTTSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETLSRFEHVDDVRGVGMLAAFTLVKNKAKRELFPDFGEIGSLCRDIFVRNNLIMDICGDHIVAAPPLVMTRAEVDEMLAVAERCLEELEQSLKARGLA。

[0033] In a preferred embodiment, the amino acid mutations in (b) are each independently selected from the group consisting of 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, Y116C, Y116D, Y116E, Y116F ... 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 S, T107Q, T107A, or T107G, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid, and preferably, (c) is a protein having a homology of 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the amino acid sequence defined in (a) or (b), and having a 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 rule for substitution and replacement is that, in general, if the properties of any amino acids are similar, the effect after the replacement will also be similar. For example, conservative amino acid replacement can occur in the above-mentioned homologous proteins. "Conservative amino acid replacement" includes, but is not limited to, the following. Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced with other hydrophobic amino acids, Hydrophobic amino acids with thick side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with thick side chains, Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains, Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0036] Those skilled in the art can also make 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 has continued to study the above active site and has found that when the active site is mutated to a different amino acid, the activity of the corresponding protein is also different, and that the transaminase activity is enhanced by a specific mutation. As a result of testing, it has been found that a protein with enhanced activity can be obtained by performing the above specific mutation in the active site. The amino acid mutation site of the transaminase protein can be flexibly selected and combined from the above mutations.

[0038] In a preferred embodiment, the mutation of the transaminase mutant comprises any 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 have been experimentally examined in the examples of the present application, and all of them have transaminase activity. Compared to the parent species having the amino acid sequence shown in SEQ ID NO: 1, they result in transaminase mutants that are more stable, more resistant to extreme environments, and have higher enzymatic activity, and that can be used for industrial scale-up production.

[0040] In a second representative embodiment of the present application, a DNA molecule encoding the transaminase mutant described above is provided.

[0041] In a third representative embodiment of the present application, a recombinant plasmid is provided in which the above-described DNA molecules are ligated.

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

[0043] In the fourth exemplary embodiment of the present application, a host cell is provided that is transformed with the above-mentioned recombinant plasmid.The host cell can be a prokaryotic cell or a eukaryotic cell.Specifically, the prokaryotic cell can be Escherichia coli, and the eukaryotic cell can be yeast.

[0044] Using the above host cells, the recombinant plasmid can be copied in the host cells, or the DNA molecule contained in the recombinant plasmid can be transcribed and translated to obtain a large amount of the transaminase mutant. Using conventional techniques, the transaminase mutant can be obtained by disrupting the host cells and purifying the protein, or by disrupting and then catalyzing with crude enzyme, or by other methods, and then catalyzing the amine compound. The host cells are non-plant-derived host cells.

[0045] In a fifth exemplary embodiment of the present application, there is provided a method for producing a chiral amine compound, comprising the step of transaminating a ketone substrate represented by formula I under the action of an amino donor using the above transaminase mutant to obtain a chiral amine compound, wherein Ar1 is selected from a first substituted aryl, a first unsubstituted aryl, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, Ar2 is selected from a second substituted aryl, a second unsubstituted aryl, a substituted or unsubstituted cycloalkyl, an alkyl or an alkylene, R is selected from H, an alkyl, an alkylene or an alkylidene, the carbon number of the alkyl, alkylene or alkylidene being selected from 1 to 5, and the alkyl, alkylene or alkylidene is selected from a substituted alkyl, alkylene, alkylidene or an unsubstituted heteroarylene. R is selected from alkyl, alkylene or alkylidene, and the alkyl, alkylene or alkylidene is linked to Ar1 and / or Ar2 to form a ring; the substituents on the first substituted aryl, substituted arylene, substituted heteroarylene, second substituted aryl or substituted alkyl, alkylene or alkylidene are each independently selected from halogen, hydroxy, amino, methyl, ethyl or -CHCHOH; and the heteroatom on the substituted heteroarylene is selected from N, O or S.

[0046] [ka]

[0047] According to the above-mentioned preparation method, the above-mentioned transaminase mutant is used to carry out amination reaction of the ketone compound represented by formula I under the action of an amino donor to obtain a chiral amine compound. The above-mentioned transaminase mutant can perform chiral catalysis on the above-mentioned ketone compound to synthesize the required highly sterically hindered chiral amine. The activity and tolerance of the transaminase are greatly improved, and it can exert catalytic action 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, the second substituted aryl or the substituted arylene are each independently selected from halogen.

[0049] In one preferred embodiment, the substituents are each independently located at any one or more of the ortho, meta or para positions of the first substituted aryl, the second substituted aryl or the 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 unsubstituted heteroarylene, Ar2 is selected from a second substituted aryl, the substituent of the second substituted aryl is selected from 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 a preferred embodiment, Ar1 is selected from a first substituted aryl or a first unsubstituted aryl, and a substituent of the first substituted aryl is selected from methyl or Cl; Ar2 is selected from an unsubstituted cycloalkyl, an alkyl or a second unsubstituted aryl, and the carbon number of the unsubstituted cycloalkyl is selected from 3 to 5, and the alkyl is selected from isopropyl or ethyl; R is selected from an alkyl, and the alkyl is selected from isopropyl, methyl or ethyl.

[0053] In a 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 an alkyl, the alkyl is selected from methyl or isopropyl, 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.

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

[0055] [ka]

[0056] In this application, we perform homology modeling on the screened mutants based on the solved three-dimensional structure of the protein (PDB: 4BA5), perform molecular docking on different large sterically hindered ketone compounds based on the model structure, and select and remodel 19 residues that may affect the catalytic activity and stability of the protein under the guidance of the above strategies to improve 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. Protein remodeling methods include saturation mutation and combinatorial mutation.

[0057] Saturation mutagenesis is a method to modify a gene encoding a target protein and obtain mutants in which the amino acid at the target site is replaced with 19 other amino acids in a short time. This method is not only a powerful tool for directed modification of proteins, but also an important means for studying the relationship between protein structure and function. Saturation mutagenesis often results in more ideal evolved organisms than single point mutations. These problems that cannot be solved by site-directed mutagenesis are exactly what saturation mutagenesis is good at. Saturation mutagenesis was constructed using whole-plasmid PCR, and the PCR products were digested with DPNI enzyme to remove the template and transformed into E. coli BL21(DE3). Since screening of mutants often requires high-throughput screening methods, we developed the following method to screen the resistance of mutants.

[0058] The following high-throughput screening method for screening the mutant library was developed.

[0059] 1. Cultivation of mutants: 300μL of LB medium was added to each well of a 96-well plate, and a single clone on the agar plate was inoculated into a deep-well 96-well plate and cultured overnight at 37℃ and 200rpm. Using Qpix, the overnight cultured bacterial solution was transferred to a 96-well plate containing 800μL of LB medium per well, and after culturing at 37℃ and 200rpm for 5h, when the OD600 of the bacterial solution in the 96-well plate reached 0.6-0.9, IPTG solution was added to the 96-well plate again using Qpix, the final concentration of IPTG in the well plate was 0.1 mM, and induction was performed overnight at 25℃ and 200rpm for about 16h. Centrifugation was performed at 4000rpm for 5min, the supernatant was discarded, and the whole cells were used for the reaction.

[0060] 2. 96-well plate high throughput screening system: 8μL of PLP mother solution (1mg / mL), 3.5μL of 6M isopropylamine hydrochloride (20eq), and 0.1M Tris-Cl 9.0 (to make up the remaining amount up to 100μL) were mixed uniformly, and the mixed sample was dispensed into a 96-well plate containing bacterial mud per well. Finally, substrate mother solution (0.3mg of substrate dissolved in methanol of different concentrations) was added, mixed uniformly, and reacted at 700rpm on a shaker at a constant temperature of 50℃ for 18h. Next, 3 volumes of methanol were added per well, centrifuged, and the supernatant sample was sent to UPLC for analysis.

[0061] The above mutants were preliminarily screened to obtain mutants with improved properties. The optimal mutants were then induced and cultured in 2L shake flasks (optimal conditions for induced expression: 25°C, induced overnight with 0.2 mM IPTG), centrifuged to obtain bacterial sludge, and the cells were sonicated to obtain crude enzyme solution. Finally, a g-grade scale reaction was performed to confirm the properties of the optimal mutants. Since the improvement effect of saturation mutagenesis at a single site is often not obvious, it is usually necessary to perform iterative saturation mutagenesis multiple times to obtain mutants with significantly improved properties.

[0062] Based on saturation mutagenesis to obtain mutants with improved activity, the beneficial amino acid sites obtained by screening can be combined to obtain mutants with better properties. The construction method of double point mutations in combination mutations is the same as that of single point mutations, which is constructed using the whole plasmid PCR method. In multiple point mutations in which two or more sites are mutated simultaneously, overlap extension PCR amplification is used to obtain mutant genes containing multiple point mutations, which are digested at both ends with restriction enzymes and then ligated into expression vectors, transformed into E. coli cells, spread on LB culture plates 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 shake flask induction culture and reaction verification were carried out in the same manner as above.

[0063] Through multiple rounds of evolution, a series of transaminase mutants were obtained, which were found to have highly improved activity and tolerance during the process of catalyzing the reaction of large sterically hindered chiral amines, and these mutants proved to be very suitable for the industrial production of large sterically hindered chiral amines.

[0064] The present invention will now be described in more detail with reference to specific examples which should not be construed as limiting the scope of protection of the present application.

[0065] In the following Examples 1-4, changes in enzyme stability are expressed in terms of conversion at various methanol concentrations and temperatures.

[0066] Example 1 Two rounds of saturation mutagenesis were carried out based on the parent SEQ ID NO: 1. The specific mutation sites are shown in the table below, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0067] 1 mL of reaction mixture 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 with 30 mg of wet bacterial slurry), and 100 mM phosphate buffer (pH 8.0). 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 above table, + indicates that the conversion rate is less than 10%, ++ indicates that the conversion rate is 10% or more and 20% or less, +++ indicates that the conversion rate is 20% or more and 30% or less, ++++ indicates that the conversion rate is 30% or more and 40% or less, +++++ indicates that the conversion rate is 40% or more and 50% or less, and ++++++ indicates that the conversion rate is 50% or more and 60% or less.

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

[0071] Each mL of reaction mixture 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 with 30 mg of wet bacterial slurry), and 100 mM phosphate buffer (pH 8.0), and 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 above table, + indicates that the conversion rate is less than 10%, ++ indicates that the conversion rate is 10% or more and 20% or less, +++ indicates that the conversion rate is 20% or more and 30% or less, ++++ indicates that the conversion rate is 30% or more and 40% or less, +++++ indicates that the conversion rate is 40% or more and 50% or less, and ++++++ indicates that the conversion rate is 50% or more and 60% or less.

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

[0075] Each mL of reaction mixture 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 with 15 mg of wet bacterial slurry), and 100 mM phosphate buffer (pH 8.0), and 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 above table, + indicates that the conversion rate is less than 10%, ++ indicates that the conversion rate is 10% or more and 20% or less, +++ indicates that the conversion rate is 20% or more and 30% or less, ++++ indicates that the conversion rate is 30% or more and 40% or less, +++++ indicates that the conversion rate is 40% or more and 50% or less, and ++++++ indicates that the conversion rate is 50% or more and 60% or less.

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

[0079] Each mL of reaction mixture 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 with 10 mg of wet bacterial slurry), and 100 mM phosphate buffer (pH 8.0), and the reaction was carried out at 50° C. for 18 hours.

[0080] [Table 4-1]

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

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

[0083] Each mL of 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 with 10 mg of wet bacterial mud) treated at 70°C for 1 h or treated with methanol, ethanol, acetonitrile, ethyl acetate, dichloromethane, methyl t-butyl ether, and n-heptane for 1 h (the control was the same amount of enzyme solution without any treatment), and 100 mM phosphate buffer (pH 8.0), and the reaction was carried out at 40°C for 18 h.

[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 above table, the relative residual activity refers to the percentage of the enzyme activity measured in an enzyme solution properly treated under extreme conditions such as high temperature and organic solvents, and the enzyme activity under optimal conditions in an enzyme solution not treated under extreme conditions. Under the same treatment conditions, a higher relative residual activity indicates a higher stability of the enzyme under these conditions. 2) In the control, * indicates that the activity is 0.1 to 0.5 times higher than that of the parent species. ** indicates that the activity is 0.5 to 1.0 times higher than that of the parent species. *** indicates that the activity is 1.0 to 1.5 times higher than that of the parent species. **** represents a 1.5- to 2-fold improvement in activity over the parent species. 3) + indicates that the relative residual activity is 10% or more and less than 30%, ++ indicates that the relative residual activity is 30% or more and less than 50%, +++ indicates that the relative residual activity is 50% or more and less than 70%, and ++++ indicates that the relative residual activity is 70% or more and less than 90%.

[0089] The above description of the embodiment reveals the following. 1) The mutants obtained in this study clearly have improved stability at high temperatures. 2) Regarding resistance to organic solvents, in addition to a clear improvement in resistance to methanol, resistance to all of the six commonly used organic solvents listed above is improved, although to varying degrees. 3) Furthermore, the above control experiments show that in addition to improving the stability of the mutants, enzyme evolution also improved the enzyme activity to various degrees.

[0090] Example 6 In a 250 mL four-neck 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 to 9.0. Next, 0.1 g of pyridoxal phosphate,

[0091] [ka] 5 g of (Substrate 1) was added, stirred uniformly, and 2 mL of an enzyme solution (0.5 g 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 temperature was raised to 50 ° C, and the reaction was stirred and reacted. After the reaction was completed, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50 mL of methyl t-butyl ether. The aqueous phase was adjusted to pH = 12, and then extracted twice with 50 mL of methyl t-butyl ether. The organic phases were combined and dried over anhydrous magnesium sulfate, and then concentrated under conditions of T < 40 ° C and P ≦ -0.06 MPa until no fraction remained. Target product

[0092] [ka] obtained.

[0093] HPLC detection showed purity >99%, de value >99%, and yield 85%.

[0094] Example 7 In a 250 mL four-neck 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 to 9.0. Next, 0.1 g of pyridoxal phosphate,

[0095] [ka] 5 g of (Substrate 2) was added, stirred uniformly, and 2 mL of an enzyme solution (0.5 g 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 temperature was raised to 50 ° C, and the reaction was stirred and reacted. After the reaction was completed, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50 mL of methyl t-butyl ether. The aqueous phase was adjusted to pH = 12, and then extracted twice with 50 mL of methyl t-butyl ether. The organic phases were combined and dried over anhydrous magnesium sulfate, and then concentrated under conditions of T < 40 ° C and P ≦ -0.06 MPa until no fraction remained. Target product

[0096] [ka] obtained.

[0097] HPLC detection showed purity >99%, de value >99%, and yield 84%.

[0098] Example 8 In a 250 mL four-neck 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.

[0099] [ka] 5 g of (Substrate 16) was added, stirred uniformly, and 2 mL of an enzyme solution (0.5 g 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 temperature was raised to 50 ° C, and the reaction was stirred and reacted. After the reaction was completed, the system was adjusted to an acidic pH of 2-3 to denature the protein. After filtration, the filtrate was extracted with 50 mL of methyl t-butyl ether. The aqueous phase was adjusted to pH = 12, and then extracted twice with 50 mL of methyl t-butyl ether. The organic phases were combined and dried over anhydrous magnesium sulfate, and then concentrated under conditions of T < 40 ° C and P ≦ -0.06 MPa until no fraction remained. Target product

[0100] [ka] obtained.

[0101] HPLC detection showed purity >99%, de value >99%, and yield 75%.

[0102] Example 9 In a 250 mL four-neck 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.

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

[0104] [ka] obtained.

[0105] HPLC detection showed 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 SEQ ID NO: 1, catalytic reactions were carried out on substrates 3 to 15 with reference to the catalytic synthesis steps of Examples 6 to 9. 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 transaminase, mutants with improved activity and stability have been obtained, enabling the synthesis of various large sterically hindered chiral amines under high temperature and high organic solvent conditions without the need for heavy metal catalysts or toxic reagents, thereby realizing green chemistry. 2) This transaminase mutant has high biocatalytic activity, stable enzyme, high concentration of reaction substrate, high product yield, and can significantly reduce wastewater, exhaust gas and solid waste, thus saving production costs.

[0108] The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. (a) a protein having the amino acid sequence set forth in SEQ ID NO:1, or (b) a protein having transaminase activity, in which at least one of the sites V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107 or L449 in the amino acid sequence of (a) has been subjected to amino acid mutation; (c) a protein having 80% or more homology to the amino acid sequence defined by either (a) or (b) and having transaminase function.

2. The amino acid mutations in (b) each independently comprise: 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, 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; selected from T107S, T107Q, T107A, or T107G; However, the letters before the number represent the original amino acid, and the letters after the number represent the mutated amino acid. The transaminase mutant described in claim 1, characterized in that (c) is a protein having a homology of 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the amino acid sequence defined in (a) or (b) and has transaminase function.

3. The transaminase mutant of claim 2, characterized in that the mutation of the transaminase mutant includes any 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.

4. A DNA molecule, characterized in that it codes for a transaminase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid comprising the DNA molecule of claim 4 ligated thereto.

6. A host cell transformed with the recombinant plasmid of claim 5.

7. A method for producing a chiral amine compound, comprising the step of carrying out a transamination reaction on a ketone substrate represented by formula I under the action of an amino donor using the transaminase mutant according to any one of claims 1 to 3 to obtain a chiral amine compound. 【Chemistry 1】 Ar1 is selected from a first substituted aryl, a first unsubstituted aryl, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, Ar2 is selected from a second substituted aryl, a second unsubstituted aryl, a substituted or unsubstituted cycloalkyl, an alkyl or an alkylene; R is selected from H, an alkyl, an alkylene, or an alkylidene, the number of carbon atoms of the alkyl, alkylene, or alkylidene being selected from 1 to 5, and the alkyl, alkylene, or alkylidene includes a substituted alkyl, a substituted alkylene, a substituted alkylidene, or an unsubstituted alkyl, an unsubstituted alkylene, or an unsubstituted alkylidene; When R is selected from alkyl, alkylene, or alkylidene, the alkyl, alkylene, or alkylidene is linked to the Ar1 and / or the 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 OH, The heteroatoms in the substituted heteroarylene are selected from N, O, or S.

8. 8. The method of claim 7, wherein the substituents of the first substituted aryl, the second substituted aryl, or the substituted arylene are each independently selected from the halogens.

9. The method according to claim 8, wherein the substituents are each independently located at any one or more of the ortho, meta, and para positions of the first substituted aryl, the second substituted aryl, or the substituted arylene.

10. 10. The method of claim 9, wherein the halogen is selected from F, Cl or Br.

11. The Ar1 is selected from the unsubstituted heteroarylenes, Ar2 is selected from the second substituted aryl, and the substituent of the second substituted aryl is selected from the halogen; R is selected from the substituted alkylenes, the number of carbon atoms of the substituted alkylenes is selected from 1 to 5, and the substituent is selected from hydroxy; The method according to claim 10, wherein the substituted alkylene is linked to Ar1 to form a ring.

12. 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 of the unsubstituted cycloalkyl is selected from 3 to 5, and the alkyl is selected from isopropyl or ethyl; 11. The method of claim 10, wherein R is selected from the alkyl group, and the alkyl group is selected from isopropyl, methyl, or ethyl.

13. 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 OH, Ar2 is selected from the second unsubstituted aryl or alkyl, and 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, and the substituent is selected from hydroxy; The method according to claim 10, wherein the substituted alkylene is linked to Ar1 to form a ring.

14. The method according to any one of claims 10 to 13, characterized in that the ketone substrate is selected from the following: 【Chemistry 2】

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