CN110982801B

CN110982801B - Transaminase mutant and construction method and application thereof

Info

Publication number: CN110982801B
Application number: CN201911377567.XA
Authority: CN
Inventors: 马富强; 张艺凡; 郭天杰; 杨广宇
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-10-30
Anticipated expiration: 2039-12-27
Also published as: CN110982801A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a transaminase mutant and a construction method and application thereof. The transaminase mutant provided by the invention comprises a plurality of mutants which are subjected to single-point mutation and combined mutation at 259, 431, 264, 269 and 381 of the amino acid sequence of wild-type transaminase (WP _0532423951.1), and compared with the wild-type transaminase (WP _0532423951.1), the mutant has longer half-life at 55 ℃, the half-life of the optimal combined mutant is about 5 times that of the wild-type transaminase (WP _0532423951.1), the heat stability is better, and the mutant is suitable for catalytically synthesizing chiral amine at higher temperature. The transaminase mutant obtained by the construction method provided by the invention has excellent stereoselectivity and catalytic activity when catalyzing and synthesizing chiral amine at higher temperature, and has better application prospect.

Description

Transaminase mutant and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a transaminase mutant and a construction method and application thereof.

Background

Chiral amine widely exists in nature, is an important intermediate for synthesizing natural products and chiral drugs, and has great economic benefit and application value when being deeply researched for asymmetric synthesis of chiral amine. Taking diabetes as an example, sitagliptin, which is the main component of the antidiabetic oral medicine Januvia, is R-type amine, in the process of synthesizing sitagliptin, the process of catalyzing an aminotransferase reaction to generate a sitagliptin key intermediate by using aminotransferase is reversible, the reaction balance between an amino group and a ketone group is promoted to move towards the generation direction of chiral amine by changing the catalytic reaction conditions, so that the synthesis efficiency of the chiral amine can be improved, and the theoretical yield can reach 100%.

One of the key enzymes catalyzing the formation of chiral amines is Transaminase (Transaminase, TA, EC 2.6.1.X), also known as Aminotransferase (Aminotransferase), which is mainly used to catalyze the transamination between amino and keto groups. Transaminases can be classified into α -transaminase and ω -transaminase according to the difference of the substrate or product, wherein the substrate or product of α -transaminase includes only α -amino acid, while the substrate and product of ω -transaminase includes keto acid, aldehyde and ketone, and has the characteristics of high stereoselectivity, etc., so that ω -transaminase can be widely applied in the field of drug synthesis. In reported research on transaminase, certain breakthrough is made on research on development of omega-transaminase mutants with good enzyme activity and high substrate stereoselectivity by using a protein engineering technology, but most of the omega-transaminase mutants have poor thermal stability and can obviously precipitate after being kept at 55 ℃ for about 2 hours, so that the industrial application of the synthesis of chiral amine catalyzed by the transaminase is greatly limited.

Common protein engineering methods include rational design (rational design) and irrational design (irrational design), wherein the rational design requires understanding of the structure and function of the protein, but because the structure-function relationship of the protein is too complicated, people still lack sufficient knowledge today, which results in low accuracy of the rational design; the key bottleneck of irrational design is high-throughput screening, and the application of irrational design is greatly limited because the establishment of a high-throughput screening method for proteins is still a difficult problem. In recent years, with the development of computer technology and bioinformatics technology, Markuss Wys et al proposed Consensus Concept which is different from rational design with Protein structure-function relationship as core, and sought a method for optimizing enzyme thermal stability from the evolutionary point of view by tracing back ancestral genes and comparing homologous sequences, such as obtaining phytase mutants with high thermal stability under the guidance of Consensus Concept (Biochim Biophys acta.2000 Dec 29; 1543(2):408 and 415; Protein Eng.2002 May; 15(5):403-11.), after which Consensus theory was widely used in the research for improving thermal stability of proteins and has been successfully guided to improve optimization of multiple proteins on thermal stability, providing a new method for optimizing Protein stability. However, the crystal structure of the transaminase is not analyzed, the transaminase is difficult to optimize through the rational design of protein engineering, and meanwhile, the related research of the transaminase based on the Consensus theory is not reported, so that the problem that the improvement of the thermal stability of the transaminase or the mutant thereof breaks through the limitation of the industrial application of the catalytic chiral amine synthesis is urgently solved.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defect of poor thermal stability of the existing transaminase, so that a transaminase mutant with good thermal stability, a construction method of the transaminase mutant and application of the transaminase mutant in catalyzing chiral amine synthesis are provided.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides a transaminase mutant, which is (a1) or (a 2):

(a1) a derived protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and has the same function with the protein shown in SEQ ID NO. 2;

(a2) a derivative protein which has one or more amino acid residues substituted for one or more positions of the amino acid sequence shown in SEQ ID No.2 and shows at least 92% homology with the protein shown in SEQ ID No. 2.

Preferably, the transaminase mutant, the mutation site of the amino acid sequence shown in SEQ ID No.2, comprises at least one of: 259 bit, 431 bit, 264 bit, 269 bit and 381 bit.

Further preferably, the transaminase mutant comprises a single point mutant of any one of the single point mutation sites in the amino acid sequence shown in SEQ ID No.2, a259F, G431T, E264A, a269E, S381E.

Further preferably, the transaminase mutant comprises a combination mutant of any one of the combination mutation sites in the amino acid sequence shown in SEQ ID No. 2G 431T/E264A, G431T/A269E and A259F/G431T/E264A.

The invention also provides a gene for coding the transaminase mutant.

The invention also provides a recombinant plasmid containing the gene.

The invention also provides a soluble protein, immobilized enzyme or engineering bacterium containing the transaminase mutant.

The invention also provides a construction method of the transaminase mutant, which comprises the following steps:

searching an amino acid sequence shown by SEQ ID NO.2 in an NCBI database, deleting a repeated identical sequence, selecting an amino acid sequence with the consistency of more than 33 percent with the amino acid sequence shown by SEQ ID NO.2, then performing multi-sequence comparison through Clustalx1.83 software, arranging the residual amino acid sequence into a fasta file, uploading the fasta file to a Consensus Maker v2.0.0 server, modifying set parameters according to needs, generating a Consensus sequence capable of being edited at a later stage by online software, and screening mutation sites related to thermal stability as follows: a259F, G431T, E264A, a269E, S381E.

The invention also provides application of the transaminase mutant in catalytic synthesis of chiral amine.

The technical scheme of the invention has the following advantages:

1. the transaminase mutant provided by the invention comprises a single-point mutant and a combined mutant, and compared with wild-type transaminase (KOF55240.1), the single-point mutant and the combined mutant have longer half-life at 55 ℃; in particular, the combination mutant showed a superimposed effect of thermal stability of the single-point mutant, with a half-life of about 5 times that of the wild-type transaminase (KOF 55240.1). Based on the above, the transaminase mutant provided by the invention has better thermal stability, and is suitable for catalytic synthesis of chiral amine at higher temperature.

2. The transaminase (KOF55240.1) gene engineering bacteria constructed by the invention can efficiently express transaminase mutants, and has the advantages of simple culture conditions, short culture period, convenient purification of expressed products and the like.

3. The construction method of the transaminase mutant is different from rational design based on the precise structure-function relationship of protein, and is based on the Consensus theory, the database is constructed, the bioinformatics technology is utilized to search, screen and compare homologous sequences, the information capable of improving the thermal stability of the transaminase is analyzed from the evolutionary point, the sequence of the transaminase family is integrated and analyzed, and the bioinformatics and crystallography methods are combined to assist, so that the novel transaminase mutant with high stability is obtained.

4. The transaminase mutant provided by the invention has excellent stereoselectivity, regioselectivity and catalytic activity when being applied to catalytic synthesis of chiral amine, and has good application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of a simulated crystal structure of wild-type transaminase (KOF55240.1) protein and the distribution of mutation sites on the crystal structure, which are provided in example 4 of the present invention.

Detailed Description

In order to facilitate understanding of the objects, technical solutions and gist of the present invention, embodiments of the present invention will be described in further detail below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, this embodiment is provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.

General description of the sources of biological materials to which the invention relates:

1. genes and primers: the optimized wild-type transaminase (KOF55240.1) gene and all primers used in the present invention were prepared synthetically by Biotech;

2. other biological materials;

the pET28a plasmid vector was purchased from Novagen;

t4 DNA ligase was purchased from NewEngland Biolabs;

PrimeSTAR Max Premix Hi enzyme, buffer from Takara;

the DpnI enzyme, DreamTaq DNA polymerase and all restriction endonucleases were purchased from Thermo;

PrimeSTAR Max polymerase from Takara;

the DNA gel recovery kit and the small plasmid extraction kit are purchased from Tiangen Biotechnology Ltd.

Example 1

This example provides a transaminase mutant, which is a derivative protein obtained by molecular modification of an amino acid sequence of a wild-type transaminase (KOF55240.1), wherein the wild-type transaminase (KOF55240.1) is a protein obtained by codon optimization of an aspartate aminotransferase (aspartate aminotransferase is a member of the ω -transaminase family) gene derived from an Achromobacter sp.dms1 strain, and the nucleic acid sequence encoding the protein is SEQ ID No.1 and the amino acid sequence is SEQ ID No. 2.

The transaminase mutants provided in this example include:

(a1) a derived protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and has the same function with the protein shown in SEQ ID NO. 2; or the like, or, alternatively,

Specifically, a certain site is selected from the amino acid sequence shown in SEQ ID No.2 for mutation, so that a plurality of single-point mutants can be obtained, the activity test is sequentially carried out, the single-point mutants with improved thermal stability and activity are screened, and the single-point mutation sites are as follows: 259, 431, 264, 269 and 381 correspond to 5 single point mutants of transaminase:

(1) the amino acid sequence shown in SEQ ID NO.2 has the amino acid sequence with the amino acid sequence A259F, wherein the 259 th tyrosine is replaced by phenylalanine;

(2) the 431 nd glycine of the amino acid sequence shown in SEQ ID NO.2 is replaced by threonine, and is marked as G431T;

(3) the 264 th glutamic acid of the amino acid sequence shown in SEQ ID NO.2 is replaced by alanine, which is marked as E264A;

(4) the 269 th alanine of the amino acid sequence shown in SEQ ID NO.2 is replaced by glutamic acid and is marked as A269E;

(5) the 381 st serine of the amino acid sequence shown in SEQ ID NO.2 is substituted by glutamic acid and is marked as S381E.

Or, selecting a plurality of mutation sites to combine in the amino acid sequence shown in SEQ ID NO.2, for example, selecting 2 mutation sites to combine from the above 5 mutation sites, to obtain the following 10 transaminase combination mutants, respectively, whose combination mutation sites are: A259F/G431T, A259F/E264A, A259F/A269E, A259F/S381E, G431T/E264A, G431T/A269A, G431T/S381E, E264A/A269E, E264A/S381E, A269E/S381E; for example, 3 mutation sites are selected from the above 5 mutation sites to be combined, so as to obtain 10 transaminase combined mutants, wherein the combined mutation sites are as follows: A259F/G431T/E264A, A259F/G431T/A269E, A259F/G431T/S381E, A259F/E264A/A269E, A259F/E264A/S381E, A259F/A269E/S381E, G431T/E264A/A269E, G431T/E264A/S381E, G431T/A269E/S381E, E264A/A269E/S381E; for example, 4 mutation sites are selected from the above 5 mutation sites to be combined, so as to obtain 5 transaminase combined mutants, wherein the combined mutation sites are as follows: A259F/G431T/E264A/A269E, A259F/E264A/A269E/S381E, A259F/G431T/A269E/S381E, A259F/G431T/E264A/S381E, G431T/E264A/A269E/S381E; selecting 5 mutation sites from the above 5 mutation sites for combination to obtain 1 transaminase combination mutant, wherein the combination mutation sites are as follows: A259F/G431T/E264A/A269E/S381E. The activity test is carried out on the obtained multiple combined mutants, and 3 combined mutants with improved thermal stability and activity are screened, wherein the mutation sites are G431T/E264A, G431T/A269E and A259F/G431T/E264A.

Example 2

This example provides a gene encoding a transaminase mutant as described in example 1:

(1) the nucleic acid sequence of the transaminase mutant with the mutation site A259F is SEQ ID NO. 3;

(2) the nucleic acid sequence of the transaminase mutant with the mutation site G431T is SEQ ID NO. 4;

(3) the nucleic acid sequence of the transaminase mutant with the mutation site E264A is SEQ ID NO. 5;

(4) the nucleic acid sequence of the transaminase mutant with the mutation site A269E is SEQ ID NO. 6;

(5) the nucleic acid sequence of the transaminase mutant with the coding mutation site of S381E is SEQ ID NO. 7;

(6) the nucleic acid sequence of the transaminase mutant with the mutation site of G431T/E264A is SEQ ID NO. 8;

(7) the nucleic acid sequence of the transaminase mutant with the mutation site of G431T/A269E is SEQ ID NO. 9;

(8) the nucleic acid sequence of the transaminase mutant with the mutation site of A259F/G431T/E264A is SEQ ID No. 10.

Example 3

This example provides a recombinant plasmid comprising the gene provided in example 2, which is a plasmid vector that can be used for stable expression of the transaminase gene integrated into host cells, such as pET28a, pUC18, pUC19, pET15b, and the like; the host cell can be selected from gram-invisible bacteria (such as Escherichia coli), gram-positive bacteria (such as Bacillus subtilis), fungi (such as Aspergillus), yeast (such as Saccharomyces cerevisiae), actinomycetes (such as Streptomyces), etc., and can be used for expressing transaminase mutant protein.

Example 4

This example provides a method for constructing a transaminase mutant, comprising the following steps:

1. cloning of wild-type transaminase (KOF55240.1) Gene

Carrying out codon optimization on a wild-type transaminase (KOF55240.1) gene by taking escherichia coli as a competent host cell to obtain an optimized wild-type transaminase (KOF55240.1) gene, wherein the nucleic acid sequence of the optimized wild-type transaminase (KOF55240.1) gene is SEQ ID No.1, and the expressed amino acid sequence of the optimized wild-type transaminase (KOF55240.1) gene is SEQ ID No. 2; using SEQ ID NO.1 as a target gene, and adopting an upstream amplification primer SEQ ID NO.11 and a downstream amplification primer SEQ ID NO.12 to amplify the target gene;

the nucleotide sequence of SEQ ID NO.11 is:

5’-TACCAGACGACGAcatTTTGACGGCCTGG-3' (underlined bases are recognition sites for restriction enzyme NdeI);

the nucleotide sequence of SEQ ID NO.12 is:

5’-CTTCCATAGCCAAggatccTTCAAGACTTTCTTCAACGA-3' (underlined bases are recognition sites for the restriction enzyme BamHI).

The amplification conditions were: amplification was performed using PrimeSTAR Max polymerase for 3min at 98 ℃, followed by 10sec at 98 ℃, 10sec at 55 ℃, 15sec at 72 ℃ for 30 cycles, and finally 10min at 72 ℃.

After the reaction is finished, detecting the PCR amplification product by 1.2% agarose gel electrophoresis to obtain a band of 1.0kb, which is consistent with the expected result. Digesting the template by using DpnI enzyme, recovering and purifying the target fragment, carrying out double digestion on the target fragment and a pET28a plasmid vector by using restriction enzymes NdeI and BamHI respectively, then carrying out ligation by using T4 DNA ligase, transforming a ligation product into an Escherichia coli E.coli BL21(DE3) electrotransformation competent cell, coating the transformed cell on a solid culture medium containing 40 mu g/mL kanamycin to screen out positive clones, extracting plasmids, and sequencing the plasmids, wherein the sequencing result shows that the gene sequence of the cloned optimized wild-type transaminase (KOF55240.1) is correct and the optimized wild-type transaminase (KOF55240.1) is correctly ligated into the pET28a plasmid vector to obtain a recombinant plasmid pET28a-KOF 55240.1.

2. Optimized expression and purification of wild-type transaminase (KOF55240.1) protein

Inoculating the engineering bacteria in the glycerin pipe into a 4mL LB liquid culture medium test tube containing 100 mug/mL kanamycin (Kan +) according to the volume ratio of 1%, and culturing at 37 ℃ and 230rpm for 11 h; then transferring the 4mL of bacterial liquid to a 1L shaking flask containing LB liquid culture medium containing 50 ug/mL kanamycin (Kan +), and culturing at 37 ℃ and 230rpm for about 2h to make OD600 reach about 0.8; then 0.1mM IPTG inducer was added, and the mixture was subjected to induction culture at 25 ℃ and 200rpm for 11-17 hours, in this example for 14 hours. And (3) centrifuging the escherichia coli thallus suspension obtained after fermentation, and performing one-step Ni-NTA affinity chromatography treatment to obtain the optimized wild-type transaminase (KOF55240.1) protein with the purity of more than 95%.

3. Construction of optimized Large-Capacity random mutation library of wild-type transaminase (KOF55240.1)

A mutation library of the optimized wild-type transaminase (KOF55240.1) was constructed by a sequential error-prone PCR (ep-PCR) method, and the mutation rate was adjusted by adjusting the concentration of magnesium ions in the PCR system.

The error-prone PCR system comprises:

DreamTaq^TM(0.05U/. mu.L) and its buffer, dATP (250. mu.M), dGTP (250. mu.M), dCTP (1050. mu.M), dTTP (1050. mu.M);

optimized wild-type transaminase (KOF55240.1) forward primer (0.4. mu.M), optimized wild-type transaminase (KOF55240.1) reverse primer (0.4. mu.M), optimized wild-type transaminase (KOF 55240.1);

pET-28a plasmid vector (0.2 ng/. mu.L), magnesium chloride (0.3-0.9 mM);

wherein the upstream primer sequence of the optimized wild-type transaminase (KOF55240.1) is SEQ ID NO.11, and the downstream primer sequence is SEQ ID NO. 12.

And (3) subpackaging the error-prone PCR system into 25 mu L/tube for error-prone PCR amplification, wherein the amplification conditions are as follows:

amplifying at 95 ℃ for 3min for 1 cycle; amplifying at 95 ℃ for 10sec, 55 ℃ for 35sec, and 72 ℃ for 1min for 30 cycles; amplification was carried out at 72 ℃ for 6min for 1 cycle. After the reaction is finished, carrying out double-enzyme cutting on the purified mutated target fragment by NdeI and BamHI, cloning the fragment to pET28a plasmid vector by adopting T4 DNA ligase, then electrically transforming the purified plasmid vector system into an escherichia coli E.coli BL21(DE3) competent cell host, inoculating the cell to be transformed into 50mL LB liquid culture medium (containing 50 mug/mL kanamycin) after recovery, culturing overnight at 37 ℃, taking the culture solution to extract plasmids to obtain mutant library plasmids, sampling and coating on a kanamycin-resistant agarose plate, and calculating the library capacity to be about 100 ten thousand; several clones were taken to determine the mutation rate, and it was found that at a magnesium ion concentration of 0.7mM, a mutation rate of approximately 1.5 mutated amino acid residues per gene on average was obtained. Plasmid transformation of the mutant gene library is transformed into a competent host escherichia coli e.colibl21(DE3), and is subjected to culture in an LB liquid medium, induction and expression, followed by screening.

4. Multiple sequence alignment and Consensus analysis of optimized wild-type transaminase (KOF55240.1) homologous proteins

(1) Entering into a Pfam database homepage (http:// Pfam. xfam. org /), inputting the amino acid SEQUENCE SEQ ID NO.2 of the optimized wild-type transaminase (KOF55240.1) into a SEQUENCE SEARCH tool for searching, and directly feeding back the amino acid SEQUENCE alignment result of the whole family of the protein by a server. In this example, the aspartate aminotransferase family shows the abundance of each amino acid at each mutation site in a histogram, and the Pfam database homepage can also automatically generate the consensus sequence of the optimized wild-type aminotransferase (KOF55240.1) protein family.

(2) Entering an NCBI protein database, inputting an amino acid sequence SEQ ID NO.2 of an optimized wild-type transaminase (KOF55240.1), finding out all protein sequences with the consistency of more than 33 percent with the amino acid sequence SEQ ID NO.2 by using a Blast tool, deleting repeated identical sequences, arranging the rest amino acid sequences into a fasta format, realizing multi-sequence comparison by using Clustalx1.83 software, and outputting comparison results in fasta, aln and dnd formats; uploading the downloaded fasta. file to a Weblogo 3(http:// Weblogo. threeplusone. com /) server, and after the set parameters are modified as required, displaying the amino acid abundance of each mutation site of the protein sequence in the multi-sequence alignment result in a column diagram form by the online software.

Uploading the fasta. file to a Consensus Maker v2.0.0(http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/Consensus. html) server, and after setting parameters are modified as required, generating a Consensus sequence which can be edited later by the online software.

(3) The amino acid sequence of the optimized wild-type transaminase (KOF55240.1), SEQ ID No.2, was compared against the family consensus sequence and the amino acid abundance map for each mutation site.

5. Optimized crystal structure analysis of wild-type transaminase (KOF55240.1) protein and selection of mutation hot spots

(1) The structure of the obtained optimized wild-type transaminase (KOF55240.1) protein crystal is analyzed by an X-ray diffraction method.

(2) Observing the crystal structure of the optimized wild-type transaminase (KOF55240.1) protein by PyMOL software, and screening out a mutant most likely to improve the thermal stability of the optimized wild-type transaminase (KOF55240.1) protein by reviewing the mutation site to be selected and the mutation form according to structural information, wherein the screening conditions are as follows:

A. the standard for judging a certain mutation site as a mutation site to be selected is as follows: the total height of most proteins of the target enzyme family with the amino acid abundance at the mutation site is higher; ② the amino acid at the mutation site is conservative amino acid; the amino acid with higher frequency of occurrence of the mutation site has larger difference of physicochemical properties with the amino acid at the mutation site of the optimized wild type transaminase (KOF55240.1) protein crystal, and the properties comprise strong and weak polarity, charge difference, steric hindrance and the like;

B. the vicinity of the active center is removed, and the amino acid residues in the embedded or semi-embedded state are removed.

After A, B two-step screening, there were 58 different sites remaining, most of which were located on the surface of the protein molecule, as shown in FIG. 1.

C. The 58 mutant forms were analyzed in detail one by one based on the crystal structure of the optimized wild-type transaminase (KOF55240.1) protein, and mutants that could improve the thermostability of the optimized wild-type transaminase (KOF55240.1) protein were selected.

The main judgment criteria are: firstly, mutation at the mutation site should eliminate the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion acting force and the like; mutations at this site should not destroy the existing stable protein structure; and thirdly, introducing a new acting force form which is beneficial to thermal stability, such as addition of hydrogen bonds, building of salt bridges, interaction of hydrophobic groups and the like, into the mutation at the mutation site.

Totally designing 5 kinds of single-point mutants, wherein the single-point mutation sites are respectively as follows: A259F, G431T, E264A, A269E and S381E, all 5 mutants were designed by the structure-assisted Consensus method.

Using a construction method similar to the single-point mutant to cumulatively combine the single-point mutants with improved thermal stability, selecting a plurality of mutation sites for combination in the amino acid sequence shown in SEQ ID NO.2, and selecting 2-5 sites from the 5 sites for mutation to respectively obtain different transaminase combined mutants:

(1) by selecting 2 mutation sites to combine, 10 transaminase combination mutants can be constructed:

A259F/G431T、A259F/E264A、A259F/A269E、A259F/S381E、G431T/E264A、G431T/A269A、G431T/S381E、E264A/A269E、E264A/S381E、A269E/S381E；

(2) by selecting 3 mutation sites to combine, 10 transaminase combination mutants can be constructed:

A259F/G431T/E264A、A259F/G431T/A269E、A259F/G431T/S381E、A259F/E264A/A269E、A259F/E264A/S381E、A259F/A269E/S381E、G431T/E264A/A269E、G431T/E264A/S381E、G431T/A269E/S381E、E264A/A269E/S381E；

(3) 4 mutation sites are selected for combination, and 5 transaminase combinatorial mutants can be obtained:

A259F/G431T/E264A/A269E、A259F/E264A/A269E/S381E、A259F/G431T/A269E/S381E、A259F/G431T/E264A/S381E、G431T/E264A/A269E/S381E；

(4) 5 mutation sites are selected for combination, and 1 transaminase combinatorial mutant can be obtained:

A259F/G431T/E264A/A269E/S381E。

respectively carrying out activity measurement on the obtained single-point mutants and the obtained combined mutants, and selecting 5 single-point mutants and 3 combined mutants with improved thermal stability and activity, wherein the mutation sites of the single-point mutants and the combined mutants are as follows:

A259F, G431T, E264A, A269E, S381E, G431T/E264A, G431T/A269E and A259F/G431T/E264A.

Test example 1

1. Characterization of the Properties of the transaminase mutants

The optimized wild-type transaminase (KOF55240.1) and various transaminase mutants provided in example 4 were subjected to a thermostability test according to a conventional transaminase activity determination method (see literature Arch Biochem Biophys.2000,373(1):182-92), specifically:

optimized wild-type transaminase (KOF55240.1) and 0.225mg/ml of each of the various transaminase mutants provided in example 4 were dissolved in 30mM (pH 7.2) phosphate buffer and heated in a water bath at 55 ℃. Taking the heating time of 0 as a starting point, and measuring the residual enzyme activity of the enzyme after the enzyme is heated in a water bath at 55 ℃ for different time periods by using an ultraviolet spectrophotometer. The water bath time at 55 ℃ when the residual enzyme activity reaches half of the initial enzyme activity is taken as the half-life value at 55 ℃.

The experimental results show that the thermal stability of the 5 single-point mutants and the 3 combined mutants is obviously improved, as shown in table 1:

TABLE 1 characterization of enzymatic Properties of wild-type transaminase (KOF55240.1), single-site mutants, and combinatorial mutants

As shown in Table 1, the transaminase mutants provided by the invention comprise single-site mutants and combined mutants, and the heat stability of the transaminase mutant at 55 ℃ is obviously improved compared with the optimized wild-type transaminase (KOF55240.1) by measuring the half-life of the optimized wild-type transaminase (KOF55240.1) and the transaminase mutant at 55 ℃; among them, the most preferred combination mutant (mutation site A259F/G431T/E264A) has about 5-fold thermal stability at 55 ℃ as compared with the optimized wild-type transaminase (KOF 55240.1). Based on the above, the transaminase mutant provided by the invention is suitable for catalyzing and synthesizing chiral amine at a higher temperature, and the industrial production process of medicines and agriculture is optimized.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

The invention name is as follows: transaminase mutant and construction method and application thereof

SEQ ID NO.1：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCGCCTATGGG

781 AGACTTGGTGAAGCAACAGCGGCTGCCTACTTCGGCGTAACGCCCGATCTCATTACTATG

841 GCAAAGGGAGTGTCGAATGCGGCTGTTCCAGCCGGGGCAGTCGCGGTAAGGCGTGAGGTG

901 CATGACGCTATCGTTAACGGTCCGCAGGGCGGAATAGAATTTTTCCACGGGTATACCTAC

961 AGTGCCCATCCTCTAGCAGCGGCTGCCGTCCTGGCAACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTTGCGCGGGCTAGAAAACTTAGCGCCCCCTTCGAAGAGGCAGCGCACTCTCTC

1081 AAGGGTGCTCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCCGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCACCTGGTGCGCGCGCTGCCGAAGCATTTCAAAAATGTTTCGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCGGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTTGAAGGTATAGGCAAGGTTCTCAAAGAGGTCGCT

SEQ ID NO.2：

MSAAKLPDLSHLWMPFTANRQFKANPRLLASAKGMYYTSFDGRQILDGTA 50

GLWCVNAGHCREEIVSAIASQAGVMDYAPGFQLGHPLAFEAATAVAGLMP 100

QGLDRVFFTNSGSESVDTALKIALAYHRARGEAQRTRLIGRERGYHGVGF 150

GGISVGGISPNRKTFSGALLPAVDHLPHTHSLEHNAFTRGQPEWGAHLAD 200

ELERIIALHDASTIAAVIVEPMAGSTGVLVPPKGYLEKLREITARHGILL 250

IFDEVITAYGRLGEATAAAYFGVTPDLITMAKGVSNAAVPAGAVAVRREV 300

HDAIVNGPQGGIEFFHGYTYSAHPLAAAAVLATLDIYRREDLFARARKLS 350

APFEEAAHSLKGAPHVIDVRNIGLVAGIELSPREGAPGARAAEAFQKCFD 400

TGLMVRYTGDILAVSPPLIVDENQIGQIFEGIGKVLKEVA

SEQ ID NO.3：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCTTCTATGGG

781 AGACTTGGTGAAGCCACAGCAGCGGCTTACTTTGGCGTAACGCCCGATCTCATTACTATG

841 GCCAAGGGAGTGTCGAATGCAGCGGTTCCAGCTGGGGCCGTCGCAGTAAGGCGTGAGGTG

901 CATGACGCGATCGTTAACGGTCCGCAGGGCGGAATAGAATTCTTTCACGGGTATACCTAC

961 AGTGCTCATCCTCTAGCCGCAGCGGCTGTCCTGGCCACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTCGCACGGGCGAGAAAACTTAGCGCTCCCTTTGAAGAGGCCGCACACTCTCTC

1081 AAGGGTGCGCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCTGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCCCCTGGTGCACGCGCGGCTGAAGCCTTCCAAAAATGTTTTGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCAGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTCGAAGGTATAGGCAAGGTTCTCAAAGAGGTCGCG

SEQ ID NO.4：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCGCCTATGGG

781 AGACTTGGTGAAGCAACAGCGGCTGCCTACTTCGGCGTAACGCCCGATCTCATTACTATG

841 GCAAAGGGAGTGTCGAATGCGGCTGTTCCAGCCGGGGCAGTCGCGGTAAGGCGTGAGGTG

901 CATGACGCTATCGTTAACGGTCCGCAGGGCGGAATAGAATTTTTCCACGGGTATACCTAC

961 AGTGCCCATCCTCTAGCAGCGGCTGCCGTCCTGGCAACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTTGCGCGGGCTAGAAAACTTAGCGCCCCCTTCGAAGAGGCAGCGCACTCTCTC

1081 AAGGGTGCTCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCCGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCACCTGGTGCGCGCGCTGCCGAAGCATTTCAAAAATGTTTCGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCGGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTTGAAACGATAGGCAAGGTTCTCAAAGAGGTCGCT

SEQ ID NO.5：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCGCCTATGGG

781 AGACTTGGTGCAGCAACAGCGGCTGCCTACTTCGGCGTAACGCCCGATCTCATTACTATG

841 GCAAAGGGAGTGTCGAATGCGGCTGTTCCAGCCGGGGCAGTCGCGGTAAGGCGTGAGGTG

901 CATGACGCTATCGTTAACGGTCCGCAGGGCGGAATAGAATTTTTCCACGGGTATACCTAC

961 AGTGCCCATCCTCTAGCAGCGGCTGCCGTCCTGGCAACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTTGCGCGGGCTAGAAAACTTAGCGCCCCCTTCGAAGAGGCAGCGCACTCTCTC

1081 AAGGGTGCTCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCCGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCACCTGGTGCGCGCGCTGCCGAAGCATTTCAAAAATGTTTCGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCGGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTTGAAGGTATAGGCAAGGTTCTCAAAGAGGTCGCT

SEQ ID NO.6：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCGCCTATGGG

781 AGACTTGGTGAAGCAACAGCGGCTGAATACTTCGGCGTAACGCCCGATCTCATTACTATG

841 GCAAAGGGAGTGTCGAATGCGGCTGTTCCAGCCGGGGCAGTCGCGGTAAGGCGTGAGGTG

901 CATGACGCTATCGTTAACGGTCCGCAGGGCGGAATAGAATTTTTCCACGGGTATACCTAC

961 AGTGCCCATCCTCTAGCAGCGGCTGCCGTCCTGGCAACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTTGCGCGGGCTAGAAAACTTAGCGCCCCCTTCGAAGAGGCAGCGCACTCTCTC

1081 AAGGGTGCTCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCCGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCACCTGGTGCGCGCGCTGCCGAAGCATTTCAAAAATGTTTCGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCGGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTTGAAGGTATAGGCAAGGTTCTCAAAGAGGTCGCT

SEQ ID NO.7：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCGCCTATGGG

781 AGACTTGGTGAAGCAACAGCGGCTGCCTACTTCGGCGTAACGCCCGATCTCATTACTATG

841 GCAAAGGGAGTGTCGAATGCGGCTGTTCCAGCCGGGGCAGTCGCGGTAAGGCGTGAGGTG

901 CATGACGCTATCGTTAACGGTCCGCAGGGCGGAATAGAATTTTTCCACGGGTATACCTAC

961 AGTGCCCATCCTCTAGCAGCGGCTGCCGTCCTGGCAACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTTGCGCGGGCTAGAAAACTTAGCGCCCCCTTCGAAGAGGCAGCGCACTCTCTC

1081 AAGGGTGCTCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCCGGAATTGAACTG

1141 GAACCGCGTGAGGGGGCACCTGGTGCGCGCGCTGCCGAAGCATTTCAAAAATGTTTCGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCGGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTTGAAGGTATAGGCAAGGTTCTCAAAGAGGTCGCT

SEQ ID NO.8：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCTTCTATGGG

781 AGACTTGGTGCTGCCACAGCAGCGGCTTACTTTGGCGTAACGCCCGATCTCATTACTATG

841 GCCAAGGGAGTGTCGAATGCAGCGGTTCCAGCTGGGGCCGTCGCAGTAAGGCGTGAGGTG

901 CATGACGCGATCGTTAACGGTCCGCAGGGCGGAATAGAATTCTTTCACGGGTATACCTAC

961 AGTGCTCATCCTCTAGCCGCAGCGGCTGTCCTGGCCACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTCGCACGGGCGAGAAAACTTAGCGCTCCCTTTGAAGAGGCCGCACACTCTCTC

1081 AAGGGTGCGCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCTGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCCCCTGGTGCACGCGCGGCTGAAGCCTTCCAAAAATGTTTTGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCAGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTCGAAACGATAGGCAAGGTTCTCAAAGAGGTCGCG

SEQ ID NO.9：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCTTCTATGGG

781 AGACTTGGTGAAGCCACAGCAGCGGAATACTTTGGCGTAACGCCCGATCTCATTACTATG

841 GCCAAGGGAGTGTCGAATGCAGCGGTTCCAGCTGGGGCCGTCGCAGTAAGGCGTGAGGTG

901 CATGACGCGATCGTTAACGGTCCGCAGGGCGGAATAGAATTCTTTCACGGGTATACCTAC

961 AGTGCTCATCCTCTAGCCGCAGCGGCTGTCCTGGCCACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTCGCACGGGCGAGAAAACTTAGCGCTCCCTTTGAAGAGGCCGCACACTCTCTC

1081 AAGGGTGCGCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCTGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCCCCTGGTGCACGCGCGGCTGAAGCCTTCCAAAAATGTTTTGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCAGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTCGAAACGATAGGCAAGGTTCTCAAAGAGGTCGCG

SEQ ID NO.10：

1 ATGTCTGCTGCCAAATTACCTGATTTGTCCCATCTTTGGATGCCCTTTACTGCAAATCGT

61 CAATTCAAGGCGAACCCACGCCTCCTAGCTTCAGCCAAAGGTATGTATTACACCTCGTTT

121 GACGGCCGACAGATTCTGGATGGAACAGCAGGGTTATGGTGTGTTAATGCGGGTCACTGC

181 CGGGAAGAGATCGTCAGTGCTATAGCCAGCCAAGCAGGCGTAATGGACTATGCGCCGGGA

241 TTCCAGTTGGGGCATCCTCTTGCTTTTGAAGCCGCAACGGCGGTGGCTGGTCTCATGCCC

301 CAAGGCCTAGATAGAGTTTTCTTTACTAACTCTGGATCCGAGTCAGTCGACACCGCCCTG

361 AAGATTGCATTAGCGTACCACAGGGCTCGTGGGGAAGCCCAGCGCACACGATTGATCGGT

421 CGGGAGAGAGGCTATCATGGAGTAGGGTTCGGTGGCATATCGGTGGGAGGGATTAGTCCA

481 AATAGGAAAACGTTTAGCGGTGCACTTCTCCCGGCGGTTGATCACCTACCTCATACTCAC

541 TCTCTGGAACATAACGCTTTCACCCGTGGCCAACCCGAGTGGGGAGCCCACTTAGCAGAC

601 GAATTGGAGCGCATCATAGCGCTTCATGATGCTTCCACAATTGCCGCAGTCATCGTAGAA

661 CCAATGGCGGGGTCAACGGGTGTGCTCGTTCCGCCTAAGGGCTACCTAGAGAAACTGCGA

721 GAAATAACTGCTCGGCACGGAATTTTATTGATCTTTGACGAGGTCATAACCTTCTATGGG

781 AGACTTGGTGCTGCCACAGCAGCGGCTTACTTTGGCGTAACGCCCGATCTCATTACTATG

841 GCCAAGGGAGTGTCGAATGCAGCGGTTCCAGCTGGGGCCGTCGCAGTAAGGCGTGAGGTG

901 CATGACGCGATCGTTAACGGTCCGCAGGGCGGAATAGAATTCTTTCACGGGTATACCTAC

961 AGTGCTCATCCTCTAGCCGCAGCGGCTGTCCTGGCCACATTAGATATTTATCGCCGAGAG

1021 GACTTGTTCGCACGGGCGAGAAAACTTAGCGCTCCCTTTGAAGAGGCCGCACACTCTCTC

1081 AAGGGTGCGCCACATGTAATCGATGTGAGGAATATAGGCCTAGTTGCTGGAATTGAACTG

1141 TCCCCGCGTGAGGGGGCCCCTGGTGCACGCGCGGCTGAAGCCTTCCAAAAATGTTTTGAC

1201 ACGGGCTTAATGGTCCGATACACTGGAGATATCTTGGCAGTATCACCCCCACTTATAGTG

1261 GACGAGAACCAGATTGGGCAAATCTTCGAAACGATAGGCAAGGTTCTCAAAGAGGTCGCG

SEQ ID NO.11：

5’-TACCAGACGACGAcatTTTGACGGCCTGG-3’

SEQ ID NO.12：

5’-CTTCCATAGCCAAggatccTTCAAGACTTTCTTCAACGA-3’

Claims

1. A transaminase mutant, characterized in that the transaminase mutant is mutated at the amino acid sequence shown in SEQ ID No.2 at the sites of a259F, G431T, E264A, a269E, S381E, G431T/E264A, G431T/a269E or a 259F/G431T/E264A.

2. A gene encoding the transaminase mutant of claim 1.

3. A recombinant plasmid comprising the gene of claim 2.

4. An immobilized or engineered enzyme comprising the transaminase mutant of claim 1.

5. A method for constructing a transaminase mutant according to claim 1, comprising the steps of:

searching an amino acid sequence shown by SEQ ID NO.2 in an NCBI database, deleting a repeated identical sequence, selecting an amino acid sequence with the consistency of more than 33 percent with the amino acid sequence shown by SEQ ID NO.2, then performing multi-sequence comparison through Clustalx1.83 software, arranging the rest amino acid sequences into fasta file, uploading the fasta file to a Consensus Makerv2.0.0 server, modifying set parameters according to needs, generating a Consensus sequence which can be edited at a later stage by online software, and screening mutation sites related to thermal stability as follows: a259F, G431T, E264A, a269E, S381E.

6. Use of the transaminase mutant of claim 1 for the catalytic synthesis of chiral amines.