Transaminase mutant and its application in biocatalytic preparation of chiral amine

Abstract

The application discloses a transaminase mutant and application thereof in biocatalytic preparation of chiral amines, and belongs to the technical field of enzyme engineering and biocatalysis. Exophiala sideris The wild-type omega-transaminase derived from Pseudomonas putida is used as a template, one or more amino acid sites are subjected to substitution mutation, and the transaminase mutant is obtained. Experiments show that the transaminase mutant has significantly improved transaminase activity and thermal stability compared with the wild-type transaminase. The application further provides a method for catalytically synthesizing chiral amine compounds and pharmaceutical intermediates by using the transaminase mutant. The method is simple in operation, mild in reaction condition, green and environment-friendly, and has a wide application prospect in the pharmaceutical industry and fine chemical production.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology, specifically relating to an engineered transaminase mutant and its application in the biocatalytic preparation of chiral amine compounds and pharmaceutical intermediates. Background Technology

[0002] Transaminases (EC 2.6.1.x) are a class of enzymes that use pyridoxal-5'-phosphate (PLP) as a coenzyme to catalyze amino transfer reactions between amino donors and ketone / aldehyde acceptors. They have wide applications in the asymmetric synthesis of chiral amines. Compared with traditional transition metal-catalyzed asymmetric synthesis methods, ω=transaminase catalysis offers advantages such as milder reaction conditions, higher stereoselectivity, no need for expensive chiral ligands, and avoidance of heavy metal contamination.

[0003] However, naturally derived ω-transaminases still face numerous technical bottlenecks in industrial applications, severely hindering their commercialization. Firstly, the substrate-binding region of wild-type ω-transaminases has a unique spatial structure, and the active pocket is highly sensitive to steric hindrance. Natural substrates are mainly small-molecule amino acids or keto acids, typically showing no or very low activity towards large-volume substrates. This is a major technical bottleneck restricting the industrial application of wild-type transaminases in the asymmetric catalytic synthesis of chiral amine compounds. Furthermore, biocatalysis in industrial production often requires overcoming relatively extreme environments, such as high temperatures, strong acids and alkalis, and organic solvents. Wild-type ω-transaminases, as catalysts, lack sufficient thermal stability and typically require relatively mild conditions for catalytic reactions. The conditions in industrial production easily lead to inactivation, further limiting their widespread application in industrial production. Therefore, developing novel transaminase mutants with higher catalytic activity and better thermal stability is of significant technical and economic importance for promoting the practical application of green biocatalysis in the pharmaceutical industry. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the present invention, through [the study of] sources [from...] Exophiala sideris By semi-rational design of wild-type transaminase (whose amino acid sequence is shown in SEQ ID NO.1), a series of beneficial mutants with significantly enhanced activity and thermostability were obtained.

[0005] Specifically, the transaminase mutant is obtained by mutating the wild-type transaminase with an amino acid sequence as shown in SEQ ID NO.1, and the mutation site includes position 53.

[0006] Furthermore, the mutation involves replacing the 53rd amino acid residue with either A or G.

[0007] Furthermore, the mutated site also includes at least one of the following sites: position 15, position 33, position 40, position 47, position 49, position 52, position 60, position 62, position 75, position 76, position 79, position 85, position 89, position 98, position 108, position 113, position 115, position 116, position 126, position 127, position 128, position 129, position 130, position 131, position 137, position 141, position 143. 146th, 148th, 149th, 150th, 169th, 171st, 181st, 186th, 194th, 200th, 204th, 209th, 214th, 218th, 223rd, 226th, 235th, 237th, 242nd, 252nd, 256th, 263rd, 267th, 272nd, 273rd, 279th, 286th, 291st, 312th.

[0008] Further, the mutation also includes at least one of the following mutation methods: replacing the 15th amino acid residue with W or Y, replacing the 33rd amino acid residue with E, replacing the 40th amino acid residue with H, S, or D, replacing the 47th amino acid residue with L, replacing the 49th amino acid residue with Y, I, L, H, M, or V, replacing the 52nd amino acid residue with I, L, or V, replacing the 60th amino acid residue with A, replacing the 62nd amino acid residue with I or N, replacing the 75th amino acid residue with L, replacing the 76th amino acid residue with S, replacing the 79th amino acid residue with F, replacing the 85th amino acid residue with L, replacing the 89th amino acid residue with P, and replacing the 98th amino acid residue with K. Alternatively, replace amino acid residue D with G, amino acid residue 113 with L, I, M, or Y, amino acid residue 115 with K, Q, or L, amino acid residue 116 with V, A, or T, amino acid residue 126 with P, K, Q, or A, amino acid residue 127 with A, T, V, M, S, N, or D, amino acid residue 128 with F or M, and amino acid residue 129 with G. Replace the following amino acid residues: 130th amino acid residue with K, R, A, or C; 131st amino acid residue with Q, A, or P; 137th amino acid residue with N; 141st amino acid residue with W, I, or N; 143rd amino acid residue with V, M, I, L, or C; 146th amino acid residue with W, L, M, I, A, or N; 148th amino acid residue with I or L; 149th amino acid residue with C; and 150th amino acid residue with E. Alternatively, replace amino acid residue 169 with V, amino acid residue 171 with R, amino acid residue 181 with F, amino acid residue 186 with F, amino acid residue 194 with H, amino acid residue 200 with F, amino acid residue 204 with P or L, amino acid residue 209 with Y or F, amino acid residue 214 with P, amino acid residue 218 with I, and amino acid residue 223 with P. The amino acid residue at position 226 is replaced with K, the amino acid residue at position 235 is replaced with E, the amino acid residue at position 237 is replaced with I, the amino acid residue at position 242 is replaced with V, the amino acid residue at position 252 is replaced with A, the amino acid residue at position 256 is replaced with E, the amino acid residue at position 263 is replaced with T, the amino acid residue at position 267 is replaced with K, the amino acid residue at position 272 is replaced with C, and the amino acid residue at position 273 is replaced with S.Replace amino acid residue 279 with C, amino acid residue 286 with G, amino acid residue 291 with N, and amino acid residue 312 with P.

[0009] Furthermore, the mutation includes at least one of the following single point mutations or combinations of mutations: .

[0010] In the amino acid mutation codes of this invention, the letters before and after the numbers are amino acid abbreviations known in the art. For example, V represents Valine, A represents Alanine, M represents Methionine, L represents Leucine, Q represents Glutamine, R represents Arginine, I represents Isoleucine, S represents Serine, H represents Histidine, E represents Glutamic acid, P represents Proline, F represents Phenylalanine, D represents Aspartic acid, Y represents Tyrosine, G represents Glycine, N represents Asparagine, C represents Cysteine, K represents Lysine, T represents Threonine, and W represents Tryptophan.

[0011] In the amino acid mutation code of this invention, the numbers are the mutation site numbers, the letters before the numbers represent the amino acids before the mutation, and the letters after the numbers represent the amino acids after the mutation. For example, H53A means that the 53rd amino acid is mutated from H to A, R126P means that the 126th amino acid is mutated from R to P, and so on.

[0012] In the amino acid combination mutation codes of this invention, "+" indicates mutations performed before and after the "+". For example, H53A+S214P+T273S represents a combination of H53A mutation, S214P mutation, and T273S mutation, and so on.

[0013] Furthermore, the transaminase mutant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0014] Preferably, the transaminase mutant has at least 85%, 90%, 93%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0015] Sequence identity can be determined using sequence alignment algorithms known in the field (such as BLASTP, FASTA, CLUSTALW, etc.), calculated using default parameters.

[0016] Based on this invention, those skilled in the art will understand that, without altering the core catalytic function and stereoselectivity of transaminase, one or more amino acids can be conservatively substituted, added, or deleted from the transaminase mutant sequence to obtain protein variants with equivalent or similar functions.

[0017] Conservative substitution refers to the substitution of amino acids within the same class, including: (1) nonpolar amino acids: alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M); (2) uncharged polar amino acids: glycine (G), serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), glutamine (Q); (3) acidic amino acids: aspartic acid (D), glutamic acid (E); (4) basic amino acids: lysine (K), arginine (R), histidine (H). Amino acid substitutions within the above categories are generally considered conservative substitutions and have little impact on protein structure and function.

[0018] The present invention also provides an isolated nucleic acid molecule that encodes any of the above-mentioned transaminase mutants.

[0019] In the nucleotide combination mutation codes of this invention, ";" indicates mutations performed before and after the ";". For example, "CAT157-159GCT;GTG178-180GCC;AGC640-642CCG;ACC817-819TCT" indicates a combination of CAT157-159GCT mutation, GTG178-180GCC mutation, AGC640-642CCG mutation, and ACC817-819TCT mutation, and so on.

[0020] The present invention also provides a recombinant expression vector containing the aforementioned nucleic acid molecule. The recombinant expression vector is constructed by operably linking the nucleic acid molecule to an expression regulatory sequence, the expression regulatory sequence including a promoter, a ribosome binding site, and a terminator.

[0021] The expression vector can be selected from any vector known in the art suitable for expressing exogenous genes in host cells, including but not limited to pET series vectors, pUC series vectors, pBAD series vectors, pQE series vectors, pGEX series vectors, etc. Preferably, the recombinant expression vector is pET28a, pET21a, pBV220, or pGEX6P.

[0022] The present invention also provides a recombinant host cell containing the recombinant expression vector, or having the nucleic acid molecule integrated into its genome.

[0023] The host cell can be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells can be selected from Escherichia coli, Bacillus subtilis, Streptomyces, etc.; eukaryotic cells can be selected from yeasts (such as Saccharomyces cerevisiae, Pichia pastoris), filamentous fungi, etc. Preferably, the host cell is Escherichia coli BL21(DE3).

[0024] The present invention further provides a method for preparing amine compounds, the method comprising the following steps: in the presence of any of the above-mentioned transaminase mutants or the above-mentioned recombinant host cells, a transamination reaction is carried out using ketone substrates and amino donors as raw materials to generate chiral amines.

[0025] Furthermore, the structure of the ketone substrate is as follows: R1 and R2 are each independently selected from C 1-6 Alkyl group, unsubstituted or substituted phenyl group, unsubstituted or substituted 5-6 membered aromatic heterocycle, LR3, LOR3, where L is selected from C 1-3 The alkylene group, R3, is selected from unsubstituted or substituted phenyl groups; or R1 and R2 are linked to form an unsubstituted or substituted 6-7 membered ring, an unsubstituted or substituted 6-7 membered ring, and a 5-6 membered aromatic heterocycle; the substituents on the phenyl group, the 5-6 membered aromatic heterocycle, the 6-7 membered ring, the 6-7 membered ring, and the 5-6 membered aromatic heterocycle are each independently selected from C1. 1-6 Alkyl, halogen, halogen-substituted C 1-6 Alkyl, C 1-6 Alkyl group, COOR4, SO2NH2, R4 is selected from C 1-6 alkyl.

[0026] Furthermore, the structure of the ketone substrate is selected from: Furthermore, the amino donor is a primary amine amino donor; the transamination reaction is carried out with the participation of a coenzyme.

[0027] Furthermore, the amino donor is isopropylamine or alanine; the coenzyme is pyridoxal phosphate.

[0028] Furthermore, the transamination reaction is carried out in a buffer solution with a pH of 7.0-10.5, a reaction temperature of 25-45°C, and a reaction time of 6-48 hours.

[0029] Furthermore, the transamination reaction is carried out in a buffer solution with a pH of 8.0±0.5, a reaction temperature of 37±3℃, and a reaction time of 17±3 hours.

[0030] Furthermore, the transaminase mutant can be used in the form of a free enzyme, an immobilized enzyme, or a whole-cell catalyst expressing the transaminase mutant.

[0031] The beneficial effects achieved by this invention are as follows: The transaminase mutant of this invention has significantly improved transaminase activity compared with wild-type transaminase, with a conversion rate of over 95%, and significantly improved thermal stability, with a Tm value that can be increased by 38°C. It shows good industrial application prospects in the green synthesis of chiral amine compounds and pharmaceutical intermediates.

[0032] The present invention will be described in detail below through specific embodiments. It should be understood that these embodiments are only for explaining the present invention and are not intended to limit the scope of the present invention. As will be known to those skilled in the art, modifications can be made to the present invention without departing from the spirit of the invention, and such modifications also fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 A schematic diagram of the reaction route for the formation of chiral amines from substrate ketones using transaminase catalysis.

[0034] Figure 2 The structural formulas of the products after reduction of substrates S1 to S8 in Example 3.

[0035] Figure 3 Liquid phase diagrams of the reaction solution after the conversion of substrates S1 to S8 in Example 3. Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] DNA fragments and protein sequences of recombinant proteins: The following examples use the transaminase with the amino acid sequence shown in SEQ ID NO:1 (nucleotide sequence SEQ ID NO:2) as the parent material for mutation experiments to obtain various variants. The amino acid sequence shown in SEQ ID NO:1 is derived from... Exophiala sideris The amino acid sequence of wild-type transaminase (esATA).

[0038] The amino acid sequence shown in SEQ ID NO: 1 is as follows: MATMDEIFAAYEKRQAVLEESSNPLSKGIAWVFGELVPLAEARIPLMDQGFMHSDLTYDVPSVWDGRFFRLDDHIARLEASCAKMRLQLPLPREEVKQILVDMVAKSEIKDAFVELIVTRGLKGVRGHTPGETFKNHLYMFVQPYVWVMDPDIQKTGGKAIIARTVRRI PPGSIDPTVKNLQWGDLTRGLFEAADRGATYPFLTDGDANLTEGSGFNVLLVKDGVIYTPDRGVLQGVTRKSCIDVAKSLGIEVRVQFVPIQMAYDADEIFMATTAGGIMPITTLDDKPIQSGKVGPITKKIWDGYWAIHYDDAYSFEIQYGASTNGTSKNGQVSGVNGH The nucleotide sequence shown in SEQ ID NO.2 is as follows: Example 1: Design of Mutation Sites This invention is derived from Exophiala sideris Wild-type ω-transaminase (SEQ ID NO.1) was used as the parental template. To obtain a mutant with significantly enhanced catalytic activity, the spatial structure of the target enzyme was first obtained using a previously resolved ω-transaminase (PDB ID: 6SNL) as a template. The substrate ketone and coenzyme pyridoxal phosphate (PLP) were docked into the enzyme's active pocket, and key amino acid residues in the substrate-binding region were analyzed. Through molecular docking and molecular dynamics simulations, several residues with potential impacts on substrate binding, catalytic transition state stability, and product release were identified. Simultaneously, multiple sequence alignment was performed to analyze evolutionary conservation and determine the flexible regions near the substrate-binding pocket and around the active site. Based on the above analysis, the present invention selects the following catalytically active sites as mutation targets: Q49, M52, H53, L56, V60, S62, F113, E115, L116, R126, G127, H128, T129, P130, G131, F141, Q143, V146, V148, D150, L190, and Y200, and superimposes other conserved and stable sites: Q15, F33, A40, M47, I75, and A7. 6. E79, M85, L89, Q98, E108, H137, M149, I169, P171, G172, L181, L186, T204, A208, N209, S214, V218, D223, I226, Q235, V237, C242, E252, Q256, A263, D267, A272, T273, A275, M279, T283, D286, S291, D312. These mutations were subjected to either saturation mutagenesis or single-site site-directed mutagenesis. After screening, multiple beneficial mutations were combined and iterated to construct a multi-site combined mutant library, ultimately obtaining highly active multiple mutants.

[0039] Example 2: Construction and Screening of Mutant Libraries Using the wild-type transaminase gene plasmid WT-esATA-pET-21a as a template, PCR amplification was performed using primer pairs containing the mutation site. The PCR product was digested with Dpn I to remove the template plasmid, then transformed into competent *E. coli* cells and plated on LB agar plates containing 100 μg / ml ampicillin, and incubated overnight at 37°C. Single colonies were picked for sequencing verification to confirm the correct introduction of the target mutation and the absence of other mutations, and a mutant library was constructed. Single colonies from the mutant library were inoculated into 96-well cell culture plates containing 150 μL of LB medium (containing 100 μg / ml ampicillin), with 8 control groups per 96-well plate; and incubated overnight in a high-throughput shaker at 37°C and 750 rpm.

[0040] 4% seed culture was inoculated into 2 mL deep-well plates containing 800 μL of fresh LB medium (containing 100 μg / mL ampicillin) and incubated in a high-throughput shaker at 37°C and 750 rpm for 5 h until OD600 reached 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 0.05 mM, and expression was induced at 25°C and 750 rpm for 18 h. After induction, the supernatant was discarded by centrifugation, and 200 μL of reaction solution was added. The mixture was thoroughly vortexed and incubated in a high-throughput shaker at 30°C for 17 h. The specific reaction system consisted of: ketone substrate, 5% DMSO, 0.1 M isopropylamine, 0.2 mM PLP, and 0.1 mol / L PB buffer (pH 7.0). After the reaction, 200 μL of acetonitrile was added, vortexed, and centrifuged. The supernatant was filtered and analyzed by HPLC.

[0041] By comparing the enzyme activity with that of wild-type esATA (SEQ ID NO.1), positive mutants with higher activity were screened. The gene sequences of the mutants were obtained after secondary screening and gene sequencing. Specific amino acid and nucleotide mutation sites are shown in Table 1.

[0042] Table 1. Mutation sites of raATA mutants Example 3: Expression and purification of mutants Transform the plasmid containing the mutant into E. coliBL21(DE3) was inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and activated overnight at 37°C. A 1% inoculum was then transferred to 80 mL of LB medium (containing 100 μg / mL ampicillin, 0.2 mM PLP) and cultured at 37°C, 220 rpm until an OD600 of 0.8–1.0 was reached. After cooling to 25°C, 0.2 mM IPTG was added to induce expression for 18 h. Cells were collected by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in buffer A (20 mM PB, 167 mM NaCl, pH 7.0, 0.02 mM PLP). Cells were disrupted by sonication (300 W, 3 s on, 5 s off, 30 min total), and centrifuged at 12000 rpm for 30 min at 4°C. A portion of the supernatant was collected and lyophilized to prepare lyophilized powder, i.e., the crude enzyme powder of the mutant.

[0043] Another portion of the supernatant was loaded onto a Ni-NTA agarose affinity chromatography column. The column was first washed with buffer A to baseline, then eluted with buffer B (20 mM PB, 167 mM NaCl, 10 mM imidazole, pH 8.0) to remove contaminating proteins. Finally, the target protein was eluted with buffer C (20 mM PB, 167 mM NaCl, 250 mM imidazole, pH 8.0). The elution peak was collected and replaced with storage buffer C (20 mM PB, 167 mM NaCl, pH 7.0) using a desalting column to obtain the purified enzyme solution. Purity was determined by SDS-PAGE, and the purified protein concentration was measured before storage at -80°C for later use. The results showed a purity greater than 90%, and the purified protein concentration was not less than 2 mg / mL.

[0044] Example 4: Enzymatic characterization of mutants 1. Enzyme activity assay Using the crude enzyme powder of the mutant as the experimental subject, the catalytic activity of the mutant was tested according to the following method. Crude enzyme activity determination reaction conditions: 0.4 mol / L, pH 8.0 sodium phosphate buffer, 0.5 M isopropylamine, 0.5 mM PLP, 5 mg / mL crude enzyme solution, substrate (one of 30 mM substrate S1, 30 mM substrate S2, 50 mM substrate S3, 10 mM substrate S4, 50 mM substrate S5, 50 mM substrate S6, 30 mM substrate S7, or 25 mM substrate S8), reacted in a high-throughput shaker at 37℃ for 17 h. After the reaction, 2 volumes of acetonitrile were added to the reaction flask, vortexed to mix, and filtered through a 0.2 μm filter membrane. The product yield was detected by HPLC, and the conversion rate was calculated by comparing the product peak area with the standard curve of the product standard. A schematic diagram of the reaction route for the conversion of a substrate ketone to a chiral amine using transaminase is shown below. Figure 1As shown, the structural formulas of the products after reduction of substrates S1~S8 are as follows: Figure 2 As shown, the HPLC chromatograms of the reaction solution after the conversion of substrates S1 to S8 are as follows: Figure 3 As shown.

[0045] The specific results are shown in Table 2.

[0046] Table 2 Comparison of activities between wild-type and mutant enzymes Activity results showed that wild-type transaminases had no activity or very low activity for the aforementioned substrate ketones; the mutants modified by saturation mutagenesis, site-directed mutagenesis and iterative combination mutagenesis acquired catalytic ability for substrates with large steric hindrance, with a significant increase in activity and a conversion rate that can be increased to over 95%, which fully demonstrates the effectiveness and innovation of the mutation method described in this invention in improving the catalytic activity of transaminases.

[0047] 2. Determination of melting temperature (Tm) Using purified enzyme from the mutant as experimental material, the melting temperature (Tm) of the ATA enzyme was determined by fluorescence method. The purified enzyme (0.5 mg / mL) was mixed with 5×SYPRO Orange dye in buffer A (20 mM PB, 167 mM NaCl, pH 7.0, 0.05 mM PLP) to a final volume of 25 μL. A real-time quantitative PCR instrument was used for gradient temperature increases (25-95℃, heating rate 1℃ / min), and changes in fluorescence intensity were monitored. The melting temperature (Tm) was obtained by fitting a curve plotted between fluorescence intensity and temperature.

[0048] Table 3. Melting temperatures (Tm) of esATA and its mutants The results showed that, compared with the wild-type M0 (Tm 42.5℃), the Tm of all mutants of this invention was increased, demonstrating improved thermal stability. Several mutants had Tm values ​​increased to 80.5℃, an increase of 38℃ compared to the wild type. These results indicate that, compared with wild-type transaminase, the mutants of this invention can be industrially produced at higher temperatures, offering greater flexibility in the production process and demonstrating promising industrial application prospects.

[0049] In summary, this invention originates from... Exophiala sideris Based on wild-type transaminase, several mutants with significantly improved activity and stability were obtained through specific mutations. These mutants have broad application prospects in the catalytic synthesis of chiral amine compounds.

Claims

1. A transaminase mutant, characterized in that, The transaminase mutant was obtained by mutating the wild-type transaminase with the amino acid sequence shown in SEQ ID NO.1, and the mutation site includes position 53.

2. The transaminase mutant according to claim 1, characterized in that, The mutation involves replacing the 53rd amino acid residue with either A or G.

3. The transaminase mutant according to claim 1 or 2, characterized in that, The mutation site also includes at least one of the following sites: position 15, position 33, position 40, position 47, position 49, position 52, position 60, position 62, position 75, position 76, position 79, position 85, position 89, position 98, position 108, position 113, position 115, position 116, position 126, position 127, position 128, position 129, position 130, position 131, position 137, position 141, position 143, position 1 46th, 148th, 149th, 150th, 169th, 171st, 181st, 186th, 194th, 200th, 204th, 209th, 214th, 218th, 223rd, 226th, 235th, 237th, 242nd, 252nd, 256th, 263rd, 267th, 272nd, 273rd, 279th, 286th, 291st, 312th.

4. The transaminase mutant according to claim 1 or 2, characterized in that, The mutation further includes at least one of the following mutation methods: replacing the 15th amino acid residue with W or Y, replacing the 33rd amino acid residue with E, replacing the 40th amino acid residue with H, S, or D, replacing the 47th amino acid residue with L, replacing the 49th amino acid residue with Y, I, L, H, M, or V, replacing the 52nd amino acid residue with I, L, or V, replacing the 60th amino acid residue with A, replacing the 62nd amino acid residue with I or N, replacing the 75th amino acid residue with L, replacing the 76th amino acid residue with S, replacing the 79th amino acid residue with F, replacing the 85th amino acid residue with L, replacing the 89th amino acid residue with P, and replacing the 98th amino acid residue with K. Alternatively, replace amino acid residue D with G, amino acid residue 113 with L, I, M, or Y, amino acid residue 115 with K, Q, or L, amino acid residue 116 with V, A, or T, amino acid residue 126 with P, K, Q, or A, amino acid residue 127 with A, T, V, M, S, N, or D, amino acid residue 128 with F or M, and amino acid residue 129 with G. Replace the following amino acid residues: 130th amino acid residue with K, R, A, or C; 131st amino acid residue with Q, A, or P; 137th amino acid residue with N; 141st amino acid residue with W, I, or N; 143rd amino acid residue with V, M, I, L, or C; 146th amino acid residue with W, L, M, I, A, or N; 148th amino acid residue with I or L; 149th amino acid residue with C; 150th amino acid residue with E or P; 169th amino acid residue with V; 171st amino acid residue with R; 181st amino acid residue with F; 186th amino acid residue with F; 194th amino acid residue with H; 200th amino acid residue with F; and 204th amino acid residue with P or L. Replace amino acid residue 209 with Y or F, amino acid residue 214 with P, amino acid residue 218 with I, amino acid residue 223 with K, amino acid residue 226 with L or V, amino acid residue 235 with E, amino acid residue 237 with I, amino acid residue 242 with V, amino acid residue 252 with A, amino acid residue 256 with E, amino acid residue 263 with T, amino acid residue 267 with K, amino acid residue 272 with C, and amino acid residue 273 with S.Replace amino acid residue 279 with C, amino acid residue 286 with G, amino acid residue 291 with N, and amino acid residue 312 with P.

5. The transaminase mutant according to claim 1, characterized in that, The mutation includes at least one of the following single point mutations or combinations of mutations: 。 6. A nucleic acid molecule, characterized in that, Its nucleotide sequence encodes the transaminase mutant as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant host cell, characterized in that, The recombinant host cell contains the recombinant expression vector of claim 7 or has the nucleic acid molecule of claim 6 integrated into its genome.

9. A method for preparing chiral amines via biocatalysis, characterized in that, The method includes the following steps: in the presence of the transaminase mutant according to any one of claims 1-5 or the recombinant host cell according to claim 8, a transamination reaction is carried out using ketone substrates and amino donors as raw materials to generate chiral amines.

10. The method according to claim 9, characterized in that, The structure of the ketone substrate is as follows: R1 and R2 are each independently selected from C 1-6 Alkyl group, unsubstituted or substituted phenyl group, unsubstituted or substituted 5-6 membered aromatic heterocycle, LR3, LOR3, where L is selected from C 1-3 The alkylene group, R3, is selected from unsubstituted or substituted phenyl groups; or R1 and R2 are linked to form an unsubstituted or substituted 6-7 membered ring, an unsubstituted or substituted 6-7 membered ring, and a 5-6 membered aromatic heterocycle; the substituents on the phenyl group, the 5-6 membered aromatic heterocycle, the 6-7 membered ring, the 6-7 membered ring, and the 5-6 membered aromatic heterocycle are each independently selected from C1. 1-6 Alkyl, halogen, halogen-substituted C 1-6 Alkyl, C 1-6 Alkyl group, COOR4, SO2NH2, R4 is selected from C 1-6 Alkyl group; the amino donor is a primary amine amino donor; the transamination reaction is carried out with the participation of a coenzyme; Preferably, the structure of the ketone substrate is selected from: The amino donor is isopropylamine or alanine; the coenzyme is pyridoxal phosphate.

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