CN118064400A

CN118064400A - S-selective omega-aminotransferase mutant with high catalytic activity, and construction method and application thereof

Info

Publication number: CN118064400A
Application number: CN202410469408.7A
Authority: CN
Inventors: 桂萍; 李晓; 王文文; 郄兴旺; 马富强; 王学峰; 陆峰; 景伟
Original assignee: Jinan Guoke Medical Engineering Technology Development Co ltd; Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Jinan Guoke Medical Engineering Technology Development Co ltd; Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2024-04-18
Filing date: 2024-04-18
Publication date: 2024-05-24
Anticipated expiration: 2044-04-18
Also published as: CN118064400B

Abstract

The invention discloses an S-selective omega-aminotransferase mutant with high catalytic activity, a construction method and application thereof, wherein the S-selective omega-aminotransferase mutant is obtained by mutating the 263 rd isoleucine of a wild omega-aminotransferase TbTA amino acid sequence shown in SEQ ID NO.1 into alanine, glycine, cysteine, methionine, valine, phenylalanine or histidine. Compared with wild type, the enzyme activity of the mutant TbTA-I263A, tbTA-I263G for catalyzing acetophenone is improved by 67.83 times and 61.26 times, and the heat stability is close to or better than that of the wild type; mutants TbTA-I263C, tbTA-I263M, tbTA-I263V, tbTA-I263F and TbTA-I263H had 11.85-fold, 8.76-fold, 3.56-fold, 3.49-fold and 2.55-fold, respectively, increased enzymatic activity.

Description

S-selective omega-aminotransferase mutant with high catalytic activity, and construction method and application thereof

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to an S-selective omega-aminotransferase mutant with high catalytic activity, and a construction method and application thereof.

Background

Omega-aminotransferase (EC 2.6.1.X, omega-TRANSAMINASE, omega-TA), a pyridoxal 5'-phosphate (pyridoxal-5' -phosphate, PLP) dependent enzyme, is capable of reversibly catalyzing the transfer of an amino group from a donor to a carbonyl acceptor, and can produce valuable chiral amines by one-step catalytic reaction under mild reaction conditions, with high theoretical yields and strict stereoselectivity. According to the difference of chiral selectivity, ω -aminotransferase can be classified into S-selectivity and R-selectivity, which respectively belong to fold i and iv of PLP-dependent enzyme, which are structurally distinct, and functionally distinct in that S-selective ω -aminotransferase catalyzes S-or L-amino donors to form S-chiral amines, and R-selective ω -aminotransferase catalyzes R-or D-amino donors to form R-chiral amines.

Natural bases of wild type ω -aminotransferase are mainly small molecular amino acids, keto acids, etc., however chiral amines having high added value functions such as pesticides or pharmaceutical intermediates usually contain a large steric hindrance group such as an aromatic group on one side or both sides of the chiral group, and for this class of compounds, natural ω -aminotransferase is usually not synthesized or is extremely inefficient, which is also a major bottleneck restricting the catalytic synthesis of high-value chiral amines by ω -aminotransferase in terms of technological breakthroughs and industrial applications.

The key factor influencing the omega-aminotransferase to catalyze the substrate with large steric hindrance is the space structure of the substrate binding region, and the substrate binding region is reasonably modified by utilizing a protein engineering technology means, so that the catalysis performance of the omega-aminotransferase on the substrate with large steric hindrance can be effectively improved, and the application range of the aminotransferase in the chiral amine green synthesis field is further widened. Therefore, it is of great importance to develop new ω -transaminases to catalyze the synthesis of high-value chiral amines and to drive their industrial application.

Disclosure of Invention

The invention aims to solve the technical problem of providing an S-selective omega-aminotransferase mutant with high catalytic activity, and a construction method and application thereof, aiming at the defects in the prior art. The invention provides an S-selective omega-aminotransferase mutant with remarkably improved catalytic activity on a large steric hindrance substrate through enzyme engineering, so as to solve the problem of low activity in the prior art when high-value chiral amine is synthesized through aminotransferase catalysis.

In order to achieve the above purpose, the invention adopts the following technical scheme: a mutant of S-selective omega-aminotransferase with high catalytic activity is obtained by mutating isoleucine (I) at position 263 of the amino acid sequence of wild-type omega-aminotransferase TbTA as shown in SEQ ID NO.1 to alanine (A), glycine (G), cysteine (C), methionine (M), valine (V), phenylalanine (F) or histidine (H). The amino acid sequence of the wild-type ω -transaminase was obtained by query and selection from NCBI database (https:// www.ncbi.nlm.nih.gov/protein/MBF 6593860.1).

Preferably, the amino acid sequence of the mutant obtained by mutating isoleucine (I) at position 263 of the amino acid sequence of wild-type ω -transaminase TbTA to alanine (A), glycine (G), cysteine (C), methionine (M), valine (V), phenylalanine (F) or histidine (H) is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8in this order.

Preferably, the S-selective ω -transaminase mutant is obtained by mutating isoleucine at amino acid sequence 263 of the wild-type ω -transaminase TbTA to alanine or glycine.

The invention also provides a gene encoding an S-selective ω -transaminase mutant having a high catalytic activity as described above.

Preferably, the nucleotide sequence of the wild type omega-aminotransferase TbTA shown as SEQ ID NO.1 is SEQ ID NO.9, and the genes for encoding the S-selective omega-aminotransferase mutant with high catalytic activity are obtained by site-directed mutagenesis based on the nucleotide sequence shown as SEQ ID NO. 9.

The present invention also provides a recombinant plasmid comprising the gene as described above.

Preferably, the plasmid vector of the recombinant plasmid is pET-28a (+).

The invention also provides a host cell comprising a gene as defined above or a recombinant plasmid.

Preferably, the host cell is a prokaryotic cell.

Preferably, the host cell is E.coli BL21 comprising the recombinant plasmid as described above.

The invention also provides a construction method of the S-selective omega-aminotransferase mutant with high catalytic activity, which comprises the following steps: culturing a host cell as described above by inducing expression of said S-selective ω -transaminase mutant; the host cells were collected for disruption and centrifugation, and the S-selective ω -transaminase mutants were isolated and purified from the supernatant.

The invention also provides a soluble protein or engineering bacterium containing the S-selective omega-aminotransferase mutant.

The invention also provides an application of the S-selective omega-aminotransferase mutant with high catalytic activity as a biocatalyst in synthesizing S-type chiral amine by catalyzing prochiral ketone compounds.

Preferably, the prochiral ketone compound is acetophenone, p-chloroacetophenone, 1-naphthacene or 2-naphthacene, and the S-type chiral amine is (S) -1-phenethylamine, (S) -1-p-chlorophenylethylamine, (S) -1- (1-naphthanyl) ethylamine or (S) -1- (2-naphthanyl) ethylamine.

Preferably, the application method comprises the following steps: adding the S-selective omega-aminotransferase mutant with high catalytic activity into a reaction system to react, so as to obtain a reaction solution; and extracting the reaction liquid to obtain the S-type chiral amine.

Preferably, the reaction system contains coenzyme, amino donor, prochiral ketone compound and buffer solution.

Preferably, the coenzyme is PLP, and the concentration of the coenzyme is 0.01-1 mmol/L.

Preferably, the amino donor is isopropylamine, L-alanine, phenethylamine or 1- (6-aminonaphthalene-2-yl) ethylamine, and the addition concentration is 2-20 mmol/L.

Preferably, the buffer solution is phosphate buffer solution, and the concentration is 0.01-0.1 mol/L.

Preferably, the reaction temperature is 37-50 ℃, the pH is 8-10, and the reaction time is 2-24 h.

Preferably, the prochiral ketone compound is acetophenone or analogues thereof, and the addition concentration is 0.2-2 mmol/L.

The beneficial effects of the invention are as follows:

The invention mutates the 263 rd isoleucine of wild type omega-aminotransferase TbTA (SEQ ID NO. 1) from thermophilic bacteria THERMACEAE BACTERIUM into alanine, glycine, cysteine, methionine, valine, phenylalanine or histidine through site-directed mutagenesis, and the obtained mutant (I263A, I263G, I263C, I263M, I263V, I263F and I263H) has obviously higher activity for catalyzing prochiral ketone to synthesize S-type chiral amine than the wild type (WT for short) before mutation, and is specifically expressed as follows: the enzyme activity of the mutant TbTA-I263A catalyzed acetophenone is improved by 67.83 times compared with that of the wild type, and the thermal stability is kept close to that of the wild type; the enzyme activity of the mutant TbTA-I263G catalyzed acetophenone is improved by 61.26 times compared with that of the wild type, and the heat stability is also obviously better than that of the wild type; mutants TbTA-I263C, tbTA-I263M, tbTA-I263V, tbTA-I263F and TbTA-I263H have 11.85-fold, 8.76-fold, 3.56-fold, 3.49-fold and 2.55-fold, respectively, increased enzymatic activity of acetophenone compared to the wild type; from the activity improvement effect, the S-selective omega-aminotransferase mutants provided by the invention, especially TbTA-I263A and TbTA-I263G, have high enzyme activity and good thermal stability for catalyzing acetophenone, and have application prospects for producing high-value chiral aromatic amine.

Drawings

FIG. 1 is a schematic diagram of the structure of the invention for modeling the ω -transaminase TbTA from thermophilic bacteria THERMACEAE BACTERIUM and for molecular docking with PMP-acetophenone; wherein K289 is a catalytic residue and I263 is a mutated target residue;

FIG. 2 is a schematic diagram of a method for detecting ω -transaminase activity used in the present invention;

FIG. 3 is a standard graph of the method for detecting ω -transaminase activity used in the present invention;

FIG. 4 is a SDS-PAGE electrophoresis of purified omega-aminotransferase TbTA Wild Type (WT) and its I263 site mutant according to the invention;

FIG. 5 is a comparison of the enzymatic activities of the omega-aminotransferase TbTA of the present invention at position I263 mutated to a different amino acid; relative enzyme activity comparison was performed with the activity of the wild type (residue I) as 100%, and "NA" indicates that no enzyme activity was detected;

FIG. 6 is a comparison of the thermostability of the highly active mutants I263A and I263G of ω -transaminase TbTA of the invention with wild-type at 37℃and 50℃respectively.

Detailed Description

The invention is further described below in connection with specific examples, but any examples should not be construed as limiting the scope or implementation of the invention. Unless otherwise indicated, all technical and scientific terms have the same meaning as commonly understood in the art to which this invention belongs.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The test methods used in the following examples are conventional methods unless otherwise specified. The material reagents and the like used in the following examples are commercially available unless otherwise specified. The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The main reagents employed in the present invention, the sources of the apparatus and the related detection, methods of operation, etc. involved therein are described in detail below.

Coli E.coli BL21 (DE 3) competent cells used in the invention were purchased from Beijing all gold biotechnology Co., ltd; primers for constructing a site-directed mutant plasmid were synthesized by Beijing qingke biotechnology Co., ltd based on the ω -transaminase wild-type plasmid of the pET-28a (+) plasmid vector; the molecular biological reagents used, such as DNA polymerase, restriction enzyme, dpnI, recombinase, etc., were purchased from Takara corporation; pyridoxal phosphate (PLP), acetophenone, p-chloroacetophenone, 1-naphthacene, and 2-naphthacene were used commercially from Sigma-Aldrich; other biochemical reagents used were purchased from Alatidine Biotechnology Co.

The PCR instrument is purchased from Siemens Feishan technology company, and the model of the enzyme-labeled instrument is Bio TEK SYNERGY H; the model of the high performance liquid chromatograph is ALLIANCE E to 2685 (Waters), and the detector is a diode array detector; the model of the chiral column used was cellophane CHIRALPAK IG (particle size 5 μm,4.6 mm X250 mm).

The following examples relate to the following media:

LB solid medium: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L agar and 50 mg/L kanamycin.

LB liquid medium: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl and 50 mg/L kanamycin.

The method for detecting ω -transaminase activity is derived from the literature (Chen et al 2020, applied Microbiology and Biotechnology 104:2999-3009) and is optimised according to the principle shown in figure 2 and specifically by the following scheme:

The fluorescence intensity of the amino donor product 1- (6-aminonaphthalen-2-yl) ethanone in the catalytic reaction is measured by an enzyme-labeling instrument by taking 1- (6-aminonaphthalen-2-yl) ethylamine as an amino donor substrate and prochiral ketone as an amino acceptor substrate, and then the molar concentration of the amino donor product 1- (6-aminonaphthalen-2-yl) ethanone is converted by a standard curve.

Enzyme activity detection system (100 μl): 0.2 mM 1- (6-aminonaphthalen-2-yl) ethylamine, 0.5 mM acetophenone, 0.1 mM PLP,10 mM phosphate buffer (pH 8.5), 10 mg/L wild type or mutant ω -transaminase.

Reaction at 37 ℃ and real-time detection of fluorescence, excitation wavelength: 357 nm, emission wavelength: 525 nm, gain: 50, continuous vibration plate 5 s, fluorescence every 2 min was detected, continuously for 2 hours. The amount (. Mu.mol) of 1- (6-aminonaphthalen-2-yl) ethanone produced per minute by the transamination reaction can be calculated from the concentration-fluorescence standard curve (FIG. 3) of 1- (6-aminonaphthalen-2-yl) ethanone.

Omega-aminotransferase activity unit (U) definition: the amount of enzyme required to catalyze the production of 1. Mu. Mol of this compound per minute was 1U at 37℃under reaction conditions of pH=8.5 with 1- (6-aminonaphthalen-2-yl) ethanone as the detection product.

Definition of ω -transaminase specific activity: number of units of enzyme activity per unit weight of protein.

Qualitative and quantitative analysis of prochiral ketone substrate and chiral amine product of catalytic reaction were performed by High Performance Liquid Chromatography (HPLC), the reversed phase system was set as in Table 1, the flow rate was set to 0.5 mL/min, the detection wavelength was set to full wavelength detection, and the sample injection amount was 10. Mu.L.

TABLE 1 reversed phase configuration of HPLC analysis of chiral amine products in the present invention

The conversion was calculated as follows:

conversion (%) = (N ₀-N₁)/N₀ ×100%;

Wherein N ₀ represents the initial molar concentration of prochiral ketone in the reaction solution, and N ₁ represents the residual molar concentration of prochiral ketone after completion of the reaction.

The optical purity of the chiral product is characterized using an enantiomeric excess value (ee) and is calculated as follows:

ee = (N_S-N_R)/(N_S+N_R) ×100%

Wherein N _S represents the molar concentration of the S-type chiral amine product in the reaction solution, and N _R represents the molar concentration of the R-type chiral amine product in the reaction solution.

Example 1: construction of plasmid for omega-aminotransferase TbTA I locus 263 saturation mutant

According to the amino acid sequence SEQ ID NO.1 of wild-type ω -aminotransferase TbTA, protein homology modeling analysis was performed on the SWISS-MODEL website (https:// swissmodel. Expasy. Org /), and the modeled structure was molecular-docked with an amino donor substrate and an amino acceptor substrate, respectively (Autodock 4.2.6 software), and the substrate binding pocket was analyzed, and the I263 site was found to be very close to the catalytic residues K289, coenzyme PLP/PMP and ketone substrate (FIG. 1), presumably affecting the catalytic activity of TbTA on the substrate.

According to the primers shown in Table 2, single-point mutation PCR was performed on the wild type plasmid pET-28a (+) -TbTA as a template to construct single-point mutation recombinant plasmids containing the coding genes of TbTA-I263 site mutated into 19 other amino acids (A/C/D/E/F/G/H/K/L/M/N/P/Q/R/S/T/V/W/Y), and the recombinant plasmids were extracted and stored after sequencing correctly.

TABLE 2 construction of TbTA-I263 site-directed mutagenesis primers

The whole plasmid single point mutation PCR system is shown in Table 3.

TABLE 3 PCR reaction system

The whole plasmid single point mutation PCR procedure was set as follows:

(1) Denaturation at 98℃for 10 sec; (2) annealing at 55 ℃ for 5 seconds; (3) extension at 72℃for 1 min; (4) repeating the steps (1) - (3), and circulating for 29 times; (5) sufficient extension at 72℃for 5 min; (6) storing the amplified product at 4 ℃.

Example 2: preparation of wild-type ω -transaminase TbTA and ω -transaminase mutants

The mutant plasmids obtained in example 1 were transformed into competent cells of E.coli BL21 (DE 3), respectively, and plated overnight for growth; then, respectively picking the monoclonal antibodies, placing the monoclonal antibodies in 15 mL LB liquid culture medium containing 0.1 mg/mL Kan ⁺, and culturing overnight; then, the mixture was transferred to 1L LB liquid medium containing 0.1 mg/mL Kan ⁺ at a ratio of 1%, and when the mixture was cultured until the OD ₆₀₀ was about 0.8, IPTG was added to 0.5mM, and the mixture was cultured at 16℃and 220 rpm for 16 hours to express the target protein.

Centrifuging the bacterial liquid, collecting bacterial mud, re-suspending with 40 mL buffer (10 mM PBS, 150 mM NaCl), and performing pressure crushing under the treatment condition of 1100 Pa and 2 min; then 12000 rpm is centrifuged for 30min, and the supernatant is filtered once by a filter membrane and placed on ice for standby; before purification, washing the nickel column with 10 column volumes of purified water, washing the nickel column with 10 column volumes of buffer solution, slowly pouring and discharging filtered supernatant, washing the nickel column with 10 column volumes of imidazole buffer solutions (10 mM PBS, 150 mM NaCl and 10 mu M PLP) containing 20 mM, 40mM, 160 mM and 500 mM in sequence, and collecting flow-through liquid, wherein the target protein is mainly in the flow-through liquid with the imidazole concentration of 160 mM; concentrating and desalting the flow-through liquid containing the high-purity target protein by using a desalting gravity column, and then replacing the flow-through liquid into 10 mM PBS buffer solution; finally, the concentration of the enzyme solution is adjusted to 5mg/mL (30% glycerol content), and the enzyme solution is preserved in a refrigerator at 20 ℃ for standby. The results of protein electrophoresis of the different mutants of wild-type ω -transaminase TbTA and I263 are shown in FIG. 4.

Example 3: enzyme activity assay for wild-type ω -transaminase TbTA and ω -transaminase mutants

The specific activities of the wild-type ω -transaminase TbTA and the ω -transaminase mutant prepared in the manner of example 2 were measured separately by taking appropriate amounts of the pure enzyme solutions. Wherein, the specific activity of the wild type omega-aminotransferase TbTA is taken as 100%, and the specific activities of the other mutants are compared with the specific activity of the wild type omega-aminotransferase, and the results are shown in Table 4 and FIG. 5.

Table 4 relative enzyme activities of wild-type ω -transaminase TbTA and ω -transaminase mutants.

"NA" means that no enzyme activity was detected.

According to the test results of Table 4, ω -transaminase mutants obtained by mutating isoleucine (I) at position 263 to alanine (A), glycine (G), cysteine (C), methionine (M), valine (V), phenylalanine (F) or histidine (H) were selected: I263A, I263G, I263C, I263M, I263V, I263F, I263H.

Example 4: high activity mutants I263A and I263G of ω -transaminase TbTA have a thermal stability at 37℃and 50℃respectively compared with the wild type

A proper amount of pure enzyme solutions of wild-type ω -transaminase TbTA (designated as WT), I263A and I263G mutants prepared according to the method of example 2 were taken, diluted to 1 mg/mL with 10 mM PBS, and incubated in water baths at 37℃and 50℃simultaneously, and small amounts of enzyme solutions were taken out to measure the activity at 0 h, 2 h, 4 h, 8h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h, respectively. The relative enzyme activities were calculated by comparing the catalytic activities at the rest of the time with the catalytic activities of 0 h as 100%, and the results are shown in FIG. 6. On the basis of greatly improving the activity, the heat stability of TbTA-I263G mutant at 37 ℃ and 50 ℃ is also obviously improved: the residual activity of TbTA-I263G after 48 h is higher than 90% at 37 ℃, the relative activity of about 80% after 120 h is incubated, and the wild type activity of TbTA is about 50% and 30% respectively at the same time. Moreover, the activity of TbTA-I263A mutant is slightly reduced at 37 ℃ and 50 ℃ to a smaller extent than that of the wild type, but the absolute activity is still obviously higher than that of the wild type in consideration of the activity improving effect. Comprehensively comparing TbTA-I263G is a relatively optimal mutant with high activity and heat stability.

Example 5: HPLC analysis of conversion rate and ee value of high activity omega-aminotransferase mutant TbTA-I263G for catalyzing acetophenone and analogues thereof to generate chiral amine

The TbTA-I263G enzyme solution prepared in example 2 was subjected to catalytic reaction according to the following system: 10 mM isopropylamine, 0.5 mM prochiral ketone (1 a, 2a, 3a and 4a as substrates respectively), 2mM PLP, 0.5 mg/mL TbTA-I263G,10% DMSO, 10 mM PBS (pH 8.0). The enzyme reaction was incubated at 50℃and 220 rpm for 24: 24 h. Centrifuging the reaction system 12000 rpm after finishing for 5min, mixing the supernatant with an equal volume of acetonitrile, and carrying out qualitative and quantitative analysis on a prochiral ketone substrate and a corresponding chiral amine product of the catalytic reaction by utilizing HPLC, wherein the reverse phase system is set as shown in table 1, the flow rate is set to be 0.5 mL/min, the detection wavelength is full wavelength, the analysis wavelength is 254 nm, and the sample injection amount is 10 mu L. The target chiral amine products were 1b, 2b, 3b and 4b, respectively, and the conversion and ee values were calculated and the results are shown in table 5. The TbTA-I263G mutant efficiently catalyzes the conversion of 4 prochiral ketones into S-chiral amines, the highest conversion rate can reach 92.8%, and the ee values are all more than 99%.

The chemical structural formulas of the prochiral ketones 1a, 2a, 3a, 4a and the target chiral amines 1b, 2b, 3b, 4b are as follows:

TABLE 5 conversion and ee measurement results

Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.

Claims

1. A high catalytic activity S-selective omega-aminotransferase mutant, which is obtained by mutating the 263 rd isoleucine of the amino acid sequence of wild-type omega-aminotransferase TbTA as shown in SEQ ID NO.1 to alanine, glycine, cysteine, methionine, valine, phenylalanine or histidine.

2. The mutant of high catalytic activity S-selective omega-aminotransferase according to claim 1, wherein the amino acid sequence of the mutant obtained by mutating isoleucine at position 263 of the amino acid sequence of wild type omega-aminotransferase TbTA to alanine, glycine, cysteine, methionine, valine, phenylalanine or histidine is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

3. The high catalytic activity S-selective ω -transaminase mutant according to claim 2, characterised in that the S-selective ω -transaminase mutant is obtained by mutating isoleucine at amino acid 263 of a wild-type ω -transaminase TbTA to alanine or glycine.

4. A gene encoding the high catalytic activity S-selective ω -transaminase mutant according to any one of claims 1 to 3.

5. The gene according to claim 4, wherein the nucleotide sequence encoding the wild-type ω -transaminase TbTA shown in SEQ ID NO.1 is SEQ ID NO.9, and the genes encoding the S-selective ω -transaminase mutants having high catalytic activity are obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO. 9.

6. A recombinant plasmid comprising the gene according to claim 4 or 5.

7. The recombinant plasmid according to claim 6, wherein the plasmid vector of the recombinant plasmid is pET-28a (+).

8. A host cell comprising the gene of claim 4 or 5 or the recombinant plasmid of claim 6 or 7.

9. The host cell of claim 8, wherein the host cell is a prokaryotic cell.

10. The host cell according to claim 9, wherein the host cell is e.coli BL21 comprising the recombinant plasmid according to claim 6 or 7.

11. A method of constructing a high catalytic activity S-selective ω -transaminase mutant according to any one of claims 1 to 3, comprising the steps of: culturing the host cell of any one of claims 8-10 by inducing expression of the S-selective ω -transaminase mutant; the host cells were collected for disruption and centrifugation, and the S-selective ω -transaminase mutants were isolated and purified from the supernatant.

12. Use of a high catalytic activity S-selective ω -transaminase mutant according to any one of claims 1 to 3 as biocatalyst for the catalytic synthesis of S-chiral amines from prochiral ketones.

13. Use according to claim 12, wherein the prochiral ketone compound is acetophenone, p-chloroacetophenone, 1-naphthaceneone or 2-naphthaceneone and the S-chiral amine is (S) -1-phenethylamine, (S) -1-p-chlorophenylethylamine, (S) -1- (1-naphtyl) ethylamine or (S) -1- (2-naphthacene) ethylamine.