Omega-transaminase mutant with high thermal stability and application of omega-transaminase mutant in synthesis of R-chiral amine

Abstract

The invention discloses an omega-transaminase mutant with high thermal stability and application of the omega-transaminase mutant in synthesis of R-chiral amine, and belongs to the technical field of biological enzyme engineering. According to the present invention, the omega-transaminase mutant is obtained by respectively mutating serine at the 132 site and arginine at the 144 site of omega-transaminase (NCBI GenBank: BAK39753.1) from Arthrobacter sp into cysteine, wherein the half-inactivation temperature of the omega-transaminase mutant is increased by 4.6 DEG C compared to the half-inactivation temperature of the wild type; and the half-life period at the temperature of 40 DEG C is 26.4 + / -0.4 min, is prolonged by 14.6 + / -0.6 min compared with that of a wild type and is 2.1 times that of the wild type, and the half-life period is remarkably prolonged. By using the high-thermal-stability omega-transaminase mutant, the synthesis steps of a chiral intermediate can be reduced, the reaction temperature can be increased, the forward reaction can be promoted, the raw material cost is low, the operation is simple and convenient, and the high-thermal-stability omega-transaminase mutant is more suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, specifically relates to enzyme engineering technology, and in particular to a highly thermally stable ω-aminotransferase mutant and application thereof in the synthesis of R-chiral amines. Background Art

[0002] Chiral amines are important intermediates for the synthesis of drugs and other fine chemicals due to their high-density structural information, hydrogen bond formation and fine biological activity. They are often used in the synthesis of chiral amine drugs, such as the diabetes drug sitagliptin, the anti-HIV drug dolutegravir, the Parkinson's drug (R)-rasagiline, the oral antihypertensive drug aliskiren, and the drug silodosin for the treatment of benign prostatic hyperplasia. According to statistics, chiral amine drugs account for about 30% of the top 200 small molecule drugs in annual sales. The application of these chiral drugs has greatly promoted the exploration of efficient synthesis of chiral amines.

[0003] ω-transaminases (ω-TAs) are attractive biocatalysts for the synthesis of optically pure chiral amines and have good application prospects in the synthesis of chiral amines. However, due to the many deficiencies of the reported wild-type transaminases in terms of substrate specificity, stability, catalytic efficiency, low activity, etc., the current demand for their industrial application is still relatively limited. With the in-depth research on the protein structure and catalytic mechanism of transaminases and the development of protein engineering technology, the research on molecular modification of transaminases using rational or semi-rational design strategies has attracted much attention.

[0004] Savile et al. studied the R-type transaminase ATA117 from Arthrobacter sp., and first modified the large pocket of the substrate binding region by site-directed saturation mutagenesis to obtain a single mutant (S223P) that exhibited certain catalytic activity for the potential chiral ketone structural analogue of sitagliptin (methyl ketone). Then, the small pocket and nearby sites of the substrate binding region were modified by combining site-directed saturation mutagenesis, combined mutagenesis and random mutagenesis techniques. After 11 rounds of mutation, the mutant ATA117 Rd11 was finally obtained, whose substrate binding region can effectively bind to the potential chiral ketone of sitagliptin, and the catalytic activity is more than 28,000 times higher than that of the wild-type enzyme. Zheng Yuguo's team reported the screening of transaminases and a new (R) selective transaminase from actinomycetes. (As-TA) has high enantioselectivity for the synthesis of (R)-3-aminobutanol, an important raw material for the preparation of dolutegravir. After optimizing the biocatalytic conditions and adopting the substrate injection-batch strategy, the substrate load increased significantly, and it had a good conversion effect at low concentrations. Summary of the invention

[0005] The object of the present invention is to provide a highly thermally stable ω-transaminase mutant and its application in the synthesis of R-chiral amines. The thermally stable ω-transaminase mutant of the present invention can be used for asymmetric synthesis of R-type chiral amines, which can reduce the synthesis steps of chiral intermediates, increase the reaction temperature, and promote the forward reaction. In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] First, the present invention provides a highly thermostable ω-aminotransferase mutant, which is obtained by mutating the serine at position 132 and the arginine at position 144 of the amino acid sequence of the ω-aminotransferase of Arthrobacter sp. to cysteine, respectively, and the mutation sites of the ω-aminotransferase mutant are: S132C / R144C. The two mutated cysteines can form a disulfide bond in the mature ω-aminotransferase mutant protein.

[0007] Preferably, the Arthrobacter sp. ω-aminotransferase is a wild type (WT), and its amino acid sequence is SEQ ID NO. 1, and the amino acid sequence of the ω-aminotransferase mutant is SEQ ID NO. 2. Specifically, the serine (S) at position 132 and the arginine (R) at position 144 in SEQ ID NO. 1 are both mutated to cysteine ​​(C).

[0008] Secondly, the present invention also provides a gene encoding the ω-aminotransferase mutant, and its nucleotide sequence is SEQ ID NO.3.

[0009] Thirdly, the present invention also provides a recombinant expression plasmid containing the above-mentioned gene encoding the ω-aminotransferase mutant.

[0010] Preferably, for the recombinant expression plasmid, the expression vector is pET-28a(+).

[0011] Fourthly, the present invention also provides a host cell containing the above-mentioned recombinant expression plasmid.

[0012] Preferably, the host cell is Escherichia coli BL21 (DE3).

[0013] Fifthly, the present invention also provides a method for constructing the expression of the ω-aminotransferase mutant, which is to transform the above-mentioned recombinant expression plasmid into E. coli BL21 (DE3) competent cells, culture them by fermentation, induce expression with IPTG, and then crush the cells and centrifuge to obtain the supernatant. Furthermore, nickel column affinity chromatography can also be used for protein purification.

[0014] Sixthly, the ω-aminotransferase mutant of the present invention can be used in catalyzing asymmetric synthesis of ketone substrates to obtain R-chiral amines.

[0015] For the above-mentioned application, preferably, the ketone substrate is at least one of 1-acetylnaphthalene and 4-hydroxy-2-butanone.

[0016] When the present invention is used to catalyze a ketone substrate to generate an R-chiral amine product, the ketone substrate is used as an amino acceptor, R-methylbenzylamine, isopropylamine or D-alanine is used as an amino donor, and an ω-transaminase mutant is used to perform an enzymatic reaction to transfer the amino group of the amino donor to the ketone substrate to generate an R-chiral amine product. During the catalytic reaction, the molar concentration of the ketone substrate is 10-100mM, and the molar concentration of the amino donor is 50-250mM. Preferably, the catalytic reaction temperature is 25-45°C, and the catalytic reaction time is 1-24h.

[0017] The invention firstly uses the three-dimensional structure model of wild-type ω-aminotransferase (NCBI GenBank: BAK39753.1) from Arthrobacter sp as a template, uses bioinformatics computing software Disulfide by Design 2.0 (http: / / cptweb.cpt.wayne.edu / DbD2) to rationally design the enzyme for introducing a disulfide bond, comprehensively considers the disulfide bond energy value, B factor value and χ3 torsion angle, designs site-directed mutagenesis primers, uses the ω-aminotransferase gene as a template, performs site-directed mutagenesis, screens a mutant containing one cysteine ​​after PCR amplification and purification, then uses the mutant as a template, performs site-directed mutagenesis to obtain a mutant containing two cysteines at the same time, transforms the mutant into a host cell, and obtains a site-directed mutagenesis library; finally, screens a ω-aminotransferase mutant with a better effect from the site-directed mutagenesis library.

[0018] The ω-aminotransferase mutant of the present invention is obtained by mutating the serine (S) at position 132 and the arginine (R) at position 144 of the ω-aminotransferase of Arthrobacter sp to cysteine ​​(C), and the half-inactivation temperature of the ω-aminotransferase mutant is 4.6°C higher than that of the wild type; the half-life at 40°C is 26.1min, which is 14.1min longer than that of the wild type, 2.1 times that of the wild type, and the thermal stability is significantly improved. The use of the high thermal stability ω-aminotransferase mutant of the present invention can reduce the synthesis steps of chiral intermediates, increase the reaction temperature, promote the forward reaction, and has low raw material cost, simple operation, and is more suitable for industrial production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the local three-dimensional structure of ω-aminotransferase after the introduction of disulfide bonds;

[0020] Figure 2 is a schematic diagram of the gene map of plasmid pET-28a(+)-S132C / R144C;

[0021] Figure 3 The relative enzyme activities of ω-aminotransferase and its mutants at different temperatures measured by the present invention;

[0022] Figure 4 The relative enzyme activities of ω-aminotransferase and its mutants measured at 40°C and different incubation times in the present invention;

[0023] Figure 5 This is a graph showing the half-inactivation temperature results of ω-aminotransferase and its mutants at different temperatures measured by the present invention.

[0024] Figure 6 This is a graph showing the results of ω-aminotransferase and its mutants catalyzing different substrates measured in the present invention. DETAILED DESCRIPTION

[0025] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0026] In the present invention, competent cells of Escherichia coli E.coli BL21 (DE3) E.coli DH5α were purchased from Quanshijin Biological Company; vector pET-28a (+) was purchased from outside. The wild-type ω-aminotransferase plasmid pET-28a (+)-ArTA was transformed into E.coli BL21 (DE3) to obtain genetically engineered bacteria E.coli BL21 (DE3) / pET-28a (+)-ArTA. Using the pET-28a (+)-ArTA plasmid of the wild-type ω-aminotransferase gene (ArTA) as a template, PCR site-directed double mutation was performed to obtain a recombinant plasmid pET-28a (+)-S132C / R144C, and then the recombinant plasmid was transformed into E.coli BL21 (DE3) to obtain a recombinant genetically engineered bacteria E.coli BL21 (DE3) / pET-28a (+)-S132C / R144C.

[0027] TB medium: 1L, Liquid A: accurately weigh 24g yeast extract, 12g tryptone, 5g glycerol, and add 900mL distilled water. Liquid B: Sterilizing phosphate with liquid A will cause the loss of components and the fermentation liquid to turn black, so it is sterilized separately. 100mL phosphate buffer, containing 0.17mM potassium dihydrogen phosphate and 0.72mM potassium hydrogen phosphate, is sterilized and used for standby use. The culture medium is prepared with deionized water.

[0028] Seed culture: Inoculate the genetically engineered bacteria at a 1% (v / v) inoculum into liquid LB medium containing 0.1 mg / mL kanamycin and culture at 37° C., 220 rpm for 12-16 h.

[0029] Fermentation and enzyme production: Add solution B to TB medium A at a ratio of 9:1, add 1% (v / v) seed solution to the mixed solution, and culture at 37°C, 220 rpm for 3.5-4.0 h. At this time, the OD of the fermentation liquid is 600 At this time, 0.2 mM IPTG was added to the fermentation broth and induced at 25°C for 16 h.

[0030] Collect bacteria: centrifuge the fermentation broth at 4°C, 8000rpm for 15min, discard the supernatant, and the lower precipitate is the bacteria.

[0031] Method for obtaining crude enzyme solution of ω-aminotransferase: resuspend the above-mentioned bacteria with 20% (v / v fermentation medium) pH 7.0 100mM potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer. Under ice bath, use 175W ultrasonic disruption (working 2s, stopping 3s) for 15min. Centrifuge the disruption solution at 4°C, 6000rpm for 10min, and take the supernatant as the crude enzyme solution.

[0032] ω-aminotransferase purification method: nickel column affinity chromatography was used for protein purification. The nickel column was equilibrated with 10 column volumes of equilibration buffer (100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5). 10 mL of crude enzyme solution filtered with a 0.45 μm microporous filter membrane was loaded onto the column at a flow rate of 0.5-1.0 mL / min. The column was washed with 10 volumes of equilibration buffer and low concentration imidazole (50 mM imidazole, 100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5), respectively, and unbound impurities were absorbed. 10 column volumes of high concentration imidazole (160 mM imidazole, 100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5) were added to elute the ω-aminotransferase protein. The eluate was collected and the imidazole was removed by ultrafiltration using a 10,000 molecular weight ultrafiltration tube. The sample was collected separately and the purity of the target protein was identified by SDS-PAGE.

[0033] Specifically, the ω-aminotransferase mutant of the present invention is used in the synthesis of chiral amines, and the method may include the following steps:

[0034] Step 1: Preparation of genetically engineered bacteria for recombinant ω-transaminase mutants

[0035] Point mutations were performed on the 132nd and 144th positions of the amino acid sequence of the wild-type ω-aminotransferase, specifically, the serine (S) at the 132nd position and the arginine (R) at the 144th position in SEQ ID NO.1 were mutated to cysteine ​​(C), and then transformed into Escherichia coli DH5α competent cells; positive transformants were selected and sequenced to obtain a recombinant expression vector; the recombinant expression vector was transferred into the Escherichia coli BL21 (DE3) strain to obtain a genetically engineered bacterium E. coli BL21 (DE3) that can induce expression of the recombinant ω-aminotransferase mutant.

[0036] / pET-28a(+)-S132C / R144C;

[0037] Step 2: Preparation of recombinant ω-aminotransferase mutants

[0038] The recombinant ω-transaminase mutant genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 12 hours to obtain a seed culture solution; the seed culture solution was inoculated into TB liquid medium containing kanamycin resistance, and the inoculation amount was 1% of the volume of the TB liquid medium containing kanamycin resistance; and then cultured at 37°C until OD 600 value is 2.0, add isopropyl-β-D-galactoside with a final concentration of 0.2mM, place at 25-28℃ for further culture for 14-24h, and collect the cells by centrifugation; use phosphate buffer to wash the collected cells once and then resuspend them to obtain a cell suspension for later use; place the suspension in an ice bath and ultrasonically disrupt it and then centrifuge it, the obtained supernatant is the recombinant ω aminotransferase mutant, and the protein content therein is detected for later use;

[0039] Step 3: Preparation of R-chiral amine

[0040] A reaction system is formed by sequentially adding a substrate with a final concentration of 10 to 100 mM, a cosolvent with a final volume percentage concentration of 5 to 50%, a cofactor with a final concentration of 0.1 to 5 mM, an amino donor with a final concentration of 50 to 250 mM, and a recombinant ω-transaminase mutant cell or a recombinant ω-transaminase mutant with a final volume percentage concentration of 5 to 25% into a reaction solution; the reaction system is reacted at 25 to 35° C. for 12 to 24 hours; after the reaction is completed, the content of the product in the reaction solution is detected; the reaction solution is then centrifuged through a membrane, semi-preparative liquid phase separation is performed, corresponding components are collected, the mobile phase is removed by rotary evaporation, and freeze-dried to obtain the target product;

[0041] The substrates selected are 1-acetylnaphthalene and 4-hydroxy-2-butanone, and 1-acetylnaphthalene, 4-hydroxy-2-butanone and the corresponding products are purchased from MacLean Reagent Company; the amino donor is R-methylbenzylamine, isopropylamine or D-alanine, all of which are purchased from Aladdin Reagent Company; the reaction solution is 50-100mM phosphate buffer with a pH value of 6.0-9.0, glycine-sodium hydroxide buffer with a pH value of 6.0-9.0, or 50-100mM sodium barbital-hydrochloric acid buffer with a pH value of 6.0-9.0; the cosolvent is dimethyl sulfoxide, N,N-dimethylformamide, methanol, acetonitrile; the cofactor is pyridoxal phosphate PLP; the amino donor is R-methylbenzylamine, isopropylamine or D-alanine.

[0042] Example 1

[0043] Design of disulfide bond mutants introduced into ω-aminotransferase (WT) from Arthrobacter sp.

[0044] Firstly, based on the crystal structure of ω-aminotransferase (from Arthrobacter sp) (PDBID: 3WWI) as a template, the bioinformatics computing software Disulfide by Design 2.0 (http: / / cptweb.cpt.wayne.edu / DbD2) was used to rationally design the introduction of disulfide bonds into the enzyme. DbD software predicted a total of 90 pairs of potential disulfide bond sites. Taking into account the potential sites with disulfide bond energy values ​​lower than or close to 2.2 kcal / mol, B factor values ​​and appropriate χ3 torsion angles, the possible potential disulfide bond site S132C / R144C was determined.

[0045] Example 2

[0046] The site-directed mutagenesis PCR method was used to sequentially perform site-directed mutagenesis on S132 and R144 of the ω-transaminase gene locus. The primers for site-directed mutagenesis are shown in Table 1, thereby obtaining the mutant S132C / R144C containing two cysteines at the same time.

[0047] Table 1 Site-directed mutagenesis primers

[0048]

[0049] Using the pET-28a(+)-ArTA plasmid containing the wild-type ω-aminotransferase gene (ArTA) as a template, a site-specific PCR amplification of position S132 was performed. The PCR amplification system was 50 μL, including: PCR SuperMix 25 μL, Forward Primer 1 μL, Reverse Primer 1 μL, plasmid template 2 μL, and purified water 21 μL. The PCR amplification program was: 95°C pre-denaturation for 2 min, 95°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 3 min, 2-4 cycles for 30 times; 72°C extension for 10 min. The PCR product was correct after electrophoresis. The obtained site-directed PCR reaction product was digested with Dpn I at 37°C for 1h to eliminate the wild template, and then transferred into competent cells E. coli DH5α. The transformation solution was coated on a LB solid plate containing kanamycin (100μg / mL) to obtain a site-directed mutation library. The mutant clones were cultured at 37°C for 12h to obtain mutant clones. The plasmids were extracted and sequenced. The sequencing results showed that the S132 position was changed to cysteine. Then, the mutant was used as a template for site-directed mutation of the R144 position. The mutation method was the same as above. After sequencing verification, the obtained plasmid was named pET-28a(+)-S132C / R144C. The plasmid map is shown in the figure. Figure 2 shown.

[0050] Example 3

[0051] The plasmid with the double mutant ω-transaminase mutant gene sequenced correctly in Example 2 was transformed into E. coli BL21 (DE3), and a single colony was picked and inoculated into a test tube with 5 mL of LB liquid medium, and cultured at 37°C and 200 rpm for 12-16 h. 2 mL of the culture medium was taken and added to 200 mL of TB medium, and cultured at 37°C and 220 rpm for 3.5-4.0 h. At this time, the OD of the fermentation liquid was 600 1.8-2.0. At this time, add 0.2mM IPTG to the fermentation broth and induce at 25℃ for 16h. Centrifuge the induced fermentation bacterial suspension at 4℃ and 8000rpm for 12-15min. Resuspend the above bacteria with 20% (v / v fermentation medium) pH7.0100mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer. In an ice bath, use 175W ultrasonic disruption for 15min. Centrifuge the disrupted solution at 4℃ and 6000rpm for 10min, and take the supernatant as the crude enzyme solution.

[0052] Nickel column affinity chromatography was used for protein purification. The nickel column was equilibrated with 10 column volumes of equilibration buffer (100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5). 10 mL of crude enzyme solution filtered with a 0.45 μm microporous filter was loaded onto the column at a flow rate of 0.5-1.0 mL / min. The column was washed with 10 volumes of equilibration buffer and low concentration imidazole (50 mM imidazole, 100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5), respectively, and unbound impurities were absorbed. 10 column volumes of high concentration imidazole (160 mM imidazole, 100 mM potassium dihydrogen phosphate and potassium dihydrogen phosphate, pH 7.5) were added to elute the ω-aminotransferase protein. The eluate was collected and the imidazole was removed by ultrafiltration using a 10,000 molecular weight ultrafiltration tube. The samples were collected separately and the purity of the target protein was identified by SDS-PAGE.

[0053] Example 4

[0054] Definition and determination method of enzyme activity: The amount of (R)-1-(1-naphthyl)ethylamine produced was quantitatively detected by HPLC, and the enzyme activity was defined based on the amount of (R)-1-(1-naphthyl)ethylamine produced. One activity unit (U) is the amount of enzyme required to catalyze the production of 1 μmol (R)-1-(1-naphthyl)ethylamine per hour at 30°C.

[0055] Reaction conditions: total volume 5mL, ketone substrate (1-acetylnaphthalene) 30mM, amino donor R-methylbenzylamine 50mM, co-solvent DMSO 15% (v / v), final concentration of 0.1mg / ml pure enzyme, PLP 1mM, dissolved in 100mM, pH 7.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer. Reaction at 30℃, 180rpm for 1h, boiling water bath for 5min to inactivate the enzyme. Liquid phase detection of product formation.

[0056] Chromatographic conditions: the liquid chromatography column is Alphasil VC-C18 column 4.6 mm × 25 cm produced by Dalian Huapu Technology Co., Ltd.; the column temperature is 30°C, the detection wavelength is 210 nm, the flow rate is 1.0 ml / min, and the injection volume is 20 μL; the mobile phase is acetonitrile (70%), water (30%) and 2-aminoethanol (0.03%); the run time is 20 min, and the product generation amount is calculated based on the standard curve of the product standard, and the ee value is determined using a chiral column.

[0057] Table 2 Determination of enzyme activity and ee value

[0058] Enzyme activity (U / mg) ee% WT 110.5 99 S132C / R144C 101.5 99

[0059] Example 5

[0060] The total volume of the reaction solution is 5 mL. The buffer solution is 100 mM, pH 7.0 potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer. The organic co-solvent DMSO 1.5 mL (10% v / v), the ketone substrate 1-acetylnaphthalene 30 mM, R-methylbenzylamine 50 mM, PLP 1 mM, the enzyme amount 10 U / mL was placed in different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C) for reaction (200 rpm), after 1 hour, it was placed in a 100°C water bath and boiled for 10 minutes, and finally placed on ice for 10 minutes, and the amount of (R)-1-(1-naphthyl)ethylamine produced was detected by high performance liquid chromatography. The results are as follows Figure 3 As shown, the optimal reaction temperatures of the wild-type ω-aminotransferase (WT) and the ω-aminotransferase mutant (S132C / R144C) are 35°C and 40°C, respectively.

[0061] Example 6

[0062] Half-life (t 1 / 2 ) refers to the time it takes for an enzyme to lose half its activity at a specific temperature, and is another important parameter for characterizing the thermal stability of an enzyme. The wild type and mutant S132C / R144C were incubated at 40°C for 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40 minutes, and then quickly placed on ice to cool for 5 minutes, and then the residual activity of the enzyme was measured. The results are shown in Figure 4 As shown, the experimental results show that the half-life of the wild-type ω-transaminase (WT) at 40°C is 12.8±0.2min, and the half-life of the ω-transaminase mutant (S132C / R144C is 26.4±0.4min, which is 2.1 times that of the wild-type, and the thermal stability is greatly improved.

[0063] Example 7

[0064] Half inactivation temperature (T50 10 ) refers to the temperature at which the enzyme loses half of its activity after being incubated at a specific temperature for a certain period of time. This is an important parameter for characterizing the thermal stability of the enzyme. The wild type and mutant S132C / R144C were kept in a water bath at 25-60°C for 10 minutes, and then quickly placed on ice for cooling. The residual specific activity of the enzyme was then measured. A graph was drawn with temperature as the horizontal axis and the ratio of the specific activity after heat treatment to that before treatment as the vertical axis to calculate the half-inactivation temperature. The results are shown in Figure 2. Figure 5 As shown, the half-inactivation temperature (T50 10 ) was 38.8±0.4℃ and the half-inactivation temperature (T50 10 ) was 43.4±0.6℃, and the temperature increased by 4.6℃.

[0065] Example 8

[0066] The wild-type ω-aminotransferase (WT) and the ω-aminotransferase mutant S132C / R144C obtained in Example 2 were used as biocatalysts, 1-acetylnaphthalene and 4-hydroxy-2-butanone were used as ketone substrates, R-(+)-methylbenzylamine was used as an amino donor, and under the action of the coenzyme PLP, the biotransformation reaction produced (R)-1-(1-naphthyl)ethylamine and (R)-3-aminobutanol, respectively.

[0067] The catalytic system and catalytic conditions are as follows: total volume 10mL, 1-acetylnaphthalene, 4-hydroxy-2-butanone 60mM, amino donor R-methylbenzylamine 150mM, DMSO 15% (v / v), ω-transaminase pure enzyme or whole cell addition (250U / mL), PLP 2.0mM, dissolved in 100mM, pH 7.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, pH adjusted to 7.5. React at 35°C, 200rpm for 24h, and inactivate the enzyme in a boiling water bath for 5min. The formation of the product was detected by liquid phase, and the results are as follows Figure 6 .

[0068] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

[0069] SEQUENCE LISTING

[0070] SEQ ID NO.1

[0071] MAFSADTSEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGYLA

[0072] SDVTYTVFHVWNGNAFRLDDHIERLFSNAESMRIIPPLTQDEVKEIALELVAKTELREAFVS

[0073] VSITRGY S STPFERDITKH R PQVYMYAVPYQWIVPFDRIRDGVHAMVAQSVRRTPRSSIDPQ

[0074] VKNFQWGDLIRAVQETHDRGFEAPLLLDGDGLLAEGAGFNVVVIKDGVVRSPGRAALPGI

[0075] TRKTVLEIAESLGHEAILADITLAELLDADEVLGCTTAGGVWPFVSVDGNPISDGVPGPITQ

[0076] MOVE TO WELNVESSSLTPVQY

[0077] SEQ ID NO.2

[0078] MAFSADTSEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGYLA

[0079] SDVTYTVFHVWNGNAFRLDDHIERLFSNAESMRIIPPLTQDEVKEIALELVAKTELREAFVS

[0080] VSITRGY C STPFERDITKH C PQVYMYAVPYQWIVPFDRIRDGVHAMVAQSVRRTPRSSIDP

[0081] QVKNFQWGDLIRAVQETHDRGFEAPLLLDGDGLLAEGAGFNVVVIKDGVVRSPGRAALP

[0082] GITRKTVLEIAESLGHEAILADITLAELLDADEVLGCTTAGGVWPFVSVDGNPISDGVPGPPIT

[0083] QSIIRRYWELNVE SSSLLTPVQY

[0084] SEQ ID NO.3

[0085] ATGGCGTTTAGCGCGGATACGAGCGAAATTGTGTATACCCATGATACCGGCCTGGATTATATTACCTATAGCGATTATGAATTAGATCCGGCGAACCCGCTGGCGGGCGGCGCGGCGTGGATTGAAGGCGCGTTTGTGCCGCCGAGCGAAGCGCGCATTAGCATTTTTGATCAAGGCTATCTGGCAAGCGATGTGACCTATACCGTGTTTCATGTGTGGAACGGCAACGCGTTTCGCCTGGATGATCATATTGAACGCCTGTTTAGCAACGCGGAAAGCATGCGCATTATTCCGCCGCTGACCCAAGATGAAGTGAAAGAAATTGCGCTGGAACTGGTGGCGAAAACCGAACTGCGCGAAGCGTTTGTGAGCGTGAGCATTACCCGCGGCTATTGTAGCACCCCGTTTGAACGCGATATTACCAAACATTGTCCGCAAGTGTATATGTATGCGGTGCCGTATCAGTGGATTGTGCCGTTTGATCGCATTCGCGATGGCGTTCACGCGATGGTGGCGCAGAGCGTGCGCCGCACCCCGCGCAGCAGCATTGATCCGCAAGTGAAAAACTTTCAGTGGGGCGATCTGATTCGCGCGGTGCAAGAAACCCATGATCGCGGCTTTGAAGCGCCGCTGTTACTGGATGGCGATGGCCTGCTGGCGGAAGGCGCAGGCTTTAACGTGGTTGTGATTAAAGATGGCGTGGTGCGCAGCCCGGGCCGCGCGGCGCTGCCGGGCATTACCCGCAAAACCGTGCTGGAAATTGCGGAAAGCCTGGGCCATGAAGCGATTCTGGCGGATATTACCCTGGCGGAACTGCTGGATGCGGATGAAGTGCTGGGCTGCACCACCGCGGGCGGCGTGTGGCCATTTGTGAGCGTTGATGGCAACCCGATTAGTGATGGTGTTCCGGGCCCGATTACGCAGAGCATTATTCGCCGCTATTGGGAACTGAACGTGGAAAGCAGTAGCCTGCTGACCCCGGTGCAGTAT

Claims

1. A highly thermostable ω-aminotransferase mutant, characterized in that: The ω-aminotransferase mutant is obtained by mutating the serine at position 132 and the arginine at position 144 of the ω-aminotransferase amino acid sequence of Arthrobacter sp. to cysteine ​​respectively, the mutation sites of the ω-aminotransferase mutant are: S132C / R144C, and the amino acid sequence of the ω-aminotransferase of Arthrobacter sp. is SEQ ID NO.

1.

2. The ω-aminotransferase mutant according to claim 1, characterized in that The amino acid sequence of the ω-aminotransferase mutant is SEQ ID NO.

2.

3. A gene encoding the ω-aminotransferase mutant according to claim 1 or 2, wherein the nucleotide sequence thereof is SEQ ID NO.

3.

4. A recombinant expression plasmid containing the gene according to claim 3.

5. The recombinant expression plasmid according to claim 4, characterized in that: The expression vector of the recombinant expression plasmid is pET-28a(+).

6. A host cell containing the gene according to claim 3 or the recombinant expression plasmid according to claim 4 or 5.

7. The host cell according to claim 6, characterized in that The host cell is Escherichia coli BL21 (DE3).

8. A method for constructing the expression of the ω-aminotransferase mutant according to claim 1 or 2, comprising transforming the recombinant expression plasmid according to claim 4 or 5 into Escherichia coli BL21 (DE3), fermenting and culturing, inducing expression with IPTG, and crushing the cells to obtain the supernatant by centrifugation.

9. Use of the ω-aminotransferase mutant according to claim 1 or 2 or the ω-aminotransferase mutant obtained by the expression construction method according to claim 8 in catalyzing ketone substrates to obtain R-chiral amines.

10. The use according to claim 9, characterized in that The ketone substrate is at least one of 1-acetylnaphthalene and 4-hydroxy-2-butanone, and the molar concentration of the ketone substrate is 10-100 mM.

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