Highly thermostable ω-aminotransferase mutants and their applications in the synthesis of R-chiral amines

Abstract

The present invention discloses a highly thermally stable ω-transaminase mutant and its application in the synthesis of R-chiral amines, belonging to the field of bioenzyme engineering technology. The ω-transaminase mutant of the present invention is derived from the genus Arthrocarpus ( Arthrobacter sp The serine at position 132 and the arginine at position 144 of the ω-transaminase derived from the wild type were mutated to cysteine, and the half-inactivation temperature of the ω-transaminase mutant was 4.6°C higher than that of the wild type; the half-life at 40°C was 26.4 ±0.4 min, which is 14.6 longer than that of the wild type. ±0.6 The use of the highly thermostable ω-transaminase mutant of the present invention can reduce the number of steps in the synthesis of chiral intermediates, increase the reaction temperature, and promote the forward reaction. Furthermore, the raw material cost is low, the operation is simple, and it is more suitable for industrial production applications.

Description

Technical Field

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

[0002] Chiral amines, due to their high density of structural information, hydrogen bond formation, and refined biological activity, are important intermediates for the synthesis of pharmaceuticals and other fine chemicals. They are commonly 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 benign prostatic hyperplasia drug silodosin. According to statistics, chiral amines account for approximately 30% of the top 200 small molecule drugs by annual sales. The application of these chiral drugs has greatly promoted the exploration of efficient chiral amine synthesis.

[0003] ω-Transaminases (ω-TAs) are highly attractive biocatalysts for the synthesis of optically pure chiral amines and hold great promise for their application in this field. However, due to numerous deficiencies in substrate specificity, stability, catalytic efficiency, and low activity, reported wild-type TAs have limited demand for industrial applications. With the in-depth study of TA protein structure and catalytic mechanism and the advancement of protein engineering technology, the molecular engineering of TAs using rational or semi-rational design strategies has attracted considerable attention.

[0004] Arthrobacter Arthrobacter sp. The R-type transaminase ATA117 from the wild-type was studied. First, the large pocket of the substrate-binding region was modified through site-directed saturation mutagenesis, resulting in a single mutant (S223P) that exhibited moderate catalytic activity toward a potential chiral ketone analog of sitagliptin (methyl ketone). Subsequently, the small pocket and nearby sites of the substrate-binding region were modified through a combination of site-directed saturation mutagenesis, combinatorial mutagenesis, and random mutagenesis. After 11 rounds of mutagenesis, the mutant ATA117 Rd11 was obtained. Its substrate-binding region effectively binds the potential chiral ketone of sitagliptin, and its catalytic activity is over 28,000-fold higher than that of the wild-type enzyme. Zheng Yuguo's team reported screening transaminases and a new (R)-selective transaminase (As-TA) from actinomycetes. This transaminase exhibited high enantioselectivity for the synthesis of (R)-3-aminobutanol, a key raw material for the preparation of dolutegravir. After optimizing biocatalytic conditions and adopting a substrate injection-batch strategy, substrate loading was significantly increased, resulting in excellent conversion at low concentrations. Summary of the Invention

[0005] The present invention aims to provide a highly thermostable ω-transaminase mutant and its use in the synthesis of R-chiral amines. The thermostable ω-transaminase mutant of the present invention can be used in the asymmetric synthesis of R-chiral amines, reducing the number of steps required to synthesize chiral intermediates, increasing the reaction temperature, and promoting the forward reaction. To achieve the above-mentioned objectives, the present invention provides the following technical solutions:

[0006] First, the present invention provides a highly thermostable ω-aminotransferase mutant, which is derived from the genus Arthrocarpus ( Arthrobacter sp. The ω-aminotransferase mutant is obtained by mutating the serine at position 132 and the arginine at position 144 in the amino acid sequence of the ω-aminotransferase to cysteine. 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 Arthrophora genus ( Arthrobacter sp. The wild-type ω-aminotransferase (WT) has an amino acid sequence of SEQ ID NO. 1, and the amino acid sequence of the mutant ω-aminotransferase 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-transaminase mutant, the nucleotide sequence of which is SEQ ID NO.3.

[0009] Thirdly, the present invention also provides a recombinant expression plasmid containing the above-mentioned gene encoding the ω-transaminase 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 E. 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 Escherichia coli E. coli The protein is obtained by fermentation in BL21 (DE3) competent cells, induction with IPTG, and then cell disruption and centrifugation to obtain the supernatant. Furthermore, nickel column affinity chromatography can be used for protein purification.

[0014] Sixth, the ω-transaminase mutant of the present invention can be used to catalyze the asymmetric synthesis reaction 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] The present invention is used to catalyze the generation of an R-chiral amine product from a ketone substrate. The enzymatic reaction is performed using a ketone substrate as an amino group acceptor and R-methylbenzylamine, isopropylamine, or D-alanine as an amino group donor. A mutant ω-aminotransferase is used to transfer the amino group of the amino group donor to the ketone substrate to generate the R-chiral amine product. During the catalytic reaction, the molar concentration of the ketone substrate is 10-100 mM, and the molar concentration of the amino group donor is 50-250 mM. The catalytic reaction temperature is preferably 25-45°C, and the catalytic reaction time is 1-24 hours.

[0017] The present invention firstly based on the Arthrobacter sp ) wild-type ω-aminase was used as a template, and the bioinformatics 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. The disulfide bond energy value, B factor value, and χ3 torsion angle were comprehensively considered to design site-directed mutagenesis primers. The ω-aminase gene was used as a template for site-directed mutagenesis. After PCR amplification and purification, a mutant containing one cysteine ​​was screened. This mutant was then used as a template for site-directed mutagenesis to obtain a mutant containing two cysteines. The mutant was then transformed into a host cell to obtain a site-directed mutagenesis library. Finally, the site-directed mutagenesis library was used to screen for ω-aminase mutants with better effects.

[0018] The ω-aminotransferase mutant of the present invention is derived from the genus Arthrocarpus ( Arthrobacter sp ) ω-aminotransferase was obtained by mutating both the serine (S) at position 132 and the arginine (R) at position 144 to cysteine ​​(C). The half-inactivation temperature of this ω-aminotransferase mutant was 4.6°C higher than that of the wild type; its half-life at 40°C was 26.1 minutes, 14.1 minutes longer than that of the wild type, and 2.1 times that of the wild type, indicating significantly improved thermal stability. The use of the highly thermostable ω-aminotransferase mutant of the present invention can reduce the number of steps in the synthesis of chiral intermediates, increase the reaction temperature, and promote the forward reaction. It also has low raw material costs and is easy to operate, making it 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-transaminase after the introduction of disulfide bonds;

[0020] Figure 2 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 for different incubation times are shown in the present invention;

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

[0024] Figure 6 This is a diagram 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 present invention to illustrate 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, Escherichia coli E. coli BL21 (DE3) competent cells E. coli DH5α was purchased from Quanshijin Biotechnology Co., Ltd.; the vector pET-28a(+) was purchased from outside. The wild-type ω-aminotransferase plasmid pET-28a(+)-ArTA was transformed into E. coli Genetically engineered bacteria were obtained from BL21 (DE3) E. coli BL21 (DE3) / pET-28a (+) -ArTA. Using the wild-type ω-aminotransferase gene (ArTA) pET-28a (+) -ArTA plasmid as a template, PCR site-directed double mutagenesis was performed to obtain the recombinant plasmid pET-28a (+) -S132C / R144C, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) obtained recombinant genetic engineering bacteria E. coli BL21(DE3) / pET-28a(+)-S132C / R144C.

[0027] TB medium: 1 L, Solution A: Accurately weigh 24 g yeast extract, 12 g tryptone, and 5 g glycerol, and add 900 mL of distilled water. Solution B: Sterilize phosphate buffer with Solution A, as sterilizing it together will result in loss of components and darken the fermentation broth, so prepare and sterilize it separately. 100 mL of phosphate buffer containing 0.17 mM potassium dihydrogen phosphate and 0.72 mM potassium hydrogen phosphate should be sterilized and used. Prepare the medium with deionized water.

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

[0029] Fermentation 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 600 At this time, 0.2 mM IPTG was added to the fermentation broth and induced at 25°C for 16 h.

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

[0031] To obtain crude ω-aminotransferase enzyme solution: Resuspend the above-mentioned bacterial cells in 20% (v / v) fermentation medium (100 mM potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer, pH 7.0). Ultrasonicate at 175 W (2 s on, 3 s off) for 15 min on ice. Centrifuge the solution at 6000 rpm for 10 min at 4°C. Collect 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 dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5). 10 mL of crude enzyme solution filtered through 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 then rinsed with 10 volumes of equilibration buffer and low concentration imidazole (50 mM imidazole, 100 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5), respectively. Unbound impurities were aspirated, and 10 column volumes of high concentration imidazole (160 mM imidazole, 100 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5) were added to elute the ω-aminotransferase protein. The eluate was collected and passed through a 10,000 molecular weight ultrafiltration tube to remove imidazole. The eluate was then collected in separate tubes 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 expressing recombinant ω-transaminase mutants

[0035] Point mutations were performed on the amino acid sequence of the wild-type ω-aminotransferase at positions 132 and 144, specifically, the serine (S) at position 132 and the arginine (R) at position 144 in SEQ ID NO.1 were mutated to cysteine ​​(C), and then transformed into Escherichia coli DH5α competent cells; positive transformants were picked and sequenced to obtain a recombinant expression vector; the recombinant expression vector was transformed into the Escherichia coli BL21 (DE3) strain to obtain a genetically engineered bacterium that can inducibly express the recombinant ω-aminotransferase mutant. E. coli BL21(DE3) / pET-28a(+)-S132C / R144C;

[0036] Step 2. Preparation of recombinant ω-transaminase mutants

[0037] The recombinant ω-transaminase mutant genetically engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C for 12 h to obtain seed culture solution. The seed culture solution was inoculated into TB liquid medium containing kanamycin resistance at an inoculum volume of 1% of the volume of the TB liquid medium containing kanamycin resistance. The bacteria were then cultured at 37°C until the OD 600 The value was 2.0, isopropyl-β-D-galactoside was added to a final concentration of 0.2 mM, and the cells were cultured at 25-28°C for 14-24 hours, and then the cells were collected by centrifugation. The collected cells were washed once with phosphate buffer and resuspended to obtain a cell suspension for later use. The suspension was ultrasonically disrupted in an ice bath and then centrifuged. The supernatant obtained was the recombinant ω transaminase mutant, and the protein content in the supernatant was determined and set aside.

[0038] Step 3: Preparation of R-chiral amine

[0039] 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% to a reaction solution; the reaction system is reacted at 25 to 35° C. for 12 to 24 hours; after the reaction, the content of the product in the reaction solution is detected; the reaction solution is then centrifuged and filtered through a membrane, and semi-preparative liquid phase separation is performed to collect the corresponding components, the mobile phase is removed by rotary evaporation, and lyophilization is performed to obtain the target product;

[0040] 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 donors are R-methylbenzylamine, isopropylamine or D-alanine, all purchased from Aladdin Reagent Company; the reaction solution is 50-100 mM 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 50-100 mM sodium barbital-hydrochloric acid buffer with a pH value of 6.0-9.0; the cosolvent is dimethyl sulfoxide, N,N-dimethylformamide, methanol, and acetonitrile; the cofactor is pyridoxal phosphate PLP; and the amino donor is R-methylbenzylamine, isopropylamine or D-alanine. Example 1

[0041] From the genus Arthrophora ( Arthrobacter sp )Design of disulfide bond-introduced mutants of ω-aminotransferase (WT).

[0042] First, according to the ω-aminotransferase (from Arthrobacter Arthrobacter sp )) crystal structure (PDB ID: 3WWI) was used as a template, and the bioinformatics 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 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 potential disulfide bond site S132C / R144C was determined. Example 2

[0043] Site-directed mutagenesis PCR was used to sequentially perform site-directed mutagenesis on S132 and R144 of the ω-aminotransferase 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.

[0044] Table 1 Site-directed mutagenesis primers

[0045]

[0046] Site-directed PCR amplification of position S132 was performed using the pET-28a(+)-ArTA plasmid containing the wild-type ω-aminotransferase gene (ArTA) as a template. The PCR amplification system (50 μL) contained: 25 μL PCR SuperMix, 1 μL Forward Primer, 1 μL Reverse Primer, 2 μL plasmid template, and 21 μL purified water. The PCR amplification program was as follows: 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 cycles, and 72°C extension for 10 min. The PCR product was confirmed to be correct by electrophoresis. The resulting site-directed PCR reaction product was digested with Dpn I at 37°C for 1 h to eliminate the wild-type template and then transformed into competent cells. E. coli DH5α, the transformation solution was plated on LB solid plates containing kanamycin (100 μg / mL) to obtain a site-directed mutation library. The mutant clones were cultured at 37°C for 12 hours to obtain mutant clones. The plasmids were extracted and sequenced. The sequencing results showed that the S132 position was converted to cysteine. The mutant was then used as a template for site-directed mutagenesis at the R144 position. The mutation method was the same as above. After sequencing verification, the resulting plasmid was named pET-28a(+)-S132C / R144C. The plasmid map is shown in Figure 2. Figure 2 shown. Example 3

[0047] The plasmid carrying the double-mutated ω-transaminase mutant gene sequenced correctly in Example 2 was transformed into E. coli In BL21 (DE3), pick a single colony and inoculate it into a test tube with 5 mL of LB liquid medium. Incubate it at 37°C and 200 rpm for 12-16 hours. Pipette 2 mL of the culture medium into 200 mL of TB medium and incubate it at 37°C and 220 rpm for 3.5-4.0 hours. At this time, the OD value of the fermentation liquid is 600 The pH value is 1.8-2.0. At this point, add 0.2 mM IPTG to the fermentation broth and induce at 25°C for 16 hours. Centrifuge the induced bacterial suspension at 8000 rpm for 12-15 minutes at 4°C. Resuspend the cells in 20% (v / v) fermentation medium, 100 mM potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer, pH 7.0. Ultrasonicate at 175 W for 15 minutes in an ice bath. Centrifuge the solution at 6000 rpm for 10 minutes at 4°C. The supernatant is the crude enzyme solution.

[0048] Protein purification was performed by nickel affinity chromatography. The nickel column was equilibrated with 10 column volumes of equilibration buffer (100 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5). 10 mL of crude enzyme solution filtered through a 0.45 μm microporous membrane was loaded onto the column at a flow rate of 0.5-1.0 mL / min. The column was then washed with 10 volumes of equilibration buffer and low concentration imidazole (50 mM imidazole, 100 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5), respectively. Unbound impurities were then aspirated. 10 column volumes of high concentration imidazole (160 mM imidazole, 100 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5) were added to elute the ω-aminotransferase protein. The eluate was collected and passed through a 10,000 molecular weight ultrafiltration tube to remove imidazole. The eluate was then collected in separate tubes and the purity of the target protein was determined by SDS-PAGE. Example 4

[0049] Definition and Determination of Enzyme Activity: Enzyme activity is determined by HPLC quantitative determination of (R)-1-(1-naphthyl)ethanamine production. One activity unit (U) is the amount of enzyme required to catalyze the production of 1 μmol (R)-1-(1-naphthyl)ethanamine per hour at 30°C.

[0050] Reaction conditions: 5 mL total volume, 30 mM ketone substrate (1-acetylnaphthyl), 50 mM amino donor R-methylbenzylamine, 15% (v / v) cosolvent DMSO, 0.1 mg / mL final concentration of pure enzyme, 1 mM PLP, dissolved in 100 mM potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer, pH 7.0. Incubate at 30°C, 180 rpm for 1 h, then inactivate the enzyme in a boiling water bath for 5 min. Product formation was detected by liquid chromatography.

[0051] Chromatographic conditions: LC column: Dalian Huapu Technology Co., Ltd. Alphasil VC-C18 column (4.6 mm × 25 cm); column temperature: 30°C, detection wavelength: 210 nm, flow rate: 1.0 ml / min, injection volume: 20 μL; mobile phase:

[0052] The reaction mixture was run for 20 min in acetonitrile (70%), water (30%), and 2-aminoethanol (0.03%). The amount of product generated was calculated based on the standard curve of the product standard. The ee value was determined using a chiral column.

[0053] Table 2 Determination of enzyme activity and ee value

[0054]

[0055] Example 5

[0056] The total volume of the reaction solution was 5 mL. The buffer was 100 mM, pH 7.0 potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer. The organic co-solvent DMSO 1.5 mL (10% v / v), ketone substrate 1-acetylnaphthalene 30 mM, R-methylbenzylamine 50 mM, PLP 1 mM, enzyme amount 10 U / mL was placed in different temperatures (25℃, 30℃, 35℃, 40℃, 45℃, 50℃) for reaction (200 rpm), after 1 hour, it was placed in a 100℃ water bath and boiled for 10 minutes, and finally placed on ice for 10 minutes. 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 wild-type ω-transaminase (WT) and ω-transaminase mutant (S132C / R144C) are 35°C and 40°C, respectively. Example 6

[0057] 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 enzyme thermal stability. 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, respectively. After the incubation period, they were quickly placed on ice to cool for 5 minutes, and then the residual enzyme activity was measured. The results are shown in Figure 2. Figure 4 As shown in the experimental results, the half-life of wild-type ω-aminotransferase (WT) at 40°C is 12.8 ±0.2 min, the half-life of ω-transaminase mutant (S132C / R144C is 26.4±0.4 min, It is 2.1 times that of the wild type, and its thermal stability is greatly improved. Example 7

[0058] Half inactivation temperature 7 degrees (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 incubated in a water bath at 25-60°C for 10 minutes, and then quickly placed on ice to cool after the incubation period. 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 before and after heat 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 ) is 38.8 ± 0.4 ℃ and the half-inactivation temperature ( T50 10 ) is 43.4 ±0.6 ℃, the temperature increased by 4.6℃. Example 8

[0059] Wild-type ω-aminotransferase (WT) and the ω-aminotransferase mutant S132C / R144C obtained in Example 2 were used as biocatalysts, with 1-acetylnaphthalene and 4-hydroxy-2-butanone as ketone substrates, and R-(+)-methylbenzylamine as an amino donor, respectively. Under the action of the coenzyme PLP, biotransformation reactions were performed to produce (R)-1-(1-naphthyl)ethylamine and (R)-3-aminobutanol, respectively.

[0060] The catalytic system and conditions are as follows: a total volume of 10 mL, 60 mM 1-acetonaphthalene and 60 mM 4-hydroxy-2-butanone, 150 mM amino donor R-methylbenzylamine, 15% (v / v) DMSO, 250 U / mL of pure ω-aminotransferase or whole cells, and 2.0 mM PLP dissolved in 100 mM, pH 7.0, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, adjusted to pH 7.5. The reaction was incubated at 35°C, 200 rpm for 24 h, and the enzyme was inactivated in a boiling water bath for 5 min. Liquid chromatography was used to detect product formation, as shown in the following table. Figure 6 .

[0061] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

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

1.

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

2.

3. A gene encoding the ω-transaminase 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, wherein 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, wherein The host cell is Escherichia coli E. coli BL21(DE3).

8. The method for expressing and constructing the ω-aminotransferase mutant according to claim 1 or 2, characterized in that: The recombinant expression plasmid according to claim 4 or 5 is transformed into Escherichia coli E. coli The enzyme was fermented and cultured in BL21 (DE3) cells, induced by IPTG, and the cells were broken and centrifuged to obtain the supernatant, which was the crude enzyme solution.

9. Use of the ω-aminotransferase mutant according to claim 1 or 2, or the ω-aminotransferase mutant obtained by the expression and construction method according to claim 8, in catalyzing the conversion of a ketone substrate to an R-chiral amine, wherein the ketone substrate is at least one of 1-acetylnaphthalene and 4-hydroxy-2-butanone, and the R-chiral amine is at least one of (R)-1-(1-naphthyl)ethylamine and (R)-3-aminobutanol.

10. The use according to claim 9, characterized in that The molar concentration of the ketone substrate is 10-100 mM.

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