An ω-transaminase mutant and its application

Abstract

The present invention discloses an ω-transaminase mutant and its application. The ω-transaminase mutant is obtained by single-point or multi-point mutations of tryptophan at position 54 and arginine at position 411 in the amino acid sequence of the wild-type ω-transaminase shown in SEQ ID NO.1. Compared with the wild-type ω-transaminase, the ω-transaminase mutant of the present invention has a high catalytic efficiency for catalyzing prochiral ketones to generate chiral amines. Among them, the conversion yield of the mutant R411A for 4-(trifluoromethyl)acetophenone is 2.39 times that of the wild-type ω-transaminase, and the enantiomeric excess (ee) of the obtained product > 99.9%.

Description

(1) Technical field

[0001] The invention belongs to the technical field of bioengineering and relates to a mutant of ω-transaminase and application thereof. (2) Background technology

[0002] Chiral amines are key intermediates in many pharmaceuticals and pesticides and have important application value. For example, the structures of the diabetes drug sitagliptin, the antibiotic penicillin, and the herbicide glufosinate-ammonium all contain chiral amine modules.

[0003] Currently, the main methods for preparing chiral amines include chemical synthesis, biological resolution, and biological asymmetric synthesis. Chemical synthesis typically requires the use of expensive metal catalysts and large amounts of organic solvents. The maximum theoretical yield of chiral amines prepared by biological resolution is 50%, resulting in low atom utilization. The theoretical yield of chiral amines prepared by biological asymmetric synthesis is 100%, offering advantages such as low cost and an environmentally friendly preparation process, making it the preferred method for preparing chiral amines.

[0004] ω-aminotransferases are a class of pyridoxal-5'-phosphate (PLP)-dependent enzymes that can be used for the asymmetric reduction of prochiral ketones to generate chiral amines with a theoretical yield of up to 100%. They have the characteristics of high stereoselectivity and regenerative cofactors. Therefore, ω-aminotransferases are widely used in the synthesis of chiral amines.

[0005] The currently reported ω-transaminases that can be used for the asymmetric reduction of prochiral ketones to prepare chiral amines still have problems such as unsatisfactory catalytic efficiency and substrate tolerance that needs to be improved. Obtaining ω-transaminases with better catalytic performance can reduce the cost of the asymmetric reduction of prochiral ketones to prepare chiral amines and enhance the industrial application potential of this enzyme. (3) Summary of the invention

[0006] The present invention aims to provide an ω-aminotransferase mutant with excellent catalytic performance and its application in the preparation of chiral amines. The present invention uses site-directed saturation mutagenesis to molecularly modify an ω-aminotransferase derived from Vitreoscilla stercoraria DSM 513, thereby obtaining an ω-aminotransferase mutant with significantly improved enzyme activity. The mutant is applied to the preparation of chiral amines, thereby reducing the cost of the asymmetric reduction of prochiral ketones to prepare chiral amines, enhancing the industrial application potential of the enzyme, and resolving the problems of unsatisfactory catalytic efficiency and improved substrate tolerance in the asymmetric reduction of prochiral ketones to prepare chiral amines using existing ω-aminotransferases.

[0007] The technical solution adopted in the present invention is:

[0008] The present invention provides a ω - transaminase mutant, which is obtained by single - point or multi - point mutation of tryptophan at position 54 and arginine at position 411 in the wild - type ω - transaminase amino acid sequence shown in SEQ ID NO.1. The nucleotide sequence of the wild - type ω - transaminase encoding gene is shown in SEQ ID NO.2.

[0009] Preferably, the ω - transaminase mutant is the amino acid sequence shown in SEQ ID NO.1 mutated into one of the following: (1) arginine at position 411 is mutated into glycine (R411G), alanine (R411A), valine (R411V), proline (R411P), isoleucine (R411I), phenylalanine (R411F), tyrosine (R411Y), tryptophan (R411W), serine (R411S), threonine (R411T), cysteine (R411C), methionine (R411M), asparagine (R411N), glutamine (R411Q), aspartic acid (R411D), glutamic acid (R411E); (2) tryptophan at position 54 is mutated into glycine (W54G), alanine (W54A), valine (W54V), cysteine (W54C), tyrosine (W54Y); (3) tryptophan at position 54 is mutated into cysteine, and arginine at position 411 is mutated into alanine (W54C / R411A), phenylalanine (W54C / R411F), tyrosine (W54C / R411Y), isoleucine (W54C / R411I), proline (W54C / R411P), valine (W54C / R411V), serine (W54C / R411S) or tryptophan (W54C / R411W); (4) tryptophan at position 54 is mutated into valine and arginine at position 411 is mutated into methionine (W54V / R411M); (5) tryptophan at position 54 is mutated into alanine and arginine at position 411 is mutated into serine (W54A / R411S); (6) tryptophan at position 54 is mutated into tyrosine, and arginine at position 411 is mutated into serine (W54Y / R411S) or alanine (W54Y / R411A).

[0010] The present invention also provides a recombinant vector containing the ω - transaminase mutant encoding gene, and the recombinant vector is based on pET28a(+).

[0011] The present invention also provides a recombinant genetically engineered bacterium containing the ω - transaminase mutant encoding gene, and the genetically engineered bacterium uses Escherichia coli E.coil BL21(DE3) as the host cell.

[0012] The present invention also provides the application of the gene encoding the ω-transaminase mutant in the preparation of the ω-transaminase mutant. The method of the application is as follows: inoculate the recombinant Escherichia coli containing the gene encoding the ω-transaminase mutant into an LB liquid medium and culture it overnight, and then transfer it to a fresh LB liquid medium for induced expression; after the induced expression ends, obtain the fermentation broth, centrifuge the fermentation broth, and collect the bacterial cells; resuspend the bacterial cells with a buffer solution, centrifuge the bacterial cells after ultrasonic disruption, and obtain the supernatant after cell disruption; pass the supernatant after cell disruption through a nickel column for protein purification to obtain the pure enzyme of the ω-transaminase mutant.

[0013] The present invention also provides the application of the ω-transaminase mutant in the catalytic synthesis of chiral amines from prochiral ketones. The method is carried out according to the following steps: using the wet bacterial cells obtained by fermentation culture of the recombinant Escherichia coli containing the gene encoding the ω-transaminase mutant (or the pure enzyme extracted after ultrasonic disruption of the cells) as a catalyst, using the prochiral ketone as an amino acceptor, isopropylamine hydrochloride as an amino donor, using pyridoxal phosphate (PLP) as a cofactor, adding dimethyl sulfoxide (DMSO) as a cosolvent, and using a potassium phosphate buffer solution with a pH of 6.5 to 8.0 (preferably pH 8.0) as a reaction medium to form a reaction system, and reacting in a shaker at 25 to 40 °C (preferably 30 °C) and 200 rpm for 0.5 - 3 h. The obtained reaction solution is separated and purified to obtain the chiral amine product. The prochiral ketones include acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, 4-nitroacetophenone, 4-methoxyacetophenone, 4-(trifluoromethyl)acetophenone, phenylbutanone, 2-(trifluoromethyl)acetophenone, 3-(trifluoromethyl)acetophenone, 2-fluoroacetophenone, or 3-fluoroacetophenone.

[0014] Preferably, in the reaction system, the added concentration of the cofactor is 0.1 to 1.0 mM, preferably 1.0 mM; the catalyst is 50 - 200 g / L based on the weight of the wet bacterial cells, preferably 100 g / L, and the catalyst is 0.1 to 0.5 mg / mL based on the protein content in the pure enzyme, preferably 0.3 mg / mL; the added concentration of the prochiral ketone is 1.0 to 10 mM, preferably 4 to 5.0 mM; the added concentration of the isopropylamine hydrochloride is 50 to 200 mM, preferably 100 mM; the final volume concentration of the dimethyl sulfoxide added is 1 to 5%, preferably 2%; and the concentration of the potassium phosphate buffer solution in the reaction system is 0.05 to 0.1 M, preferably 0.05 M.

[0015] Preferably, the wet bacterial cells are prepared as follows: recombinant Escherichia coli containing a gene encoding a ω-transaminase mutant is inoculated into an LB liquid culture medium containing 50 μg / mL kanamycin (Kan), and cultured overnight at 37°C and 200 rpm to obtain a seed solution; the seed solution is transferred to a fresh LB liquid culture medium containing 50 μg / mL kanamycin (Kan) at an inoculum concentration of 1% by volume, and fermented at 37°C and 200 rpm to an optical density at 600 nm of between 0.6 and 0.8, IPTG is added at a final concentration of 0.2 mM, and the expression is induced in a shaker at 25°C and 200 rpm for 12 hours, followed by centrifugation of the fermentation broth, and the resulting precipitate is washed three times with physiological saline to collect the wet bacterial cells.

[0016] Preferably, the pure enzyme is prepared as follows: the wet cell culture is resuspended in buffer A (0.05 M potassium phosphate, 0.5 M NaCl, 0.02 M imidazole, pH 8.0), ultrasonically disrupted for 20 min at 360 W, working for 3 s, and resting for 7 s, and then centrifuged at 9000 rpm for 10 min. The supernatant is collected to obtain a crude enzyme solution containing the ω-aminotransferase mutant;

[0017] The crude enzyme solution was separated and purified by Ni-NTA 6FF affinity chromatography. The purification process was as follows: first, the nickel column was equilibrated with buffer A, and then the crude enzyme solution was naturally passed through the nickel column with a sample volume of 3 column volumes; buffer A was continued to be used to elute the impurities not bound to the nickel column with an elution volume of 6 column volumes, and then buffer B (0.05M potassium phosphate, 0.5M NaCl, 0.5M imidazole, pH 8.0) and buffer A were mixed to form an eluent containing different concentrations of imidazole (50, 100, 250mM) for gradient elution, with each gradient elution lasting 6 column volumes to elute the recombinant protein bound to the nickel column, and the effluent corresponding to the eluents with imidazole concentrations of 100mM and 250mM was collected and concentrated by ultrafiltration (penetration molecular weight of 10kDa), and the concentrate was taken to obtain pure enzyme solution.

[0018] When the prochiral ketone in the present invention is acetophenone, the chiral amine is (S)-α-phenylethylamine; when the prochiral ketone is 4-fluoroacetophenone, the chiral amine is (S)-1-[4-fluorophenyl]ethylamine; when the prochiral ketone is 4-chloroacetophenone, the chiral amine is (S)-1-[4-chlorophenyl]ethylamine; when the prochiral ketone is 4-bromoacetophenone, the chiral amine is (S)-1-[4-bromophenyl]ethylamine; when the prochiral ketone is 4-nitroacetophenone, the chiral amine is (S)-1-[4-nitrophenyl]ethylamine; when the prochiral ketone is 4-methoxyacetophenone, the chiral amine is (S)-1-[4-methoxyphenyl]ethylamine; when the prochiral ketone is 4-(trifluoromethyl)acetophenone, the chiral amine is (S)-1-[4-(trifluoromethyl)phenyl]ethylamine; when the prochiral ketone is phenylbutanone, the chiral amine is (S)-1-phenylbutylamine; when the prochiral ketone is 2-(trifluoromethyl)acetophenone, the chiral amine is (S)-1-[2-(trifluoromethyl)phenyl]ethylamine; when the prochiral ketone is 3-(trifluoromethyl)acetophenone, the chiral amine is (S)-1-[3-(trifluoromethyl)phenyl]ethylamine; when the prochiral ketone is 2-fluoroacetophenone, the chiral amine is (S)-1-[2-fluorophenyl]ethylamine; when the prochiral ketone is 3-fluoroacetophenone, the chiral amine is (S)-1-[3-fluorophenyl]ethylamine.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] Compared with the wild-type ω-transaminase, the ω-transaminase mutant of the present invention has a high catalytic efficiency for catalyzing the formation of chiral amines from prochiral ketones. Among them, the conversion yield of the mutant R411A catalyzing 4-(trifluoromethyl)acetophenone is 2.39 times that of the wild-type ω-transaminase, and the enantiomeric excess (ee) of the obtained product > 99.9%. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the reaction process of ω-transaminase catalyzing the formation of chiral amines from prochiral ketones.

[0022] Figure 2 : SDS-PAGE electrophoresis pattern of the protein of wild-type ω-transaminase, where M: Marker, 1: flow-through solution, 2: 20 mM imidazole eluate, 3: 50 mM imidazole eluate, 4: 100 mM imidazole eluate, 5: 250 mM imidazole eluate.

[0023] Figure 3 : Optimal reaction pH value of wild-type ω-transaminase.

[0024] Figure 4 : Optimal reaction temperature of wild-type ω-transaminase.

[0025] Figure 5 : Alanine scanning result diagram.

[0026] Figure 6 : SDS-PAGE electrophoresis diagram of protein of ω-transaminase mutant R411A, where M: Marker, 1: Perfusion liquid, 2: 20 mM imidazole eluate, 3: 50 mM imidazole eluate, 4: 100 mM imidazole eluate, 5: 250 mM imidazole eluate.

[0027] Figure 7 : Optimal reaction pH value of ω-transaminase mutant R411A.

[0028] Figure 8 : Optimal reaction temperature of ω-transaminase mutant R411A.

[0029] Figure 9 : Gas chromatography detection results of 4-(trifluoromethyl)acetophenone and its product. (V) Specific implementation manners

[0030] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0031] The culture media involved in the following embodiments are as follows:

[0032] LB liquid culture medium: Yeast powder 5.0 g / L, Tryptone 10.0 g / L, NaCl 10.0 g / L, and the solvent is water.

[0033] LB solid culture medium: Yeast powder 5.0 g / L, Tryptone 10.0 g / L, NaCl 10.0 g / L, Agar powder 10.0 g / L, and the solvent is water.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Standard reaction conditions for ω-transaminase enzyme activity determination: 0.3 mg / mL (calculated based on protein content) pure enzyme, 1 mM pyridoxal phosphate (PLP), 100 mM isopropylamine hydrochloride, 5 mM 4-(trifluoromethyl)acetophenone, 2% (v / v) dimethyl sulfoxide (DMSO), 0.05 M potassium phosphate buffer (pH 8.0), and the total volume of the reaction system is 0.5 mL. After reacting for 30 min at 30 °C and 200 rpm, the reaction is quenched with 4 M NaOH, 1 mL of ethyl acetate containing 4.4 mM dodecane as an internal standard is added for extraction, 250 μL of the upper organic phase is taken, 5 μL of acetic anhydride and 5 μL of pyridine are added to derivatize the product, and gas phase analysis is performed on the product derivative, and the enzyme activity is calculated according to the gas phase detection results.

[0036] Enzyme activity definition: Under the above standard reaction conditions, the amount of enzyme required to catalyze the production of 1 mM (S)-1-[4-(trifluoromethyl)phenyl]ethylamine from 4-(trifluoromethyl)acetophenone per minute is defined as one enzyme activity unit (U).

[0037] Specific enzyme activity definition: The number of enzyme activity units per unit weight of protein.

[0038] Qualitative and quantitative analysis by gas chromatography: The internal standard method was used to detect the contents of the product and the residual substrate after the reaction.

[0039] The gas chromatography conditions are as follows: Agilent 7820A gas chromatograph, Varian CP-Chirasil-Dex CB chiral capillary gas chromatography column (25 m × 0.25 mm × 0.25 μm), FID detector. The carrier gas is high-purity nitrogen with a flow rate of 2 mL / min, the injection port temperature is 250 °C, the detector temperature is 300 °C, the temperature programming: hold at 70 °C for 2 min, increase to 120 °C at 20 °C / min, hold for 0 min, increase to 160 °C at 10 °C / min, hold for 4 min, the injection volume is 1 μL, and the split ratio is 15:1.

[0040] The calculation formula for the product yield is as follows:

[0041] Yield (%) = C p / C0 × 100%

[0042] In the formula, C p is the concentration of the chiral amine of the product after the reaction, and C0 is the initial concentration of the prochiral ketone.

[0043] The optical purity of the product is characterized by the ee value, and the calculation formula is as follows:

[0044]

[0045] In the formula, C R and C S are the molar concentrations of the R-configuration and S-configuration chiral amines, respectively.

[0046] Example 1: Induced expression and purification of wild-type ω-transaminase

[0047] The coding gene of the wild-type ω-transaminase derived from Vitreoscilla stercoraria DSM 513 (NZ_KB908020.1, whose sequence is shown in SEQ ID NO.3) from the NCBI database was codon-optimized, and the synthetic gene fragment (the nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1) was inserted between the Nde I and Hind III restriction sites of the pET28a(+) expression vector, and then transferred into E. coli BL21(DE3) to obtain the recombinant E. coli E. coli BL21(DE3)-pET28a(+)-VsTA.

[0048] The recombinant E. coli was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin (Kan), and cultured overnight at 37 °C and 200 rpm to obtain the recombinant E. coli seed solution. The seed solution was transferred to 100 mL of fresh LB liquid medium containing 50 μg / mL kanamycin (Kan) at an inoculation amount of 1% (v / v), and fermented at 37 °C and 200 rpm until the optical density at 600 nm was between 0.6 and 0.8. Then, IPTG with a final concentration of 0.2 mM was added, and the mixture was placed in a shaker at 25 °C and 200 rpm for induction expression for 12 h. The fermentation broth was centrifuged to collect the cells, which were washed 3 times with physiological saline. The collected recombinant E. coli cells were resuspended in buffer A (0.05 M potassium phosphate, 0.5 M NaCl, 0.02 M imidazole, pH 8.0), and sonicated for 20 min under the conditions of 360 W, working for 3 s and intermittent for 7 s, and then centrifuged at 9000 rpm for 10 min. The supernatant was taken to obtain the crude enzyme solution containing the wild-type ω-transaminase.

[0049] The crude enzyme solution was separated and purified by a Ni-NTA 6FF affinity chromatography column (nickel column, purchased from Shanghai Sangon Biotech Co., Ltd.). The purification process was as follows: First, the nickel column was equilibrated with buffer A, and then the crude enzyme solution was allowed to flow through the nickel column naturally. The sample loading volume was 3 column volumes, and the flow-through was collected. The electrophoresis pattern is shown in Figure 2 lane 1 in Figure 2 Then, buffer B (0.05 M potassium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 8.0) and buffer A were mixed to form elution solutions containing different concentrations of imidazole (50, 100, 250 mM) for gradient elution. Each gradient was eluted with 6 column volumes to elute the recombinant protein bound to the nickel column. The eluents of each gradient were collected separately, and the electrophoresis pattern is shown in Figure 2In the middle lanes 3, 4, and 5, the effluents corresponding to the eluates with imidazole concentrations of 100 mM and 250 mM collected were concentrated by ultrafiltration (with a molecular weight cut-off of 10 kDa), and the concentrated solution was taken to obtain a pure enzyme solution of wild-type ω-transaminase. The pure enzyme solution was detected using a Bradford method protein quantification detection reagent (purchased from Shanghai Shanggong Biotech Co., Ltd.), and the protein concentration was 1.7 mg / mL. The pure enzyme solution obtained by gradient elution was subjected to SDS-PAGE electrophoresis analysis, and the analysis results are shown in Figure 2 .

[0050] As can be seen from Figure 2 , the pure enzyme solution obtained by gradient elution showed a relatively thick single band at around 52.2 kDa, and there were fewer impurity proteins, indicating good separation effect after purification by nickel column.

[0051] The sequence of SEQ ID NO.1 is as follows:

[0052] MTNTTLNATANHLHPFSDNQQLKEKGVRVITKADGIYIYDDAGNKIIDGMAGL

[0053] WCVNIGYGRKELADIARDQMNELAYYNTFFKTSHPAVINLSAKLASIAPAGFNHVF

[0054] YTGSGSESVDSMIRMVRHYWDSVGKPSKKTLIGRWNGYHGSTIGGVSMGGMKG

[0055] MHKQGGFPIEGIVHIEQPWFYGLHEEGETAEQFGIRAANWLEEKILEIGADNVAAFV

[0056] GEPIQGAGGVIIPPSTYWPRIQEICQKYDILLVADEVICGFGRTGEWFGHETFGFQPDI

[0057] FTTAKGLSSGYLPIGAVFVGDKVVEGILKGGDFNHGFTYAGHPVAAAVALKNVEIL

[0058] DDEGIIKNVHDKTGPYMQAKWREMLSQFKYVDDVRGVGLIQGFTLVKNKATREM

[0059] FPNEGETGTMLRDIFFSNNLIMRACGDHIVASPPLTITKEEIDLMLATAEKCLQEFET

[0060] AMDSKLGA。

[0061] Example 2: Optimal reaction pH value and reaction temperature of wild-type ω-transaminase

[0062] (1) Determination of the optimal reaction pH value: Take 88.2 μL of the wild-type ω-transaminase pure enzyme solution prepared according to the method of Example 1, and place it in 0.05 M buffer systems with different pH values (the pH value range of potassium dihydrogen phosphate-potassium hydrogen phosphate buffer is 6.0 - 8.0; the pH value range of boric acid-sodium borate buffer is 8.0 - 9.0; the pH value range of glycine-sodium hydroxide buffer is 9.0 - 10.0). After incubating in a water bath at 30 °C for 5 min, measure the enzyme activity under standard reaction conditions to investigate the effect of different reaction pH values on the enzyme activity. The results are as Figure 3 shown. The optimal reaction pH value of this wild-type ω-transaminase is the potassium dihydrogen phosphate-potassium hydrogen phosphate buffer at pH 8.0.

[0063] (2) Determination of the optimal reaction temperature: Take 88.2 μL of the wild-type ω-transaminase pure enzyme solution prepared according to the method of Example 1, place it in 0.05 M potassium dihydrogen phosphate-potassium hydrogen phosphate buffer at pH 8.0, and incubate in a water bath at different temperatures (20, 25, 30, 37, 45 °C) for 5 min. Then measure the enzyme activity under standard reaction conditions to investigate the effect of different temperatures on the activity of wild-type ω-transaminase, so as to determine the optimal reaction temperature for this enzyme to catalyze. The results are as Figure 4 shown. The optimal reaction temperature of this wild-type ω-transaminase is 30 °C.

[0064] Example 3: Preparation of ω-transaminase mutants

[0065] (1) Selection of mutation sites

[0066] Use the SWISS-MODEL online software to perform homology modeling on the amino acid sequence SEQ ID NO.1 of wild-type ω-transaminase to obtain the model of ω-transaminase. Then use the AutoDock 4.2 software to perform molecular docking of the protein model of ω-transaminase with the substrate molecule. According to the docking results, select a total of ten sites, namely F16, L53, W54, F82, Y147, S150, G314, F315, T316, and R411, for alanine scanning. The primers used for alanine scanning are shown in Table 1. The results of alanine scanning are as Figure 5 . From Figure 5 it can be seen that only when the two sites W54 and R411 are mutated to alanine, the activity is still retained. Therefore, these two sites are selected for the next single-site saturation mutation and combinatorial mutation. <�

[0067] (2) Construction of mutants

[0068] According to the primers shown in Table 1, the plasmid pET28a(+)-VsTA constructed in Example 1 was used as a template for whole-plasmid PCR to construct a single mutation recombinant plasmid containing the gene encoding the ω-transaminase mutant, as shown in Table 2; according to the primers shown in Table 1, the single mutation recombinant plasmid was used as a template for whole-plasmid PCR to obtain a combined mutation recombinant plasmid with double mutations, as shown in Table 2.

[0069] Table 1 Primers

[0070]

[0071]

[0072]

[0073] The whole plasmid PCR reaction system was as follows: 0.5 μL upstream primer, 0.5 μL downstream primer, 0.25 μL template, 10 μL PrimeSTARMax Premix (2X), and 8.75 μL ddH2O.

[0074] The PCR amplification conditions for the whole plasmid were as follows: (1) 98°C pre-denaturation for 2 min. (2) 98°C denaturation for 10 s. (3) 55°C annealing for 15 s. (4) 72°C extension for 1 min. Repeat steps (2) to (4) for 35 cycles, followed by a final extension at 72°C for 10 min. The PCR amplification product was stored at 4°C.

[0075] The PCR amplification products were detected by 1% agarose gel electrophoresis. After the detection, methylation template digestion enzyme (Dpn I) was added to the PCR amplification products, and the reaction was carried out at 37°C for 2 hours. The PCR amplification products treated with Dpn I were transformed into Escherichia coli BL21 (DE3), and the transformation products were spread on LB solid medium and cultured at 37°C overnight. Samples of transformants were picked from the LB solid medium and sent for sequencing.

[0076] Example 4: Mutant activity detection

[0077] Pick single colonies of the single mutants and double mutants constructed in Example 3, and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin (Kan) respectively. Culture overnight at 37 °C and 200 rpm to obtain recombinant Escherichia coli seed solutions. Transfer the seed solutions to 100 mL of fresh LB liquid medium containing 50 μg / mL kanamycin (Kan) according to the inoculation amount of 1% (v / v). Ferment and culture at 37 °C and 200 rpm until the optical density at 600 nm is between 0.6 and 0.8. Add IPTG with a final concentration of 0.2 mM, and place it in a shaker at 25 °C and 200 rpm for induction expression for 12 h. Centrifuge the fermentation broth to collect cells, and wash them 3 times with physiological saline. Resuspend the collected recombinant Escherichia coli cells in buffer A (0.05 M potassium phosphate, 0.5 M NaCl, 0.02 M imidazole, pH 8.0), and prepare the mutant pure enzyme solution according to the method of Example 1.

[0078] Take a certain amount of the wild-type ω-transaminase pure enzyme solution and the mutant pure enzyme solution prepared according to the method of Example 1, and detect their specific enzyme activities respectively. Among them, taking the specific enzyme activity of the wild-type ω-transaminase as 100%, the relative enzyme activities of the other ω-transaminase mutants are calculated compared with it, and the calculation results are shown in Table 2.

[0079] Table 2 Relative enzyme activities of wild-type ω-transaminase and ω-transaminase mutants

[0080]

[0081] - Enzyme activity not detected

[0082] As can be seen from Table 2, the specific enzyme activities of most ω-transaminase mutants are significantly higher than that of the wild-type ω-transaminase. Among them, the specific enzyme activity of the ω-transaminase mutant R411A is the highest, which is 2.39 times that of the wild-type ω-transaminase. The protein concentration of the pure enzyme solution of the ω-transaminase mutant R411A is 2.3 mg / mL. The protein SDS-PAGE electrophoresis pattern of the ω-transaminase mutant R411A is shown in Figure 6 , where M: Marker, 1: Flow-through solution, 2: 20 mM imidazole eluate, 3: 50 mM imidazole eluate, 4: 100 mM imidazole eluate, 5: 250 mM imidazole eluate.

[0083] Example 5: Optimal reaction pH value and reaction temperature of ω-transaminase mutant R411A

[0084] (1) Determination of the optimal reaction pH value: Take 65.2 μL of the pure enzyme solution of the ω-transaminase mutant R411A prepared according to the method of Example 4, and place it in 0.05 M buffer systems with different pH values (the pH value range of potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer is 6.0 - 8.0; the pH value range of boric acid - sodium borate buffer is 8.0 - 9.0; the pH value range of glycine - sodium hydroxide buffer is 9.0 - 10.0). After incubating in a water bath at 30 °C for 5 min, measure the enzyme activity under standard reaction conditions to investigate the effect of different reaction pH values on the enzyme activity. The results are as Figure 7 shown. The optimal reaction pH value of this ω-transaminase mutant R411A is the potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer at pH 8.0. (2) Determination of the optimal reaction temperature: Take 65.2 μL of the pure enzyme solution of the ω-transaminase mutant R411A prepared according to the method of Example 4, place it in 0.05 M potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer at pH 8.0, and after incubating in a water bath at different temperatures (20, 25, 30, 37, 45 °C) for 5 min, measure the enzyme activity under standard reaction conditions to determine the effect of different temperatures on the enzyme activity and thus determine the optimal reaction temperature catalyzed by this enzyme. The results are as Figure 8 shown. The optimal reaction temperature of this ω-transaminase mutant R411A is 30 °C.

[0085] Example 6: Kinetic parameters of wild-type ω-transaminase and ω-transaminase mutant R411A

[0086] The initial velocity of the ω-transaminase reaction at different substrate concentrations was measured by the enzyme activity assay method. The concentrations of the substrate 4-(trifluoromethyl)acetophenone were 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 2.0 mM, and 4.0 mM respectively. Substitute the corresponding enzymatic reaction rates at different substrate concentrations into the Michaelis equation, and use the Lineweaver Burk double-reciprocal method to plot a graph to calculate V max , K m , and then calculate the turnover number K cat according to the calculation formula of the turnover number K cat , so as to calculate the catalytic efficiency K cat / K m .

[0087] The calculation formula for the turnover number K cat is as follows:

[0088]

[0089] [E] is the molar concentration of the enzyme

[0090] Table 3 Kinetic parameters of wild-type ω-transaminase and ω-transaminase mutant R411A

[0091]

[0092] As can be seen from Table 3, both the maximum reaction rate and catalytic efficiency of the ω-transaminase mutant R411A are significantly improved compared with the wild-type ω-transaminase. Among them, the catalytic efficiency K cat / K m has increased by 69.3%.

[0093] Example 7: Catalytic Activity of ω-Transaminase Mutant R411A towards Different Substrates

[0094] E. coli BL21(DE3)-pET28a(+)-VsTA-R411A constructed according to the method of Example 3 was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin (Kan), and cultured overnight at 37 °C and 200 rpm to obtain a recombinant E. coli seed solution. The seed solution was transferred to 100 mL of fresh LB liquid medium containing 50 μg / mL kanamycin (Kan) according to an inoculation amount of 1% (v / v), and fermented and cultured at 37 °C and 200 rpm until the optical density at 600 nm was between 0.6 and 0.8. IPTG with a final concentration of 0.2 mM was added, and the mixture was placed in a shaker at 25 °C and 200 rpm for induction expression for 12 h. The fermentation broth was centrifuged, washed 3 times with physiological saline, and wet bacterial cells were collected.

[0095] Reaction system for ω-transaminase mutant R411A to catalyze different substrates: 0.1 g / mL E. coli BL21(DE3)-pET28a(+)-VsTA-R411A (WCW) wet bacterial cells, 1 mM pyridoxal phosphate (PLP), 100 mM isopropylamine hydrochloride, 4 mM different prochiral ketones, 2% (v / v) dimethyl sulfoxide (DMSO), glycine-sodium hydroxide buffer (0.05 M, pH 10.6). After reacting at 30 °C and 200 rpm for 2 h in a total volume of 1 mL of the reaction system, the reaction was quenched with 4 M NaOH. 2 mL of ethyl acetate containing 4.4 mM dodecane as an internal standard was added for extraction. 250 μL of the upper organic phase was taken, and 5 μL of acetic anhydride and 5 μL of pyridine were added to derivatize the product. The product yield and enantiomeric excess value (ee) were detected by gas chromatography. The gas chromatography detection results of 4-(trifluoromethyl)acetophenone, (S)-1-[4-(trifluoromethyl)phenyl]ethylamine, (R)-1-[4-(trifluoromethyl)phenyl]ethylamine standards and dodecane are as Figure 9 shown.

[0096] Table 4 Catalytic Activity of ω-Transaminase Mutant R411A towards Different Substrates

[0097]

[0098]

[0099] - No product detected

[0100] As can be seen from Table 4, the ω-transaminase mutant R411A has good reaction yields and high selectivity for a variety of substrate ketones. Therefore, the ω-transaminase mutant R411A is an excellent catalyst for the preparation of chiral amines.

Claims

1. A ω-transaminase mutant, characterized in that, The ω-transaminase mutant is one in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) the arginine at position 411 is mutated into alanine, valine, proline, isoleucine, tyrosine, tryptophan, glycine, phenylalanine, methionine, asparagine, aspartic acid, glutamic acid, serine, threonine, cysteine or glutamine; (2) the tryptophan at position 54 is mutated into glycine, alanine, valine, cysteine or tyrosine; (3) the tryptophan at position 54 is mutated into cysteine, and the arginine at position 411 is mutated into alanine, phenylalanine, tyrosine, isoleucine, proline, valine, serine or tryptophan; (4) the tryptophan at position 54 is mutated into valine and the arginine at position 411 is mutated into methionine; (5) the tryptophan at position 54 is mutated into alanine and the arginine at position 411 is mutated into serine; (6) the tryptophan at position 54 is mutated into tyrosine, and the arginine at position 411 is mutated into serine or alanine.

2. A coding gene for the ω-transaminase mutant according to claim 1.

3. A recombinant genetically engineered bacterium containing the coding gene for the ω-transaminase mutant according to claim 1.

4. Use of the ω-transaminase mutant according to claim 1 in the synthesis of chiral amines from prochiral ketones, characterized in that, The application is as follows: using the wet cell bodies obtained by fermentation culture of recombinant Escherichia coli containing the coding gene for the ω-transaminase mutant or the pure enzyme extracted after ultrasonic disruption of the wet cell bodies as a catalyst, using prochiral ketone as an amino acceptor, isopropylamine hydrochloride as an amino donor, using pyridoxal phosphate as a cofactor, adding dimethyl sulfoxide as a cosolvent, using a potassium phosphate buffer solution with a pH of 6.5 - 8.0 as a reaction medium to form a reaction system, reacting in a shaker at 25 - 40 °C and 200 rpm for 0.5 - 3 h, and obtaining a chiral amine product after separation and purification of the obtained reaction solution; the prochiral ketone is 4-(trifluoromethyl)acetophenone.

5. The application according to claim 4, characterized in that When the catalyst used is the wet cell bodies obtained by fermentation culture of recombinant Escherichia coli with the coding gene for the ω-transaminase mutant in which the arginine at position 411 is mutated into alanine or the pure enzyme extracted after ultrasonic disruption of the wet cell bodies, the prochiral ketone is acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, 4-nitroacetophenone, 4-(trifluoromethyl)acetophenone, 3-(trifluoromethyl)acetophenone or 2-fluoroacetophenone.

6. The application according to claim 4, characterized in that In the reaction system, the added concentration of the cofactor is 0.1 - 1.0 mM; the catalyst is 50 - 200 g / L based on the weight of the wet cell bodies, and the catalyst is 0.1 - 0.5 mg / mL based on the protein content in the pure enzyme; the added concentration of the prochiral ketone is 1.0 - 10 mM; the added concentration of the isopropylamine hydrochloride is 50 - 200 mM; the final volume concentration of the added dimethyl sulfoxide is 1 - 5%.

7. The application according to claim 4, wherein The wet bacterial cells are prepared as follows: Recombinant Escherichia coli containing the encoding gene of ω-transaminase mutant is inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C and 200 rpm to obtain a seed solution; The seed solution is transferred to fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of volume concentration of 1%, and fermented at 37 °C and 200 rpm until the optical density value at 600 nm is between 0.6 and 0.

8. IPTG with a final concentration of 0.2 mM is added, and the mixture is placed in a shaker at 25 °C and 200 rpm for induction expression for 12 h. Subsequently, the fermentation broth is centrifuged, and the obtained precipitate is washed 3 times with physiological saline to collect wet bacterial cells.

8. The application according to claim 4, wherein The pure enzyme is prepared as follows: The wet bacterial cells are resuspended in buffer A, and ultrasonicated for 20 min under the conditions of 360 W, working for 3 s and intermittent for 7 s, then centrifuged at 9000 rpm for 10 min, and the supernatant is taken to obtain a crude enzyme solution containing ω-transaminase mutant. The crude enzyme solution is separated and purified by a Ni-NTA6FF affinity chromatography column. The purification process is as follows: First, the nickel column is equilibrated with buffer A, then the crude enzyme solution naturally flows through the nickel column, and the sample loading amount is 3 column volumes; Buffer A is continuously used to elute the impurity proteins not bound to the nickel column, and the elution volume is 6 column volumes. Then, gradient elution is carried out successively with an eluent composed of a mixture of buffer B and buffer A containing 50 - 250 mM concentration of imidazole, and each gradient is eluted for 6 column volumes to elute the recombinant protein bound to the nickel column. The effluent corresponding to the eluent with imidazole concentrations of 100 mM and 250 mM is collected, ultrafiltered and concentrated, and the concentrated solution is taken to obtain a pure enzyme solution; Buffer A: 0.05 M potassium phosphate, 0.5 M NaCl, 0.

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