Ketoreductase mutant and application thereof in synthesis of (R)-methyl o-chloromandelate

Abstract

The invention provides a ketoreductase mutant, the amino acid sequence of the ketoreductase mutant is obtained by mutating a sequence as shown in SEQ ID No.1 in a sequence table, and the mutation comprises at least one mutation of amino acids at the 24 , 25 , 116 , 193 , 234 , 238 and 245 in the sequence as shown in SEQ ID No.1 in the sequence table. The invention also provides a corresponding nucleic acid, a recombinant expression vector, a genetically engineered bacterium and application of the genetically engineered bacterium in synthesis of (R)-methyl o-chloromandelate. The ketoreductase mutant can catalyze asymmetric reduction reaction with high-concentration methyl o-chlorobenzoylformate as a substrate to synthesize (R)-methyl o-chloromandelate, the yield is high, the requirement for 99% + stereoselectivity is met, and the ketoreductase mutant has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, relates to a ketoreductase mutant, and further relates to a nucleic acid encoding the ketoreductase mutant, an expression vector comprising the ketoreductase mutant, a preparation method and application thereof, and specifically relates to an application thereof in catalytic synthesis of (R)-methyl o-chloromandelate. Background Art

[0002] Ketoreductase has important applications in the field of biocatalysis. It generates corresponding chiral alcohols by catalyzing the reduction reaction of ketones and is widely used in industries such as medicine, agriculture and chemical industry. Traditional chemical synthesis methods often require multi-step reactions and strict conditions, usually with low yields and more by-products. Therefore, the use of enzyme-catalyzed methods to synthesize high-purity, highly selective target products has gradually become a research hotspot. (R)-methyl o-chloromandelate is an important intermediate of the best-selling antiplatelet drug (S)-clopidogrel and is widely used in organic synthesis and drug development. However, the traditional method for synthesizing (R)-methyl o-chloromandelate has the problems of complex steps, harsh conditions and environmental unfriendliness. In order to improve synthesis efficiency and environmental friendliness, the development of efficient, stable and highly stereoselective ketoreductase has become the key. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the invention provides a ketoreductase mutant, through directed evolution and rational design, the catalytic performance, stereoselectivity and thermal stability of the enzyme, pH stability are improved. The mutant can be used as a substrate to efficiently catalyze the synthesis of (R)-o-chloromandelate methyl ester with catalysis of o-chlorobenzoylmethyl formate, and the product has high optical purity, and has environmental friendliness and is easy to amplify. In addition, the present invention also provides nucleic acids encoding the above-mentioned ketoreductase mutant and vectors for expressing the enzyme, providing technical support for industrial production. This enzyme mutant has shown significant superiority in the preparation of chiral compounds, providing new approaches for green chemistry and sustainable development.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] The first object of the present invention is to provide a ketoreductase mutant, whose amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 1 in the sequence listing, and the mutation includes at least one mutation of the amino acids at positions 24, 25, 116, 193, 234, 238, and 245 in the sequence shown in SEQ ID No. 1 in the sequence listing.

[0006] Preferably, the mutation includes at least one of the following mutations to the sequence shown in SEQ ID No. 1 in the sequence listing: I238G, I238T, F25P, I234G, V24G, F25Y, H116I, W193Y and E245L.

[0007] In the present invention, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutant amino acids.

[0008] Preferably, the mutation includes at least two of the following mutations of the sequence shown in SEQ ID No. 1 in the sequence listing: F25P or F25Y, I238G or I238T, W193Y and E245L;

[0009] Preferably, the mutation includes two or three of the following mutations to the sequence shown in SEQ ID No. 1 in the sequence listing: F25P or F25Y, I238G or I238T, W193Y and E245L.

[0010] Preferably, the mutation is selected from any one of the following mutations of the sequence shown in SEQ ID No. 1 in the sequence table: I238G / F25Y, I238T / F25Y, I238G / F25P, I238T / F25P, I238G / W193Y, I238T / W193Y, I238G / E245L, I238T / E245L, I238G / F25Y / W193Y, I238T / F25Y / W193Y 3Y, I238G / F25Y / E245L, I238T / F25Y / E245L, I238G / F25P / W193Y, I238T / F25P / W193Y , I238G / F25P / E245L, I238T / F25P / E245L, I238G / W193Y / E245L, I238T / W193Y / E245L;

[0011] Preferably, the mutation is the following mutation of the sequence shown in SEQ ID No. 1 in the sequence listing: I238T / W193Y.

[0012] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned ketoreductase mutant;

[0013] Preferably, the nucleotide sequence is shown in the sequence listing SEQ ID No.2.

[0014] The third object of the present invention is to provide a recombinant expression vector comprising the above nucleotide sequence.

[0015] The fourth object of the present invention is to provide a genetically engineered bacterium comprising the above-mentioned recombinant expression vector;

[0016] Preferably, the genetically engineered bacteria is a recombinant strain obtained by connecting the above nucleotide sequence to a vector to obtain a recombinant expression vector, and then introducing it into a host bacterium for inducing expression, wherein the host bacterium includes Escherichia coli, Bacillus subtilis or yeast;

[0017] Preferably, the Escherichia coli is the Escherichia coli BL21 (DE3) strain.

[0018] The fifth object of the present invention is to provide the use of the above-mentioned ketoreductase mutant, nucleotide sequence, recombinant expression vector, and genetically engineered bacteria in catalyzing the synthesis of (R)-methyl o-chloromandelate.

[0019] Preferably, methyl o-chlorobenzoylformate is used as a substrate and a ketoreductase mutant is used as a catalyst, the reaction is carried out at a pH value of 5.5-7.5, preferably a pH value of 6.5, and the reaction temperature is 20-40° C.; preferably, the reaction temperature is 30° C.;

[0020] Preferably, the concentration of methyl o-chlorobenzoylformate is 1-4 mol / L; preferably, the concentration of methyl o-chlorobenzoylformate is 3.2 mol / L;

[0021] Preferably, the protein content of the ketoreductase mutant is 30-40 g / L; preferably, the protein content of the ketoreductase mutant is 40 g / L;

[0022] Preferably, the reaction time is 1-36 h; preferably, the reaction time is 16 h.

[0023] More preferably, methyl o-chlorobenzoylformate, NADP+, a pH buffer solution, a ketoreductase mutant, GDH, and glucose are mixed and reacted.

[0024] The sixth object of the present invention is to provide a method for synthesizing (R)-methyl o-chloromandelate, using methyl o-chlorobenzoylformate as a substrate and the above-mentioned ketoreductase mutant as a catalyst, reacting at a pH value of 5.5-7.5, preferably a pH value of 6.5, and a reaction temperature of 20-40° C., preferably a reaction temperature of 30° C.;

[0025] Preferably, the concentration of methyl o-chlorobenzoylformate is 1-4 mol / L; preferably, the concentration of methyl o-chlorobenzoylformate is 3.2 mol / L;

[0026] Preferably, the protein content of the ketoreductase mutant is 30-40 g / L; preferably, the protein content of the ketoreductase mutant is 40 g / L;

[0027] Preferably, the reaction time is 1-36 h; preferably, the reaction time is 16 h.

[0028] More preferably, methyl o-chlorobenzoylformate, NADP+, a pH buffer solution, a ketoreductase mutant, GDH, and glucose are mixed and reacted.

[0029] The ketoreductase mutant of the invention can catalyze the asymmetric reduction reaction of high-concentration methyl o-chlorobenzoylformate as a substrate to synthesize (R)-methyl o-chloromandelate, has high yield and meets the requirement of 99%+ stereoselectivity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 This is an asymmetric synthetic route for (R)-methyl o-chloromandelate.

[0032] Figure 2 Schematic diagram of the relative activity of MF-KR at different pH values.

[0033] Figure 3 Schematic diagram of the relative activity of MF-KR-M6 at different pH values.

[0034] Figure 4 Schematic diagram of the relative activities of MF-KR and MF-KR-M6 at different temperatures.

[0035] Figure 5 Schematic diagram of pH stability of MF-KR and MF-KR-M6 at the optimum temperature and the optimum pH 6.5.

[0036] Figure 6 Schematic diagram of the thermal stability of MF-KR.

[0037] Figure 7 Schematic diagram of the thermal stability of MF-KR-M6.

[0038] Figure 8 The product of (R)-methyl o-chloromandelate was generated by MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate. 1 H NMR chromatogram.

[0039] Fig. 9 The product of (R)-methyl o-chloromandelate was generated by MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate. 13 C NMR chromatogram.

[0040] Fig.10This is the HPLC diagram of MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate to generate (R)-methyl o-chloromandelate product. DETAILED DESCRIPTION

[0041] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0042] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. In addition, except for the special instructions in this example, the terms and processes involved in this example are understood according to the general cognition and conventional methods in the prior art.

[0043] Raw materials of the present invention:

[0044] pET28a vector: Novagen, catalog number: 69864-3.

[0045] Escherichia coli BL21 (DE3): Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CD601-02.

[0046] LB medium: 10 g tryptone, 5 g yeast extract, 5 g NaCl per liter, dilute to 1 L with deionized water. Steam sterilize under high pressure for 20 min.

[0047] TB medium: 12 g tryptone, 24 g yeast extract, 4 mL 87% glycerol, 100 mL phosphate buffer solution (pH 6.5), dilute to 1 L with deionized water. Steam sterilize under high pressure for 20 min.

[0048] Example 1 Construction of recombinant bacteria

[0049] 1. Construction of wild-type recombinant expression vector

[0050] The ketoreductase protein sequence from Mesobacillus foraminis (GenBank Accession Number: WP_121613048.1) was obtained from NCBI, as shown in SEQ ID NO.1, and the DNA sequence encoding the enzyme was obtained after codon optimization, as shown in SEQ ID NO.2, which was synthesized by Beijing Qingke Biotechnology Co., Ltd. and inserted into the two restriction endonuclease sites of Nde I and EcoR I introduced. Using pET-28a(+) as an expression vector, the constructed recombinant plasmid was named pET-28a(+)-MF-KR, and MF-KR plasmid dry powder was prepared.

[0051] The amino acid sequence of ketoreductase is as follows (SEQ ID NO.1):

[0052]

[0053] The nucleic acid sequence encoding the above ketoreductase amino acid sequence (codon optimized) is as follows (SEQ ID NO.2):

[0054]

[0055]

[0056] 2. Construction of mutant recombinant expression vector

[0057] Using the MF-KR unmutated enzyme plasmid as a template, PCR amplification was performed using the corresponding primers, KOD, and DNA polymerase. The site-directed mutagenesis PCR reaction system is shown in Table 1, the PCR reaction procedure is shown in Table 2, the mutation sites of each mutant are shown in Table 3, and the primers for some mutants are shown in Table 4. After verification by agarose gel electrophoresis, DpnⅠ restriction enzyme was added to the PCR reaction product, and the template was removed by standing at 37°C in a sterile environment for 30 minutes to obtain a mutant plasmid dissolved in sterile water.

[0058] Table 1 PCR reaction system

[0059] PCR reaction system Volume / μL Sterile water 22 KODOneTMPCRMasterMix 25 Plasmids 1 Upstream primer 1 Downstream primer 1

[0060] Table 2 PCR reaction program

[0061]

[0062] Example 2 Plasmid transformation

[0063] The ketoreductase plasmid powder (4 μg) obtained in Example 1 or the ketoreductase plasmid powder (4 μg) obtained in Example 2 was added to 100 μL of sterile water and shaken to mix well. 5 μL was added to 50 μL of E. coli BL21 (DE3) competent cells, flicked to mix well, placed on ice for 30 minutes, placed in a 42°C water bath for heat shock for 45 seconds, and added to 500 μL of sterile, non-resistant LB liquid culture medium after ice bathing for 2 minutes. After mixing, place on a 37°C shaker for activation for 1 hour. After centrifugation at 10000 rpm for 1 minute, 300 μL of supernatant was taken out, the remaining liquid was mixed with the bacterial sediment, 50 μL was applied to the LB solid culture medium plate containing kanamycin, and cultured in a 37°C incubator for 16 hours.

[0064] Example 3 Preparation of recombinant bacteria

[0065] A single colony was selected from the plate obtained in Example 2 and placed in 10 mL of LB liquid medium containing kanamycin, and cultured at 37°C and 180 rpm for 16 h to obtain a seed solution. The seed solution was then inoculated into 50 mL of TB medium (inoculation volume 1%), the medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm until the bacterial solution OD reached 0. 600nm 0.6-0.8, add IPTG with a final concentration of 0.5 μmol / L, continue shaking culture at 20°C and 180 rpm for 20-24 h, and collect the bacteria by centrifugation at 5000 rcf for 15 min.

[0066] The recombinant bacterial cells were washed and resuspended twice with 0.9% physiological saline, broken with a homogenizer, centrifuged at 12000 rpm for 15 min, and the supernatant was collected as the crude enzyme solution. The protein concentration was measured according to the Bradford method.

[0067] Example 4 Enzyme activity determination

[0068] Prepare the enzyme activity detection reaction system: 850μL of 100mmol / L phosphate-sodium phosphate buffer solution, 10mmol / L methyl o-chlorobenzoylformate, 20μL of the protein solution to be tested (protein content is about 0.1mg), final NADPH concentration of 1mmol / L, and react at 30℃ for 3min. The change in absorbance before and after the reaction is measured at 340nm. One unit activity (U) is defined as the amount of enzyme required to catalyze the conversion of 1μmol of substrate in 1 minute.

[0069] Calculation formula of specific enzyme activity:

[0070] Among them, U: enzyme activity; mg: protein content.

[0071] Example 5 Preliminary screening of ketoreductase mutants

[0072] The mutant recombinant expression vector was constructed according to the method of Example 1, and the final reaction product was transformed into Escherichia coli BL21 (DE3) according to the methods of Examples 2 and 3, and finally the mutant crude enzyme solution was obtained, and its relative activity was measured using Example 4. The results are shown in Table 3. Finally, six optimal single-point mutants were screened out: MF-KR-m3, MF-KR-m4, MF-KR-m8, MF-KR-m17, MF-KR-m21, and MF-KR-m26.

[0073] Table 3 Preliminary screening of ketoreductase mutants

[0074]

[0075]

[0076] Table 4 Primer design for some mutants

[0077]

[0078] Example 6 Ketoreductase mutant rescreening

[0079] After screening out the six single mutants with the best activity, they were combined and their enzyme activities were measured respectively as shown in Example 4. The results are shown in Table 5.

[0080] Table 5 Ketoreductase mutant rescreening

[0081]

[0082]

[0083] As shown in Table 5, the best mutant is MF-KR-M6.

[0084] Example 7 Asymmetric Synthesis of (R)-Methyl o-chloromandelate

[0085] Synthesis route see Figure 1, methyl o-chlorobenzoylformate (3.2 mol / L), NADP+ (0.5 mmol / L), 5 mL of 100 mmol / L phosphate-sodium phosphate buffer solution, ketoreductase mutants 1-18 (MF-KR-M1 to MF-KR-M18) (40 g / L), GDH (5 g / L), and glucose (4.8 mol / L) were added to a 10 mL round-bottom flask. The reaction mixture was stirred at 30 ° C for 18-24 h, and 1 mol / L Na2CO3 was added during the reaction to maintain the pH at about 6-7. After the reaction was completed, it was extracted three times with n-hexane, and the extracts were combined and filtered using a 0.45 μm filter membrane. The yield, configuration and ee value of the asymmetric reduction reaction product were determined by high performance liquid chromatography. The chromatographic conditions were: Chiralcel OD-H column, mobile phase volume ratio: n-hexane / isopropanol = 95 / 5, flow rate 1 mL / min, and detection wavelength 254 nm.

[0086] Figure 1 This is an asymmetric synthetic route for (R)-methyl o-chloromandelate.

[0087] Table 6 Ketoreductase mutant rescreening

[0088]

[0089]

[0090] As shown in Table 6, the best mutant is MF-KR-M6

[0091] Example 8 Exploration of the optimal pH for wild type and optimal mutant

[0092] Take an appropriate amount of enzyme solution and incubate it in buffer solutions of different pH values ​​(4.0-10.0) for 10 min. Then, at 30°C, measure the enzyme activity in buffer solutions of different pH values ​​according to the method in Example 4. The enzyme activity under the optimal pH condition is used as the control (100%). Figure 2 As shown, the MF-KR-M6 results are as follows Figure 3 shown.

[0093] Figure 2 Schematic diagram of the relative activity of MF-KR at different pH values.

[0094] Figure 3 Schematic diagram of the relative activity of MF-KR-M6 at different pH values.

[0095] Depend on Figure 2 and 3It can be seen that the optimum pH of MF-KR and MF-KR-6 is 6.5, among which MF-KR maintains more than 85% of its catalytic activity at pH 6-7, and MF-KR-M6 maintains more than 85% of its catalytic activity at pH 5.5-7.5, with a wider pH spectrum.

[0096] Example 9 Exploration of the Optimal Temperature of the Wild Type and the Optimal Mutant

[0097] Take appropriate amount of enzyme solution, incubate at 10-50℃ for 10min, and then measure enzyme activity at different temperatures at the optimum pH according to the method in Example 4. The enzyme activity at the optimum temperature is used as the control (100%). The effect of temperature on the enzyme activity of the combined mutant MF-KR-M6 is investigated. The results are as follows: Figure 4 shown.

[0098] Figure 4 Schematic diagram of the relative activities of MF-KR and MF-KR-M6 at different temperatures.

[0099] Depend on Figure 4 It can be seen that with the increase of temperature, the activities of MF-KR and MF-KR-M6 showed a trend of first increasing and then decreasing, and the optimum temperature of MF-KR and MF-KR-M6 was 30℃.

[0100] Example 10 Study on pH stability of wild type and optimal mutant

[0101] At the optimum temperature (30°C), take an appropriate amount of enzyme solution and incubate it in a buffer solution with an optimum pH of 6.5 for a certain period of time. According to the method in Example 4, take samples at regular intervals to measure the residual enzyme activity, and use the enzyme activity after incubation for 0 hours as a control. Figure 5 shown.

[0102] Figure 5 Schematic diagram of pH stability of MF-KR and MF-KR-M6 at the optimum temperature and the optimum pH 6.5.

[0103] Depend on Figure 5 It can be seen that with the increase of incubation time, the enzyme activity gradually decreased, but the relative enzyme activity of MF-KR-M6 was always higher than that of MF-KR. After 18 hours, MF-KR had 90% activity, while MF-KR-M6 had 92% activity after 24 hours. It can be seen that compared with the wild type, the combined mutant MF-KR-M6 showed better stability.

[0104] Example 11 Study on the thermal stability of wild type and optimal mutant

[0105] At the optimum pH (pH 6.5), take an appropriate amount of enzyme solution and incubate at 30-50°C for a certain period of time. According to the method in Example 4, take samples regularly to measure the residual enzyme activity, and take the enzyme activity after incubation for 0 hours as the control (100%). Figure 6 As shown, the MF-KR-M6 results are as follows Figure 7 shown.

[0106] Figure 6 Schematic diagram of the thermal stability of MF-KR.

[0107] Figure 7 Schematic diagram of the thermal stability of MF-KR-M6.

[0108] Depend on Figure 6 and Figure 7 It can be seen that the thermal stability of MF-KR and MF-KR-M6 is poor at 50℃ and 40℃, but the thermal stability of both is good at 30℃. After incubation for 60h, MF-KR retains 60% of the initial activity, and after incubation for 72h, MF-KR-M6 retains 63.2% of the initial activity.

[0109] Example 12 Preparation of (R)-methyl o-chloromandelate at different substrate concentrations

[0110] Synthesis route see Figure 1 , the substrate methyl o-chlorobenzoylformate was added into a 10mL round-bottom flask according to the concentration of (1mol / L, 2mol / L, 3mol / L, 3.2mol / L, 3.5mol / L, 4mol / L) and NADP+ (0.5mmol / L), 100mmol / L pH 6.5 phosphate-sodium phosphate buffer solution 5mL, ketoreductase mutant 6 (MF-KR-M6) (30g / L, 40g / L), GDH (5g / L), glucose (1.5 times the molar concentration of methyl o-chlorobenzoylformate). The reaction mixture was stirred at 30°C for 36h, and 1mol / L Na2CO3 was added during the reaction to maintain the pH at about 6-7. After the reaction was completed, it was extracted three times with n-hexane, the extracts were combined, and filtered using a 0.45μm filter membrane. The yield, configuration and ee value of the asymmetric reduction product were determined by high performance liquid chromatography. The chromatographic conditions were: Chiralcel OD-H column, mobile phase volume ratio: n-hexane / isopropanol = 95 / 5, flow rate 1 mL / min, detection wavelength 254 nm.

[0111] Table 7 Preparation of (R)-methyl o-chloromandelate at different substrate concentrations

[0112] MF-KR-M6 enzyme concentration (g / L) Substrate concentration mol / L Yield (%) Time (h) Conversion rate (%) 30 1 99 2 90 30 2 99 6 99 30 3 99 15 99 40 3 99 12 99 30 4 75 36 99 40 3 99 12 99 40 4 79 36 99 40 3.2 99 16 99 40 3.5 94 36 99

[0113] It can be seen from Table 7 that when 3.2 mol / L substrate is added, MF-KR-6 can catalyze the production of optically pure R-methyl o-chloromandelate within 18 h.

[0114] Example 13: Scale-up preparation of (R)-methyl o-chloromandelate

[0115] Synthesis route see Figure 1 , add substrate methyl o-chlorobenzoylformate (3.2 mol / L), NADP+ (0.5 mmol / L), 50 mL of 100 mmol / L pH 6.5 phosphate-sodium phosphate buffer solution, ketoreductase mutant 6 (MF-KR-M6) (40 g / L), GDH (5 g / L), and glucose (4.8 mol / L) into a 250 mL round-bottom flask. Stir the reaction mixture at 30 °C for 20 h, and add 1 mol / L Na2CO3 during the reaction to maintain the pH at about 6-7. After the reaction is completed, extract with n-hexane three times, combine the extracts, and filter with a 0.45 nm filter membrane. The yield, configuration, and ee value of the asymmetric reduction product were determined by high performance liquid chromatography. Chromatographic conditions: Chiralcel OD-H column, mobile phase volume ratio: n-hexane / isopropanol = 95 / 5, flow rate 1 mL / min, detection wavelength 254 nm. Nuclear magnetic chromatography ( 1 H Spectrum Figure 8 , 13 C Spectrum Fig. 9 ) and high performance liquid chromatography ( Fig.10 ) verification, (R)-methyl o-chloromandelate was obtained with a yield of 99% and an ee value of 99%. 1 H NMR (CDCl3, 600MHz): 7.30–7.17 (m, 2H), 5.57 (d, J = 3.2Hz, 1H), 3.75 (d, J = 1.9Hz, 3H), 3.67 (d, J = 4.8Hz, 1H).

[0116] Figure 8 The product of (R)-methyl o-chloromandelate was generated by MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate. 1 H NMR chromatogram.

[0117] Fig. 9 The product of (R)-methyl o-chloromandelate was generated by MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate. 13 C NMR chromatogram.

[0118] Fig.10 This is the HPLC diagram of MF-KR-6 in Example 13 using methyl o-chlorobenzoylformate as substrate to generate (R)-methyl o-chloromandelate product.

[0119] The ketoreductase mutant catalyzes an asymmetric reduction reaction of methyl o-chlorobenzoylformate as a substrate to synthesize (R)-methyl o-chloromandelate, has a high yield and meets the requirement of 99%+ stereoselectivity, and has broad application prospects.

[0120] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0121] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A ketoreductase mutant, characterized in that: The amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 1 in the sequence list, wherein the mutation includes at least one mutation of the amino acids at positions 24, 25, 116, 193, 234, 238, and 245 in the sequence shown in SEQ ID No. 1 in the sequence list.

2. The ketoreductase mutant according to claim 1, characterized in that: The mutations include at least one of the following mutations of the sequence shown in SEQ ID No. 1 in the sequence listing: I238G, I238T, F25P, I234G, V24G, F25Y, H116I, W193Y and E245L.

3. The ketoreductase mutant according to claim 2, characterized in that: The mutations include at least two of the following mutations of the sequence shown in SEQ ID No. 1 in the sequence listing: F25P or F25Y, I238G or I238T, W193Y and E245L; Preferably, the mutation includes two or three of the following mutations to the sequence shown in SEQ ID No. 1 in the sequence listing: F25P or F25Y, I238G or I238T, W193Y and E245L.

4. The ketoreductase mutant according to claim 3, characterized in that: The mutation is selected from any one of the following mutations of the sequence shown in SEQ ID No. 1 in the sequence table: I238G / F25Y, I238T / F25Y, I238G / F25P, I238T / F25P, I238G / W193Y, I238T / W193Y, I238G / E245L, I238T / E245L, I238G / F25Y / W193Y, I238T / F25Y / W193Y 93Y, I238G / F25Y / E245L, I238T / F25Y / E245L, I238G / F25P / W193Y, I238T / F25P / W193Y , I238G / F25P / E245L, I238T / F25P / E245L, I238G / W193Y / E245L, I238T / W193Y / E245L; Preferably, the mutation is the following mutation of the sequence shown in SEQ ID No. 1 in the sequence listing: I238T / W193Y.

5. A nucleotide sequence encoding the ketoreductase mutant according to any one of claims 1 to 4; Preferably, the nucleotide sequence is shown in the sequence listing SEQ ID No.

2.

6. A recombinant expression vector comprising the nucleotide sequence of claim 5.

7. A genetically engineered bacterium comprising the recombinant expression vector according to claim 6; Preferably, the genetically engineered bacteria is a recombinant strain obtained by connecting the nucleotide sequence of claim 5 to a vector to obtain a recombinant expression vector, and then introducing it into a host bacterium for inducing expression, wherein the host bacterium includes Escherichia coli, Bacillus subtilis or yeast; Preferably, the Escherichia coli is the Escherichia coli BL21 (DE3) strain.

8. Use of the ketoreductase mutant according to any one of claims 1 to 4, the nucleotide sequence according to claim 5, the recombinant expression vector according to claim 6, and the genetically engineered bacteria according to claim 7 in catalyzing the synthesis of (R)-methyl o-chloromandelate.

9. The use according to claim 8, characterized in that: Taking methyl o-chlorobenzoylformate as substrate and ketoreductase mutant as catalyst, the reaction is carried out at a pH value of 5.5-7.5, preferably a pH value of 6.5, and a reaction temperature of 20-40° C.; preferably a reaction temperature of 30° C.; Preferably, the concentration of methyl o-chlorobenzoylformate is 1-4 mol / L; preferably, the concentration of methyl o-chlorobenzoylformate is 3.2 mol / L; Preferably, the protein content of the ketoreductase mutant is 30-40 g / L; preferably, the protein content of the ketoreductase mutant is 40 g / L; Preferably, the reaction time is 1-36 h; preferably, the reaction time is 16 h. More preferably, methyl o-chlorobenzoylformate, NADP+, a pH buffer solution, a ketoreductase mutant, GDH, and glucose are mixed and reacted.

10. A method for synthesizing (R)-methyl o-chloromandelate, characterized in that: Using methyl o-chlorobenzoylformate as a substrate and the ketoreductase mutant according to any one of claims 1 to 4 as a catalyst, the reaction is carried out at a pH value of 5.5-7.5, preferably a pH value of 6.5, and a reaction temperature of 20-40° C., preferably a reaction temperature of 30° C.; Preferably, the concentration of methyl o-chlorobenzoylformate is 1-4 mol / L; preferably, the concentration of methyl o-chlorobenzoylformate is 3.2 mol / L; Preferably, the protein content of the ketoreductase mutant is 30-40 g / L; preferably, the protein content of the ketoreductase mutant is 40 g / L; Preferably, the reaction time is 1-36 h; preferably, the reaction time is 16 h. More preferably, methyl o-chlorobenzoylformate, NADP+, a pH buffer solution, a ketoreductase mutant, GDH, and glucose are mixed and reacted.

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