Ketoreductase mutants and their use in the synthesis of (r)-o-chloromandelic acid methyl ester

Abstract

The present application provides a ketoreductase mutant, the amino acid sequence of which is mutated from the sequence shown in the sequence table SEQ ID No. 1, 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 the sequence table SEQ ID No. 1. The present application also provides the corresponding nucleic acid, the recombinant expression vector, the genetically engineered bacteria and the application thereof in the synthesis of (R)-o-chloromandelic acid methyl ester. The ketoreductase mutant of the present application can catalyze the asymmetric reduction reaction of high-concentration methyl o-chlorobenzoylformate as the substrate to synthesize (R)-o-chloromandelic acid methyl ester, the yield is high and meets the requirement of 99%+ stereoselectivity, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and relates to ketone reductase mutants, as well as a nucleic acid encoding the above-mentioned ketone reductase mutant, an expression vector containing the above-mentioned ketone reductase mutant, a preparation method and an application, specifically its application in the catalytic synthesis of (R)-o-chloromandelate methyl ester. Background Technology

[0002] Ketone reductases have important applications in biocatalysis, catalyzing the reduction of ketones to produce corresponding chiral alcohols, and are widely used in pharmaceuticals, agriculture, and chemicals. Traditional chemical synthesis methods often require multiple steps and stringent conditions, typically resulting in low yields and numerous byproducts. Therefore, the use of enzymatic catalysis to synthesize high-purity, highly selective target products has gradually become a research hotspot. Methyl (R)-o-chloromandelate, as an important intermediate for the best-selling antiplatelet drug (S)-clopidogrel, has wide applications in organic synthesis and drug development. However, traditional methods for synthesizing (R)-o-chloromandelate suffer from complex steps, demanding conditions, and environmental unfriendliness. To improve synthetic efficiency and environmental friendliness, the development of efficient, stable, and highly stereoselective ketone reductases is crucial. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a ketone reductase mutant that, through directed evolution and rational design, improves the enzyme's catalytic performance, stereoselectivity, thermal stability, and pH stability. This mutant can efficiently catalyze the synthesis of (R)-o-chloromandelate methyl ester using methyl o-chlorobenzoylformate as a substrate, producing a product with high optical purity, environmental friendliness, and ease of scale-up. Furthermore, this invention provides the nucleic acid encoding the aforementioned ketone reductase mutant and a vector for expressing the enzyme, providing technical support for industrial production. This enzyme mutant demonstrates significant advantages in the preparation of chiral compounds, offering a new pathway for green chemistry and sustainable development.

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

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

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

[0007] In this invention, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

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

[0009] Preferably, the mutation includes two or three of the following mutations in the sequence shown in SEQ ID No. 1: 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: I238G / F25Y, I238T / F25Y, I238G / F25P, I238T / F25P, I238G / W193Y, I238T / W193Y, I238G / E245L, I238T / E245L, I238G / F25Y / W193Y, I238T / F25Y / W19 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 performed on the sequence shown in SEQ ID No. 1 of the sequence listing: I238T / W193Y.

[0012] A second objective of this invention is to provide a nucleotide sequence encoding the aforementioned ketone reductase mutant;

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

[0014] A third objective of this invention is to provide a recombinant expression vector containing the above-described nucleotide sequence.

[0015] A fourth objective of this invention is to provide genetically engineered bacteria comprising the above-described recombinant expression vector;

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

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

[0018] The fifth objective of this invention is to provide the application of the above-mentioned ketone reductase mutant, nucleotide sequence, recombinant expression vector, and genetically engineered bacteria in the catalytic synthesis of (R)-o-chloromandelate.

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

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

[0021] Preferably, the protein content of the ketone reductase mutant is 30-40 g / L; more preferably, the protein content of the ketone reductase mutant is 40 g / L.

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

[0023] As a further preferred option, methyl o-chlorobenzoylformate, NADP+, pH buffer solution, ketone reductase mutant, GDH, and glucose are mixed and reacted.

[0024] The sixth object of the present invention is to provide a method for synthesizing (R)-o-chloromandelic acid methyl ester, using o-chlorobenzoylformate methyl ester as a substrate, the above-mentioned ketone reductase mutant as a catalyst, and carrying out the reaction under conditions of pH 5.5-7.5, preferably pH 6.5, and at a reaction temperature of 20-40°C, preferably 30°C.

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

[0026] Preferably, the protein content of the ketone reductase mutant is 30-40 g / L; more preferably, the protein content of the ketone reductase mutant is 40 g / L.

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

[0028] As a further preferred option, methyl o-chlorobenzoylformate, NADP+, pH buffer solution, ketone reductase mutant, GDH, and glucose are mixed and reacted.

[0029] The ketone reductase mutant of the present invention can catalyze the asymmetric reduction reaction of high concentration of methyl o-chlorobenzoylformate as substrate to synthesize (R)-methyl o-chloromandelate with high yield and meet the requirement of 99%+ stereoselectivity, and has broad application prospects. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0031] Figure 1 The asymmetric synthetic route for (R)-o-chloromandelic acid methyl ester.

[0032] Figure 2 This is a schematic diagram showing the relative activity of MF-KR at different pH values.

[0033] Figure 3 This is a schematic diagram showing the relative activity of MF-KR-M6 at different pH values.

[0034] Figure 4 This is a schematic diagram showing the relative activity of MF-KR and MF-KR-M6 at different temperatures.

[0035] Figure 5 This is a schematic diagram showing the pH stability of MF-KR and MF-KR-M6 at the optimal temperature and optimal pH 6.5.

[0036] Figure 6 This is a schematic diagram of the thermal stability of MF-KR.

[0037] Figure 7 This is a schematic diagram of the thermal stability of MF-KR-M6.

[0038] Figure 8 This refers to the product (R)-o-chloromandelate methyl ester generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13. 1 H NMR chromatogram.

[0039] Figure 9 This refers to the product (R)-o-chloromandelate methyl ester generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13. 13 C NMR chromatogram.

[0040] Figure 10This is a high-performance liquid chromatography (HPLC) diagram of the (R)-methyl o-chloromandelate product generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0042] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent companies. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged. Furthermore, unless otherwise specified in this embodiment, the terms and processes involved in this embodiment can be understood in accordance with general knowledge and conventional methods in the prior art.

[0043] Raw materials for this invention:

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

[0045] Escherichia coli BL21(DE3): Beijing TransGen Biotech Co., Ltd., Product No.: CD601-02.

[0046] LB medium: 10g tryptone, 5g yeast extract, 5g NaCl, and deionized water to a final volume of 1L. Autoclave for 20 minutes.

[0047] TB medium: 12g tryptone, 24g yeast extract, 4mL 87% glycerol, 100mL phosphate buffer (pH 6.5) per liter, and bring the volume to 1L with deionized water. Autoclave for 20min.

[0048] Example 1 Construction of recombinant bacteria

[0049] I. Construction of Wild-Type Recombinant Expression Vectors

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

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

[0052]

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

[0054]

[0055]

[0056] II. Construction of mutant recombinant expression vectors

[0057] Using the MF-KR plasmid without mutant enzyme 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 some mutant primers 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 incubation at 37°C for 30 min under sterile conditions, yielding mutant plasmids dissolved in sterile water.

[0058] Table 1 PCR reaction system

[0059] PCR reaction system Volume / μL sterile water 22 KODOne™ PCRMasterMix 25 plasmid 1 upstream primer 1 Downstream primer 1

[0060] Table 2 PCR reaction procedures

[0061]

[0062] Example 2 Plasmid Transformation

[0063] Add 4 μg of ketone reductase plasmid powder obtained in Example 1 or 4 μg of ketone reductase plasmid powder obtained in Example 2 to 100 μL of sterile water and shake to mix. Take 5 μL and add it to 50 μL of E. coli BL21(DE3) competent cells, gently tumble to mix, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 s, incubate on ice for 2 min, then add 500 μL of sterile, antibiotic-free LB liquid medium, mix well, and incubate at 37°C on a shaker for 1 h. After that, centrifuge at 10000 rpm for 1 min, take 300 μL of supernatant, mix the remaining liquid with the bacterial sediment, take 50 μL and spread it on LB solid medium plates containing kanamycin, and incubate at 37°C for 16 h.

[0064] Example 3: Preparation of Recombinant Bacteria

[0065] Single colonies were selected from the plates obtained in Example 2 and transferred to 10 mL of LB liquid medium containing kanamycin. The culture was incubated at 37°C and 180 rpm for 16 h with shaking to obtain a seed culture. The seed culture was then inoculated into 50 mL of TB medium (1% inoculum) containing a final concentration of 50 μg / mL kanamycin. The culture was incubated at 37°C and 180 rpm with shaking until the bacterial growth rate reached OD500. 600nm Add 0.6-0.8 μmol / L of IPTG to a final concentration of 0.5 μmol / L, and continue incubation at 20°C and 180 rpm for 20-24 h with shaking. Collect the cells by centrifugation at 5000 rcf for 15 min.

[0066] The recombinant bacterial cells were washed and resuspended twice with 0.9% physiological saline. After resuspending, the cells were homogenized and centrifuged at 12,000 rpm for 15 min. The supernatant was collected as the crude enzyme solution. The protein concentration was determined according to the Bradford method.

[0067] Example 4 Enzyme Activity Assay

[0068] The enzyme activity assay reaction system was prepared as follows: 850 μL of 100 mmol / L phosphate-sodium phosphate buffer, 10 mmol / L methyl o-chlorobenzoylformate, 20 μL of the test protein solution (protein content approximately 0.1 mg), and a final NADPH concentration of 1 mmol / L. The reaction was carried out at 30 °C for 3 min. The change in absorbance before and after the reaction was measured at 340 nm. One unit of activity (U) was defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate in 1 minute.

[0069] Formula for calculating specific enzyme activity:

[0070] Where U: enzyme activity; mg: protein content.

[0071] Example 5: Initial screening of ketoreductase mutants

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

[0073] Table 3 Initial screening of ketoreductase mutants

[0074]

[0075]

[0076] Table 4. Primer Design for Some Mutants

[0077]

[0078] Example 6: Rescreening of Ketoreductase Mutants

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

[0080] Table 5. Secondary screening of ketone reductase mutants

[0081]

[0082]

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

[0084] Example 7: Asymmetric Synthesis of (R)-O-chloromandelate

[0085] See the synthetic route. Figure 1Methyl o-chlorobenzoylformate (3.2 mol / L), NADP+ (0.5 mmol / L), 5 mL of 100 mmol / L phosphate-sodium phosphate buffer, ketone reductase 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, with 1 mol / L Na₂CO₃ added during the reaction to maintain the pH at approximately 6-7. After the reaction was complete, the mixture was extracted three times with n-hexane, and the extracts were combined and filtered through a 0.45 μm filter membrane. The yield, configuration, and ee value of the asymmetric reduction reaction products were determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: Chiralcel OD-H column, mobile phase volume ratio: n-hexane / isopropanol = 95 / 5, flow rate: 1 mL / min, detection wavelength: 254 nm.

[0086] Figure 1 The asymmetric synthetic route for (R)-o-chloromandelic acid methyl ester.

[0087] Table 6. Second Screening of Ketoreductase Mutants

[0088]

[0089]

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

[0091] Example 8: Investigation of the optimal pH for wild-type and best mutant

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

[0093] Figure 2 This is a schematic diagram showing the relative activity of MF-KR at different pH values.

[0094] Figure 3 This is a schematic diagram showing the relative activity of MF-KR-M6 at different pH values.

[0095] Depend on Figure 2 and 3It is known that the optimal pH for both MF-KR and MF-KR-6 is 6.5. MF-KR maintains more than 85% catalytic activity at pH 6-7, while MF-KR-M6 maintains more than 85% catalytic activity at pH 5.5-7.5, exhibiting a wider pH spectrum.

[0096] Example 9: Investigation of the optimal temperature for wild-type and optimal mutant

[0097] Take an appropriate amount of enzyme solution and incubate it for 10 min within the range of 10–50 °C. Then, following the method in Example 4, measure the enzyme activity at different temperatures under the optimal pH, using the enzyme activity at the optimal temperature as a control (100%). Investigate the effect of temperature on the enzyme activity of the combinatorial mutant MF-KR-M6. The results are as follows: Figure 4 As shown.

[0098] Figure 4 This is a schematic diagram showing the relative activity of MF-KR and MF-KR-M6 at different temperatures.

[0099] Depend on Figure 4 It can be seen that as the temperature increases, the activity of both MF-KR and MF-KR-M6 shows a trend of first increasing and then decreasing. The optimal temperature for both MF-KR and MF-KR-M6 is 30℃.

[0100] Example 10: Investigation of pH stability in wild-type and optimal mutant strains

[0101] At the optimal temperature (30℃), an appropriate amount of enzyme solution was taken and incubated in a buffer solution with an optimal pH of 6.5 for a certain period of time. Following the method in Example 4, samples were taken periodically to measure the remaining enzyme activity, with enzyme activity after 0 hours of incubation used as a control. The results are as follows: Figure 5 As shown.

[0102] Figure 5 This is a schematic diagram showing the pH stability of MF-KR and MF-KR-M6 at the optimal temperature and optimal pH 6.5.

[0103] Depend on Figure 5 It can be seen that the enzyme activity gradually decreased with increasing incubation time, but the relative enzyme activity of MF-KR-M6 remained higher than that of MF-KR. After 18 hours, MF-KR retained 90% activity, while MF-KR-M6 still retained 92% activity after 24 hours. Therefore, the hybrid mutant MF-KR-M6 exhibits better stability compared to the wild type.

[0104] Example 11: Investigation of the thermal stability of wild-type and optimal mutant

[0105] At the optimal pH (pH 6.5), an appropriate amount of enzyme solution was taken and incubated at 30–50°C for a certain period of time. Following the method in Example 4, samples were taken periodically to measure the remaining enzyme activity, with the enzyme activity after 0 hours of incubation serving as a control (100%). MF-KR results are as follows: Figure 6 As shown, the MF-KR-M6 results are as follows: Figure 7 As shown.

[0106] Figure 6 This is a schematic diagram of the thermal stability of MF-KR.

[0107] Figure 7 This is a schematic diagram of the thermal stability of MF-KR-M6.

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

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

[0110] See the synthetic route. Figure 1 Methyl o-chlorobenzoylformate was added to 10 mL round-bottom flasks at concentrations of 1 mol / L, 2 mol / L, 3 mol / L, 3.2 mol / L, 3.5 mol / L, and 4 mol / L, respectively, along with NADP+ (0.5 mmol / L), 5 mL of 100 mmol / L pH 6.5 phosphate-sodium phosphate buffer, ketoreductase mutant 6 (MF-KR-M6) (30 g / L and 40 g / L), GDH (5 g / L), and glucose (1.5 times the molar concentration of methyl o-chlorobenzoylformate). The reaction mixture was stirred at 30 °C for 36 h, with 1 mol / L Na₂CO₃ added during the reaction to maintain the pH at approximately 6-7. After the reaction was complete, the mixture was extracted three times with n-hexane, and the extracts were combined and filtered through a 0.45 μm filter membrane. The yield, configuration, and ee value of the products of the asymmetric reduction reaction 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.

[0111] Table 7. Preparation of (R)-o-chloromandelic acid methyl ester 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] As shown in Table 7, when 3.2 mol / L of substrate is added, MF-KR-6 can catalyze the formation of optically pure R-o-chloromandelate methyl ester within 18 h.

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

[0115] See the synthetic route. Figure 1 The 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, ketoreductase mutant 6 (MF-KR-M6) (40 g / L), GDH (5 g / L), and glucose (4.8 mol / L) were added to a 250 mL round-bottom flask. The reaction mixture was stirred at 30 °C for 20 h, and 1 mol / L Na2CO3 was added during the reaction to maintain the pH at approximately 6-7. After the reaction was complete, the mixture was extracted three times with n-hexane, and the extracts were combined and filtered through a 0.45 nm filter membrane. The yield, configuration, and ee value of the asymmetric reduction reaction product were determined by high-performance liquid chromatography (HPLC). 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 resonance (NMR) was used to determine the product. 1 H spectrum Figure 8 , 13 C spectrum Figure 9 ) and high performance liquid chromatography (HPLC) Figure 10 Verification yielded (R)-o-chloromandelic acid methyl ester 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 This refers to the product (R)-o-chloromandelate methyl ester generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13. 1 H NMR chromatogram.

[0117] Figure 9 This refers to the product (R)-o-chloromandelate methyl ester generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13. 13 C NMR chromatogram.

[0118] Figure 10 This is a high-performance liquid chromatography (HPLC) diagram of the (R)-methyl o-chloromandelate product generated by MF-KR-6 using methyl o-chlorobenzoylformate as a substrate in Example 13.

[0119] The ketone reductase mutant of the present invention catalyzes the asymmetric reduction reaction of methyl o-chlorobenzoylformate as a substrate to synthesize (R)-o-chloromandelate methyl ester, with high yield and meeting the requirement of 99%+ stereoselectivity, and has broad application prospects.

[0120] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within 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 describe the various possible combinations separately.

Claims

1. A ketoreductase mutant characterized in that: The amino acid sequence is mutated from the sequence shown in the sequence table SEQ ID No. 1, and the mutation is the following mutation of the sequence shown in the sequence table SEQ ID No. 1: W193Y.

2. Nucleotide encoding the ketoreductase mutant of claim 1.

3. Recombinant expression vector comprising the nucleotide of claim 2.

4. Genetically engineered bacteria comprising the recombinant expression vector of claim 3.

5. The genetically engineered bacteria according to claim 4, characterized in that: The genetically engineered bacteria are recombinant strains obtained by linking the nucleotide of claim 2 to a vector to obtain a recombinant expression vector, and then introducing the recombinant expression vector into a host bacteria for induced expression, wherein the host bacteria include Escherichia coli, Bacillus subtilis, or yeast. 6.The genetically engineered bacteria according to claim 5, characterized in that: The Escherichia coli is Escherichia coli BL21 (DE3) strain.

7. The use of the ketoreductase mutant of claim 1, the nucleotide of claim 2, the recombinant expression vector of claim 3, the genetically engineered bacterium of any one of claims 4-6 in catalyzing the synthesis of (R)-methyl o-chloro mandelate, characterized in that: R - the ketoreductase mutant of claim 1, the nucleotide of claim 2, the recombinant expression vector of claim 3, the genetically engineered bacterium of any one of claims 4-6. The methyl o-chlorobenzoylformate is used as the substrate in the catalytic synthesis.

8. Use according to claim 7, characterized in that: The reaction is carried out at a pH value of 5.5-7.5, and the reaction temperature is 20-40 ℃.

9. Use according to claim 8, characterized in that: The reaction is carried out at a pH value of 6.5, and the reaction temperature is 30 ℃.

10. Use according to claim 8, characterized in that: The concentration of the methyl o-chlorobenzoylformate is 1-4 mol / L.

11. Use according to claim 10, characterized in that: The concentration of the methyl o-chlorobenzoylformate is 3.2 mol / L.

12. The use according to claim 8, characterized in that: The protein content of the ketoreductase mutant is 30-40 g / L.

13. Use according to claim 12, characterized in that: The protein content of the ketoreductase mutant is 40 g / L.

14. The use according to claim 8, characterized in that: The reaction time is 1-36 h.

15. Use according to claim 14, characterized in that: The reaction time is 16 h.

16. The use according to any one of claims 8 to 15, characterized in that: The methyl o-chlorobenzoylformate, NADP+, pH buffer solution, ketoreductase mutant, GDH, and glucose are mixed for reaction.

17. A synthesis ( R The method for producing methyl 1-o-chloromandelate is characterized by: The reaction is carried out at a pH value of 5.5-7.5, and the reaction temperature is 20-40 ℃.

18. The method of claim 17, wherein: The reaction is carried out at a pH value of 6.5, and the reaction temperature is 30 ℃.

19. The method of claim 17, wherein: The concentration of the methyl o-chlorobenzoylformate is 1-4 mol / L.

20. The method of claim 19, wherein: The concentration of the methyl o-chlorobenzoylformate is 3.2 mol / L.

21. The method of claim 17, wherein: The protein content of the ketoreductase mutant is 30-40 g / L.

22. The method of claim 21, wherein: The protein content of the ketoreductase mutant is 40 g / L.

23. The method of claim 17, wherein: The reaction time is 1-36 h.

24. The method of claim 23, wherein: The reaction time is 16 h.

25. The method of any one of claims 17-24, wherein: The methyl o-chlorobenzoylformate, NADP+, pH buffer solution, ketoreductase mutant, GDH, and glucose are mixed for reaction.

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