Improved ketoreductase and application thereof in preparation of montelukast sodium intermediate

Abstract

The invention belongs to the field of enzyme catalysis, and particularly relates to optimization of an improved carbonyl reductase sequence, construction of engineering bacteria and application of the engineering bacteria in montelukast sodium intermediate catalysis. The invention provides carbonyl reductase and an engineering bacterium. According to the strain, (E)-2-(3-(3-(2-(7-chloro-2-quinolyl) vinyl) phenyl)-3-carbonyl propyl) methyl benzoate is used as a starting substrate, under the condition of carbonyl reductase, efficient synthesis of the product S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl) vinyl) phenyl)-3-hydroxy propyl) methyl benzoate is achieved, and the problems that in a traditional method, the concentration of a substrate is low, the yield is high, and the like are solved. According to the method, the problems of high production cost, long conversion time and the like are solved, the substrate conversion rate is greater than or equal to 99.6%, the molar yield reaches 95%, and the chiral purity of the product reaches 100%.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalytic synthesis, and in particular relates to an application of a ketoreductase mutant in the synthesis of a key chiral intermediate of montelukast sodium, S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester. Background Art

[0002] Montelukast sodium, chemical name is (+)-1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-quinolyl)-vinyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetic acid monosodium salt, it is a leukotriene receptor antagonist, a highly effective, low-toxic, safe antiasthmatic and antiallergic drug. S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester is the key chiral intermediate for the preparation of Montelukast sodium, and its structural formula is as follows:

[0003] Traditional chemical synthesis of this intermediate mainly uses chemical reduction of diisopine camphenyl chloroborane and Grignard reaction, which not only requires two steps of reaction, but also has harsh reaction conditions (low temperature of minus 20°C) and produces a large amount of organic waste liquid. The main characteristics of bioenzyme catalysis are mildness and specificity of reaction, and it has been used as an important means to construct the chiral center of compounds. Using bioenzyme catalysis technology, the chiral intermediate of montelukast sodium can be prepared in one step, which significantly simplifies the operation steps of intermediate IV and reduces production costs.

[0004] CN104326976B uses carbonyl reductase from Suzhou Hanzy Biotechnology Co., Ltd. to catalyze the preparation of the intermediate, but faces the problems of low substrate concentration, long enzyme catalysis time (25-60 h), and the process still requires two-step reactions. The pharmaceutical industry still needs to continue to work hard to find ketoreductases with high catalytic activity and better reaction systems to reduce production costs.

[0005] CN101889081A provides an artificially modified carbonyl reductase with improved properties compared to the naturally occurring wild-type carbonyl reductase. Also provided are polynucleotides encoding the artificially modified carbonyl reductase, host cells capable of expressing the artificially modified carbonyl reductase, and a method for synthesizing S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester using the artificially modified carbonyl reductase. The catalytic substrate concentration is ≥20g / L.

[0006] CN104293850A proposes that (E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester is catalyzed by carbonyl reductase from Sigma to obtain S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, the catalytic system contains 66% toluene, and coenzyme and hydrogen peroxide are added, the yield can reach 91%-97%, and the catalytic substrate concentration is about 33.3 g / L.

[0007] CN118421578A uses a carbonyl reductase derived from a modified Lactobacillus lentus to catalyze the intermediate (E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester. The catalytic substrate is 100 g / L, the bacterial dosage is 100 g / L (the catalyst dosage is 1 kg / kg), and the substrate conversion rate reaches more than 99.0% after 24 hours of catalysis. However, the use of tetrahydrofuran and 0.1 mol / L triethanolamine solution in the reaction brings environmental pressure, and the high dosage of the bio-enzyme catalyst brings production cost pressure. Summary of the invention

[0008] In view of the shortcomings of the current chemical synthesis process of montelukast sodium, the present invention provides a ketoreductase mutant and a ternary solvent reaction system. Compared with the wild-type ketoreductase, the conversion rate is significantly improved, the process steps of montelukast sodium are shortened, and the chiral purity of the product is improved, less waste liquid is generated, and it meets the "green chemistry" standard.

[0009] Purpose of the invention: To address the shortcomings of the existing ketoreductase-catalyzed preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, such as long reaction time, low conversion rate and low chiral purity, a ketoreductase mutant and a ternary solvent reaction system are provided, which can significantly improve the catalytic activity and the chiral purity of the product. Technical Solution

[0010] The present invention is based on Fermented Lachancea The ketoreductase evolved from the wild-type ketoreductase of the strain and its encoding gene are used to catalyze the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester (intermediate IV), and the reaction formula is as follows:

[0011] The present invention provides an improved ketoreductase, namely a ketoreductase mutant, wherein the enzyme activity and stereoselectivity of the ketoreductase mutant are higher than those of the wild-type ketoreductase, and the conversion rate can reach more than 99% after a reaction of 12 hours.R The optical purity of the )-type product can reach 100%.

[0012] The technical solution of the present invention is a ketoreductase mutant, whose amino acid sequence is a mutant of the amino acid sequence shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 2), and the mutation sites include R79S / W139G / L186M / S239P.

[0013] The amino acid sequence of the ketoreductase mutant of the present invention is as shown in SEQ ID NO: 1, and the nucleotide sequences of the corresponding encoding genes are as shown in SEQ ID NO: 2.

[0014] According to another aspect of the present invention, a recombinant plasmid is provided, the recombinant plasmid contains the nucleotide sequence of any one of the above genes, and further, the plasmid is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+ ), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C, etc.

[0015] According to another aspect of the present invention, a host cell is provided, wherein the host cell contains any of the above-mentioned recombinant plasmids, and the host cell includes a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is preferably an Escherichia coli BL21 (DE3) cell. According to another aspect of the present invention, a ketoreductase mutant is provided for use in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, comprising: in the presence of the ketoreductase mutant, (E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester is used as a substrate, and S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester is obtained by asymmetric catalytic hydrogenation.

[0016] Specifically, in the use of the above-mentioned ketoreductase mutant in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, the temperature is preferably controlled at 35-40°C.

[0017] Specifically, in the use of the above-mentioned ketoreductase mutant in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, the asymmetric catalytic hydrogenation reaction temperature is controlled at 35-45° C., preferably 35-40° C. When the reaction temperature is lower than 35° C. or higher than 45° C., the enzyme-catalyzed reaction rate will be reduced.

[0018] Beneficial effects: The technical solution of the present invention uses a molecular biological method of random mutation to mutate the ketoreductase gene on the basis of the wild-type ketoreductase gene sequence, thereby changing the amino acid sequence of the enzyme and realizing changes in the enzyme structure and function; and then obtains a single-site mutant or a double-site mutant or a three-site or four-site mutant ketoreductase mutant having the above-mentioned site by a directed evolution screening method. Through mutation, screening, and optimization, the above-mentioned four-site mutated ketoreductase mutant is finally obtained to catalyze (E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester to prepare S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester. Compared with the wild-type ketoreductase parent and the single mutant or double mutant ketoreductase, the ketoreductase mutant with mutations at all three amino acid sites has a significantly higher enzyme activity than the wild-type ketoreductase (Example 5), and has high stereoselectivity and catalytic activity. The optimal mutant showed that the unit production capacity (substrate concentration) was increased to 200 g / L in a reaction system of toluene-isopropanol-water ternary solvent, the enzyme dosage was reduced to 1g enzyme solution / g (equivalent to 0.2g wet bacteria / g), the substrate conversion rate could reach 99.5% after 12 hours of reaction, the chemical purity of the obtained product was higher than 99.0% and the optical purity reached 100%, and the molar yield was not less than 95%. Compared with the enzyme catalysis process reported in the existing publicly published literature, the present invention provides a feasible green technology solution with higher unit production capacity and higher substrate catalysis efficiency.

[0019] Specific implementation method: The present invention is further described below in conjunction with specific implementation examples, but the protection scope of the present invention is not limited thereto: The high performance liquid chromatography method adopted in the present invention is used to quantitatively detect S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, and the specific conditions are as follows:

Chiral purity

[0020]

Chemical Purity

[0021]

[0022] Example 1: Obtaining Fermented LacEancea wild-type ketoreductase parent recombinant plasmid Obtained from NCBI GenBank nucleic acid database L. fermentedThe ketoreductase parent gene of the strain was codon-optimized and commissioned to be artificially synthesized into the pET28a(+) expression plasmid by a service provider, and then transformed into E. coli BL21(DE3) competent cells, spread on LB agar plates containing 50 mg / L kanamycin, and cultured at 37°C overnight. Several single colonies were selected and added to LB medium (containing 50 mg / L kanamycin), and after being cultured at 37°C overnight, the recombinant plasmid was extracted using a plasmid extraction kit, and PCR and sequencing verification were performed to obtain the recombinant plasmid of the wild-type ketoreductase parent. Example 2: Random mutagenesis of the wild-type ketoreductase parent gene

[0023] According to Example 1, Fermented Lachancea The ketoreductase parent gene recombinant plasmid of the strain was used as a template. Fermented Lachancea The ketoreductase parent gene of the strain was designed and the primers at both ends were synthesized using Primer 5.0 (Table 1). The linear gene fragment containing a large number of base mutations was obtained using error-prone PCR technology (materials and concentrations are shown in Table 2, and reaction conditions are shown in Table 3). The above PCR products and pET28a(+) expression plasmid were digested with enzymes, recovered by gel excision, ligated and transformed into Escherichia coli BL21(DE3) competent cells, spread on LB agar plates containing 50 mg / L kanamycin, and cultured at 37°C overnight.

[0024]

[0025] Example 3: Cloning and expression of ketoreductase mutants In order to facilitate the cloning, expression and identification of ketoreductase mutants, compatible restriction endonuclease sites were designed at the 5' and 3' ends of the gene. I am and Xho I The target gene and pET28a(+) (other expression plasmids that can express proteins in E. coli can also be used) are digested with restriction endonucleases and DNA is recovered by gel excision. The recovered target gene and plasmid are ligated with T4 DNA ligase, and the ligation product is transformed into E. coli BL21(DE3) competent cells. The transformed competent cells are then spread on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight.

[0026] Pick a single colony grown on the above culture dish and inoculate it into LB liquid medium containing 50 mg / L kanamycin, shake and culture at 37°C overnight, collect the bacteria for plasmid extraction, PCR identification and double enzyme digestion identification, name the correct recombinant plasmid pET28a(+)-AN, and perform subsequent induced expression in Escherichia coli containing the correct recombinant plasmid. Transfer the above bacterial liquid into 500 mL LB liquid medium containing 100 mg / L kanamycin, shake and culture at 37°C until OD reaches 0. 600 =0.6-0.8, add IPTG to a final concentration of 0.05-0.5 mM (preferably 0.2 mM), induce expression at 22-28°C (preferably 25°C) for 12-16 h (preferably 15 h), remove the bacterial solution, and collect the bacteria by centrifugation at 6000 × g for 20 min. Example 4: Initial screening of ketoreductase mutants

[0027] According to the contents described in Example 2 and Example 3, the monoclonal colonies on the above LB agar medium were picked and inoculated into a 96-deep-well plate, 1 mL of LB medium containing 50 mg / L kanamycin sulfate was pre-added to each well, and the culture was shaken at 37°C and 220 rpm for 3 h, and then a certain amount of isopropyl-β-D-thiogalactoside (IPTG, final concentration of 0.2 mM) was added, and the culture was induced at 25°C and 220 rpm for 15 h. The bacterial cells were collected by centrifugation at 6000 × g for 20 min, and the supernatant was discarded and resuspended in 0.1 mol / L sodium phosphate buffer (pH 7.5). 0.1 g of substrate, 0.2 g of isopropanol, and 3 mg of NAD were added. The total reaction volume was 1.0 ml, and the reaction was carried out at 37°C for 12 h. 1 ml of methanol was added to terminate the reaction. After shaking and centrifugation, the supernatant was taken out and sent to HPLC to detect the conversion rate. Example 5: Rescreening of ketoreductase mutants

[0028] (1) Preparation of whole cells of ketoreductase mutants The mutant strains with higher enzyme activity than the parent strain in Example 4 were inoculated at 0.1% inoculum into 500 mL of LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 220 rpm for 5-6 h. A certain amount of isopropyl-β-D-thiogalactoside (IPTG, final concentration of 0.2 mM) was added, and the culture was induced at 25°C and 220 rpm for 15 h. The bacteria were collected by centrifugation at 6000 × g.

[0029] (2) Ketoreductase catalytic reaction 4.0 g of substrate, 16.3 g of isopropanol, 3.8 g of toluene, 12 g of 0.1 mol / L sodium phosphate buffer (pH 7.5), 5 mg of NADP coenzyme and 4 g of ketoreductase mutant whole cells (wet weight) were added to a 50 mL reaction bottle. After reacting at 37°C for 12 h, the conversion rate and chiral purity of the crude product were analyzed by HPLC.

[0030] (3) Conversion rate and chiral purity determination of ketoreductase hydrogenation reaction The reaction system described in (2) was treated with methanol, filtered through a membrane, and directly injected into HPLC for analysis; the reaction system described in (2) was post-treated to obtain a white powdery solid, dissolved in ethanol, diluted with a mobile phase, filtered through a membrane, and directly injected into HPLC for analysis. The mutants with catalytic activity better than the parent were selected for sequencing and the mutation sites were analyzed. The catalytic activity and stereoselectivity of R79S / W139G / L186M / S239P (SEQ ID NO: 1, the nucleotide sequence of the corresponding coding gene is SEQ ID NO: 2) were significantly improved compared with the parent and double mutants of this scheme. The reaction results are shown in Table 4.

[0031]

[0032] The above results indicate that the R79S / W139G / L186M / S239P mutant is the optimal enzyme mutant, and its catalytic efficiency for substrate is significantly higher than that of the wild-type ketoreductase parent, and the product has higher chiral purity and catalytic activity. Example 6: Preparation of R79S / W139G / L186M / S239P mutant enzyme solution

[0033] Transfer the glycerol culture to 50 ml LB liquid medium containing 100 mg / L kanamycin sulfate at a 0.1% inoculum and culture at 37°C overnight; transfer the above bacterial solution to multiple bottles of 500 mL LB liquid medium containing 100 mg / L kanamycin and culture at 37°C with shaking until OD reaches 0. 600 =0.6-0.8, add IPTG to a final concentration of 0.05-0.5 mM (preferably 0.2 mM), and induce expression at 22-28°C (preferably 25°C) for 12-16 h (preferably 15 h), then take out the bacterial solution and collect the bacteria by centrifugation at 6000×g for 20 min. Resuspend the bacteria in 0.1 mol / L sodium phosphate buffer (pH 7.5) according to the bacterial concentration (200 g / L), and break the bacteria with a high-pressure homogenizer. After breaking, centrifuge the crude enzyme solution (10000×g, 20 min) and take the supernatant, which is the R79S / W139G / L186M / S239P mutant enzyme solution. Example 7: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0034] Add 10.0 g of the main raw material (intermediate III), 25 mL of isopropanol, and 0.5 mL of toluene to five 250 mL reaction bottles, respectively, and stir until dissolved. Continue to add 10 mL of drinking water, 10 g of R79S / W139G / L186M / S239P mutant enzyme solution, and 10 mg of NADP coenzyme. Set the reaction temperature to 25~30℃, 30~35℃, 35~40℃, 40~45℃, and 45~50℃ for five reaction gradients, control the temperature and stir the reaction for 15h, and the substrate conversion rates are 88.52%, 92.51%, 99.78%, 98.87%, and 94.52%, respectively. The reaction temperature is determined to be 35~45℃, especially 35~40℃. The best reaction temperature is 35~45℃. Example 8: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0035] Add 10.0 g of the main raw material (intermediate III), 25 mL of isopropanol and 0.5 mL of toluene to a 200 mL reaction bottle and stir until dissolved. Continue to add 10 mL of drinking water, 10 g of R79S / W139G / L186M / S239P mutant enzyme solution and 10 mg of NADP coenzyme. Raise the temperature to 35-40°C, control the temperature and stir the reaction for 12-15 hours, the substrate conversion rate is 99.72%, and the chiral purity of the crude product is 99.97%. Example 9: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0036] Add 20.0 g of the main raw material (intermediate III), 50 mL of isopropanol and 1 mL of toluene to a 500 mL reaction bottle and stir until dissolved. Continue to add 20 mL of drinking water, 20 g of R79S / W139G / L186M / S239P mutant enzyme solution, and 15 mg of NADP coenzyme. Raise the temperature to 35-40°C, control the temperature and stir the reaction for 12-15 hours. A large amount of solid precipitates as the reaction proceeds. The final substrate conversion rate is 99.67%, and the chiral purity of the crude product is 99.98%. Example 10: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0037] Add 50.0 g of the main raw material (intermediate III), 125 mL of isopropanol and 25 mL of toluene to a 1000 mL reaction bottle and stir until dissolved. Continue to add 50 mL of drinking water, 50 g of R79S / W139G / L186M / S239P mutant enzyme solution, and 30 mg of NADP coenzyme. Raise the temperature to 35-40°C, control the temperature and stir the reaction for 12-13 hours. A large amount of solid precipitates as the reaction proceeds, and the final substrate conversion rate is 99.52%.

[0038] Preparation of crude product: After the reaction is completed, cool to 25-30°C. Filter and rinse the filter cake with 50 mL of isopropanol. Control the temperature of the filter cake at 40°C~45°C and vacuum dry for 4~6 hours (vacuum degree ≥0.09MPa). Take a sample and measure the moisture content ≤5.0% before collecting the material to obtain the crude intermediate of montelukast sodium. The chiral purity (optical purity) of the crude product reaches 100%.

[0039] Purification: Add 250 mL of isopropanol into a 1000 mL three-necked flask, start stirring, add 50 mL of toluene, add the crude product of montelukast sodium intermediate IV into the flask, heat to 75-80°C, and stir for 10-20 minutes. Filter the liquid while it is hot. Heat the liquid to 70-75°C, drip 200 mL of drinking water; after the addition is complete, cool to 15-20°C for 1-1.5 hours, and stir for 60-70 minutes. Centrifuge and rinse the filter cake with 50 mL of isopropanol. Control the temperature at 40°C~45°C and vacuum dry for 3~6 hours (vacuum degree ≥0.09MPa), take a sample to measure the moisture content ≤4.0%, and collect the material to obtain 47.25 g of the product, with a molar yield of 94.5% and a purity (chemical purity) of 99.43%.

[0040] The results showed that the ketoreductase mutant shown in SEQ ID NO:1 could achieve a conversion rate of more than 99.52% in the enzyme catalysis process, i.e., in a 200 g / L substrate reaction system, and a reaction time of 12 h, and the chiral purity of the product reached 100%. The screened ketoreductase mutant showed extremely high stereoselectivity and efficiency in the enzymatic preparation of methyl S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoate.

[0041] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An improved ketoreductase mutant, characterized in that The amino acid sequence of the reductase mutant is a mutation of the wild-type amino acid sequence shown in SEQ ID NO: 3, wherein the mutation sites include the 79th R mutation to S, the 139th W mutation to G, the 186th L mutation to M, and the 239th S mutation to P.

2. The ketoreductase mutant according to claim 1, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO: 1, and the nucleotide sequences of the corresponding encoding genes are shown in SEQ ID NO:

2.

3. A recombinant plasmid containing the gene encoding the ketoreductase mutant according to any one of claims 1 or 2.

4. The recombinant plasmid according to claim 3, characterized in that The plasmids are pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pE T-41b(+), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C, etc.

5. A host cell containing the recombinant plasmid according to claim 4, characterized in that The host cell includes a prokaryotic cell or a eukaryotic cell.

6. The host cell according to claim 5, characterized in that The prokaryotic cells were Escherichia coli BL21 (DE3) cells.

7. Use of the ketoreductase mutant according to claim 1 in catalysis of montelukast sodium intermediates.

8. The use according to claim 7, characterized in that Using montelukast sodium intermediate (E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester as substrate, S-(E)-2-(3-(3-(2-(7-chloro-2-quinolyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester was obtained by asymmetric catalysis in the presence of a ketoreductase mutant.

9. The use according to claim 7, characterized in that The asymmetric catalytic reaction temperature is 35°C-45°C using a montelukast sodium intermediate in the presence of a ketoreductase mutant.

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