Improved ketoreductase and its use in the preparation of montelukast sodium intermediates

Abstract

The application belongs to the field of enzyme catalysis, and particularly relates to optimization of an improved carbonyl reductase sequence, construction of an engineering bacterium and application of the engineering bacterium in catalysis of a montelukast sodium intermediate. The application provides a carbonyl reductase and an engineering bacterium, wherein the bacterium takes (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)vinyl)phenyl)-3-oxopropyl)benzoic acid methyl ester as a starting substrate, realizes efficient synthesis of a product S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)vinyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester in the presence of the carbonyl reductase, and solves problems of low substrate concentration, high production cost and long conversion time in a traditional method, wherein a substrate conversion rate is greater than or equal to 99.6%, a molar yield reaches 95%, and a product chiral purity reaches 100%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biocatalytic synthesis, and particularly relates to application of a ketoreductase mutant in synthesis of a key chiral intermediate S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester of montelukast sodium. BACKGROUND

[0002] Montelukast sodium, chemically named (+)-1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetic acid monosodium salt, is a leukotriene receptor antagonist and is a highly effective, low-toxicity and safe antiasthmatic and antiallergic drug. S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester is a key chiral intermediate for preparing montelukast sodium, and its structural formula is as follows:

[0003]

[0004] Traditional chemical synthesis for preparing the intermediate mainly involves diisopinocampheylchloroborane chemical reduction and Grignard reaction, which not only needs two steps of reaction, but also has harsh reaction conditions (low temperature of minus 20 DEG C) and produces a large amount of organic waste liquid. The main features of biocatalysis are reaction mildness and specificity, and biocatalysis has been used as an important means for constructing chiral centers of compounds. By using biocatalysis technology, the chiral intermediate of montelukast sodium can be prepared in one step, so that the operation steps of the intermediate IV are significantly simplified and the production cost is reduced.

[0005] CN104326976B uses a carbonyl reductase from Suzhou Hanmei Biotechnology Co., Ltd. to catalyze the preparation of the intermediate, and faces the problems of low substrate concentration, long enzyme catalysis time (25-60 h) and two-step reaction process. It is still necessary for the pharmaceutical industry to continue to seek ketoreductase with high catalytic activity and a more optimal reaction system to reduce production cost.

[0006] CN101889081A provides an artificially engineered carbonyl reductase having improved properties compared to naturally occurring wild-type carbonyl reductases. Also provided are polynucleotides encoding the artificially engineered carbonyl reductases, host cells capable of expressing the artificially engineered carbonyl reductases, and methods of using the artificially engineered carbonyl reductases to synthesize S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester. The catalytic substrate concentration is greater than or equal to 20 g / L.

[0007] CN104293850A proposed (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) vinyl) phenyl)-3- carbonylpropyl) methyl benzoate is obtained by Sigma Company's carbonyl reductase catalysis S- (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) vinyl) phenyl)-3-hydroxypropyl) methyl benzoate, the catalytic system contains 66% of toluene, and the coenzyme and hydrogen peroxide are added, and the yield can reach 91%-97%, and the catalytic substrate concentration is about 33.3 g / L.

[0008] CN118421578A uses the improved carbonyl reductase derived from the similar brock lactobacillus to catalyze the intermediate (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) vinyl) phenyl)-3- carbonylpropyl) methyl benzoate, and the substrate conversion rate reaches more than 99.0% under the conditions of 100 g / L of catalytic substrate, 100 g / L of bacterial body dosage (1 kg / kg of catalyst dosage) and 24 h of catalysis time. However, the use of tetrahydrofuran and 0.1 mol / L triethanolamine solution in the reaction brings environmental pressure, and the high dosage of biological enzyme catalyst brings production cost pressure. SUMMARY

[0009] In view of the shortcomings of the current montelukast sodium chemical synthesis process, the present application provides a ketoreductase mutant and a ternary solvent reaction system, which can significantly improve the conversion rate compared with the wild-type ketoreductase, shorten the process steps of montelukast sodium, and improve the chiral purity of the product, produce less waste liquid, and meet the "green chemistry" standard.

[0010] The purpose of the application is to overcome the shortcomings of long reaction time, low conversion rate and low chiral purity of the existing ketoreductase catalytic preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) vinyl) phenyl)-3-hydroxypropyl) methyl benzoate, and to provide a ketoreductase mutant and a ternary solvent reaction system, which can significantly improve the catalytic activity and the chiral purity of the product.

[0011] Technical scheme

[0012] The present application is based on Lachancea fermentati The ketoreductase mutant and its encoding gene evolved from the wild-type ketoreductase of the strain are used to catalyze the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) vinyl) phenyl)-3-hydroxypropyl) methyl benzoate (intermediate IV), and the reaction formula is as follows:

[0013]

[0014] The application provides an improved ketoreductase, i.e. a ketoreductase mutant, which has higher enzyme activity and stereoselectivity than a wild-type ketoreductase, and a conversion rate of 99% or more can be reached after 12 hours of reaction, R and the optical purity of the -type product can reach 100%.

[0015] The technical scheme of the application is a ketoreductase mutant, the amino acid sequence of which is a mutant of the amino acid sequence shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding coding gene is shown in SEQ ID NO: 2), and the mutation sites include R79S / W139G / L186M / S239P.

[0016] The amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO: 1, and the nucleotide sequence of the corresponding coding gene is shown in SEQ ID NO: 2.

[0017] According to another aspect of the present application, there is provided a recombinant plasmid comprising the nucleotide sequence of any one of the above-mentioned 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.

[0018] According to another aspect of the present application, there is provided a host cell comprising any one of the above-mentioned recombinant plasmids, and the host cell comprises a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is preferably Escherichia coli BL21(DE3) cell. According to another aspect of the present application, there is also provided a use of the ketoreductase mutant in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, comprising: obtaining S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester by asymmetric catalytic hydrogenation with (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-oxopropyl)benzoic acid methyl ester as a substrate in the presence of the ketoreductase mutant.

[0019] Specifically, the application of the above ketoreductase mutant in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester is preferably controlled at a temperature of 35-40℃.

[0020] Specifically, the application of the above ketoreductase mutant in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester is controlled at an asymmetric catalytic hydrogenation reaction temperature of 35-45℃, preferably 35-40℃. If the reaction temperature is lower than 35℃ or higher than 45℃, the enzyme catalytic reaction speed will be reduced.

[0021] Beneficial effects: The technical scheme of the present application mutates the ketoreductase gene by using the molecular biology method of random mutation on the basis of the wild-type ketoreductase gene sequence, thereby changing the amino acid sequence of the enzyme to realize the change of enzyme structure and function; and then the method of directed evolution screening is used to obtain the ketoreductase mutant with unit point mutation or double site mutation or three site or four site mutation in the above-mentioned sites. Through mutation, screening and optimization, the ketoreductase mutant with four site mutation is finally obtained to catalyze (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-carbonylpropyl)benzoic acid methyl ester to prepare S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester. Compared with the wild-type ketoreductase and the single mutant or double mutant ketoreductase, the enzyme activity of the ketoreductase mutant with mutation of three amino acid sites is obviously higher than that of the wild-type ketoreductase (Example 5), and the ketoreductase mutant has high stereoselectivity and catalytic activity. In the reaction system of toluene-isopropyl alcohol-water ternary solvent, the optimal mutant shows that the unit production capacity (substrate concentration) is increased to 200 g / L, the enzyme dosage is reduced to 1 g of enzyme liquid / g (equivalent to 0.2 g of wet mycelium / g), the substrate conversion rate can reach 99.5% after 12 h of reaction, the chemical purity of the obtained product is higher than 99.0%, the optical purity reaches 100%, and the molar yield is not less than 95%. Compared with the enzyme catalytic process reported in the existing published literature, the present application provides a feasible green technical scheme with higher unit production capacity and higher substrate catalytic efficiency. DETAILED DESCRIPTION The present application will be further described below in combination with specific implementation examples, but the protection scope of the present application is not limited to this:

[0022] The high performance liquid chromatography method used in the present application is used for quantitative detection of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)benzoic acid methyl ester, and the specific conditions are as follows:

[0023]

Chiral purity

[0024]

Chemical purity

[0025]

[0026] Example 1: Obtain Lachancea fermentati Wild-type ketoreductase strain of the parent recombinant plasmid

[0027] Obtained from NCBI GenBank nucleic acid database L. fermentatiThe ketoreductase gene of the strain was codon-optimized and the full-length gene was artificially synthesized by a service provider into a pET28a(+) expression plasmid. The recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells, which were coated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight. Several single colonies were selected and cultured in LB medium (containing 50 mg / L kanamycin) at 37°C overnight. The recombinant plasmid was extracted using a plasmid extraction kit, and PCR and sequencing were performed to verify the wild-type ketoreductase gene.

[0028] Example 2: Random mutation of the wild-type ketoreductase gene

[0029] According to Example 1, the wild-type ketoreductase gene was codon-optimized and the full-length gene was artificially synthesized by a service provider into a pET28a(+) expression plasmid. The recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells, which were coated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight. Several single colonies were selected and cultured in LB medium (containing 50 mg / L kanamycin) at 37°C overnight. The recombinant plasmid was extracted using a plasmid extraction kit, and PCR and sequencing were performed to verify the wild-type ketoreductase gene. Lachancea fermentati The ketoreductase gene of the strain was codon-optimized and the full-length gene was artificially synthesized by a service provider into a pET28a(+) expression plasmid. The recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells, which were coated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight. Several single colonies were selected and cultured in LB medium (containing 50 mg / L kanamycin) at 37°C overnight. The recombinant plasmid was extracted using a plasmid extraction kit, and PCR and sequencing were performed to verify the wild-type ketoreductase gene. Lachancea fermentati The ketoreductase gene of the strain was codon-optimized and the full-length gene was artificially synthesized by a service provider into a pET28a(+) expression plasmid. The recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells, which were coated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight. Several single colonies were selected and cultured in LB medium (containing 50 mg / L kanamycin) at 37°C overnight. The recombinant plasmid was extracted using a plasmid extraction kit, and PCR and sequencing were performed to verify the wild-type ketoreductase gene.

[0030]

[0031] Example 3: Cloning and expression of ketoreductase mutants

[0032] To facilitate the cloning, expression and identification of ketoreductase mutants, compatible restriction enzyme sites were designed at the 5' and 3' ends of the gene. The target gene and pET28a(+) (or other expression plasmids that can express proteins in E. coli) were simultaneously digested and DNA gel recovered using the appropriate restriction enzymes. Nde I and Xho I The recovered target gene and plasmid were subjected to ligation reaction using T4 DNA ligase. The ligation product was transformed into E. coli BL21(DE3) competent cells, which were then coated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C overnight.

[0033] The single colony grown on the above culture dish was inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured at 37°C overnight. After the bacterial cells were collected, plasmid extraction, PCR identification and double enzyme digestion identification were performed, and the correct recombinant plasmid was named pET28a(+)-A-N. The E. coli containing the correct recombinant plasmid was used for subsequent induction expression. The above bacterial liquid was transferred into 500 mL LB liquid medium containing 100 mg / L kanamycin, and cultured at 37°C until the OD 600 = 0.6-0.8, and then IPTG was added to a final concentration of 0.05-0.5 mM (preferably 0.2 mM). After induction expression at 22-28°C (preferably 25°C) for 12-16 h (preferably 15 h), the bacterial liquid was removed, and the bacterial cells were collected by centrifugation at 6000 x g for 20 min.

[0034] Example 4: Preliminary screening of ketoreductase mutants

[0035] According to the contents described in Examples 2 and 3, the single colony on the above LB agar medium was inoculated into a 96-deep well plate, 1 mL of LB medium containing 50 mg / L kanamycin sulfate was added to each well, and the culture was incubated at 37°C and 220 rpm for 3 h. Then a certain amount of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the culture was induced at 25°C and 220 rpm for 15 h. The bacterial cells were collected by centrifugation at 6000 x g for 20 min, and then the supernatant was discarded. The bacterial cells were resuspended in 0.1 mol / L sodium phosphate buffer (pH 7.5), 0.1 g of substrate, 0.2 g of isopropyl alcohol, 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. Then 1 ml of methanol was added to terminate the reaction, and after shaking and centrifugation, the supernatant was taken for HPLC detection of the conversion rate.

[0036] Example 5: Secondary screening of ketoreductase mutants

[0037] (1) Preparation of whole cells of ketoreductase mutants

[0038] The mutant strains with enzyme activity higher than that of the parent in Example 4 were inoculated into 500 mL LB medium containing 50 mg / L kanamycin at an inoculation amount of 0.1%, and cultured at 37°C and 220 rpm for 5-6 h. Then a certain amount of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the culture was induced at 25°C and 220 rpm for 15 h. The bacterial cells were collected by centrifugation at 6000 x g.

[0039] (2) Catalytic reaction of ketoreductase

[0040] 50 mL reaction bottle is added with substrate 4.0 g, isopropyl alcohol 16.3 g, toluene 3.8 g, 0.1 mol / L sodium phosphate buffer (pH 7.5) 12 g, NADP coenzyme 5 mg and ketoreductase mutant whole cell (wet weight) 4 g. After 37°C reaction for 12 h, HPLC is used to analyze the conversion rate and the chiral purity of the crude product.

[0041] (3) Conversion rate and chiral purity determination of ketoreductase hydrogenation reaction

[0042] The reaction system in (2) is treated with methanol, filtered with a membrane and then analyzed by HPLC direct injection; the reaction system in (2) is subjected to post-treatment to obtain white powdery solid, which is dissolved in ethanol and then diluted with a mobile phase, filtered with a membrane and then analyzed by HPLC direct injection. The mutants with better catalytic activity than the parent are sequenced, the mutation sites are analyzed, and the catalytic activity and stereoselectivity of R79S / W139G / L186M / S239P (SEQ ID NO: 1, the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 2) are significantly higher than those of the parent and the double mutant of the present scheme, and the reaction results are shown in Table 4.

[0043]

[0044] The above results show that the R79S / W139G / L186M / S239P mutant is the optimal enzyme mutant, and its catalytic efficiency on the substrate is significantly higher than that of the wild-type ketoreductase parent, and the product has higher chiral purity and catalytic activity.

[0045] Example 6: Preparation of R79S / W139G / L186M / S239P mutant enzyme solution

[0046] The glycerol bacteria are inoculated in 50 ml of LB liquid medium containing 100 mg / L kanamycin at an inoculation amount of 0.1%, and cultured at 37°C overnight; the bacterial liquid is inoculated in multiple 500 mL LB liquid medium containing 100 mg / L kanamycin, and cultured at 37°C until the OD 600 = 0.6-0.8, IPTG is added to a final concentration of 0.05-0.5 mM (preferably 0.2 mM), and induction expression is carried out at 22-28°C (preferably 25°C) for 12-16 h (preferably 15 h), then the bacterial liquid is taken out, centrifuged at 6000xg for 20 min to collect the bacterial cells. The bacterial cells are resuspended in 0.1 mol / L sodium phosphate buffer (pH 7.5) according to the concentration (200 g / L), and the bacterial cells are broken by a high-pressure homogenizer. After breaking, the crude enzyme solution is centrifuged (10000xg, 20 min) and the supernatant is taken, which is the R79S / W139G / L186M / S239P mutant enzyme solution.

[0047] Example 7: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0048] Into five 250 mL reaction bottles, main raw material (intermediate III) 10.0 g, isopropyl alcohol 25 mL and toluene 0.5 mL were added and stirred until dissolved. Then, drinking water 10 mL, R79S / W139G / L186M / S239P mutant enzyme solution 10 g and NADP coenzyme 10 mg were added. The reaction temperature was set at 25-30°C, 30-35°C, 35-40°C, 40-45°C and 45-50°C, respectively, and the temperature was controlled to stir for 15 h. The substrate conversion rates were 88.52%, 92.51%, 99.78%, 98.87% and 94.52%, respectively. The reaction temperature was determined to be 35-45°C, and the best temperature was 35-40°C.

[0049] Example 8: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0050] In a 200 mL reaction bottle, main raw material (intermediate III) 10.0 g, isopropyl alcohol 25 mL and toluene 0.5 mL were added and stirred until dissolved. Then, drinking water 10 mL, R79S / W139G / L186M / S239P mutant enzyme solution 10 g and NADP coenzyme 10 mg were added. The temperature was raised to 35-40°C, and the temperature was controlled to stir for 12-15 h. The substrate conversion rate was 99.72%, and the chiral purity of the crude product was 99.97%.

[0051] Example 9: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0052] In a 500 mL reaction bottle, main raw material (intermediate III) 20.0 g, isopropyl alcohol 50 mL and toluene 1 mL were added and stirred until dissolved. Then, drinking water 20 mL, R79S / W139G / L186M / S239P mutant enzyme solution 20 g and NADP coenzyme 15 mg were added. The temperature was raised to 35-40°C, and the temperature was controlled to stir for 12-15 h. A large amount of solid precipitated during the reaction. The final substrate conversion rate was 99.67%, and the chiral purity of the crude product was 99.98%.

[0053] Example 10: Application of R79S / W139G / L186M / S239P mutant in montelukast sodium intermediate

[0054] In a 1000 mL reaction bottle, the main raw material (intermediate III) 50.0 g, isopropyl alcohol 125 mL and toluene 25 mL were added and stirred until dissolved. Then 50 mL of drinking water, 50 g of R79S / W139G / L186M / S239P mutant enzyme solution, 30 mg of NADP coenzyme were added. The temperature was raised to 35-40℃, and the reaction was stirred for 12-13 h. A large amount of solid precipitated during the reaction, and the final substrate conversion rate was 99.52%.

[0055] Crude product preparation: after the reaction was completed, the temperature was lowered to 25-30℃. The filter cake was filtered and rinsed with 50 mL of isopropyl alcohol. The filter cake was vacuum dried at 40℃-45℃ for 4-6 h (vacuum degree ≥0.09 MPa), and the sample was taken to measure the moisture content ≤5.0% to obtain the crude montelukast sodium intermediate, and the chiral purity (optical purity) of the crude product reached 100%.

[0056] Refining: isopropyl alcohol 250 mL was added to a 1000 mL three-necked flask, and the stirring was started. Toluene 50 mL was added, and the crude montelukast sodium intermediate IV was added to the flask. The temperature was raised to 75-80℃, and the stirring was continued for 10-20 min. The liquid was filtered while hot. The liquid was heated to 70-75℃, and 200 mL of drinking water was added dropwise. After the addition was completed, the temperature was lowered to 15-20℃ in 1-1.5 h, and the stirring was continued for 60-70 min. The centrifugation was performed, and the filter cake was rinsed with 50 mL of isopropyl alcohol. The vacuum drying was performed at 40℃-45℃ for 3-6 h (vacuum degree ≥0.09 MPa), and the sample was taken to measure the moisture content ≤4.0% to obtain the product 47.25 g, the molar yield was 94.5%, and the related purity (chemical purity) was 99.43%.

[0057] The results show that the ketoreductase mutant represented by SEQ ID NO: 1 has a conversion rate of more than 99.52% and a chiral purity of 100% in the enzyme catalysis process, i.e. a 200 g / L substrate reaction system, and the reaction is continued for 12 h. The ketoreductase mutant screened in the preparation of S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl)-3-hydroxypropyl) benzoic acid methyl ester by enzyme method has high stereoselectivity and high efficiency.

[0058] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. An improved ketone reductase mutant, characterized in that, The amino acid sequence of the ketone reductase mutant is shown in SEQ ID NO: 1, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO:

2.

2. A recombinant plasmid containing the gene encoding the ketone reductase mutant as described in claim 1.

3. The recombinant plasmid according to claim 2, characterized in that, The plasmids mentioned 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-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, and 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 IV, pCold-GST or pTrcHis C.

4. A host cell containing the recombinant plasmid of claim 3, characterized in that, The host cells include prokaryotic cells or eukaryotic cells.

5. The host cell according to claim 4, characterized in that, The prokaryotic cells mentioned are Escherichia coli BL21(DE3) cells.

6. The application of the ketone reductase mutant according to claim 1 in the catalysis of montelukast sodium intermediates, characterized in that, Using methyl montelukast sodium intermediate (E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)vinyl)phenyl)-3-carbonylpropyl)benzoate as a substrate, S-(E)-2-(3-(3-(2-(7-chloro-2-quinolinyl)vinyl)phenyl)-3-hydroxypropyl)benzoate was obtained via asymmetric catalysis in the presence of a ketone reductase mutant.

7. The application according to claim 6, characterized in that, The asymmetric catalytic reaction temperature is 35℃-45℃.

Images