Carbonyl reductase mutant and application thereof in preparation of diltiazem key intermediate

Abstract

The invention relates to a carbonyl reductase mutant and application thereof in preparation of a diltiazem key intermediate, and belongs to the technical field of bioengineering. In particular to a carbonyl reductase mutant which is derived from Candida parapsilosas and has obviously improved specific activity, a coding nucleic acid of the carbonyl reductase mutant, and a recombinant expression vector and a recombinant expression transformant containing the gene of the recombinant carbonyl reductase mutant. The invention also relates to an application of the carbonyl reductase mutant as a catalyst in preparation of a diltiazem key intermediate 2-chloro-3-(4-methoxyphenyl)-3 (S)-methyl hydracrylate. Compared with a maternal enzyme, the carbonyl reductase mutant disclosed by the invention has the advantage that the specific activity of the chiral hydroxyl compound 2-chloro-3 (S)-hydroxy-3-phenylpropionic acid methyl ester prepared by catalyzing asymmetric reduction is greatly improved. The chiral hydroxyl compound can further generate a cardiovascular drug diltiazem chiral intermediate, namely (2R, 3S)-p-methoxyphenyl glycidyl acid methyl ester through intramolecular cyclization, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to a carbonyl reductase mutant derived from Candida parapsilosis with significantly improved specific activity, a coding nucleic acid, a recombinant expression vector and a recombinant expression transformant containing the recombinant carbonyl reductase mutant gene, and application of the carbonyl reductase mutant as a catalyst in preparing 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester, a key intermediate of diltiazem. Background Art

[0002] Diltiazem is a calcium channel blocker and a very important cardiovascular drug. The drug was first developed by Tanabe Corporation in Japan in the 1870s and has been widely used around the world. Currently, the incidence and mortality of cardiovascular diseases rank first and are still rising, which has seriously affected human health. The demand for drugs to treat such diseases is increasing, and the importance of synthesizing intermediates for this drug is self-evident.

[0003] The chemical synthesis of diltiazem has a long route, harsh reaction conditions, low raw material utilization, high production energy consumption and other costs. In comparison, the biocatalytic method has many advantages such as mild reaction conditions and environmental friendliness. (2R,3S)-p-methoxyphenyl glycidyl acid methyl ester [(2R,3S)-MPGM] is a key chiral intermediate for the synthesis of diltiazem. In 1993, Tanabe Company screened a product from Salmonella marcescens that can efficiently catalyze the separation of racemates to prepare a high optical purity product (2R,3S)-MPGM (J.Ferment.Bioeng.1993,75:93-98) and applied it to industrial production. In the same year, Takeji Shibatani et al. also discovered a variety of microorganisms that can catalyze the reduction of the substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonyl propionate. The enzymatic reduction product can further cyclize to generate optically active MPGM, but the activity of strains with high stereoselectivity is extremely low (US 005204248A). Following the research routes in the two directions, there have been subsequent studies on a variety of high-activity and high-stereoselective lipases, as well as a variety of wild strains and recombinant carbonyl reductases that catalyze the asymmetric reduction of the substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonyl propionate.

[0004] The above two research ideas have their own advantages, but also limitations. The theoretical yield of the lipase splitting method is only 50%, and there are problems such as waste of raw materials and difficulty in utilizing by-products, and the production cost is high. Although the enzymatic asymmetric synthesis method has the obvious advantage of a theoretical yield of 100%, the catalyst activity is low and cannot meet industrial needs. Improving the catalytic activity of reductase and breaking the limitations of asymmetric synthesis is the most feasible solution for the chemical-enzymatic preparation of diltiazem.

[0005] In recent years, recombinant carbonyl reductase has been mostly used for the enzymatic reduction reaction of the substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionic acid methyl ester. In 2021, the research group of the inventor of this application was the first to report the acquisition of carbonyl reductase CpKR from Candida parapsilosis through gene mining, and for the first time achieved a significant increase in substrate loading and space-time yield (Mol. Catal. 2021, 510: 111670). In 2023, the inventor of this application achieved remarkable results through computationally assisted thermal stability modification of carbonyl reductase CpKR and obtained mutant CpKR M2 (ACS Catal. 2023, 13: 7407-7416). In 2022, the carbonyl reductase LfSDR1 reported by Chen Fener et al. catalyzed the reduction of 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropanoic acid methyl ester, with a substrate loading of 413 mM, but there was a serious substrate inhibition problem (Chem. Commun. 2022, 58: 9010-9013). In 2024, another carbonyl reductase SSCR reported by Chen Fener et al. M2 (Biotechnol. J. 2024, 19: e2300250), its substrate loading capacity has been greatly improved, but there are still problems such as low specific activity of the enzyme, and there are still certain limitations in production. Summary of the invention

[0006] Aiming at the problems of low specific activity and poor stability of carbonyl reductase used for catalytic synthesis of 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester in the prior art, the present invention provides a carbonyl reductase mutant and its application in preparing a key intermediate of diltiazem.

[0007] More specifically, the present invention obtains a highly thermally stable carbonyl reductase mutant CpKR in the early stage of modification. M2On the basis of (see patent CN113174377B), the activity is improved by a directed evolution strategy to provide a mutant with significantly improved specific activity and better stability, the carbonyl reductase mutant gene, a recombinant expression vector containing the gene and a recombinant expression transformant, and the recombinant carbonyl reductase mutant is used to efficiently catalyze the synthesis of 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] One of the technical solutions adopted by the present invention:

[0010] Provided is a carbonyl reductase mutant. The carbonyl reductase mutant is a carbonyl reductase CpKR obtained in the early stage of the laboratory. M2 A derivative protein with improved carbonyl reducing activity obtained by substituting one or more amino acids of the parent protein.

[0011] Wherein, the carbonyl reductase CpKR M2 The nucleic acid sequence is shown in the sequence listing SEQ ID No.1, and the amino acid sequence is shown in the sequence listing SEQ ID No.2.

[0012] During the study, the carbonyl reductase CpKR M2 The nucleic acid sequence of M2 .

[0013] In the present invention, the substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonyl propionate is reacted with the carbonyl reductase CpKR containing the cofactor NADPH. M2 The crystal structure of the present invention is molecularly docked to locate the key residues near the substrate and cofactor NADPH binding sites, and single-point saturation mutation is performed based on this; on the basis of activity screening, the mutant amino acid residues of the mutants with improved activity are combined to provide a variety of carbonyl reductase mutants with significantly improved activity, and the carbonyl reductase is selected from the protein with the following amino acid sequence:

[0014] (1) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0015] (2) replacing the phenylalanine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0016] (3) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine, and replacing the phenylalanine at position 240 with threonine;

[0017] (4) the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, and the isoleucine at position 172 is replaced by leucine;

[0018] (5) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine;

[0019] (6) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamic acid at position 102 is replaced by lysine;

[0020] (7) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the asparagine at position 199 is replaced by glutamine;

[0021] (8) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan;

[0022] (9) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the leucine at position 270 is replaced by valine;

[0023] (10) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine;

[0024] (11) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by alanine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine;

[0025] (12) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the alanine at position 68 is replaced by glycine;

[0026] (13) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan;

[0027] (14) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the leucine at position 270 is replaced by valine;

[0028] (15) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine;

[0029] (16) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine;

[0030] (17) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine;

[0031] (18) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine;

[0032] (19) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the glutamine at position 19 is replaced by tyrosine, and the leucine at position 270 is replaced by valine;

[0033] (20) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine;

[0034] (21) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine;

[0035] (22) In the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine.

[0036] The second technical solution adopted by the present invention is:

[0037] Also provided is an isolated nucleic acid, which is a nucleic acid molecule encoding the carbonyl reductase mutant.

[0038] The method for preparing the nucleic acid of the present invention is a conventional method in the art, and the method preferably comprises:

[0039] A DNA molecule encoding a carbonyl reductase mutant is obtained by gene cloning technology; or a DNA molecule encoding a carbonyl reductase mutant is obtained by artificial sequence total synthesis.

[0040] The third technical solution adopted by the present invention is:

[0041] Provided is a recombinant expression vector comprising the carbonyl reductase mutant nucleic acid sequence of the invention.

[0042] It can be constructed by connecting the carbonyl reductase mutant gene nucleic acid sequence of the present invention to various suitable vectors through conventional methods in the art.

[0043] The vector can be any conventional vector in the art, preferably a plasmid, and more preferably plasmid pET-28a(+). The carbonyl reductase mutant gene can be operably linked to the downstream of a regulatory sequence suitable for expression in the selected vector to achieve constitutive or inducible expression of the carbonyl reductase mutant.

[0044] The fourth technical solution adopted by the present invention is:

[0045] A recombinant expression transformant comprising the carbonyl reductase mutant nucleic acid of the present invention.

[0046] The recombinant expression vector containing the carbonyl reductase mutant nucleic acid sequence of the present invention is transformed into a host cell to obtain the recombinant expression transformant.

[0047] The host cell can be any conventional host cell in the art that can stably replicate the recombinant expression vector and effectively express the carbonyl reductase mutant gene. The host cell is preferably Escherichia coli BL21 (DE3).

[0048] The fifth technical solution adopted by the present invention is:

[0049] A method for preparing the recombinant carbonyl reductase mutant. The method for preparing the recombinant carbonyl reductase of the present invention is preferably: culturing the recombinant expression transformant as described above, and isolating and obtaining the recombinantly expressed carbonyl reductase.

[0050] The culture medium used for culturing the recombinant expression transformant can be any culture medium in the art that can grow the transformant and produce the recombinant carbonyl reductase of the present invention. The culture method and culture conditions are not particularly limited and can be appropriately selected according to the conventional knowledge in the art based on factors such as the host cell type and the culture method, as long as the transformant can grow and produce the recombinant carbonyl reductase. The specific operation of culturing the recombinant expression transformant can be carried out according to conventional operations in the art.

[0051] For example, 20 μL of the glycerol bacterial culture of the recombinant carbonyl reductase mutant was inoculated into a 4 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin, and cultured for 12 h at 37°C and 200 rpm in a shaker. 500 μL of the test tube bacterial culture was inoculated into a 500 mL shake flask containing 50 mL LB liquid culture medium, and cultured for 3-4 h at 37°C and 200 rpm in a shaker until the OD 600 When the concentration reaches 0.5-1.0, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0mM (preferably 0.2mM) to induce enzyme production, and continue to culture at 16°C for 24h. Use a centrifuge to collect the bacteria. Use 10mL potassium phosphate buffer (100mM, pH 6.0) to resuspend the bacteria, ultrasonically disrupt them in an ice water bath for 15min to obtain a crude enzyme solution. The LB liquid culture medium formula is: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0052] The protein in the present invention has a histidine tag (His-Tag) at the N-terminus, so the protein can be purified using a Ni NTA Beads6FF type nickel column. The enzyme purification buffers are:

[0053] Solution A: 20 mM PBS buffer, 500 mM NaCl, 35 mM imidazole, 2 mM β-mercaptoethanol.

[0054] Solution B: 20 ​​mM PBS buffer, 500 mM NaCl, 160 mM imidazole, 2 mM β-mercaptoethanol.

[0055] Solution C: 20 mM PBS buffer, 150 mM NaCl, 1 mM dithiothreitol.

[0056] Use 5-10 column volumes of solution A to wash away the impurities with weak binding ability to the nickel column, and then use 3-5 column volumes of solution B to elute the target protein adsorbed by the nickel column, and collect the eluate. After the eluate is centrifuged to less than 0.5 mL using an ultrafiltration tube, add solution C to replace and remove imidazole, repeat three times, obtain pure enzyme, freeze it in liquid nitrogen, and store it in a -80℃ refrigerator for later use.

[0057] The specific activity of the enzyme was determined by spectrophotometry. The activity measurement system was 1 ml, containing 970 μL of potassium phosphate buffer (100 mM, pH 6.0), 10 μL of substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionic acid methyl ester (100 mM, dissolved in dimethyl sulfoxide), 10 μL of coenzyme NADPH (10 mmol / L, dissolved in KPB) and 10 μL of incubated enzyme solution. The reaction temperature was 35 ° C, and the change in absorbance at 340 nm was detected. The enzyme activity was calculated according to the enzyme activity definition formula and Lambert-Beer law.

[0058] The present invention also provides a method for detecting the thermal stability of the recombinant carbonyl reductase mutant. The present invention detects the melting temperature of the enzyme by circular dichroism (CD). The melting temperature refers to the reaction temperature corresponding to half of the protein unfolding under certain conditions, which is used to characterize thermodynamic stability. Specifically, the purified protein sample is used, diluted to 0.5 mg / mL and added to the sample pool of the circular dichroism spectrometer, and the temperature program is set from 20°C to 80°C, rising at a rate of 2°C / min, scanning the absorbance value changes of the protein in the wavelength range of 180-260nm, and then the melting temperature T is obtained by data analysis using Global 3 software. m .

[0059] The sixth technical solution adopted by the present invention is:

[0060] A carbonyl reductase mutant catalyst is provided, which is any one of the following forms:

[0061] (1) culturing the recombinant expression transformant and isolating resting cells containing the carbonyl reductase mutant;

[0062] (2) freeze-dried cells obtained by freeze-drying the resting cells described in form (1);

[0063] (3) disrupting the resting cells of form (1) to obtain a cell disrupted liquid containing the carbonyl reductase mutant;

[0064] (4) A freeze-dried enzyme powder obtained by freeze-drying the cell disrupted liquid described in form (3).

[0065] After the cell culture is completed, the precipitated bacterial cells are collected by centrifugation, which are the resting cells of the recombinant expression transformant; the obtained cells are suspended in potassium phosphate buffer, ultrasonically disrupted, the disrupted liquid is centrifuged, and the supernatant is collected to obtain the cell disrupted liquid of the recombinant carbonyl reductase. The resting cells and the cell disrupted liquid are freeze-dried respectively to obtain freeze-dried cells and freeze-dried enzyme powder.

[0066] The seventh technical solution adopted by the present invention is:

[0067] The carbonyl reductase mutant (CpKR mutant) or the carbonyl reductase mutant catalyst of the present invention is used to catalyze the asymmetric reduction of substrate carbonyl compounds to prepare diltiazem key intermediates.

[0068] Carbonyl reductase belongs to the oxidoreductase class and requires NADPH as an electron donor to generate NADP + NADPH is very expensive, so in the enzyme catalysis process, a coenzyme recycling system is usually coupled to use one of the dehydrogenases such as glucose dehydrogenase GDH, formate dehydrogenase FDH, isopropanol dehydrogenase IPADH, etc. to convert NADP + Under the reaction conditions, it is reduced to NADPH to reduce the cost of raw materials.

[0069] The carbonyl compound is selected from methyl 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionate.

[0070] The reduction product of the carbonyl compound is 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester, and the 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester is subjected to intramolecular cyclization to synthesize the key chiral intermediate (2R, 3S)-MPGM for the synthesis of diltiazem.

[0071] Specifically, the catalytic reaction conditions are: substrate concentration is 100-600 mM, coenzyme NADP + The concentration is 0.1-2.0 mM, glucose is 150-900 mM, the dosage of glucose dehydrogenase GDH is 2-15 U / mL, the reaction temperature is 20-45° C., the pH is 4-9, and the reaction time is 0.5-24 h.

[0072] During the reaction, samples were taken intermittently, and the amount of the product was analyzed by liquid chromatography. The analysis conditions were as follows: the chromatographic column was a Daicel liquid phase analysis chromatographic column Chiral OJ-H (25 cm × 4.6 mm, 5 μm), the mobile phase was n-hexane / isopropanol (70 / 30, v / v), the flow rate was 0.5 mL / min, the column temperature was 35°C, and the detection wavelength was 254 nm.

[0073] Compared with the prior art, the present invention has significant advantages:

[0074] The recombinant carbonyl reductase mutant of the present invention has the significant advantages of high catalytic activity and good thermal stability in the application of catalyzing the asymmetric reduction of the substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonyl propionate to prepare 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate methyl ester. The reduction product 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate methyl ester can be efficiently synthesized into the key chiral intermediate (2R, 3S)-MPGM for the synthesis of diltiazem through intramolecular cyclization, with low production cost, suitable for industrial application, and has good industrial development and application prospects in the production process of drug intermediates such as diltiazem. DETAILED DESCRIPTION

[0075] Each reaction or detection condition described in the content of the present invention can be combined or changed according to the common sense in the art, and can be verified by experiments. The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0076] The content of the present invention is further described below through specific embodiments.

[0077] The sources of materials in the following examples are:

[0078] The expression plasmid pET-28a(+) was purchased from Novagen, and the plasmid pET-28a(+)-CpKR M2 Previously constructed by this lab.

[0079] Escherichia coli BL21 (DE3) competent cells and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0080] The restriction endonuclease Dpn I is a commercially available product from New England Biolabs (NEB).

[0081] Fast PCR polymerase PrimeSTAR MAX was purchased from Takara

[0082] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0083] Example 1 Construction of carbonyl reductase CpKR mutant

[0084] Carbonyl reductase CpKR M2As the starting mutant, according to its crystal structure with the coenzyme NADPH, the molecular docking was performed using Autodock Vina to obtain the docking conformation. A total of 60 amino acid residues around the substrate and NADPH were selected, and primers were designed to construct the NNK degenerate codon saturation mutant library. The plasmid pET-28a(+)-CpKR M2 As a PCR template, use the fast PCR polymerase PrimeSTAR MAX for PCR amplification. After the PCR product is verified by agarose gel electrophoresis analysis, the restriction endonuclease Dpn I is added and digested at 37°C for 2h. The digestion products are placed in the same centrifuge tube in equal proportions and mixed, transformed into E.coliBL21 (DE3) competent cells, and spread on LB solid culture medium plates containing 50μg / mL kanamycin, and cultured at 37°C for 12-14h.

[0085] Use a toothpick to pick a single colony on the plate into a 96-well shallow plate containing 200 μL LB liquid medium. The shallow plate is cultured at 30°C and 220 rpm for 14 to 16 hours. Take 10 μL of bacterial solution and add it to a deep-well plate containing 290 μL TB autoinduction medium. Culture at 30°C for 2.5 hours, then cool to 16°C and culture for 24 hours. Add 95 μL of 50% glycerol to the remaining shallow-well plate bacterial solution and store it in a -80°C refrigerator for later use. Centrifuge the 96-well deep-well plate at 4000 rpm and 4°C for 15 minutes, discard the supernatant, and freeze it in a -80°C refrigerator for 1 hour for later use.

[0086] Example 2 Screening of carbonyl reductase CpKR mutants

[0087] Take out the 96-well deep-well plate from the -80℃ refrigerator, add 200μL 2g / L lysozyme to each well, shake at 30℃ for 2h, continue to incubate at 35℃ for 4h, and centrifuge for use. Add the following reaction system to the new 96-well ELISA plate: 10μL substrate (100mM, dissolved in dimethyl sulfoxide), 10μL enzyme solution from the deep-well plate centrifugation, 10μL NADPH (10mM, KPB dissolved), 120μL KPB (100mM, pH 6.0). At 35℃, the change in absorbance at 340nm within 10min was detected by an ELISA instrument. The mutants with improved activity were inoculated into shake flasks for amplification and culture, and the enzyme activity of the mutants was screened again using an ultraviolet spectrophotometer. Finally, the mutants with improved activity were purified and screened again. Finally, mutants with improved activity and good stability were obtained.

[0088] Table 1 provides a list of carbonyl reductase CpKR mutants disclosed in the present invention with improved specific activity and good stability. In the following table, the sequence numbers refer to a series of sequences following Table 1. M2In comparison, one plus sign “+” indicates that the mutant protein has a 1- to 5-fold increase in specific activity; two plus signs “++” indicate that the mutant protein has a 5- to 25-fold increase in specific activity; three plus signs “+++” indicate that the mutant protein has a 25- to 50-fold increase in specific activity; and four plus signs “++++” indicate that the mutant protein has a more than 50-fold increase in specific activity.

[0089] Table 1. List of carbonyl reductase CpKR mutant sequences and corresponding specific activity improvements

[0090]

[0091]

[0092] The amino acid sequence of the carbonyl reductase mutant is one of the following sequences:

[0093] (1) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0094] (2) replacing the phenylalanine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0095] (3) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine, and replacing the phenylalanine at position 240 with threonine;

[0096] (4) the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, and the isoleucine at position 172 is replaced by leucine;

[0097] (5) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine;

[0098] (6) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamic acid at position 102 is replaced by lysine;

[0099] (7) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the asparagine at position 199 is replaced by glutamine;

[0100] (8) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan;

[0101] (9) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the leucine at position 270 is replaced by valine;

[0102] (10) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine;

[0103] (11) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by alanine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine;

[0104] (12) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the alanine at position 68 is replaced by glycine;

[0105] (13) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan;

[0106] (14) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the leucine at position 270 is replaced by valine;

[0107] (15) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine;

[0108] (16) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine;

[0109] (17) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine;

[0110] (18) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine;

[0111] (19) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the glutamine at position 19 is replaced by tyrosine, and the leucine at position 270 is replaced by valine;

[0112] (20) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine;

[0113] (21) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine;

[0114] (22) In the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine.

[0115] Example 3 Inducible expression and protein purification of recombinant carbonyl reductase CpKR mutant

[0116] The recombinant expression transformant expressing the carbonyl reductase CpKR mutant obtained in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured on a shaker at 37°C for 12 h. Then, the inoculum was inoculated into 100 mL LB medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum, and cultured on a shaker at 37°C and 200 rpm. When the culture OD 600When the p-value reached 0.6, IPTG was added to induce the cells at a final concentration of 0.2 mM. After culturing at 16°C for 24 h, the culture solution was centrifuged and the cell precipitate was collected to obtain the mutant transformant cells. The obtained cells were resuspended in potassium phosphate buffer (100 mM, pH 6.0), ultrasonically disrupted in an ice water bath, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant carbonyl reductase CpKR mutant.

[0117] All purification experiments were completed using nickel affinity self-packed columns. The buffer used in the purification process was Solution A: 20mM PBS buffer, 500mM NaCl, 35mM imidazole, 2mM β-mercaptoethanol. Solution B: 20mM PBS buffer, 500mM NaCl, 160mM imidazole, 2mM β-mercaptoethanol. Solution C: 20mM PBS buffer, 150mM NaCl, 1mM dithiothreitol.

[0118] The Ni column (bed volume 2ml) was pre-equilibrated with 5-10 column volumes of liquid A, and the crude enzyme solution was filtered through a filter membrane and then loaded (load volume 10ml). 5-10 column volumes of liquid A were used to wash away the impurities with weak binding ability to the nickel column, and then 3-5 column volumes of liquid B were used to elute the target protein adsorbed by the nickel column, and the eluate was collected. The eluate was centrifuged to less than 0.5mL with an ultrafiltration tube, and then liquid C was added to replace and remove imidazole. This was repeated three times, and finally concentrated to 0.5mL to obtain pure enzyme, which was quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use.

[0119] Example 4 Melting temperature (T m ) determination

[0120] The purified enzyme solution obtained in Example 3 was diluted to 0.5 mg / mL and added to the circular dichroism spectrometer sample pool. The temperature was set to rise from 20°C to 80°C at a rate of 2°C / min. The absorbance value change of the protein in the wavelength range of 180-260 nm was detected, and then the melting temperature T was obtained by data analysis using Global 3 software. m The results are shown in Table 2.

[0121] Table 2 Melting temperatures of carbonyl reductase CpKR mutants

[0122]

[0123] Example 5 Whole cell catalytic reduction of 100 mM substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionic acid methyl ester

[0124] In a potassium phosphate buffer (100 mM, pH 6.0) containing 100 mM substrate (24.2 g / L, 10% DMSO) and 150 mM glucose (27 g / L), 5 g / L of the recombinant expression transformant (pET-28a(+)-CpKR M23 ) wet cells, 10 U / mL glucose dehydrogenase freeze-dried enzyme powder and 0.5 mM NADP + , potassium phosphate buffer is supplemented to make the final reaction system 10mL. The reaction is carried out at 35°C under magnetic stirring, and 1M potassium carbonate solution is added by automatic potentiometric titrator to control the pH at 6.0. After 8 hours of reaction, two volumes of ethyl acetate are added for extraction, and the extract is added with anhydrous sodium sulfate and dried overnight. After filtering with a 0.22μm oil filter membrane, it is measured by high performance liquid chromatography: the substrate conversion rate is greater than 99% within 5h, and the content of 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester is greater than 99%.

[0125] Example 6 Whole cell catalytic reduction of 400 mM substrate 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionic acid methyl ester

[0126] In a potassium phosphate buffer (100 mM, pH 6.0) containing 400 mM substrate (100 g / L, 10% DMSO) and 600 mM glucose (108 g / L), 5 g / L of the recombinant expression transformant (pET-28a(+)-CpKR M23 ) wet cells, 50 U / mL glucose dehydrogenase freeze-dried enzyme powder and 0.5 mM NADP + , potassium phosphate buffer is added to make the final reaction system 10mL. The reaction is carried out at 35°C under magnetic stirring, and 1M potassium carbonate solution is added by automatic potentiometric titrator to control the pH at 6.0. After 24 hours of reaction, two volumes of ethyl acetate are added for extraction, and the extract is dried overnight by adding anhydrous sodium sulfate, filtered with a 0.22μm oil filter membrane, and analyzed by high performance liquid chromatography to determine: the substrate conversion rate is greater than 99%, and the content of 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester is greater than 99%.

[0127] Example 7 Enzymatic Preparation of Methyl 2-Chloro-3-(4-methoxyphenyl)-3(S)-Hydroxypropionate

[0128] The reaction solution obtained after the whole cell catalytic reduction reaction as in Example 5 above was extracted twice with equal volumes of ethyl acetate, the extracts were combined, the solvent was removed by vacuum distillation, and the product was separated by rapid silica gel column (petroleum ether: ethyl acetate = 8:1), and finally 230 mg of the product was obtained with a separation yield of 94%.

[0129] Comparative Example 1

[0130] In 10 mL of potassium phosphate buffer (100 mM, pH 6.0) containing 400 mM substrate (100 g / L) and 600 mM glucose (108 g / L), 50 g / L of the recombinant expression transformant (pET-28a(+)-CpKR M2 @E.coli BL21(DE3)) and 5g / L recombinant expression transformant (pET-28a(+)-CpKR M23 @E.coli BL21(DE3)), 50U / mL lyophilized glucose dehydrogenase powder and 0.5mM NADP + , potassium phosphate buffer was added to make the final reaction system 10mL. The reaction was carried out at 35°C under magnetic stirring, and 1M potassium carbonate solution was added by automatic potentiometric titrator to control the pH at 6.0. After 24 hours of reaction, two volumes of ethyl acetate were added for extraction, and the extract was dried overnight by adding anhydrous sodium sulfate, filtered with a 0.22μm oil filter membrane, and analyzed by high performance liquid chromatography to obtain: CpKR M2 The substrate conversion rate was 31.5%, CpKR M23 The substrate conversion rate is greater than 99%, and the content of 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester in both products is greater than 99%.

[0131] The sequence information involved in the present invention is as follows:

[0132] SEQ ID No.1:

[0133] Encoding carbonyl reductase CpKR M2 The nucleotide sequence of the nucleic acid

[0134] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCATGTCATCAGAAACTGTTGTATTCGTCAGTGGTGCTACTGGATTCATTGCTCAACAAATTGTCAAAACCCTGCTTGAAGCTGGTTACAAAACTATCGGCTCAGTGAGATCAGAGGAAAAAGGAAAGTACTTAAAATCATTGATCGAGTCTGCTGGACTCAATTCTAATCTTTTCAATTATGTCATTGTGAAGGACATTGCAGCCAAAGGTGCATTTAACGAAGCTTTGCAAGCCCATCCGGAGGTGACAGTGTTTTTACACACTGCATCTCCTGCTACATTTGAAATTCATGATGTTGAGAAGGAATTGTTAAAACCGGCCATTGAGGGTACCATTAATGCACTTAACGCAATTACCGTGTATGGTAAAAACGTTCAAAGGGTTGTCATCACATCGTCTTATGCTGCGGTTGCTGGTTTTGCAAATTTGGCTACGCCTGGTAAAGAAGTAAATGAAGAATCGTGGAACCCAATCACATACGAGCAGGCTTTGGAAAATCCCTTTCTTGGTTACATTGGATCAAAGAAATTTGCTGAAAAGGCAGTATGGAACTACATCGAAGAAAAGAAGCCAAAATGGGATGTCACTTTTGTGAATCCTGCATTTGTTTTGGGACCACAAGCTTTTGCCGTTAGAGACAAGTCCAAGTTGAACGCATCAAATGAAATCATCAACAGTCTCTTGACTGCAAACAAGACAAAAGTGGAGCCTCAACAATTTGTTGGATACTTTATTGATGTCAGAGATGTCGCAAAAGCCCATCTCATTGCTTTTGAGAAGAATGAAACTGTGGGCCAGAGATTGCTTTTGGCAAATGCACCTTTTTCCTCCGCTGGGATCTTGGACATTATTGAAAAAGATTTCCCTGAATTGAAATCTTCATTACCAAAGTTGGATAAGTCAAAAGCACCAAAGTTTGAGGAAACTGAAAGTGTCGTAAACAATGAAAAGACGAGAAGGATTTTGGGTTTCAAATTCATTGATTTGAAAAAGTCGGTTGATGACACTATTAAGCAGTTGGTGTAA

[0135] SEQ ID No.2:

[0136] Carbonyl reductase CpKR M2 amino acid sequence of

[0137] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSETVVFVSGATGFIAQQIVKTVLEAGYKTIGSVRSEEKGKYLKSLIESAGLNSNLFNYVIVKDIGAKGAFNEALQAHPEVTVFLHTASPATFEIHDVEKELLKPAIEGTINALNAVTVYGKNVQRVVITSSYAAVAGFANLATPGKEVNEESWNPITYEQALENPFLGYIGSKKLAEKTVWNYIEEKKPKWDVTFVNPAFVLGPQAFAVRDKSKLNASNEIINSLLTANKTKVEPQQFVGYFIDVRDVAKAHLIAFEKNETVGQRLLLANAPFSSAGILDIIEKDFPNLKSELPKLDKSKSPKFEETESVVNNEKTRRILGFKFIDLKKSVDDTIKQLV

[0138] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that: The carbonyl reductase is selected from proteins having the following amino acid sequence: (1) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine; (2) replacing the phenylalanine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with threonine; (3) replacing the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 with threonine, and replacing the phenylalanine at position 240 with threonine; (4) the valine at position 203 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, and the isoleucine at position 172 is replaced by leucine; (5) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine; (6) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamic acid at position 102 is replaced by lysine; (7) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the asparagine at position 199 is replaced by glutamine; (8) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan; (9) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the leucine at position 270 is replaced by valine; (10) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine; (11) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by alanine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, and the alanine at position 273 is replaced by glutamine; (12) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, and the alanine at position 68 is replaced by glycine; (13) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the tyrosine at position 243 is replaced by tryptophan; (14) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the leucine at position 270 is replaced by valine; (15) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, and the glutamine at position 19 is replaced by tyrosine; (16) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine; (17) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine; (18) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine; (19) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the glutamine at position 19 is replaced by tyrosine, and the leucine at position 270 is replaced by valine; (20) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the leucine at position 270 is replaced by valine; (21) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the tyrosine at position 243 is replaced by tryptophan, and the glutamine at position 19 is replaced by tyrosine; (22) In the amino acid sequence shown in SEQ ID No. 2, the valine at position 203 is replaced by threonine, the phenylalanine at position 240 is replaced by threonine, the isoleucine at position 172 is replaced by leucine, the phenylalanine at position 197 is replaced by threonine, the alanine at position 273 is replaced by glutamine, the asparagine at position 199 is replaced by glutamine, the leucine at position 270 is replaced by valine, and the glutamine at position 19 is replaced by tyrosine.

2. An isolated nucleic acid, characterized in that The nucleic acid is a nucleic acid molecule encoding the carbonyl reductase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Comprising the nucleic acid as described in claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector as described in claim 3.

5. A carbonyl reductase mutant catalyst, characterized in that: It is any of the following forms: (1) culturing the recombinant expression transformant according to claim 4, and isolating resting cells containing the carbonyl reductase mutant according to claim 1; (2) freeze-dried cells obtained by freeze-drying the resting cells described in form (1); (3) disrupting the resting cells described in form (1) to obtain a cell disrupted liquid containing the carbonyl reductase mutant described in claim 1; (4) A freeze-dried enzyme powder obtained by freeze-drying the cell disrupted liquid described in form (3).

6. Use of the carbonyl reductase mutant according to claim 1 or the carbonyl reductase mutant catalyst according to claim 5 for catalyzing the asymmetric reduction of carbonyl compounds.

7. The use according to claim 6, characterized in that: The carbonyl reductase catalyzes the asymmetric reduction of carbonyl compounds in the presence of the coenzyme NADPH, wherein NADPH is catalyzed by glucose dehydrogenase to convert glucose into NADP + The conversion reaction realizes regeneration.

8. The use according to claim 6, characterized in that: The carbonyl compound is selected from methyl 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionate.

9. The use according to claim 8, characterized in that: The reduction product of the carbonyl compound is 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester, The 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester is used to synthesize the key chiral intermediate (2R, 3S)-MPGM for the synthesis of diltiazem through intramolecular cyclization.

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