A carbonyl reductase mutant and its application in preparing a key intermediate of diltiazem

Abstract

The present invention relates to a carbonyl reductase mutant and its application in preparing a key intermediate of diltiazem, belonging to the field of bioengineering technology. Specifically, it relates to a carbonyl reductase mutant derived from Candida parapsilosis with significantly improved specific activity, its encoding nucleic acid, a recombinant expression vector and a recombinant expression transformant containing the recombinant carbonyl reductase mutant gene, and the use of the carbonyl reductase mutant as a catalyst in preparing the key intermediate of diltiazem, 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionic acid methyl ester. Compared with the parent enzyme, the carbonyl reductase mutant of the present invention catalyzes asymmetric reduction to prepare the chiral hydroxy compound 2-chloro-3(S)-hydroxy-3-phenylpropionic acid methyl ester. The chiral hydroxy compound can further generate the cardiovascular drug diltiazem chiral intermediate methyl glycidyl (2R, 3S)-p-methoxyphenyl glycidyl ester through intramolecular cyclization, which has good industrial application prospects.

Description

Technical Field

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

[0002] Diltiazem, a calcium channel blocker, is a very important cardiovascular drug. First developed by Tanabe Corporation in Japan in the 1870s, it has subsequently become widely used worldwide. Cardiovascular disease currently ranks first in morbidity and mortality, and continues to rise, severely impacting human health. The increasing demand for drugs to treat these diseases has necessitated the development of synthetic intermediates for this drug.

[0003] Chemical synthesis of diltiazem involves long pathways, demanding reaction conditions, low raw material utilization, and high production energy costs. In comparison, biocatalysis offers numerous advantages, including mild reaction conditions and environmental friendliness. (2R,3S)-p-methoxyphenyl glycidyl methyl ester [(2R,3S)-MPGM] is a key chiral intermediate in the synthesis of diltiazem. In 1993, Tanabe Co., Ltd. identified a compound from Salmonella marcescens that efficiently catalyzes the resolution of racemates to produce a high-optical-purity product (2R,3S)-MPGM (J. Ferment. Bioeng. 1993, 75:93-98), which was then applied to industrial production. That same year, Takeji Shibatani et al. also discovered a variety of microorganisms capable of catalyzing the reduction of the substrate methyl 2-chloro-3-(4-methoxyphenyl)-3-oxopropionate. The enzymatic reduction product could further cyclize to produce optically active MPGM, but strains with high stereoselectivity had extremely low activity (US 005204248A). Following these two research directions, subsequent research has led to the development of a variety of highly active and stereoselective lipases, as well as the development of a variety of wild-type strains and recombinant carbonyl reductases that catalyze the asymmetric reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxopropionate.

[0004] Both of these research approaches have their advantages, but also limitations. The lipase-mediated resolution method has a theoretical yield of only 50%, with issues such as raw material waste and difficulty in utilizing byproducts, resulting in high production costs. While the enzymatic asymmetric synthesis method offers the distinct advantage of a 100% theoretical yield, its catalyst activity is relatively low, failing to meet industrial requirements. Improving the catalytic activity of reductases and overcoming the limitations of asymmetric synthesis is the most feasible approach to the chemo-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-carbonylpropionate. 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 time-space 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-carbonylpropionic 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's research group M2 (Biotechnol. J. 2024, 19: e2300250), its substrate loading capacity has been greatly improved, but there are still problems such as low enzyme specific activity, and there are still certain limitations in production. Summary of the Invention

[0006] Aiming at the problems in the prior art of low specific activity and poor stability of carbonyl reductase used for catalytic synthesis of methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate, the present invention provides a carbonyl reductase mutant and its use in the preparation of 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 the transformation. M2On the basis of the invention (see patent CN113174377B), the activity of the carbonyl reductase 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 are provided. The recombinant carbonyl reductase mutant is used to efficiently catalyze the synthesis of methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate.

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

[0009] One of the technical solutions adopted in 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 It is 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 the PCR product was connected to the plasmid pET-28a(+) and named pET-28a(+)-CpKR 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 Molecular docking was performed on the crystal structure of the present invention to locate the key residues near the substrate and cofactor NADPH binding sites, and single-point saturation mutagenesis was performed based on this. Based on the activity screening, the mutated amino acid residues of the mutants with improved activity were combined to provide a variety of carbonyl reductase mutants with significantly improved activity. The carbonyl reductase was 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 present invention.

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

[0043] The vector can be any conventional vector in the art, preferably a plasmid, 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 preferably comprises: 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 conventional knowledge in the art based on factors such as the host cell type and culture method, as long as the transformant can grow and produce the recombinant carbonyl reductase. The specific operations for culturing the recombinant expression transformant can be carried out according to conventional operations in the art.

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

[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 Beads 6FF 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] Wash away weakly bound nickel-column-bound contaminants with 5-10 column volumes of Solution A. Elute the target protein adsorbed to the nickel column with 3-5 column volumes of Solution B and collect the eluate. Ultracentrifuge the eluate to less than 0.5 mL, then add Solution C to displace and remove the imidazole. Repeat this process three times to obtain pure enzyme. Quick-freeze in liquid nitrogen and store in a -80°C freezer until ready for use.

[0057] Enzyme specific activity was determined spectrophotometrically. The assay system consisted of 1 mL of 970 μL of potassium phosphate buffer (100 mM, pH 6.0), 10 μL of the substrate methyl 2-chloro-3-(4-methoxyphenyl)-3-oxopropionate (100 mM, dissolved in dimethyl sulfoxide), 10 μL of the coenzyme NADPH (10 mmol / L, dissolved in KPB), and 10 μL of the incubated enzyme solution. The reaction temperature was 35°C, and the absorbance change at 340 nm was measured. Enzyme activity was calculated according to the enzyme activity definition formula and the 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 at a rate of 2°C / min. The absorbance value change of the protein in the wavelength range of 180-260nm is scanned, and then the data is analyzed by Global 3 software to obtain the melting temperature T m .

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

[0060] Provided is a carbonyl reductase mutant catalyst, which is in 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) 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 to obtain the resting cells of the recombinant expression transformant. The resulting cells are suspended in potassium phosphate buffer, disrupted by ultrasonication, and the disrupted liquid is centrifuged. The supernatant is collected to obtain a cell disrupted liquid containing the recombinant carbonyl reductase. The resting cells and the cell disrupted liquid are then freeze-dried 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 a substrate carbonyl compound to prepare a key intermediate of diltiazem.

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

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

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

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

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

[0074] The recombinant carbonyl reductase mutant described herein exhibits significant advantages in high catalytic activity and good thermal stability in the asymmetric reduction of the substrate methyl 2-chloro-3-(4-methoxyphenyl)-3-carbonylpropionate to produce methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate. The reduced product, methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate, undergoes intramolecular cyclization to efficiently synthesize (2R,3S)-MPGM, a key chiral intermediate in the synthesis of diltiazem. This method offers low production costs, is suitable for industrial application, and has promising prospects for industrial development and application in the production of pharmaceutical intermediates such as diltiazem. DETAILED DESCRIPTION

[0075] The various reaction or detection conditions described in the present invention may be combined or modified according to common knowledge in the art and may be verified by experiments. The technical solutions and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, the scope of protection 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 scope of protection of the present invention.

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

[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 laboratory.

[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 was a commercial 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, molecular docking was performed using Autodock Vina based on the crystal structure of the coenzyme NADPH 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 PCR amplification was performed using the fast PCR polymerase PrimeSTAR MAX as a PCR template. After verification by agarose gel electrophoresis, the PCR product was digested with the restriction endonuclease Dpn I at 37°C for 2 hours. The digestion products were transferred to the same centrifuge tube in equal proportions and mixed thoroughly. The cells were then transformed into competent E. coli BL21 (DE3) cells and plated onto LB solid medium plates containing 50 μg / mL kanamycin. The cells were incubated at 37°C for 12–14 hours.

[0085] Use a toothpick to pick a single colony from the plate and transfer it to a 96-well shallow-well plate containing 200 μL of LB liquid medium. Incubate the shallow-well plate at 30°C, 220 rpm, and shake for 14-16 hours. Transfer 10 μL of the bacterial culture to a deep-well plate containing 290 μL of TB autoinduction medium. Incubate at 30°C for 2.5 hours, then cool to 16°C and incubate for 24 hours. Add 95 μL of 50% glycerol to the remaining shallow-well plate and store at -80°C until needed. Centrifuge the 96-well deep-well plate at 4000 rpm, 4°C for 15 minutes, discard the supernatant, and quickly freeze at -80°C for 1 hour until needed.

[0086] Example 2 Screening of carbonyl reductase CpKR mutants

[0087] Remove the 96-well deep-well plate from the -80°C freezer and add 200 μL of 2g / L lysozyme to each well. Shake at 30°C for 2 hours, incubate at 35°C for another 4 hours, and then centrifuge for later use. Add the following reaction system to a new 96-well microtiter plate: 10 μL of substrate (100 mM, dissolved in dimethyl sulfoxide), 10 μL of enzyme supernatant from the previous deep-well plate centrifugation, 10 μL of NADPH (10 mM, dissolved in KPB), and 120 μL of KPB (100 mM, pH 6.0). Monitor absorbance changes at 340 nm for 10 minutes at 35°C using a microplate reader. Mutants with improved activity are inoculated into shake flasks for scale-up and re-screened for enzyme activity using a UV spectrophotometer. Finally, mutants with improved activity are purified and re-screened. Ultimately, mutants with enhanced activity and excellent stability are obtained.

[0088] Table 1 provides a list of carbonyl reductase CpKR mutants disclosed in the present invention with improved specific activity and good stability. M2In comparison, one plus sign “+” indicates that the specific activity of the mutant protein is increased by 1 to 5 times; two plus signs “++” indicate that the specific activity of the mutant protein is increased by 5 to 25 times; three plus signs “+++” indicate that the specific activity of the mutant protein is increased by 25 to 50 times; four plus signs “++++” indicate that the specific activity of the mutant protein is increased by more than 50 times.

[0089] Table 1. Sequences of carbonyl reductase CpKR mutants 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 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 a final concentration of 0.2 mM for induction. After culturing at 16°C for 24 hours, the culture medium was centrifuged and the cell pellet was collected to obtain the mutant transformant cells. The resulting 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 performed using nickel affinity self-packed columns. The following buffers were used: Solution A: 20 mM PBS, 500 mM NaCl, 35 mM imidazole, 2 mM β-mercaptoethanol; Solution B: 20 ​​mM PBS, 500 mM NaCl, 160 mM imidazole, 2 mM β-mercaptoethanol; and Solution C: 20 mM PBS, 150 mM NaCl, 1 mM dithiothreitol.

[0118] A Ni column (bed volume 2 ml) was pre-equilibrated with 5-10 column volumes of Solution A. The crude enzyme solution was filtered through a membrane and loaded (sample volume 10 ml). Contaminants with weak binding to the nickel column were washed away with 5-10 column volumes of Solution A. The target protein adsorbed by the nickel column was then eluted with 3-5 column volumes of Solution B, and the eluate was collected. The eluate was centrifuged using an ultrafiltration tube to less than 0.5 ml, after which Solution C was added to displace and remove the imidazole. This was repeated three times, and the enzyme was finally concentrated to 0.5 ml to obtain pure enzyme. The enzyme was quickly frozen in liquid nitrogen and stored in a -80°C freezer until ready for 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 cell. 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. The data was then analyzed using Global 3 software to obtain the melting temperature T. 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-oxopropionic 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 lyophilized glucose dehydrogenase powder, and 0.5 mM NADP + The potassium phosphate buffer was added to a final volume of 10 mL. The reaction was carried out at 35°C under magnetic stirring. 1 M potassium carbonate solution was added via an automatic potentiometric titrator to maintain the pH at 6.0. After 8 hours of reaction, extraction was performed with twice the volume of ethyl acetate. The extract was then dried overnight over anhydrous sodium sulfate and filtered through a 0.22 μm oil-based filter. High-performance liquid chromatography revealed a substrate conversion greater than 99% within 5 hours, and a methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate content greater than 99%.

[0125] Example 6 Whole Cell Catalytic Reduction of 400 mM Substrate 2-Chloro-3-(4-methoxyphenyl)-3-oxopropionic 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 lyophilized glucose dehydrogenase powder and 0.5 mM NADP + The potassium phosphate buffer was added to a final volume of 10 mL. The reaction was carried out at 35°C under magnetic stirring. A 1 M potassium carbonate solution was added via an automatic potentiometric titrator to control the pH at 6.0. After 24 hours of reaction, extraction was performed with twice the volume of ethyl acetate. The extract was dried overnight over anhydrous sodium sulfate and filtered through a 0.22 μm oil-based filter membrane. High-performance liquid chromatography analysis revealed a substrate conversion greater than 99% and a methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate content 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 and the solvent was removed by distillation under reduced pressure. The product was separated by rapid silica gel column (petroleum ether:ethyl acetate=8:1) to finally obtain 230 mg of the product with an isolation 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 recombinant expression transformant (pET-28a(+)-CpKR M2 @E.coli BL21(DE3)) and 5g / L of recombinant expression transformant (pET-28a(+)-CpKR M23 @E.coli BL21(DE3)), 50U / mL lyophilized glucose dehydrogenase enzyme powder and 0.5mM NADP + The potassium phosphate buffer was added to make the final reaction volume 10 mL. The reaction was carried out at 35°C under magnetic stirring. 1M potassium carbonate solution was added by an automatic potentiometric titrator to control the pH at 6.0. After 24 hours of reaction, two volumes of ethyl acetate were added for extraction. The extract was dried overnight by adding anhydrous sodium sulfate, filtered through a 0.22 μm oil filter membrane, and analyzed by high performance liquid chromatography to determine the CpKR M2 The substrate conversion rate was 31.5%, CpKR M23 The substrate conversion rate is greater than 99%, and the content of methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate 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 intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 according to claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to 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) 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 reduce glucose and 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 methyl 2-chloro-3-(4-methoxyphenyl)-3(S)-hydroxypropionate, 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.

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