Carbonyl reductase mutant and application thereof in preparation of chiral alcohol

Abstract

The invention discloses a carbonyl reductase mutant and application thereof in preparation of chiral alcohol, and relates to the technical field of biological pharmacy and biochemical engineering. The carbonyl reductase mutant is obtained by mutating at least one site in SEQ ID NO: 2: the 119th site, the 180th site, the 183rd site, the 185th site, the 219th site, the 227th site and the 236th site. The invention further provides application of the carbonyl reductase mutant in catalyzing asymmetric reduction reaction of carbonyl compounds to prepare chiral alcohol and a preparation method of (R)-4-chromogen alkanol compounds. The carbonyl reductase mutant is constructed, the (R)-4-chromogen alkanol compound is prepared through a biological preparation way by adopting the carbonyl reductase mutant as a catalyst, and the method is environment-friendly, simple and convenient to operate, easy to industrially amplify, high in substrate concentration, wide in substrate universality and high in product yield, and has a very good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biopharmaceutical and biochemical technology, specifically relating to a carbonyl reductase mutant and its application in the preparation of chiral alcohols. Background Technology

[0002] ( R 4-Chromophenols are widely found in natural products and pharmaceuticals, and have become important chiral building blocks. In recent years, fluorinated compounds have found wide applications in pharmaceuticals, agrochemicals, and functional materials. Among them ( R )-5,7-difluoro-3,4-dihydro-2 H -1-Benzofuran-4-ols play an important role in the pharmaceutical field. For example, these compounds are effective in treating gastroesophageal reflux disease.

[0003] Currently synthesized ( R The chemical methods for the preparation of 3,4-dihydro-2H-1-benzopyran-4-one compounds mainly include: (1) Existing technology one uses ruthenium catalyst and formic acid as hydrogen donor to perform asymmetric reduction of 3,4-dihydro-2H-1-benzopyran-4-one compounds, which can obtain highly selective products, but the preparation process is complicated and requires the use of the precious metal ruthenium in the reaction, resulting in high cost; (2) Existing technology two uses (dimethyl sulfide) trihydroboron and ( R Asymmetric reduction of 5,7-difluoro-3,4-dihydro-2H-1-benzopyran-4-one with 2-methyl-cbs-oxazolium borane yielded a product with an ee value >90%. S The method yields 5,7-difluoro-3,4-dihydro-2H-1-benzopyran-4-ol, but the process is cumbersome, especially as it requires more than one equivalent of expensive and difficult-to-recycle chiral reagents and flammable and irritating borane reagents. In addition, the chiral side chains prepared by this method have low ee values.

[0004] Bioreductase reductase methods have gained widespread attention due to their green and environmentally friendly nature. Existing technology three uses 5,7-difluoro-3,4-dihydro-2H-1-benzopyran-4-one as a raw material, reducing it to obtain racemic 5,7-difluoro-3,4-dihydro-2H-1-benzopyran-4-ol, which is then stereoselectively acylated using Novozym lipase (Novozym 435) and vinyl acetate to obtain a product with an ee value >99%. R )-5,7-fluoro-3,4-dihydro-2H-1-benzopyran-4-ol. The Novozymes lipase used in this method is scarce and expensive. Its enzyme reaction system uses ester or ether-based low-polarity, low-boiling-point organic solvents, which easily inactivate the protein-structured enzyme, resulting in a theoretical yield of only 50% and poor atom economy. Furthermore, the substrate concentration is low, which cannot meet industrial requirements. Regarding the asymmetric synthesis of carbonyl reductase (…R 4-chromanol compounds have not been reported, and therefore, there is a need to excavate carbonyl reductases with superior performance to meet the industrial needs of high substrate concentration, high catalytic reaction efficiency, high yield and simple operation. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art, and provide a carbonyl reductase mutant and its application in preparing chiral alcohol.

[0006] The technical solution of the present application is as follows: In a first aspect, the present application provides a carbonyl reductase mutant, which is obtained by mutating at least one of the following positions in the amino acid sequence shown as SEQ ID NO: 2: position 119, position 180, position 183, position 185, position 219, position 227 and position 236.

[0007] Preferably, the mutation at the 119th position is A119I or A119T or A119E or A119V or A119L or A119W or A119F or A119Q; the mutation at the 180th position is N180L or N180T or N180F or N180R or N180E or N180H or N180Y; the mutation at the 183rd position is Q183A or Q183F or Q183T or Q183D or Q183L; the mutation at the 185th position is Q185L or Q185I or Q185S or Q185A or Q185M; the mutation at the 219th position is Y219L or Y219T or Y219V or Y219A or Y219K or Y219H or Y219I; the mutation at the 227th position is F227S or F227A or F227P or F227L or F227Q or F227H; the mutation at the 236th position is W236F or W236V or W236T or W236P or W236I or W236L or W236M or W236A or W236H.

[0008] Preferably, the carbonyl reductase mutant is selected from any one of the following (1)~(24): (1) replacing alanine at the 119th position with isoleucine and replacing tryptophan at the 236th position with phenylalanine in the amino acid sequence shown as SEQ ID NO: 2; i.e. A119I / W236F; (2) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by tryptophan, the tyrosine at position 219 is replaced by alanine, and the phenylalanine at position 227 is replaced by serine; i.e. A119W / Y219A / F227S; (3) the asparagine at position 180 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by arginine, the glutamine at position 185 is replaced by methionine, and the tryptophan at position 236 is replaced by alanine; i.e. N180R / Q185M / W236A; (4) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by phenylalanine, the glutamine at position 183 is replaced by aspartic acid, and the phenylalanine at position 227 is replaced by leucine; i.e. A119F / Q183D / F227L (5) the asparagine at position 180 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by glutamic acid, the tyrosine at position 219 is replaced by lysine, the phenylalanine at position 227 is replaced by proline, and the tryptophan at position 236 is replaced by alanine; i.e. N180E / Y219K / F227P / W236A; (6) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by isoleucine, the asparagine at position 180 is replaced by threonine, the tyrosine at position 219 is replaced by leucine, and the tryptophan at position 236 is replaced by valine; i.e. A119I / Q183T / Y219L / W236V; (7) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by glutamic acid, the asparagine at position 180 is replaced by phenylalanine, the glutamine at position 185 is replaced by leucine, the phenylalanine at position 227 is replaced by serine, and the tryptophan at position 236 is replaced by proline; i.e. A119E / N180F / Q185L / F227S / W236P; (8) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by threonine, the glutamine at position 183 is replaced by alanine, the tyrosine at position 219 is replaced by threonine, and the tryptophan at position 236 is replaced by threonine; i.e. A119T / Q183A / Y219T / W236T; (9) the alanine at position 119 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by glutamine, the asparagine at position 180 is replaced by histidine, the glutamine at position 183 is replaced by alanine, and the phenylalanine at position 227 is replaced by isoleucine; i.e. A119Q / N180H / Q183A / F227I; (10) the alanine at position 119, the glutamine at position 183, the glutamine at position 185, and the tyrosine at position 219 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by valine, threonine, methionine, and leucine, respectively; i.e. A119V / Q183T / Q185M / Y219L; (11) the asparagine at position 180, the glutamine at position 183, the glutamine at position 185, the phenylalanine at position 227, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by leucine, alanine, isoleucine, alanine, and isoleucine, respectively; i.e. N180L / Q183A / Q185I / F227A / W236I; (12) the asparagine at position 180, the glutamine at position 183, and the glutamine at position 185 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by leucine, threonine, and serine, respectively; i.e. N180L / Q183T / Q185S; (13) the alanine at position 119, the glutamine at position 183, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by isoleucine, alanine, and histidine, respectively; i.e. A119I / Q183A / W236H; (14) the alanine at position 119, the glutamine at position 183, the tyrosine at position 219, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by histidine, alanine, histidine, and leucine, respectively; i.e. A119H / Q183A / Y219H / W236L; (15) the alanine at position 119, the asparagine at position 180, the glutamine at position 185, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by leucine, phenylalanine, alanine, and methionine, respectively; i.e. A119L / N180F / Q185A / W236M; (16) the alanine at position 119, the asparagine at position 180, the glutamine at position 183, the tyrosine at position 219, and the phenylalanine at position 227 of the amino acid sequence set forth in SEQ ID No. 2 are replaced by leucine, threonine, alanine, valine, and proline, respectively; i.e. A119L / N180T / Q183A / Y219V / F227P; (17) the alanine at position 119, the asparagine at position 180, the glutamine at position 183, the glutamine at position 185, the phenylalanine at position 227, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by phenylalanine, tyrosine, leucine, alanine, valine, and leucine, respectively; i.e., A119F / N180Y / Q183L / Q185A / F227V / W236L; (18) the alanine at position 119, the asparagine at position 180, the glutamine at position 185, the phenylalanine at position 227, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by glutamine, alanine, isoleucine, glutamine, and leucine, respectively; i.e., A119Q / N180A / Q185I / F227Q / W236L; (19) the alanine at position 119, the asparagine at position 185, the tyrosine at position 219, the phenylalanine at position 227, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by glutamic acid, alanine, isoleucine, histidine, and isoleucine, respectively; i.e., A119E / Q185A / Y219I / F227H / W236I; (20) the alanine at position 119, the asparagine at position 180, the tyrosine at position 219, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by isoleucine, threonine, leucine, and isovaline, respectively; i.e., A119I / N180T / Y219L / W236V; (21) the alanine at position 119, the glutamine at position 183, the glutamine at position 185, the phenylalanine at position 227, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by valine, threonine, isoleucine, alanine, and isoleucine, respectively; i.e., A119V / Q183T / Q185I / F227A / W236I; (22) the asparagine at position 180, the asparagine at position 183, and the asparagine at position 185 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by leucine, alanine, and serine, respectively; i.e., N180L / Q183A / Q185S; (23) the alanine at position 119, the glutamine at position 183, the glutamine at position 185, and the tryptophan at position 236 of the amino acid sequence set forth in SEQ ID NO: 2 are replaced by isoleucine, phenylalanine, alanine, and leucine, respectively; i.e., A119I / Q183F / Q185A / W236L; (24) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with leucine, asparagine at position 180 with tyrosine, glutamine at position 185 with isoleucine, phenylalanine at position 227 with proline, and tryptophan at position 236 with leucine; that is, A119L / N180Y / Q185I / F227P / W236L.

[0009] Preferably, the nucleotide sequence of the gene encoding the carbonyl reductase mutant is shown in SEQ ID NO:1.

[0010] In a second aspect, the present invention provides a carbonyl reductase catalyst, the catalyst comprising any one of the following forms: 1) a transformed somatic cell expressing the carbonyl reductase mutant or its frozen stem cells; 2) a lysate of the transformed somatic cell expressing the carbonyl reductase mutant or its lyophilized enzyme powder.

[0011] Thirdly, the present invention provides the application of the carbonyl reductase mutant or the catalyst described herein in the preparation of chiral alcohols by catalyzing the asymmetric reduction reaction of carbonyl compounds.

[0012] Preferably, the chiral alcohol comprises ( R )-4-chromobolols, the ( R )-4-chromoalkanols have the following structures: R is selected from at least one of halogen, C1-C8 alkyl or cycloalkyl, thienyl, furanyl, naphthyl and pyridyl.

[0013] Preferably, the carbonyl compound is selected from any one of the following compounds 2a to 2y: .

[0014] Preferably, the asymmetric reduction reaction is carried out in a solvent in the presence of NADPH. Further, NADPH catalyzes the reaction of glucose and NADP by glucose dehydrogenase (GDH). + It is produced by a transformation reaction.

[0015] Fourthly, the present invention provides a ( R The preparation method of 4-chromoalkanols includes the following steps: The carbonyl reductase mutant or the catalyst is mixed with glucose dehydrogenase, and then mixed with a carbonyl compound, solvent, hydrogen donor, and cofactor to carry out an asymmetric reduction reaction, yielding ( R )-4-chromopropanols.

[0016] Preferably, the method comprises the following steps: S1, preparing a first engineered bacterium containing the carbonyl reductase mutant and a second engineered bacterium containing glucose dehydrogenase; S2, inducing expression and purification of the first and second engineered bacteria respectively to obtain cell supernatant containing the carbonyl reductase mutant and cell supernatant containing glucose dehydrogenase; S3, mixing the cell supernatant containing the carbonyl reductase mutant and the cell supernatant containing glucose dehydrogenase, and then mixing with a carbonyl compound (the structural formula of which is shown as compound 2 in the reaction formula), a solvent, a hydrogen donor and a coenzyme to perform an asymmetric reduction reaction to obtain a (R)-4-chromanemethanol compound (the structural formula of which is shown as compound 1 in the reaction formula). R

[0017] The reaction formula is as follows: Further, the solvent is a mixed solvent of a phosphate buffer and a cosolvent; the volume ratio of the phosphate buffer to the cosolvent is 2:1 to 19:1 (preferably 9:1); the concentration of the phosphate buffer is 50 to 300 mM, and the pH is 6.0 to 8.0; the cosolvent is selected from one or more of a high dielectric constant solvent, an aromatic solvent, a nonpolar solvent and a polar solvent; the high dielectric constant solvent is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide and N,N-dimethylacetamide; the aromatic solvent is selected from one or more of benzene, toluene, ethylbenzene, chlorobenzene and bromobenzene; the nonpolar solvent is n-hexane and / or cyclohexane; and the polar solvent is selected from one or more of acetonitrile, ethyl acetate, dichloromethane, 1,2-dichloroethane, methanol, ethanol and isopropanol. Preferably, the solvent is a mixed solvent of DMSO and phosphate buffer, and the volume ratio is 10:90.

[0018] Further, the hydrogen donor is selected from one or more of glucose and isopropanol, and the coenzyme is selected from one or more of NADP+ and NAD+.

[0019] Further, in the reaction system, the concentration of the carbonyl compound is 10 to 1000 mM, the amount of the mutant or the catalyst is 0.01 g wet weight / L to 25 g wet weight / L, the reaction temperature is 20 to 40°C, and the pH is 6.0 to 8.0.

[0020] The present application has at least one of the following beneficial effects: The present application constructs an engineered bacterium containing a carbonyl reductase Km SDR and its mutant, or an engineered bacterium containing a carbonyl reductase Km SDR and its mutant, and applies it to catalytic reduction of chromanone compounds, thereby obtaining a (R)-4-chromanemethanol compound (such as (R)-4-chromanemethanol). R (R)-4-chromanemethanol​R The production of the (-)-5,7-difluoro-3,4-dihydro-2H-1-benzopyran-4-ol compound provides a new biological preparation approach. Compared with other preparation methods, the obtained carbonyl reductase-containing mutant and glucose dehydrogenase (GDH)-containing engineered bacteria prepared by using the method of the present application have the advantages of environmental friendliness, simple operation, easy industrial amplification, etc., and can obtain hydroxyl compounds with wide substrate universality, high substrate concentration, high stereoselectivity, high yield, and have good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For the gene engineering bacteria induced expression in the preferred embodiment 2 of the present application Km SDR cell supernatant protein SDS-PAGE electrophoresis diagram. Wherein, M: protein marker, 1: gene engineering induced expression of Km SDR cell supernatant protein SDS-PAGE electrophoresis diagram. Wherein, M: protein marker, 1: gene engineering induced expression of

[0022] Figure 2 For the racemic product in the preferred embodiment 15 of the present application R High performance liquid chromatogram of (-)-4-chroman-ol compound 1r.

[0023] Figure 3 For the racemic product in the preferred embodiment 15 of the present application R High performance liquid chromatogram of (-)-4-chroman-ol compound 1r.

[0024] Figure 4 For the racemic product in the preferred embodiment 15 of the present application R Nuclear magnetic hydrogen spectrum of (-)-4-chroman-ol compound 1r.

[0025] Figure 5 For the racemic product in the preferred embodiment 15 of the present application R Nuclear magnetic carbon spectrum of (-)-4-chroman-ol compound 1r.

[0026] Figure 6 For the racemic product in the preferred embodiment 15 of the present application R Nuclear magnetic fluorine spectrum of (-)-4-chroman-ol compound 1r. DETAILED DESCRIPTION

[0027] In order to make the technical problems solved by the present application, the technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0028] Example 1 Carbonyl reductase Km Construction of SDR mutant 1. Obtain carbonyl reductase KmSDR The carbonyl reductase in this embodiment Km SDR is derived from Max Kluyveromyces yeast ( Kluyveromyces marxianus After searching for the homology of carbonyl reductases reported in the literature, the inventors synthesized carbonyl reductases through Sangon Biotech (Shanghai) Co., Ltd. Km SDR gene sequence, carbonyl reductase Km The nucleotide sequence of the gene encoding SDR is shown in SEQ ID NO:1, carbonyl reductase. Km The amino acid sequence of SDR is shown in SEQ ID NO:2.

[0029] 2. Obtain carbonyl reductase Km SDR mutant Carbonyl reductase was constructed using a coherent analysis method. Km SDR mutation library: Km The SDR protein sequence (SEQ ID NO: 2) was used as a probe for sequence alignment, preferably selecting different protein sequences from thermophilic sources with more than 30% homology. Through ClustalX2 sequence alignment and Espript cognition analysis, a series of non-conserved residues were selected for single-point mutation. Corresponding mutation primers were designed, and plasmid pET28a- Km SDR (synthesized by a gene synthesis company) was used as a template, and PCR amplification was performed using high-fidelity polymerase (T8 DNA Polymerase). The PCR reaction conditions were as follows: 0.5 ~ 20 ng of template, 25 μL of PCR premix (2×T8 High-Fidelity Master Mix), 2.0 μL each of a pair of mutant primers (10 μM), and sterile ddH2O were added to a final volume of 50 μL in a 50 μL PCR reaction system. The PCR reaction program was as follows: (1) 98 ℃ pre-denaturation for 2 min, (2) 98 ℃ denaturation for 10 s, (3) Tm + 3 ~ 5 ℃ annealing for 10 s, (4) 72 ℃ extension for 90 s. Steps (1) ~ (3) were performed for a total of 28 cycles, and the PCR products were stored at 4 ℃. After the PCR products were verified by agarose gel electrophoresis, restriction endonuclease was added. Dpn I was digested at 37 °C for 2 h. The digestion products were then converted to... E.coli BL21(DE3) competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated statically at 37 ℃ for approximately 12 h. The resulting single colonies were then picked and transferred to LB tubes containing 50 μg / mL kanamycin, and cultured with shaking at 37 ℃ for 12 h. Plasmids were extracted and DNA sequencing was performed for verification. Confirmed mutant strains were then further fermented.

[0030] The carbonyl reductase obtained in this example Km The SDR mutant is any one of the invention contents (1) ~ (24).

[0031] Table 1 Carbonyl reductase Km Primers used for construction of SDR mutant Example 2 Construction of genetically engineered bacteria and induction of expression The laboratory-preserved pET28a-GDH plasmid (wherein the amino acid sequence of GDH is shown as SEQ ID NO: 95, and the nucleotide sequence of GDH is shown as SEQ ID NO: 96) and the plasmid pET28a-SDR of the correct mutant strain in Example 1 were used to transform the expression host BL21 (DE3) by the method of Example 1. Km SDR was transformed into the expression host respectively E. coli BL21 (DE3), the positive transformants appeared on the LB solid culture medium containing kanamycin were inoculated into 3 mL LB liquid medium containing 100 μg / mL kanamycin, and cultured overnight at 37 ℃, 200 rpm; 1% of the inoculum was inoculated into 2 L of 500 mL of the above LB liquid medium, and cultured at 37 ℃, 200 rpm for 4 ~ 5 h; when the culture OD reached about 0.6 ~ 0.8, IPTG was added to a final concentration of 0.1 mmol / L, and the culture was induced overnight at 16 ℃, 100 rpm. The bacterial cells were collected by centrifugation at 9,000 rpm, which could be directly used as catalyst or freeze-dried or resuspended in 20 mL sterile water, ultrasonically broken, and the supernatant was collected by centrifugation at 15,000 rpm, which was the crude enzyme solution, thereby obtaining the GDH crude enzyme solution and the carbonyl reductase mutant crude enzyme solution respectively.

[0032] The crude enzyme solution containing the carbonyl reductase mutant was purified using a Ni-NTA column: The column was equilibrated by washing with 5 column volumes of binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.0), followed by loading of the sample. Unbound protein was washed away with 10 column volumes of binding buffer, and the target protein was eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.0). The eluent was desalted and concentrated using a 30 kDa ultrafiltration tube (Amicon Ultra 15, Millipore) to obtain recombinant carbonyl reductase (carbonyl reductase mutant) with a purity of over 90%. The SDS-PAGE of the recombinant carbonyl reductase (carbonyl reductase mutant) is shown below. Figure 1 As shown.

[0033] Example 3 Km The SDR mutant (KmSDR-A119I / W236F) catalyzes the reduction of substrate 2a at 100 mmol / L. The reaction formula is as follows: To 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.0) containing 100 mmol / L substrate 2a (15.1 g / L), methanol (10% v / v), and 200 mmol / L glucose (36.2 g / L), 10 g / L of the solution obtained according to Example 2 was added. Km SDR-A119I / W236F crude enzyme solution, 50 mg / L GDH crude enzyme solution obtained in Example 2, and 0.1 mmol / L NADP+ were added. The reaction was carried out at 30 °C with magnetic stirring, and the pH was maintained at 7.0 during the reaction by adding 1.0 mol / L sodium phosphate solution. After the reaction was completed, the mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 1a.

[0034] Analysis by chiral HPLC (Chiracel® IF, cyclohexane / isopropanol as mobile phase) showed an ee value >99% and a dr value >60:1. 1 H NMR (400 MHz, CDCl3) δ 6.46 – 6.35 (m, 2H), 4.99 (s, 1H), 4.33 – 4.20(m, 2H), 2.24 (s, 1H), 2.09 – 1.94 (m, 2H). 19F NMR (376 MHz, CDCl3) δ -109.40,-117.10. 13 C NMR (101 MHz, CDCl3) δ 109.04 (d, J = 24.0 Hz), 100.22 (dd, J =24.3, 3.6 Hz), 95.98 (t, J = 26.2 Hz), 62.00, 56.98 (d, J = 4.4 Hz).

[0035] Example 4 Km SDR mutant ( Km SDR-A119I / Q183T / Y219L / W236V) catalytically reduced substrate 2b at a concentration of 500 mmol / L. The reaction formula is as follows: To 10 mL of sodium phosphate buffer (50 mmol / L, pH 6.5) containing 300 mM substrate 2b (50.4 g / L), acetonitrile (5% v / v), and 900 mM glucose (164 g / L), 10 g / L of the solution obtained according to Example 2 was added. Km SDR-A119I / Q183T / Y219L / W236V crude enzyme solution, 100 mg / L GDH crude enzyme solution obtained in Example 2, and 0.1 mmol / L NADP + The reaction was carried out at 20 °C with magnetic stirring, and the pH was maintained at 6.5 by adding 0.5 mol / L sodium carbonate solution during the reaction. After the reaction was completed, equal volumes of ethyl acetate were added for extraction twice, the extracts were combined, anhydrous sodium sulfate was added and dried overnight, filtered and evaporated to dryness to obtain compound 1b.

[0036] Analysis by chiral HPLC (Chiracel® AD-H, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 49:1. 1 H NMR (400 MHz, CDCl3) δ 7.30-7.32 (m, 1H), 6.96 – 7.02 (m, 1H), 6.49 – 6.52 (m, 1H), 5.23 – 4.74 (m, 1H), 4.33 – 4.37 (m, 1H), 4.17 – 4.22(m, 1H), 2.72 – 2.73 (d, 1H), 2.17 – 2.23 (m, 1H), 1.98 – 2.04 (m, 1H). 9F NMR (376 MHz, CDCl3) δ -108.3. 13 C NMR (101 MHz, CDCl3) δ 162.94, 160.97, 156.54(d, J = 8.1 Hz), 127.64 (d, J = 8.3 Hz), 123.77 (d, J = 4.1 Hz), 110.21 (d, J= 22.4 Hz), 101.61 (d, J = 25.8 Hz), 64.53 (d, J = 104.5 Hz), 32.20.

[0037] Example 5 Km SDR mutant ( Km SDR-A119Q / N180H / Q183A / F227I) catalytically reduced 100 mmol / L substrate for 2 days. The reaction formula is as follows: To 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.0) containing 100 mM substrate 2d (22.6 g / L), methanol (15% v / v), and 120 mM glucose (21.6 g / L), 1 g / L of the solution obtained according to Example 2 was added. Km SDR-A119Q / N180H / Q183A / F227I crude enzyme solution, 100 mg / L GDH crude enzyme solution obtained in Example 2, and 0.1 mmol / L NADP. + The reaction was carried out at 20 °C with magnetic stirring, and the pH was maintained at 7.0 during the reaction by adding 1.0 mol / L sodium carbonate solution. After the reaction was completed, the mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 1d.

[0038] Analysis by chiral HPLC (Chiracel® IJ-H, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 98% and a dr value > 53:1. 1 H NMR (400 MHz, CDCl3) δ 7.33 (d, 2H), 7.16 (dd, J = 1.5, 1H), 4.93(m, 1H), 4.34 (m, 1H), 4.19 (m, 1H), 2.73 (d, 1H), 2.20 (m, 1H), 2.01 (m,1H). 13C NMR (101 MHz, CDCl3) δ 155.19, 127.67, 126.33, 125.40, 120.66, 114.70, 65.27, 63.92, 32.03.

[0039] Example 6 Km SDR mutant ( Km SDR-A119T / Q183A / Y219T / W236T) catalytically reduces 2g of substrate at 500 mmol / L. The reaction formula is as follows: To a 10 mL sodium phosphate buffer (300 mmol / L, pH 6.5) containing 2 g (81 g / L) of 500 mM substrate, methanol (15% v / v), and 2000 mM glucose (180 g / L), 20 g / L of the solution obtained according to Example 2 was added. Km SDR-A119T / Q183A / Y219T / W236T crude enzyme solution, 5 g / L GDH crude enzyme solution obtained in Example 2, and 0.05 mmol / L NADP+. The reaction was carried out at 25 °C with magnetic stirring, and the pH was maintained at 6.5 during the reaction by adding 0.5 mol / L sodium carbonate solution. After the reaction was completed, equal volumes of ethyl acetate were added for extraction twice, the extracts were combined, anhydrous sodium sulfate was added and dried overnight, filtered and evaporated to dryness to obtain 1 g of the compound.

[0040] Analysis by chiral HPLC (Chiracel® OJ, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value = 25:1. 1 H NMR (400 MHz, CDCl3) δ 7.22 – 7.13 (m, 1H), 7.11 – 6.98 (m, 2H), 5.09 – 4.71 (m, 1H), 4.39 (m, 1H), 4.32 – 3.96 (m, 1H), 3.03 (d, J = 6.0 Hz,1H), 2.28 (m, 1H), 2.11 (m, 1H). 19 F NMR (376 MHz, CDCl3) δ -109.3. 13C NMR (100MHz, CDCl3) δ 152.98, 150.98, 143.94 (d, J = 12.4 Hz), 130.26 (d, J = 6.7Hz), 127.71, 115.14, 66.15 (d, J = 3.6 Hz), 65.46 (d, J = 4.5 Hz), 29.54.

[0041] Example 7 Km SDR mutant ( Km SDR-A119E / N180F / Q185L / F227S / W236P) catalytically reduced 1000 mmol / L of substrate 2j The reaction formula is as follows: To 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.5) containing 1 M substrate 2j (182.2 g / L), dichloromethane (20% v / v), and 1.5 M glucose (270 g / L), 25 g / L of the solution obtained according to Example 2 was added. Km SDR-A119E / N180F / Q185L / F227S / W236P crude enzyme solution, 20 g / L GDH crude enzyme solution obtained in Example 2, and 0.1 mmol / L NADP. + The reaction was carried out at 35 °C with magnetic stirring, and the pH was maintained at 7.5 during the reaction by adding 0.5 mol / L sodium carbonate solution. After the reaction was completed, the mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 1j.

[0042] Analysis by chiral HPLC (Chiracel® AS-H, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 49:1. 1 H NMR (400 MHz, CDCl3) δ 7.43 – 7.05 (m, 2H), 6.84 (m, J = 8.0,1H), 5.10 (m, 1H), 4.34 (m, 1H), 4.19 (m, 1H), 2.89 (d, 1H), 2.67 – 2.31 (m,1H), 2.19 (m,1H). 13 C NMR (100 MHz, CDCl3) δ 155.36, 132.02, 129.04, 125.09, 123.83, 112.06, 64.10, 63.95, 32.05.

[0043] Example 8 Km SDR mutant ( Km SDR-A119F / Q183D / F227L) catalytically reduced 2L of substrate at a concentration of 500 mmol / L. The reaction formula is as follows: To a 10 mL sodium phosphate buffer (150 mmol / L, pH 7.5) containing 2 L of 500 mM substrate (81.0 g / L), ethanol (15% v / v), and 900 mM glucose (164 g / L), 50 g / L of the solution obtained according to the method in Example 2 was added. Km SDR-A119F / Q183D / F227L crude enzyme solution, 10 g / L GDH crude enzyme solution obtained in Example 2, and 0.25 mmol / L NADP + The reaction was carried out at 35 °C with magnetic stirring, and the pH was maintained at 7.5 during the reaction by passing a 4 mol / L sodium carbonate solution. After the reaction was completed, the mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered, and evaporated to dryness to give compound 1L.

[0044] Analysis by chiral HPLC (Chiracel® OD-H, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 90:1. 1 H NMR (400 MHz, CDCl3) δ 7.20 (m, 1H), 7.01 – 6.84 (m, 1H), 6.74 (d, 1H), 5.07 – 4.67 (m, 1H), 4.34 (m, 1H), 4.19 (m, 1H), 2.73 (d, 1H), 2.31(s, 3H), 2.21 (m, 1H), 2.03 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 154.68, 138.44, 126.56, 124.94, 123.66, 112.36, 65.37, 63.91, 31.98, 21.52.

[0045] Example 9 Km SDR mutant ( Km SDR-A119V / Q183T / Q185I / F227A / W236I) catalytically reduces 800 mmol / L of substrate 2n. The reaction formula is as follows: In 10 mL of sodium phosphate buffer (200 mmol / L, pH 6.0) containing 800 mM of substrate 2n (161.6 g / L), isopropanol (30% v / v) and 1 M glucose (216 g / L), 20 g / L of the crude enzyme solution of SDR-A119V / Q183T / Q185I / F227A / W236I obtained according to the procedure of Example 2 was added. Km SDR-A119V / Q183T / Q185I / F227A / W236I crude enzyme solution, 3 g / L GDH crude enzyme solution obtained in Example 2 and 0.01 mmol / L of NADP + The reaction was carried out at 25 °C under magnetic stirring and the pH was controlled at 6.0 by means of a 0.5 mol / L sodium carbonate solution. After the reaction was completed, an equivalent amount of ethyl acetate was added and the extraction was carried out twice. The extracts were combined, anhydrous sodium sulfate was added and left to dry overnight. The mixture was filtered and the solvent was evaporated under reduced pressure to obtain compound 1n.

[0046] The ee value was > 99% and the dr value was > 98:1 by analysis by chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase). 1 H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 2.5 Hz, 1H), 7.07 (m, 1H), 6.86(d, J = 8.6 Hz, 1H), 4.96 – 4.76 (m, 1H), 4.32 (m, 1H), 4.17 (m, 1H), 2.97(d, J = 6.0 Hz, 1H), 2.33 (d, J = 0.7 Hz, 3H), 2.32 – 2.25 (m, 1H), 2.12 (m,1H). 13 C NMR (100 MHz, CDCl3) δ 152.55, 131.10, 128.97, 127.50, 127.22, 112.46,65.37, 63.03, 29.68, 21.33。

[0047] Example 10 Km SDR mutant (A119I / Q183A / W236H) catalyzing the reduction of 400 mmol / L of substrate 2p Km SDR-A119I / Q183A / W236H) catalyzing the reduction of 400 mmol / L of substrate 2p The reaction scheme is as follows: In 10 mL of sodium phosphate buffer (100 mmol / L, pH 6.5) containing 400 mM of substrate 2p (70.4 g / L), acetonitrile (10% v / v) and 500 mM glucose (90 g / L), 30 g / L of the crude enzyme solution of SDR-A119I / Q183A / W236H obtained according to the procedure of Example 2 was added.Km SDR-A119I / Q183A / W236H crude enzyme, 2 g / L of GDH crude enzyme obtained in Example 2 and 0.15 mmol / L of NADP + The reaction was carried out at 20 °C under magnetic stirring, and the pH was controlled at 6.5 by using 0.5 mol / L sodium carbonate solution during the reaction. After the reaction, equal amount of ethyl acetate was added and extracted twice. The combined extract was dried over anhydrous sodium sulfate overnight, filtered and concentrated to give compound 1p.

[0048] The ee value was >99% and the dr value was >45:1 by chiral HPLC (Chiracel® IF, cyclohexane / isopropanol as mobile phase) analysis. 1 H NMR (400 MHz, CDCl3) δ 7.14 (d, 1H), 6.72 (s, 1H), 5.06 - 4.74 (m, 1H), 4.34 (m, 1H), 4.19 (m, 1H), 2.97 (d, 1H), 2.37 (s, 3H), 2.20 (s, 3H), 2.12 - 2.24 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 152.38, 137.39, 130.88, 127.66, 124.53, 112.68, 65.47, 63.91, 29.68, 19.60, 19.22.

[0049] Example 11 Km SDR mutants (Example 2) Km SDR-N180L / Q183A / Q185S reduced 90 mmol / L of substrate 2r The reaction scheme is as follows: In 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.5) containing 90 mM of substrate 2r (16.6 g / L), 1,2-dichloroethane (10% v / v) and 180 mM of glucose (32.4 g / L), 20 g / L of SDR-N180L / Q183A / Q185S crude enzyme obtained in Example 2 and 0.03 mmol / L of NADP were added. Km SDR-N180L / Q183A / Q185S reduced 90 mmol / L of substrate 2r +The reaction was carried out at 30 °C under magnetic stirring, and the pH was controlled at 7.5 by 2 mol / L sodium carbonate solution during the reaction. After the reaction, equal amount of ethyl acetate was added for extraction twice, the extract was combined, anhydrous sodium sulfate was added for drying overnight, filtered and rotary evaporated to give compound 1r.

[0050] The ee value was >99% and the dr value was >98:1 by chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase) analysis. 1 H NMR (400 MHz, CDCl3) δ 6.82 (m, J = 12.1, 2.1 Hz, 1H), 6.50 (dd, J= 12.1, 2.2 Hz, 1H), 5.07 (m, J = 6.8, 5.9, 4.8, 3.9 Hz, 1H), 4.36 (m, J =11.2, 6.5, 3.8 Hz, 1H), 4.21 (m, J = 11.3, 6.5, 3.8 Hz, 1H), 3.04 (d, J = 5.9Hz, 1H), 2.33 (m, J = 14.0, 6.4, 3.8 Hz, 1H), 2.15 (m, J = 13.9, 6.6, 3.8 Hz,1H). 13 C NMR (100 MHz, CDCl3) δ δ 163.19 (d, J = 15.0 Hz), 161.92 – 160.80 (m),159.09 (d, J = 12.2 Hz), 157.40 (dd, J = 7.9, 6.4 Hz), 110.47 (dd, J = 13.9,4.9 Hz), 100.16 (dd, J = 26.6, 24.4 Hz), 99.49 (dd, J = 25.8, 5.0 Hz), 64.03,31.89 (d, J = 3.1 Hz)。

[0051] Example 12 Km SDR mutant (A119I / Q183F / Q185A / W236L) catalyzed the reduction of 50 mol / L of substrate 2u Km SDR mutant (A119I / Q183F / Q185A / W236L) catalyzed the reduction of 50 mol / L of substrate 2u The reaction scheme is as follows: In 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.0) containing 50 mM of substrate 2u (9.4 g / L), ethyl acetate (33% v / v) and 60 mM of glucose (10.8 g / L), 1.23 g / L of the SDR-A119I / Q183F / Q185A / W236L crude enzyme solution obtained according to the method of Example 2 was added. Km SDR-A119I / Q183F / Q185A / W236L crude enzyme solution, 10 mg / L GDH crude enzyme solution obtained in Example 2 and 0.12 mmol / L of NADP + The reaction was carried out at 40 °C under magnetic stirring, and the pH was controlled at 6.0 by using 0.5 mol / L potassium carbonate solution during the reaction. After the reaction, equal amount of ethyl acetate was added for extraction twice, the extract was combined, anhydrous sodium sulfate was added for drying overnight, and the filtrate was rotary evaporated to obtain compound 1u.

[0052] The ee value was ≥98%, and the dr value was >89:1 by chiral HPLC (Chiracel® IA, cyclohexane / isopropanol as mobile phase) analysis. 1 H NMR (400 MHz, CDCl3) δ 7.99 – 7.87 (m, 1H), 7.84 (d, J = 1.4 Hz,1H), 7.06 (t, J = 1.9 Hz, 1H), 7.03 (s, 1H), 5.02 – 4.73 (m, 1H), 4.34 (ddd,J = 11.4, 6.6, 3.8 Hz, 1H), 4.19 (ddd, J = 11.2, 6.6, 3.8 Hz, 1H), 2.97 (d, J= 6.0 Hz, 1H), 2.30 (ddt, J = 14.2, 6.4, 3.9 Hz, 1H), 2.12 (dtd, J = 14.1,6.6, 3.8 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ δ 155.41, 155.01, 145.85, 123.97,123.71, 121.07, 106.98, 96.89, 64.52, 64.02, 29.99。

[0053] Example 13 Km SDR mutant (A119L / N180F / Q185A / W236M) catalyzing the reduction of 200 mmol / L of substrate 2w Km SDR-A119L / N180F / Q185A / W236M) catalyzing the reduction of 200 mmol / L of substrate 2w The reaction formula is as follows: In 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.0) containing 200 mM of substrate 2w (37.4 g / L), ethanol (20% v / v) and 300 mM glucose (54 g / L), 8 g / L of SDR-A119L / N180F / Q185A / W236M crude enzyme solution obtained in the method of Example 2 was added. Km SDR-A119L / N180F / Q185A / W236M crude enzyme solution, 100 mg / L of GDH crude enzyme solution obtained in Example 2 and 0.01 mmol / L of NADP + The reaction was carried out at 30 °C under magnetic stirring, and the pH was controlled at 7.0 by 0.5 mol / L sodium carbonate solution during the reaction. After the reaction, equal amount of ethyl acetate was added for extraction twice, the extracted solution was combined, anhydrous sodium sulfate was added for drying overnight, and the filtrate was rotary evaporated to obtain compound 1w.

[0054] The ee value was ≥99%, and the dr value was >99:1 by chiral HPLC (Chiracel® IB, cyclohexane / isopropanol as mobile phase) analysis. 1 H NMR (400 MHz, CDCl3) δ 8.03 (t, J = 5.6 Hz, 1H), 7.50 – 7.06 (m,1H), 6.85 (s, 1H), 5.17 – 4.71 (m, 1H), 4.35 (ddd, J = 11.2, 6.6, 3.8 Hz,1H), 4.20 (ddd, J = 11.2, 6.4, 3.8 Hz, 1H), 3.64 (dt, J = 5.5, 0.7 Hz, 2H),2.97 (d, J = 5.9 Hz, 1H), 2.30 (ddt, J = 14.2, 6.4, 3.9 Hz, 1H), 2.12 (dtd, J= 14.1, 6.6, 3.8 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ 153.96, 152.07, 146.97,129.34, 125.93, 125.44, 104.63, 64.43, 64.07, 38.01, 29.62。

[0055] Example 14 Km SDR mutants ( Km SDR-A119E / Q185A / Y219I / F227H / W236I) catalyzed the reduction of 300 mmol / L of substrate 2y The reaction formula is as follows: To 10 mL of sodium phosphate buffer (150 mmol / L, pH 7.0) containing 300 mM substrate 2y (59.4 g / L), dichloromethane (10% v / v), and 320 mM glucose (57.6 g / L), 10 g / L of the solution obtained according to Example 2 was added. Km SDR-A119E / Q185A / Y219I / F227H / W236I crude enzyme solution, 1 g / L GDH crude enzyme solution obtained in Example 2, and 0.03 mmol / L NADP. + The reaction was carried out at 30 °C with magnetic stirring, and the pH was maintained at 7.0 during the reaction by adding 1.5 mol / L sodium carbonate solution. After the reaction was completed, the mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 1y.

[0056] Analysis by chiral HPLC (Chiracel® OJ-H, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 53:1. 1 H NMR (400 MHz, CDCl3) δ 7.89 (m, 1H), 7.85 – 7.79 (m, 1H), 7.74(m, 1H), 7.51 (m, 2H), 7.42 (d, 1H), 4.89 (m, 1H), 4.34 (m, 1H), 4.19 (m,1H), 2.97 (d, 1H), 2.34 (m, 1H), 2.16 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ153.22, 133.42, 128.46, 128.12, 126.72, 126.44, 126.14, 126.11, 125.37,107.57, 64.96, 63.91, 29.68.

[0057] Example 15 Km SDR mutant ( Km SDR-A119L / N180Y / Q185I / F227P / W236L) catalytically reduces 1 mol / L of substrate 2r The reaction formula is as follows: To 10 mL of sodium phosphate buffer (300 mmol / L, pH 6.5) containing 1 mol / L substrate 2r (196 g / L), methanol (30% v / v), and 2 mol / L glucose (360 g / L), 50 g / L of the solution obtained according to Example 2 was added. Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km Km KmSDR-A119L / N180Y / Q185I / F227P / W236L crude enzyme solution, 25 g / L GDH crude enzyme solution obtained in Example 2 and 0.3 mmol / L NADP + The reaction was carried out at 30 °C under magnetic stirring, and the pH was controlled at 6.5 by 4 mol / L sodium carbonate solution during the reaction. After the reaction, equal amount of ethyl acetate was added to extract twice, the extract was combined, anhydrous sodium sulfate was added to dry overnight, filtered and rotary evaporated to obtain compound 1r.

[0058] The substrate conversion rate was 96% measured by high performance liquid analysis, R ) 1r content was 97%. m.p. = 88.2 ~ 93.4 °C; 1 H NMR (400 MHz, CDCl3) δ 6.46 – 6.35 (m, 2H), 4.99 (s, 1H), 4.33 –4.20 (m, 2H), 2.24 (s, 1H), 2.09 – 1.94 (m, 2H). 19 F NMR (376 MHz, CDCl3) δ -109.40, -117.32. 13 C NMR (101 MHz, CDCl3) δ 109.04 (d, J = 24.0 Hz), 100.22 (dd, J = 24.3, 3.6 Hz), 95.98 (t, J = 26.2 Hz), 62.00, 56.98 (d, J = 4.4 Hz). [α] D 25 =+153.46 (c = 1.0, MeOH).

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is mutated at at least one of the following positions in the amino acid sequence shown as SEQ ID NO: 2: position 119, position 180, position 183, position 185, position 219, position 227, and position 236. 2.The carbonyl reductase mutant of claim 1, wherein, the mutation at the position 119 is A119I or A119T or A119E or A119V or A119L or A119W or A119F or A119Q; the mutation at the position 180 is N180L or N180T or N180F or N180R or N180E or N180H or N180Y; the mutation at the position 183 is Q183A or Q183F or Q183T or Q183D or Q183L; the mutation at the position 185 is Q185L or Q185I or Q185S or Q185A or Q185M; the mutation at the position 219 is Y219L or Y219T or Y219V or Y219A or Y219K or Y219H or Y219I; the mutation at the position 227 is F227S or F227A or F227P or F227L or F227Q or F227H; the mutation at the position 236 is W236F or W236V or W236T or W236P or W236I or W236L or W236M or W236A or W236H. 3.The carbonyl reductase mutant of claim 1, wherein the carbonyl reductase mutant is selected from any one of (1) to (24): (1) A119I / W236F; (2) A119W / Y219A / F227S; (3) N180R / Q185M / W236A; (4) A119F / Q183D / F227L; (5) N180E / Y219K / F227P / W236A; (6) A119I / Q183T / Y219L / W236V; (7) A119E / N180F / Q185L / F227S / W236P; (8) A119T / Q183A / Y219T / W236T; (9) A119Q / N180H / Q183A / F227I; (10) A119V / Q183T / Q185M / Y219L; (11) N180L / Q183A / Q185I / F227A / W236I; (12) N180L / Q183T / Q185S; (13) A119I / Q183A / W236H; (14) A119H / Q183A / Y219H / W236L; (15) A119L / N180F / Q185A / W236M; (16) A119L / N180T / Q183A / Y219V / F227P; (17) A119F / N180Y / Q183L / Q185A / F227V. ​ (18) A119Q / N180A / Q185I / F227Q / W236L; (19) A119E / Q185A / Y219I / F227H / W236I; (20) A119I / N180T / Y219L / W236V; (21) A119V / Q183T / Q185I / F227A / W236I; (22) N180L / Q183A / Q185S; (23) A119I / Q183F / Q185A / W236L; (24) A119L / N180Y / Q185I / F227P / W236L.

4. The carbonyl reductase mutant according to claim 1, wherein The nucleotide sequence of the gene encoding the carbonyl reductase mutant is shown in SEQ ID NO:

1.

5. A carbonyl reductase catalyst characterized in that, The catalyst includes any one of the following forms: 1) transformant cells expressing the carbonyl reductase mutant of any one of claims 1-4 or lyophilized cells thereof; 2) a broken solution of transformant cells expressing the carbonyl reductase mutant of any one of claims 1-4 or lyophilized enzyme powder thereof.

6. Use of the carbonyl reductase mutant of any one of claims 1-4 or the catalyst of claim 5 in catalyzing the asymmetric reduction reaction of a carbonyl compound to prepare a chiral alcohol.

7. Use according to claim 6, characterized in that, The chiral alcohols include ( R )-4-chromobolols, the ( R )-4-chromoalkanols have the following structures: ； R is selected from at least one of halogen, C1-C8 alkyl or cycloalkyl, thienyl, furanyl, naphthyl and pyridyl.

8. Use according to claim 6, characterized in that, The carbonyl compound is selected from any one of the following compounds 2a-2y: 。 9. A method of treating a subject having a disease or disorder associated with abnormal cell proliferation comprising administering to the subject a therapeutically effective amount of a compound of claim 1. R A method for producing a 4-chromanol compound, characterized by, comprising the following steps: The carbonyl reductase mutant according to any one of claims 1 to 4 or the catalyst according to claim 5 is mixed with glucose dehydrogenase, and then mixed with a carbonyl compound, solvent, hydrogen donor and cofactor to carry out an asymmetric reduction reaction to obtain ( R )-4-chromopropanols.

10. The preparation method of claim 9, wherein, The solvent is a mixed solvent of a phosphate buffer and a cosolvent; wherein the volume ratio of the phosphate buffer to the cosolvent is 2:1-19:1; the concentration of the phosphate buffer is 50-300 mM, and the pH is 6.0-8.0; the cosolvent is selected from at least one of a high dielectric constant solvent, an aromatic solvent, a non-polar solvent and a polar solvent; In the reaction system, the concentration of the carbonyl compound is 10-1000 mM, the amount of the carbonyl reductase mutant is 0.01 g wet weight / L-25 g wet weight / L, the reaction temperature is 20-40°C, and the pH is 6.0-8.0.

Images