A carbonyl reductase mutant and its use in preparing chiral alcohol

Abstract

The application discloses a carbonyl reductase mutant and application thereof in preparation of chiral alcohol, and relates to the technical field of biopharmacy and bio-chemical industry. The carbonyl reductase mutant is obtained by mutation of at least one site in SEQ ID NO:2, i.e. the 119th site, the 180th site, the 183rd site, the 185th site, the 219th site, the 227th site and the 236th site. The application also provides application of the carbonyl reductase mutant in preparation of chiral alcohol by catalyzing asymmetric reduction reaction of carbonyl compound and a preparation method of (R)‑4‑chrysantheol. R The application constructs the carbonyl reductase mutant, and adopts the same as a catalyst to prepare (R)‑4‑chrysantheol by a biological preparation route. R The method is environment-friendly, simple in operation, easy to be industrialized, high in substrate concentration, wide in substrate universality, high in product yield, and has a 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 No research reports have been published on 4-chromoalkanols. Therefore, there is a need to develop high-performance carbonyl reductases to meet the industrial requirements of high substrate concentration, high catalytic efficiency, high yield and simple operation. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a carbonyl reductase mutant and its application in the preparation of chiral alcohols.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a carbonyl reductase mutant obtained by mutation at least one of the following sites in the amino acid sequence shown in SEQ ID NO:2: site 119, site 180, site 183, site 185, site 219, site 227 and site 236.

[0008] Preferably, the mutation at the 119th site is A119I, A119T, A119E, A119V, A119L, A119W, A119F, or A119Q;

[0009] The mutation at the 180th site is N180L or N180T or N180F or N180R or N180E or N180H or N180Y;

[0010] The mutation at the 183rd site is Q183A, Q183F, Q183T, Q183D, or Q183L;

[0011] The mutation at the 185th site is Q185L, Q185I, Q185S, Q185A, or Q185M.

[0012] The mutation at position 219 is Y219L, Y219T, Y219V, Y219A, Y219K, Y219H, or Y219I;

[0013] The mutation at position 227 is either F227S, F227A, F227P, F227L, F227Q, or F227H.

[0014] The mutation at position 236 is W236F or W236V or W236T or W236P or W236I or W236L or W236M or W236A or W236H.

[0015] Preferably, the carbonyl reductase mutant is selected from any one of the following (1) to (24):

[0016] (1) Replace the alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with isoleucine and the tryptophan at position 236 with phenylalanine; that is, A119I / W236F;

[0017] (2) Replace the alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with tryptophan, replace the tyrosine at position 219 with alanine, and replace the phenylalanine at position 227 with serine; that is, A119W / Y219A / F227S.

[0018] (3) Replace the asparagine at position 180 of the amino acid sequence shown in SEQ ID NO:2 with arginine, the glutamine at position 185 with methionine, and the tryptophan at position 236 with alanine; that is, N180R / Q185M / W236A.

[0019] (4) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, glutamine at position 183 with aspartic acid, and phenylalanine at position 227 with leucine; that is, A119F / Q183D / F227L

[0020] (5) Replace the asparagine at position 180 of the amino acid sequence shown in SEQ ID NO:2 with glutamic acid, the tyrosine at position 219 with lysine, the phenylalanine at position 227 with proline, and the tryptophan at position 236 with alanine; that is, N180E / Y219K / F227P / W236A;

[0021] (6) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with isoleucine, glutamine at position 183 with threonine, tyrosine at position 219 with leucine, and tryptophan at position 236 with valine; that is, A119I / Q183T / Y219L / W236V;

[0022] (7) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid, asparagine at position 180 with phenylalanine, glutamine at position 185 with leucine, phenylalanine at position 227 with serine, and tryptophan at position 236 with proline; that is, A119E / N180F / Q185L / F227S / W236P;

[0023] (8) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with threonine, glutamine at position 183 with alanine, tyrosine at position 219 with threonine, and tryptophan at position 236 with threonine; that is, A119T / Q183A / Y219T / W236T;

[0024] (9) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with glutamine, asparagine at position 180 with histidine, glutamine at position 183 with alanine, and phenylalanine at position 227 with isoleucine; that is, A119Q / N180H / Q183A / F227I;

[0025] (10) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with valine, glutamine at position 183 with threonine, glutamine at position 185 with methionine, and tyrosine at position 219 with leucine; that is, A119V / Q183T / Q185M / Y219L;

[0026] (11) Replace asparagine at position 180 of the amino acid sequence shown in SEQ ID No. 2 with leucine, glutamine at position 183 with alanine, glutamine at position 185 with isoleucine, phenylalanine at position 227 with alanine, and tryptophan at position 236 with isoleucine; that is, N180L / Q183A / Q185I / F227A / W236I;

[0027] (12) Replace the asparagine at position 180 of the amino acid sequence shown in SEQ ID No. 2 with leucine, the glutamine at position 183 with threonine, and the glutamine at position 185 with serine; that is, N180L / Q183T / Q185S;

[0028] (13) Replace the alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine, replace the glutamine at position 183 with alanine, and replace the tryptophan at position 236 with histidine; that is, A119I / Q183A / W236H;

[0029] (14) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID No. 2 with histidine, glutamine at position 183 with alanine, tyrosine at position 219 with histidine, and tryptophan at position 236 with leucine; that is, A119H / Q183A / Y219H / W236L;

[0030] (15) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with leucine, asparagine at position 180 with phenylalanine, glutamine at position 185 with alanine, and tryptophan at position 236 with methionine; that is, A119L / N180F / Q185A / W236M;

[0031] (16) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with leucine, asparagine at position 180 with threonine, glutamine at position 183 with alanine; tyrosine at position 219 with valine, and phenylalanine at position 227 with proline; that is, A119L / N180T / Q183A / Y219V / F227P;

[0032] (17) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, asparagine at position 180 with tyrosine, glutamine at position 183 with leucine, glutamine at position 185 with alanine, and phenylalanine at position 227 with valine; that is, A119F / N180Y / Q183L / Q185A / F227V;

[0033] (18) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with glutamine, asparagine at position 180 with alanine, glutamine at position 185 with isoleucine, phenylalanine at position 227 with glutamine, and tryptophan at position 236 with leucine; that is, A119Q / N180A / Q185I / F227Q / W236L;

[0034] (19) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with glutamic acid, glutamine at position 185 with alanine, tyrosine at position 219 with isoleucine, phenylalanine at position 227 with histidine, and tryptophan at position 236 with isoleucine; that is, A119E / Q185A / Y219I / F227H / W236I;

[0035] (20) Replace the alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with isoleucine, the asparagine at position 180 with threonine, the tyrosine at position 219 with leucine, and the tryptophan at position 236 with valine; that is, A119I / N180T / Y219L / W236V;

[0036] (21) Replace alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with valine, glutamine at position 183 with threonine, glutamine at position 185 with isoleucine, phenylalanine at position 227 with alanine, and tryptophan at position 236 with isoleucine; that is, A119V / Q183T / Q185I / F227A / W236I;

[0037] (22) Replace the asparagine at position 180 of the amino acid sequence shown in SEQ ID NO:2 with leucine, the glutamine at position 183 with alanine, and the glutamine at position 185 with serine; that is, N180L / Q183A / Q185S;

[0038] (23) Replace the alanine at position 119 of the amino acid sequence shown in SEQ ID NO:2 with isoleucine, the glutamine at position 183 with phenylalanine, the glutamine at position 185 with alanine, and the tryptophan at position 236 with leucine; that is, A119I / Q183F / Q185A / W236L;

[0039] (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.

[0040] Preferably, the nucleotide sequence of the gene encoding the carbonyl reductase mutant is shown in SEQ ID NO:1.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Preferably, the carbonyl compound is selected from any one of the following compounds 2a to 2y:

[0045] .

[0046] 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.

[0047] Fourthly, the present invention provides a ( R The preparation method of 4-chromoalkanols includes the following steps:

[0048] 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.

[0049] Preferably, the method includes 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 purifying the first and second engineered bacteria respectively to obtain cell supernatants containing the carbonyl reductase mutant and cell supernatants containing glucose dehydrogenase; S3, mixing the cell supernatants containing the carbonyl reductase mutant and the cell supernatants containing glucose dehydrogenase, and then mixing them with a carbonyl compound (whose structural formula is shown in compound 2 in the reaction formula), a solvent, a hydrogen donor, and a cofactor to carry out an asymmetric reduction reaction to obtain ( R )-4-chromoalkanols (the structural formula of which is shown in compound 1 in the reaction formula).

[0050] The reaction formula is shown below:

[0051] Further, the solvent is a mixture of phosphate buffer and co-solvent; wherein the volume ratio of the phosphate buffer to the co-solvent 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 co-solvent is selected from one or more of high dielectric constant solvents, aromatic solvents, nonpolar solvents, and polar solvents; 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; 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 mixture of DMSO and phosphate buffer with a volume ratio of 10:90.

[0052] Furthermore, the hydrogen donor is selected from one or more of glucose and isopropanol, and the cofactor is selected from one or more of NADP+ and NAD+.

[0053] Furthermore, in the reaction system, the concentration of the carbonyl compound is 10 ~ 1000 mM, the amount of the mutant or the catalyst is 0.01 g wet weight / L ~ 25 g wet weight / L, the reaction temperature is 20 ~ 40 ℃, and the pH is 6.0 ~ 8.0.

[0054] This invention has at least one of the following beneficial effects:

[0055] This invention constructs a carbonyl reductase. Km SDR and its mutants, or by constructing carbonyl reductases Km Engineered bacteria of SDR and its mutants were used to catalytically reduce chromone compounds, for ( R )-4-chromoprotanols (such as ( R This invention provides a novel biosynthetic route for the production of 5,7-fluoro-3,4-dihydro-2H-1-benzopyran-4-ols. Compared to other methods, the carbonyl reductase mutant and glucose dehydrogenase (GDH) engineered bacteria prepared using this method are environmentally friendly, easy to operate, and readily scalable for industrial application. Furthermore, it yields hydroxyl compounds with broad substrate versatility, high substrate concentration, high stereoselectivity, and high yield, demonstrating promising prospects for industrial applications. Attached Figure Description

[0056] Figure 1 The preferred embodiment of the present invention, induced expression by genetically engineered bacteria, is shown in Example 2. KmSDS-PAGE electrophoresis image of proteins in SDR cell supernatant. Where M: protein marker, 1: protein expressed in a genetically engineered manner. Km SDR cell lysis supernatant.

[0057] Figure 2 The racemic product in the preferred embodiment 15 of the present invention ( R High performance liquid chromatogram of 1r, a 4-chromoprotanol compound.

[0058] Figure 3 In the preferred embodiment 15 of the present invention ( R High performance liquid chromatogram of 1r, a 4-chromoprotanol compound.

[0059] Figure 4 In the preferred embodiment 15 of the present invention ( R 1H NMR spectrum of 4-chromoalkanol compound 1r.

[0060] Figure 5 In the preferred embodiment 15 of the present invention ( R 1R NMR spectrum of 4-chromoalkanol compound 1r.

[0061] Figure 6 In the preferred embodiment 15 of the present invention ( R 1r NMR fluorine spectrum of 4-chromoprotanol compound 1r. Detailed Implementation

[0062] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0063] Example 1 Carbonyl reductase Km Construction of SDR mutants

[0064] 1. Obtaining carbonyl reductase Km SDR

[0065] 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. KmThe amino acid sequence of SDR is shown in SEQ ID NO:2.

[0066] 2. Obtain carbonyl reductase Km SDR mutant

[0067] 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.

[0068] The carbonyl reductase obtained in this embodiment Km The SDR mutant is any one of the invention contents (1) to (24).

[0069] Table 1 Carbonyl reductases Km Primers used in the construction of SDR mutants

[0070]

[0071] Example 2 Construction and Induced Expression of Genetically Engineered Bacteria

[0072] The pET28a-GDH plasmid (wherein the amino acid sequence of GDH is shown in SEQ ID NO:95 and the nucleotide sequence of GDH is shown in SEQ ID NO:96) and the plasmid pET28a- of the correct mutant strain in Example 1 were used. Km SDRs convert the expression host E. coli BL21 (DE3), a positive transformant appearing on LB solid medium plates containing kanamycin, was inoculated into 3 mL of LB liquid medium containing 100 μg / mL kanamycin and cultured overnight at 37 °C and 200 rpm. Then, 1% of the transformant was inoculated into 2 L of the same 500 mL LB liquid medium and cultured for another 4–5 h at 37 °C and 200 rpm. When the OD of the culture reached approximately 0.6–0.8, IPTG was added to a final concentration of 0.1 mmol / L, and the culture was induced overnight at 16 °C and 100 rpm. The bacterial cells were collected by centrifugation at 9,000 rpm. The cells could be used directly as a catalyst, or the stem cells could be frozen using a freeze dryer, or the cells could be resuspended in 20 mL of sterile water, sonicated, and the supernatant collected by centrifugation at 15,000 rpm. This supernatant was the crude enzyme solution, thus yielding crude GDH enzyme solution and crude carbonyl reductase mutant enzyme solution, respectively.

[0073] 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.

[0074] Example 3 Km The SDR mutant (KmSDR-A119I / W236F) catalyzes the reduction of substrate 2a at 100 mmol / L.

[0075] The reaction formula is as follows:

[0076] 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.

[0077] 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). 19 F 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).

[0078] Example 4 Km SDR mutant ( Km SDR-A119I / Q183T / Y219L / W236V) catalytically reduced substrate 2b at a concentration of 500 mmol / L.

[0079] The reaction formula is as follows:

[0080] 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.

[0081] 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). 9 F 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.

[0082] Example 5 Km SDR mutant ( Km SDR-A119Q / N180H / Q183A / F227I) catalytically reduced 100 mmol / L substrate for 2 days.

[0083] The reaction formula is as follows:

[0084] 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.

[0085] 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). 13 C NMR (101 MHz, CDCl3) δ 155.19, 127.67, 126.33, 125.40, 120.66, 114.70, 65.27, 63.92, 32.03.

[0086] Example 6 Km SDR mutant ( Km SDR-A119T / Q183A / Y219T / W236T) catalytically reduces 2g of substrate at 500 mmol / L.

[0087] The reaction formula is as follows:

[0088] 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. KmSDR-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.

[0089] 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. 13 C 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.

[0090] Example 7 Km SDR mutant ( Km SDR-A119E / N180F / Q185L / F227S / W236P) catalytically reduced 1000 mmol / L of substrate 2j

[0091] The reaction formula is as follows:

[0092] 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. KmSDR-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.

[0093] 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.

[0094] Example 8 Km SDR mutant ( Km SDR-A119F / Q183D / F227L) catalytically reduced 2L of substrate at a concentration of 500 mmol / L.

[0095] The reaction formula is as follows:

[0096] 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.

[0097] 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.

[0098] Example 9 Km SDR mutant ( Km SDR-A119V / Q183T / Q185I / F227A / W236I) catalytically reduces 800 mmol / L of substrate 2n.

[0099] The reaction formula is as follows:

[0100] To 10 mL of sodium phosphate buffer (200 mmol / L, pH 6.0) containing 800 mM substrate 2n (161.6 g / L), isopropanol (30% v / v), and 1 M glucose (216 g / L), 20 g / L of the solution obtained according to the method in 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 NADP. + The reaction was carried out at 25 °C with magnetic stirring, and the pH was maintained at 6.0 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 1n.

[0101] Analysis by chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 98:1. 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.

[0102] Example 10 Km SDR mutant ( Km SDR-A119I / Q183A / W236H) catalyzes the reduction of 400 mmol / L substrate 2p

[0103] The reaction formula is as follows:

[0104] To a 10 mL sodium phosphate buffer (100 mmol / L, pH 6.5) containing 400 mM substrate 2p (70.4 g / L), acetonitrile (10% v / v), and 500 mM glucose (90 g / L), 30 g / L of the solution obtained according to Example 2 was added. Km SDR-A119I / Q183A / W236H crude enzyme solution, 2 g / L GDH crude enzyme solution obtained in Example 2, and 0.15 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, 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 1p.

[0105] Analysis by chiral HPLC (Chiracel® IF, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 45:1. 1H 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.

[0106] Example 11 Km SDR mutant ( Km SDR-N180L / Q183A / Q185S) reduces 90 mmol / L of substrate 2r

[0107] The reaction formula is as follows:

[0108] To 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.5) containing 90 mM substrate 2r (16.6 g / L), 1,2-dichloroethane (10% v / v), and 180 mM glucose (32.4 g / L), 20 g / L of the solution obtained according to Example 2 was added. Km SDR-N180L / Q183A / Q185S crude enzyme solution, 8 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 7.5 by passing a 2 mol / L sodium carbonate solution during the reaction. 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 1r.

[0109] Analysis by chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 98:1. 1H 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).

[0110] Example 12 Km SDR mutant ( Km SDR-A119I / Q183F / Q185A / W236L) catalytically reduces 50 mol / L substrate 2u

[0111] The reaction formula is as follows:

[0112] To 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.0) containing 50 mM substrate 2u (9.4 g / L), ethyl acetate (33% v / v), and 60 mM glucose (10.8 g / L), 1.23 g / L of the solution obtained according to 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 NADP. +The reaction was carried out at 40 °C with magnetic stirring, and the pH was maintained at 6.0 during the reaction by adding 0.5 mol / L potassium 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 1u.

[0113] Analysis by chiral HPLC (Chiracel® IA, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 98% and a dr value > 89:1. 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 155.41, 155.01, 145.85, 123.97, 123.71, 121.07, 106.98, 96.89, 64.52, 64.02, 29.99.

[0114] Example 13 Km SDR mutant ( Km SDR-A119L / N180F / Q185A / W236M) catalytically reduces 200 mmol / L substrate by 2 weeks.

[0115] The reaction formula is as follows:

[0116] To a 10 mL sodium phosphate buffer (100 mmol / L, pH 7.0) containing 200 mM substrate (2 w, 37.4 g / L), ethanol (20% v / v), and 300 mM glucose (54 g / L), 8 g / L of the solution obtained according to the method in Example 2 was added. Km SDR-A119L / N180F / Q185A / W236M crude enzyme solution, 100 mg / L GDH crude enzyme solution obtained in Example 2, and 0.01 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 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 give 1 w of the compound.

[0117] Analysis by chiral HPLC (Chiracel® IB, cyclohexane / isopropanol as mobile phase) showed an ee value ≥ 99% and a dr value > 99:1. 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.

[0118] Example 14 Km SDR mutant ( Km SDR-A119E / Q185A / Y219I / F227H / W236I) catalytically reduces 300 mmol / L of substrate 2y

[0119] The reaction formula is as follows:

[0120] 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. KmSDR-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.

[0121] 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.

[0122] Example 15 Km SDR mutant ( Km SDR-A119L / N180Y / Q185I / F227P / W236L) catalytically reduces 1 mol / L of substrate 2r

[0123] The reaction formula is as follows:

[0124] 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 SDR-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 with magnetic stirring, and the pH was maintained at 6.5 by passing a 4 mol / L sodium carbonate solution during the reaction. 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 1r.

[0125] High-performance liquid chromatography analysis showed that the substrate conversion rate was 96%. R ) The 1r content is 97%. mp = 88.2 ~ 93.4 ℃; 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, Its amino acid sequence is obtained by mutating the sequence shown in SEQ ID NO:2, and the mutation method is selected from any of the following: (1) Replace A in the 119th position with I and W in the 236th position with F; that is, A119I / W236F; (2) Replace A in the 119th position with F, Q in the 183rd position with D, and F in the 227th position with L; that is, A119F / Q183D / F227L; (3) Replace A in the 119th position with I, Q in the 183rd position with T, Y in the 219th position with L, and W in the 236th position with V; that is, A119I / Q183T / Y219L / W236V; (4) Replace A in the 119th position with E, N in the 180th position with F, Q in the 185th position with L, F in the 227th position with S, and W in the 236th position with P; that is, A119E / N180F / Q185L / F227S / W236P; (5) Replace A in the 119th position with T, Q in the 183rd position with A, Y in the 219th position with T, and W in the 236th position with T; that is, A119T / Q183A / Y219T / W236T; (6) Replace A in the 119th position with Q, N in the 180th position with H, Q in the 183rd position with A, and F in the 227th position with I; that is, A119Q / N180H / Q183A / F227I; (7) Replace A in the 119th position with I, Q in the 183rd position with A, and W in the 236th position with H; that is, A119I / Q183A / W236H; (8) Replace A in the 119th position with L, N in the 180th position with F, Q in the 185th position with A, and W in the 236th position with M; that is, A119L / N180F / Q185A / W236M; (9) Replace A in the 119th position with E, Q in the 185th position with A, Y in the 219th position with I, F in the 227th position with H, and W in the 236th position with I; that is, A119E / Q185A / Y219I / F227H / W236I; (10) Replace A in the 119th position with V, Q in the 183rd position with T, Q in the 185th position with I, F in the 227th position with A, and W in the 236th position with I; that is, A119V / Q183T / Q185I / F227A / W236I; (11) Replace N in the 180th position with L, Q in the 183rd position with A, and Q in the 185th position with S; that is, N180L / Q183A / Q185S; (12) Replace A in the 119th position with I, Q in the 183rd position with F, Q in the 185th position with A, and W in the 236th position with L; that is, A119I / Q183F / Q185A / W236L; (13) Replace A in the 119th position with L, N in the 180th position with Y, Q in the 185th position with I, F in the 227th position with P, and W in the 236th position with L; that is, A119L / N180Y / Q185I / F227P / W236L.

2. A carbonyl reductase catalyst, characterized in that, The catalyst comprises any one of the following forms: 1) Transformed somatic cells expressing the carbonyl reductase mutant of claim 1 or frozen stem cells thereof; 2) A lysate of the transformed cells expressing the carbonyl reductase mutant of claim 1, or a lyophilized enzyme powder prepared therefrom.

3. The application of the carbonyl reductase mutant of claim 1 or the catalyst of claim 2 in the asymmetric reduction reaction of carbonyl compounds to prepare chiral alcohols, characterized in that, The carbonyl compound is selected from any one of the following compounds: 2a, 2b, 2d, 2g, 2j, 2l, 2n, 2p, 2r, 2u, 2w, or 2y: ； The chiral alcohol is ( R )-4-chromobolols, the ( R )-4-chromoalkanols have the following structures: ； R is selected from at least one of halogen, C1 alkyl, furanyl or naphthyl.

4. A kind of ( R The method for preparing 4-chromoalkanol compounds is characterized by, Includes the following steps: The carbonyl reductase mutant of claim 1 and glucose dehydrogenase are mixed, and then mixed with the carbonyl compound of claim 3, as well as a solvent, a hydrogen donor and a cofactor, to carry out an asymmetric reduction reaction to obtain ( R )-4-chromoprotanols; The ( R )-4-chromoalkanols have the following structures: ； R is selected from at least one of halogen, C1 alkyl, furanyl or naphthyl.

5. The preparation method according to claim 4, characterized in that, The solvent is a mixture of phosphate buffer and co-solvent; wherein the volume ratio of the phosphate buffer to the co-solvent is 2:1 to 19:1; the concentration of the phosphate buffer is 50 to 300 mM, and the pH is 6.0 to 8.0; the co-solvent is selected from non-polar solvents or polar solvents. 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 ℃, and the pH is 6.0 ~ 8.0.

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