Carbonyl reductase mutant and application thereof in preparation of (2R, 3R)-2-fluoro-3-hydroxy ester compound

Abstract

The invention discloses a carbonyl reductase mutant and an application of the carbonyl reductase mutant in preparation of a (2R, 3R)-2-fluoro-3-hydroxyl ester compound. In particular to a preparation method for preparing the (2R, 3R)-2-fluoro-3-hydroxy ester compound by using carbonyl reductase derived from Zygosaccharomyces bayer and free enzyme or recombinant expression transformant of a mutant of which the enzyme activity and the stability are remarkably improved as a catalyst through asymmetric reduction by using the carbonyl reductase derived from Zygosaccharomyces bayer and the free enzyme or the recombinant expression transformant of the mutant as the catalyst. Compared with other methods for preparing (2R, 3R)-2-fluoro-3-hydroxy ester compounds, the enzyme and the technical method thereof disclosed by the invention have the advantages that the theoretical yield is up to 100%, the reaction condition is mild, the enzyme is environment-friendly, the operation is simple and convenient, the industrial amplification is easy, the substrate concentration is high, and the like, and the enzyme and the technical method thereof can be used for preparing (2R, 3R)-2-fluoro-3-hydroxy ester compounds. The compound has a good application prospect in the (3R, 3R)-2-fluoro-3-hydroxy ester compound.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular, relates to a carbonyl reductase mutant and its application in the preparation of (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds. Background Art

[0002] β -Hydroxyester compounds are widely present in natural products and drugs, and have developed into one of the important chiral building blocks. The introduction of fluorine atoms causes fluorinated compounds to exhibit special physical and chemical properties that are different from non-fluorinated compounds. In recent years, fluorinated compounds have been widely used in drugs, agricultural chemicals and functional materials. α -fluorine β -Hydroxyester compounds play an important role in the medical field. For example, these compounds have anti-tumor effects (WO Patent 2,000,069,846), inhibit aromatase ( J. Pharm. Pharmacol . 2010, 62,1717-1728) and other important biological activities.

[0003] Currently synthesized α -fluorine β The chemical methods for -hydroxy ester compounds mainly include: (1) JT Welch et al. used aldehydes to react with lithium enolate reagents to obtain α -fluorine β -hydroxy ester compounds, but a chemical amount of base is required in the reaction, which has poor atom economy; (2) CFBarbas et al. used aldehydes to react with monofluoro substituted acetone to obtain α -fluorine β -hydroxy ester compounds, but the reaction time is long; (3) Professor Chen Qingyun et al. used TMS-OTf under reflux conditions or CuCl in the toxic HMPA solvent to react aldehydes with fluorinated enol silyl ethers to obtain α -fluorine β -Hydroxy ester compounds, the reaction conditions are harsh. (4) Professor Jia Zhenhua et al. used 0.5 mol% CsF to obtain α -fluorine β -hydroxy ester compounds, but the reaction requires a chemical amount of CsF, which has poor atom economy; (5) Professor Zhong Weihui et al. obtained the transition metal Ir and chiral ligands under a hydrogen pressure of 0.5 MPa. α -fluorine β -hydroxy ester compounds, the reaction conditions require high pressure and the use of transition metals.

[0004] The bioreductase method has attracted wide attention due to its green and environmentally friendly nature. European Journal of Organic Chemistry, 2019, 25 , 4080-4084) reported that various commercial carbonyl reductases catalyze the reduction α -fluorine β -ketoester compounds, selectively generate (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds, ee The highest value can reach 56%, but the catalytic substrate concentration is not at the gram level, which is far from meeting the needs of industry. Therefore, it is necessary to explore carbonyl reductases with superior performance to meet the industrial needs of high substrate concentration, high catalytic reaction efficiency, high yield and simple operation. Summary of the invention

[0005] The present invention aims to provide a carbonyl reductase mutant and its use in the preparation of (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a carbonyl reductase mutant, wherein the mutant is a protein with an amino acid sequence as shown in SEQ ID NO: 2 (derived from Zygosaccharomyces bailii Zygosaccharomyces parabailii The carbonyl reductase ZPAR of which the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1, including nucleotide sequences with a similarity of greater than 80% thereto) is mutated, wherein the mutant is selected from any one of the following (1) to (19): (1) replacing the threonine at position 26 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, and replacing the tyrosine at position 297 with valine; (2) the tyrosine at position 29 of the amino acid sequence as shown in SEQ ID NO: 2 is replaced by tryptophan, the asparagine at position 158 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by phenylalanine; (3) replacing the valine at position 63 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, the serine at position 123 with threonine, and the tyrosine at position 207 with alanine; (4) the glutamine at position 94 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by phenylalanine, the serine at position 91 is replaced by threonine, and the tryptophan at position 298 is replaced by isoleucine; (5) In the amino acid sequence of SEQ ID NO: 2, the serine at position 91 is replaced by aspartic acid, the serine at position 123 is replaced by arginine, the glutamine at position 158 is replaced by asparagine, and the tryptophan at position 298 is replaced by alanine; (6) in the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the valine at position 63 is replaced by threonine, the arginine at position 94 is replaced by isoleucine, and the tyrosine at position 297 is replaced by valine; (7) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the glutamine at position 93 is replaced by valine, the tyrosine at position 207 is replaced by isoleucine, the tryptophan at position 298 is replaced by phenylalanine, and the tyrosine at position 246 is replaced by valine; (8) The tyrosine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is replaced by proline, the serine at position 123 is replaced by valine, the asparagine at position 158 is replaced by alanine, and the tyrosine at position 207 is replaced by threonine; (9) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by glutamine, the glutamine at position 158 is replaced by valine, the tyrosine at position 207 is replaced by alanine, and the tryptophan at position 298 is replaced by leucine; (10) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine, the serine at position 123 is replaced by aspartic acid, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by leucine; (11) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the valine at position 63 is replaced by phenylalanine, the serine at position 123 is replaced by isoleucine, the tyrosine at position 207 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by isoleucine; (12) The serine at position 123 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by serine; (13) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by isoleucine, the tyrosine at position 207 is replaced by alanine, and the tyrosine at position 297 is replaced by isoleucine; (14) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by histidine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (15) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (16) In the amino acid sequence of SEQ ID NO: 2, the valine at position 63 is replaced by leucine, the serine at position 123 is replaced by threonine, the tyrosine at position 207 is replaced by phenylalanine, the tyrosine at position 297 is replaced by valine, and the tryptophan at position 298 is replaced by proline; (17) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by phenylalanine, the valine at position 63 is replaced by valine, the arginine at position 94 is replaced by leucine, the tyrosine at position 297 is replaced by alanine, and the tryptophan at position 298 is replaced by valine; (18) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by glutamine, the valine at position 63 is replaced by phenylalanine, the arginine at position 94 is replaced by isoleucine, the tyrosine at position 297 is replaced by glutamine, and the tryptophan at position 298 is replaced by leucine; (19) The tryptophan at position 298 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by alanine.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the mutant.

[0008] In a third aspect, the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0009] In a fourth aspect, the present invention provides a carbonyl reductase catalyst, wherein the catalyst is in any of the following forms: 1) transformant cells (such as recombinant bacteria) or lyophilized cells thereof expressing the mutant; 2) A disrupted liquid of a transformant cell (such as a recombinant bacterium) expressing the mutant or a freeze-dried enzyme powder thereof.

[0010] In a fifth aspect, the present invention provides the use of the mutant or the catalyst in catalyzing the asymmetric reduction reaction of carbonyl compounds to prepare chiral hydroxyl compounds.

[0011] Preferably, the carbonyl compound is (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds, the structure is as follows:

[0012] Among them, R 1 R is selected from halogen such as fluorine, chlorine and bromine, C1-C8 alkyl or cycloalkyl, thienyl, furanyl, naphthyl, pyridyl, etc.; 2 Selected from alkyl or cycloalkyl, monosubstituted or polysubstituted.

[0013] Furthermore, the carbonyl compound is selected from any one of the following compounds 1a to 1y:

[0014] Furthermore, the asymmetric reduction reaction is carried out in a solvent in the presence of NADPH.

[0015] Furthermore, NADPH is catalyzed by glucose dehydrogenase (GDH) to convert glucose and NADP into + The conversion reaction is generated.

[0016] The specific steps of preparation are: (1) Preparing a first engineered bacterium containing carbonyl reductase or a mutant thereof and a second engineered bacterium containing glucose dehydrogenase; (2) preparing resting cell suspensions of two engineered bacteria respectively; (3) preparing a cell supernatant containing carbonyl reductase or its mutant and a cell supernatant containing glucose dehydrogenase respectively; (4) The two cell supernatants are mixed, and then mixed with compound (II), a solvent, a hydrogen donor and a cofactor to perform an asymmetric reduction reaction to obtain compound (I).

[0017] The reaction formula is shown in formula ①: Formula ① Further, the solvent is a mixed solvent of phosphate buffer and a 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 100-250 mM, pH 5.0-7.5; The co-solvent is selected from one or more of a high dielectric constant solvent, an aromatic solvent, a non-polar solvent, a polar solvent, etc.; The high dielectric constant solvent is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, etc.; The aromatic solvent is selected from one or more of benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, etc.; The non-polar 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, isopropanol and the like.

[0018] Preferably, the solvent is a mixed solvent of methanol and phosphate buffer in a volume ratio of 10:90.

[0019] Furthermore, in the reaction system, the concentration of the carbonyl compound is 100-2000 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°C, and the pH is 5.0-7.5.

[0020] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention constructs an engineered bacterium containing carbonyl reductase ZPAR and its mutants, or constructs an engineered bacterium containing carbonyl reductase ZPAR and its mutants and glucose dehydrogenase GDH, and applies the engineered bacterium to catalytically reduce carbonyl compounds to obtain chiral hydroxyl compounds (such as (2 R ,3 R The present invention provides a new biological preparation method for the production of 2-fluoro-3-hydroxy ester compounds. Compared with other preparation methods, the engineered bacteria containing carbonyl reductase ZPAR mutant and glucose dehydrogenase GDH prepared by the method of the present invention have the advantages of being environmentally friendly, simple to operate, and easy to scale up industrially, and can obtain hydroxy compounds with wide substrate universality, high substrate concentration, high stereoselectivity, and high yield, and has a good industrial application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 ZPAR and ZPAR expressed by genetically engineered bacteria in a preferred embodiment of the present invention W298A SDS-PAGE electrophoresis of cell supernatant protein. Wherein, M: protein marker, 1: supernatant of ZPAR cell lysate induced by genetic engineering, 1: ZPAR induced by genetic engineering W298A Cell disruption supernatant.

[0022] Figure 2 The HPLC chromatogram of the racemic product of 2-fluoro-3-hydroxy-3-propionic acid methyl ester (2a) in a preferred embodiment of the present invention is shown.

[0023] Figure 3 In the preferred embodiment of the present invention (2 R , 3 R )-2-fluoro-3-hydroxy-3-propionic acid methyl ester (2a).

[0024] Figure 4 In the preferred embodiment of the present invention (2R , 3 R )-2-fluoro-3-hydroxy-3-propionic acid methyl ester (2a).

[0025] Figure 5 In the preferred embodiment of the present invention (2 R , 3 R )-2-fluoro-3-hydroxy-3-propionic acid methyl ester (2a).

[0026] Figure 6 In the preferred embodiment of the present invention (2 R , 3 R )-2-fluoro-3-hydroxy-3-propionic acid methyl ester (2a).

[0027] Figure 7 In the preferred embodiment of the present invention, the carbonyl reductase ZPAR W298A The optimum temperature (a) and the effect of temperature on the reductase ZPAR W298A Effect of stability (b).

[0028] Figure 8 In the preferred embodiment of the present invention, the carbonyl reductase ZPAR W298A The optimum pH (a) of carbonyl reductase ZPAR W298A pH stability (b). DETAILED DESCRIPTION

[0029] The present invention is aimed at the current α -fluorine β -Hydroxyester compounds have high energy consumption and low product yield. Through gene mining and protein engineering, a novel Zygosaccharomyces parabailii ) and its mutants with improved enzyme activity and significantly improved stability, its gene, recombinant expression vector containing the gene and recombinant expression transformant, and the use of the recombinant carbonyl reductase to efficiently catalyze the synthesis of (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds. The present invention utilizes carbonyl reductase to catalyze the reduction α -fluorine β -Ketoester compounds, high efficiency and high stereoselectivity ( ee >99%, >49:1 dr ) to generate (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds. Compared with the existing synthesis methods, this method has the advantages of mild reaction conditions, high reaction yield, and low cost. The space-time yield can reach 400 g·L -1 ·d -1 , and has good industrial application value.

[0030] The present invention adopts the following technical solution: The present invention provides a method for preparing a yeast derived from Zygosaccharomyces bayeris Zygosaccharomyces parabailii ) carbonyl reductase and its free enzyme or recombinant expression transformant with significantly improved enzyme activity and stability as catalyst for asymmetric reduction preparation of chiral hydroxyl compounds, especially in the preparation of (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds.

[0031] The present invention provides a carbonyl reductase ZPAR. The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO: 2. The carbonyl reductase can reduce the compound (II) in formula ① with high selectivity. The present invention uses bioinformatics to analyze and predict carbonyl reductases that may have obvious reducing activity on compound (II), clones and expresses its gene, and constructs recombinant Escherichia coli. By measuring the activity and selectivity of the recombinantly expressed carbonyl reductase on compound (II), a series of candidate carbonyl reductases are screened, and finally a carbonyl reductase with better catalytic performance and higher selectivity is obtained, which is derived from Zygosaccharomyces bailii ( Zygosaccharomyces parabailii )'s carbonyl reductase ZPAR.

[0032] The present invention also provides a variety of carbonyl reductase mutants, which can also be called recombinant carbonyl reductase. The present invention molecularly modifies the amino acid sequence of the carbonyl reductase ZPAR and provides a variety of carbonyl reductase mutants, wherein the carbonyl reductase mutant is a derivative protein with improved stability formed by replacing one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2, and is selected from the proteins corresponding to the following amino acid sequences: (1) replacing the threonine at position 26 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, and replacing the tyrosine at position 297 with valine; (2) the tyrosine at position 29 of the amino acid sequence as shown in SEQ ID NO: 2 is replaced by tryptophan, the asparagine at position 158 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by phenylalanine; (3) replacing the valine at position 63 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, the serine at position 123 with threonine, and the tyrosine at position 207 with alanine; (4) the glutamine at position 94 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by phenylalanine, the serine at position 91 is replaced by threonine, and the tryptophan at position 298 is replaced by isoleucine; (5) In the amino acid sequence of SEQ ID NO: 2, the serine at position 91 is replaced by aspartic acid, the serine at position 123 is replaced by arginine, the glutamine at position 158 is replaced by asparagine, and the tryptophan at position 298 is replaced by alanine; (6) in the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the valine at position 63 is replaced by threonine, the arginine at position 94 is replaced by isoleucine, and the tyrosine at position 297 is replaced by valine; (7) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the glutamine at position 93 is replaced by valine, the tyrosine at position 207 is replaced by isoleucine, the tryptophan at position 298 is replaced by phenylalanine, and the tyrosine at position 246 is replaced by valine; (8) The tyrosine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is replaced by proline, the serine at position 123 is replaced by valine, the asparagine at position 158 is replaced by alanine, and the tyrosine at position 207 is replaced by threonine; (9) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by glutamine, the glutamine at position 158 is replaced by valine, the tyrosine at position 207 is replaced by alanine, and the tryptophan at position 298 is replaced by leucine; (10) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine, the serine at position 123 is replaced by aspartic acid, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by leucine; (11) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the valine at position 63 is replaced by phenylalanine, the serine at position 123 is replaced by isoleucine, the tyrosine at position 207 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by isoleucine; (12) The serine at position 123 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by serine; (13) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by isoleucine, the tyrosine at position 207 is replaced by alanine, and the tyrosine at position 297 is replaced by isoleucine; (14) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by histidine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (15) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (16) In the amino acid sequence of SEQ ID NO: 2, the valine at position 63 is replaced by leucine, the serine at position 123 is replaced by threonine, the tyrosine at position 207 is replaced by phenylalanine, the tyrosine at position 297 is replaced by valine, and the tryptophan at position 298 is replaced by proline; (17) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by phenylalanine, the valine at position 63 is replaced by valine, the arginine at position 94 is replaced by leucine, the tyrosine at position 297 is replaced by alanine, and the tryptophan at position 298 is replaced by valine; (18) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by glutamine, the valine at position 63 is replaced by phenylalanine, the arginine at position 94 is replaced by isoleucine, the tyrosine at position 297 is replaced by glutamine, and the tryptophan at position 298 is replaced by leucine; (19) The tryptophan at position 298 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by alanine.

[0033] The present invention also provides a recombinant expression transformant comprising the carbonyl reductase gene or the carbonyl reductase mutant gene or its recombinant expression vector. The recombinant expression transformant can be prepared by transforming the above recombinant expression vector into a host cell.

[0034] In the present invention, homologues of SEQ ID NO: 1 also refer to promoter mutants. The promoter of the carbonyl reductase gene can be changed by replacing, inserting or lacking one or more nucleotides. The expression level of the carbonyl reductase can be increased by changing the sequence of the promoter or using a more effective promoter from a different source, and these changes have no negative impact on the function of the enzyme. The recombinant expression vector is obtained by cloning the carbonyl reductase ZPAR or its mutant nucleic acid into various expression vectors by conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, phages or viral vectors, etc., preferably pET28a.

[0035] The present invention also provides a recombinant expression transformant comprising the carbonyl reductase gene or the carbonyl reductase mutant gene or its recombinant expression vector. The recombinant expression transformant can be prepared by transforming the above recombinant expression vector into a host cell.

[0036] The host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the gene of carbonyl reductase ZPAR or its mutant carried by it can be effectively expressed. The host cell is preferably Escherichia coli, and more preferably: Escherichia coli E. coli BL21(DE3). The recombinant expression vector was transformed into Escherichia coli E. coli BL21 (DE3), the preferred recombinant expression transformant of the present invention can be obtained. For example, the recombinant expression vector pET28a-ZPAR is transformed into Escherichia coli E. coli BL21(DE3) was used to obtain recombinant E. coli E. coli BL21(DE3) / pET28a-ZPAR.

[0037] Prepare the resting cell suspension of the co-expressing engineered bacteria, specifically by inoculating the engineered bacteria into a culture medium containing kanamycin, activating it on a shaker, and expanding the culture until the OD 600 When the value reaches 0.8-1.2, the inducer is added, the culture is continued, the cells are collected by centrifugation, and the cells are resuspended with buffer to obtain a suspension of resting cells (first resting cells).

[0038] Furthermore, the inducer is IPTG, and the concentration of the inducer is 0.05 mM-0.8 mM; the culture conditions after adding the inducer are: the culture temperature is 15 ~ 25 ° C, and the culture time is 8-24 h.

[0039] Further, two cell supernatants are prepared, specifically in the following steps: ultrasonically disrupting the first resting cell suspension, centrifuging, and obtaining a first cell supernatant or a second cell supernatant, wherein the carbonyl reductase ZPAR cells are the first cells, and the glucose dehydrogenase cells are the second cells.

[0040] Furthermore, in the mixture of two cell supernatants, the volume ratio of the first cell supernatant to the second cell supernatant is 20:1-1:2, preferably 10:1-8:1.

[0041] Furthermore, in the asymmetric redox reaction, the concentration of compound (II) is 1.0-300 g / L, the cell concentration of the first resting cell suspension is 0.1 g wet weight / L-25 g wet weight / L, the cell concentration of the second resting cell suspension is 0.1 g wet weight / L-25 g wet weight / L, the concentration of the hydrogen donor (glucose) is 5-750 g / L, and the concentration of the cofactor (NADP + ) at a concentration of 0-0.5 mM.

[0042] During the asymmetric reduction reaction, on the one hand, carbonyl reductase ZPAR or its dominant mutant catalyzes the reduction of compound (II) to asymmetric synthesis of compound (I); on the other hand, glucose dehydrogenase (GDH) oxidizes glucose to gluconolactone, while consuming the oxidized coenzyme factor NADP. + , regenerating the reduced coenzyme factor NADPH, which will provide hydrogen to the substrate and be oxidized to the oxidized coenzyme factor NADP + , forming a closed loop of coenzyme factor consumption and regeneration, promoting the main reaction.

[0043] In the asymmetric reduction reaction, the reaction temperature is 20-40°C, and the pH of the reaction buffer is 5.0-7.5. Preferably, the reaction temperature is 20-25°C, and the pH of the reaction buffer is 6.5-7.5. The buffer concentration is 100-250 mM.

[0044] In the asymmetric reduction reaction, the solvent is a mixed solvent of phosphate buffer and a co-solvent, wherein the co-solvent is selected from one or more of high dielectric constant solvents: dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, aromatic solvents: benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, non-polar solvents: n-hexane, cyclohexane, and polar solvents: acetonitrile, ethyl acetate, dichloromethane, 1,2-dichloroethane, methanol, ethanol, and isopropanol.

[0045] The preferred solvent is a mixed solvent of methanol and phosphate buffer in a volume ratio of 10:90.

[0046] During the entire reaction process, after the reaction was completed by GC detection of the substrate being completely consumed, extraction was performed with an equal volume of ethyl acetate for 3-4 times, the organic phases were combined, washed with saturated sodium bicarbonate twice, washed with water once, washed with saturated brine once, dried over anhydrous sodium sulfate, and the organic solvent was removed by reduced pressure distillation to obtain the target product.

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0048] Example 1 Construction of genetically engineered bacteria and induced expression Transform the expression host with plasmid pET28a-ZPAR or its mutants E. coli BL21 (DE3), when positive transformants appear on the LB solid medium plate containing kanamycin, inoculate them into 3 mL of LB liquid medium containing 100 μg / mL kanamycin, and culture them at 37 ℃, 200 rpm overnight; inoculate 1% of the inoculum into 1 L of the above LB medium, and continue to culture at 37 ℃, 200 rpm for 4-5 hours. When the OD of the culture reaches about 0.6 - 0.8, add IPTG with a final concentration of 0.1 mmol / L, and induce the culture at 16 ℃, 100 rpm overnight. Collect the cells by centrifugation at 5 000 rpm, resuspend the cells with 10 mL of sterile water, and ultrasonically break them. Collect the supernatant by centrifugation at 20 000 rpm, which is the crude enzyme solution.

[0049] The crude enzyme solution was purified using a Ni-NTA column: After 5 column volumes of the equilibrated column were washed with Binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.0), the sample was loaded, and the unbound proteins in the column were washed 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 eluate was desalted and concentrated using a 30 kDa ultrafiltration tube (Amicon Ultra 15, Millipore), and finally a recombinant carbonyl reductase with a purity of more than 90% was obtained. SDS-PAGE showed that Figure 1 shown.

[0050] Example 2 Determination of carbonyl reductase activity and HPLC analysis of enzyme catalysis products The reaction system is as follows: Substrate (100mM): 5 μL Crude enzyme solution (15% v / v): 2 μL NADPH (2 mM): 40 μL Phosphate buffer (100 mM, pH 7.0): 153 μL The definition of enzyme activity is as follows: in the above reaction system, the enzyme activity that catalyzes the oxidation of 1 μmol of reduced coenzyme NADPH within 1 min is defined as one enzyme activity unit, represented by U.

[0051] The enzyme activity calculation formula is as follows:

[0052] Wherein, A2: absorbance at 340 nm after 1 min of reaction; A1: absorbance at 340 nm at the beginning of reaction; V: total volume of reaction solution, mL; 6220: molar absorption coefficient, L / mol / cm; L: optical path length, cm.

[0053] In a 60 mL reaction system, (sodium phosphate buffer (PBS) (100 mM, pH 7.0) was added 10 mL of crude carbonyl reductase enzyme solution, 1 mL of GDH, 10 mM aryl- α -fluorine β -Ketoester (Compound II), 20 mM glucose solution, 0.2 mM NADP + , 10% (v / v) methanol. React at 30 °C, 520 rpm for 12 h. Add NaCl until supersaturated, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate, filter, spin dry, and use normal liquid chromatography to determine the conversion rate and enantioselectivity. Among them, carbonyl reductase ZPAR or its mutant asymmetric reduction of aromatic- α -fluorine- β -The reaction formula of ketoester is shown in formula ①.

[0054] Detection conditions: chromatographic column: Chiracel® IC (250 mm×4.6 mm), n-hexane: isopropanol = 90:10 (volume ratio), flow rate: 1.0 mL / min, 220 nm UV.

[0055] Example 3 Construction of carbonyl reductase ZPAR mutant The mutant library of carbonyl reductase ZPAR was constructed by consensus analysis: the ZPAR protein sequence was used as a probe for sequence alignment, and different protein sequences with a homology of more than 30% from thermophilic sources were selected. A series of non-conserved residues were selected for single-point mutations by ClustalX2 sequence alignment and Espript consensus analysis. The corresponding mutant primers were designed, and plasmid pET28a-ZPAR 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 2×T8 High-Fidelity Master Mix, 2.0 μL of each pair of mutant primers (10 μM), and sterile ddH2O were added to 50 μL. PCR reaction procedure: (1) pre-denaturation at 98 ℃ for 2 min, (2) denaturation at 98 ℃ for 10 s, (3) T m+ 3 ~ 5 ℃ annealing for 10 s. (3) 72 ℃ extension for 90 s. Steps (1) to (3) were repeated for 28 cycles. The PCR product was stored at 4 ℃. After the PCR product was verified by agarose gel electrophoresis, restriction endonucleases were added. Dpn I was digested at 37 °C for 2 h. The digestion product was transformed into E. coli BL21(DE3) competent cells, and spread on LB solid medium plates containing 50 μg / mL kanamycin, and place in a 37 ℃ incubator for static culture for about 12 hours. The obtained monoclonal colonies were picked into LB test tubes containing 50 μg / mL kanamycin, and cultured at 37 ℃ for 12 hours. The plasmids were extracted for DNA sequencing verification, and the correct mutant strains were confirmed for further fermentation culture.

[0056] The mutant is selected from any one of the following (1) to (19): (1) replacing the threonine at position 26 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, and replacing the tyrosine at position 297 with valine; (2) the tyrosine at position 29 of the amino acid sequence as shown in SEQ ID NO: 2 is replaced by tryptophan, the asparagine at position 158 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by phenylalanine; (3) replacing the valine at position 63 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, the serine at position 123 with threonine, and the tyrosine at position 207 with alanine; (4) the glutamine at position 94 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by phenylalanine, the serine at position 91 is replaced by threonine, and the tryptophan at position 298 is replaced by isoleucine; (5) In the amino acid sequence of SEQ ID NO: 2, the serine at position 91 is replaced by aspartic acid, the serine at position 123 is replaced by arginine, the glutamine at position 158 is replaced by asparagine, and the tryptophan at position 298 is replaced by alanine; (6) in the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the valine at position 63 is replaced by threonine, the arginine at position 94 is replaced by isoleucine, and the tyrosine at position 297 is replaced by valine; (7) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the glutamine at position 93 is replaced by valine, the tyrosine at position 207 is replaced by isoleucine, the tryptophan at position 298 is replaced by phenylalanine, and the tyrosine at position 246 is replaced by valine; (8) The tyrosine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is replaced by proline, the serine at position 123 is replaced by valine, the asparagine at position 158 is replaced by alanine, and the tyrosine at position 207 is replaced by threonine; (9) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by glutamine, the glutamine at position 158 is replaced by valine, the tyrosine at position 207 is replaced by alanine, and the tryptophan at position 298 is replaced by leucine; (10) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine, the serine at position 123 is replaced by aspartic acid, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by leucine; (11) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the valine at position 63 is replaced by phenylalanine, the serine at position 123 is replaced by isoleucine, the tyrosine at position 207 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by isoleucine; (12) The serine at position 123 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by serine; (13) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by isoleucine, the tyrosine at position 207 is replaced by alanine, and the tyrosine at position 297 is replaced by isoleucine; (14) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by histidine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (15) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (16) In the amino acid sequence of SEQ ID NO: 2, the valine at position 63 is replaced by leucine, the serine at position 123 is replaced by threonine, the tyrosine at position 207 is replaced by phenylalanine, the tyrosine at position 297 is replaced by valine, and the tryptophan at position 298 is replaced by proline; (17) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by phenylalanine, the valine at position 63 is replaced by valine, the arginine at position 94 is replaced by leucine, the tyrosine at position 297 is replaced by alanine, and the tryptophan at position 298 is replaced by valine; (18) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by glutamine, the valine at position 63 is replaced by phenylalanine, the arginine at position 94 is replaced by isoleucine, the tyrosine at position 297 is replaced by glutamine, and the tryptophan at position 298 is replaced by leucine; (19) The tryptophan at position 298 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by alanine.

[0057] Example 4 Inducible expression and purification of recombinant carbonyl reductase ZPAR and its mutants The recombinant expression transformant obtained in Example 2 E. coli BL21(DE3) / pET28a-ZPAR was inoculated into LB medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C for 12 h. Then, the inoculum was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculum and cultured at 37°C and 200 rpm. When the OD of the culture solution reached 600 When it reached 0.6, IPTG was added at a final concentration of 0.1 mmol / L for induction. After induction at 16°C for 18 hours, the culture medium was centrifuged at 8500 rpm, the cell precipitate was collected and washed with physiological saline to obtain resting cells, which were freeze-dried to obtain lyophilized cells with a specific activity of 80 U / mg DCW.

[0058] The resting cells were suspended in 100 mL of potassium phosphate buffer (100 mmol / L, pH 6.0), ultrasonically disrupted in an ice-water bath, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant carbonyl reductase ZPAR and its mutants. The obtained crude enzyme solution was analyzed by polyacrylamide gel electrophoresis, and the recombinant carbonyl reductase ZPAR and its mutants existed in a soluble form.

[0059] Example 5 Characterization of the enzymatic properties of carbonyl reductase ZPAR Determination of the optimal temperature: According to the standard reaction in the above example, the enzyme activity was measured at 20, 25, 30, 35, 40, 45, and 50 °C. Each reaction was carried out in parallel and the highest enzyme activity was taken as 100%. The relative enzyme activity at other different temperatures was calculated to determine the optimal reaction temperature of the enzyme.

[0060] Effect of temperature on enzyme stability: According to the standard reaction in the above example, the enzyme solution was placed in a water bath at 20, 25, 30, 35, and 40 ℃. After keeping warm for different periods of time, the residual enzyme activity was measured. Each reaction was set up in 3 parallels. The highest enzyme activity measured was taken as 100%. The relative enzyme activity under various temperature conditions was calculated to examine the thermal stability of the enzyme at different temperatures.

[0061] Determination of the optimal pH: According to the standard reaction in the above example, the enzyme activity was determined at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0 at 30°C. The highest enzyme activity was taken as 100%, and the relative enzyme activity at other different pH values ​​was calculated. Each reaction was set up in 3 parallels to explore the effect of different temperatures on enzyme stability.

[0062] Effect of pH on enzyme stability: At 4 ℃, the enzyme solution was placed in a reaction system of pH 4.0, 5.0, 6.0, and 7.0. The enzyme solution was taken out after different periods of time and the residual enzyme activity was determined. Each reaction was set up in 3 parallels. The highest enzyme activity measured was taken as 100%. The relative enzyme activity under various pH conditions was calculated to determine the pH stability of the enzyme.

[0063] Effect of metal ions on enzyme activity: According to the standard reaction in the above example, 5 mM of different metal ions (Fe 3+ 、Al 3+ , Mn 2+ , Cu 2+ Mg 2+ , Fe 2+ 、Co 2+ , Ca 2+ 、Na + , K + 、Ag + ) Under the optimal reaction conditions, the samples were placed for different time periods, and the residual enzyme activity was measured. The sample enzyme activity without metal ions was taken as 100%, and three parallels were set for each reaction to investigate the effect of metal ions on enzyme activity. Table 1 shows the effect of metal ions on the reductase ZPAR.

[0064] Table 1 Effects of metal ions on reductase ZPAR

[0065] Example 6 Determination of substrate specificity of carbonyl reductase To detect the carbonyl reductase ZPAR W298A To investigate the substrate specificity of mutant 19, 100 mM phosphate solution at pH 6.0 was used to prepare each substrate at a final concentration of 2.5 mM. The enzyme activity at different concentrations was determined as described above. K cat / K m As shown in Table 2, the substrate specificity of the enzyme was explored by detecting the catalytic activity of the enzyme for different types of prochiral ketone substrates, and the activity detection conditions were the same as those for substrate 1a.

[0066] Table 2 Kinetic parameters of carbonyl reductase for different substrates

[0067] Carbonyl reductase ZPAR W298A The HPLC chromatogram of asymmetric synthesis 2a is shown in Figure 2 .

[0068] (2 R , 3 R The HPLC chromatogram of methyl 2-fluoro-3-hydroxy-3-propionate (2a) is shown in Figure 3 .

[0069] (2 R , 3 R The H NMR spectrum of methyl 2-fluoro-3-hydroxy-3-propionate (2a) is shown in Figure 4 .

[0070] (2 R , 3 R The C NMR spectrum of methyl 2-fluoro-3-hydroxy-3-propionate (2a) is shown in Figure 5 .

[0071] (2 R , 3 R The NMR fluorine spectrum of methyl 2-fluoro-3-hydroxy-3-propionate (2a) is shown in Figure 6 .

[0072] Carbonyl reductase ZPAR W298A The optimum temperature and the effect of temperature on the reductase ZPAR W298A The effect of stability is shown in Figure 7 (a and b).

[0073] Carbonyl reductase ZPAR W298A Optimal pH of carbonyl reductase ZPAR W298A The pH stability of Figure 8 (a and b).

[0074] Example 7 ZPAR catalytic reduction of 100 mmol / L substrate 1a The reaction formula is as follows:

[0075] In 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.0) containing 100 mmol / L substrate 1a (19.6 g / L), methanol (10% v / v) and 200 mmol / L glucose (36.2 g / L), 1 g / L of ZPAR wet cells, 50 mg / L of GDH wet cells and 0.1 mmol / L of NADP were added. + The reaction was carried out at 25 °C under magnetic stirring. During the reaction, the pH was controlled at 6.0 by adding 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® OD-H, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value = 92%, dr Value = 8:1. 1 H NMR (400 MHz, CDCl3) δ 7.37-7.31 (m,5H), 5.10-4.97 (m, 2H), 3.69 (s, 3H), 3.36 (s, 1H). 19 F NMR (376 MHz, CDCl3) δ-197.9. 13 C NMR (100 MHz, CDCl3) δ 168.3 (d, J = 23.3 Hz), 137.7 (d, J = 2.9 Hz),128.6, 128.5, 126.8 (d, J = 1.3 Hz), 91.2 (d, J = 190.5 Hz), 73.6 (d, J = 22.1 Hz),52.4.

[0076] Example 8 ZPAR Y29W / N158F / W298P Catalytic reduction of 500 mmol / L substrate 1b The reaction formula is as follows:

[0077] 1 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 5.5) containing 500 mM substrate 1b (107 g / L), methanol (5% v / v) and 1 M glucose (216 g / L). Y29W / N158F / W298P Wet cells, 50 mg / L GDH wet cells and 0.1 mmol / L NADP +The reaction was carried out at 25 °C under magnetic stirring. During the reaction, the pH was controlled at 5.5 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® OD-H, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value >49:1. 1 H NMR (400 MHz, CDCl3) δ 7.40-7.34 (m, 2H), 7.10-7.03 (m, 2H), 5.14-4.96 (m, 2H), 3.75 (s, 3H), 2.92 (d, J = 4.2 Hz, 1H). 19 F NMR (376 MHz, CDCl3) δ -113.1, -198.0. 13 C NMR (100 MHz, CDCl3) δ 168.2 (d, J = 23.2Hz), 162.9 (d, J = 247.2 Hz), 133.3, 128.6 (dd, J = 8.3, 1.4 Hz), 115.5 (d, J =21.6 Hz), 90.9 (d, J = 191.4 Hz), 73.0 (d, J = 22.2 Hz), 52.6.

[0078] Example 9 ZPAR V63L / S123T / Y207A Catalytic reduction of 500 mmol / L substrate 1j The reaction formula is as follows:

[0079] 1.3 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 7.0) containing 1 M substrate 1j (226.2 g / L), methanol (20% v / v) and 1.5 M glucose (270 g / L). V63L / S123T / Y207A Wet cells, 200 mg / L GDH wet cells and 0.1 mmol / L NADP +The reaction was carried out at 35 °C under magnetic stirring. During the reaction, the pH was controlled at 7.5 by adding 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® OD-H, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value >49:1. 1 H NMR (400 MHz, CDCl3) δ 7.31-7.25 (m,1H), 6.95-6.92 (m, 2H), 6.88-6.85 (m, 1H), 5.12-4.98 (m, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.39 (d, J = 4.1 Hz, 1H). 19 F NMR (376 MHz, CDCl3) δ -197.6. 13 CNMR (100 MHz, CDCl3) δ 168.3 (d, J = 23.4 Hz), 159.6, 139.3 (d, J = 2.9 Hz),129.5, 119.0, 114.2, 112.2 (d, J = 1.3 Hz), 91.1 (d, J = 190.7 Hz), 73.5 (d, J =22.3 Hz), 55.2, 52.5.

[0080] Example 10 ZPAR S91D / S123R / Q158N / W298A Catalytic reduction of 500 mmol / L substrate 1m The reaction formula is as follows:

[0081] 0.8 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 5.0) containing 800 mM substrate 1m (168 g / L), methanol (15% v / v) and 1 M glucose (216 g / L). S91D / S123R / Q158N / W298A Wet cells, 50 mg / L GDH wet cells and 0.05 mmol / L NADP +The reaction was carried out at 20 °C under magnetic stirring. During the reaction, the pH was controlled at 5.0 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value >35:1. 1 H NMR (400 MHz, CDCl3) δ 7.36-7.27 (m,5H), 5.10-5.08 (m, 1H), 5.01 (dd, J = 34.4, 4.6 Hz, 1H), 4.13 (q, J = 7.1 Hz,2H), 3.72 (s, 1H), 1.14 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ -198.1. 13 C NMR (100 MHz, CDCl3) δ 167.8 (d, J = 23.5 Hz), 137.7 (d, J = 3.0 Hz), 128.4,128.3, 126.8 (d, J = 1.3 Hz), 91.2 (d, J = 190.6 Hz), 73.5 (d, J = 21.9 Hz), 61.7,13.9.

[0082] Example 12 ZPAR T26L / S123I / Y207F / W298I Catalytic reduction of 500 mmol / L substrate 1n The reaction formula is as follows:

[0083] 0.6 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.0) containing 800 mM substrate 1n (161.6 g / L), methanol (30% v / v) and 1 M glucose (216 g / L). T26L / S123I / Y207F / W298I Wet cells, 300 mg / L GDH wet cells and 0.01 mmol / L NADP +The reaction was carried out at 25 °C under magnetic stirring. During the reaction, the pH was controlled at 6.0 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® IC, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value >98:1. 1 H NMR (400 MHz, CDCl3) δ 7.31 (dd, J =5.1, 1.2 Hz, 1H), 7.06 (d, J = 3.5 Hz, 1H), 7.03-6.97 (m, 1H), 5.35 (dd, J =19.1, 4.5 Hz, 1H), 5.12 (dd, J = 48.4, 4.5 Hz, 1H), 3.74 (s, 3H). 19 F NMR (376MHz, CDCl3) δ -199.4. 13 C NMR (100 MHz, CDCl3) δ 168.0 (d, J = 23.1 Hz), 140.4(d, J = 2.3 Hz), 126.8, 126.1, 126.0 (d, J = 1.3 Hz), 90.9 (d, J = 191.6 Hz), 69.8(d, J = 22.3 Hz), 52.7.

[0084] Example 13 ZPAR T26L / Y207A / Y297F / W298L Catalytic reduction of 900 mmol / L substrate for 1s The reaction formula is as follows:

[0085] 1.3 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 7.5) containing 900 mM substrate 1s (181.8 g / L), methanol (10% v / v) and 1.62 M glucose (291.6 g / L). T26L / Y207A / Y297F / W298L Wet cells, 40 mg / L GDH wet cells and 0.03 mmol / L NADP +The reaction was carried out at 25 °C under magnetic stirring. During the reaction, the pH was controlled at 7.5 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® IA, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value>97:1. 1 H NMR (400 MHz, CDCl3) δ 5.05(dd, J = 54.1, 8.1 Hz, 1H), 4.33 (dd, J = 6.3, 3.6 Hz, 1H), 4.19 (dtd, J =14.6, 8.2, 6.3 Hz, 1H), 3.83 (s, 2H), 1.89 (dtd, J = 8.2, 5.9, 3.7 Hz, 1H), 1.77 – 1.59 (m, 2H), 1.58 – 1.45 (m, 2H), 1.46 – 1.27 (m, 6H). 13 C NMR (100MHz, CDCl3) δ 170.23 (d, J = 22.9 Hz), 91.25 (d, J = 190.5 Hz), 73.90 (d, J =20.3 Hz), 52.56 (d, J = 5.2 Hz), 40.01 (d, J = 6.4 Hz), 29.49 (d, J = 2.4Hz), 27.75, 26.72.

[0086] Example 14 ZPAR V63L / S123T / Y207F / Y297V / W298P Catalytic reduction of 2 mol / L substrate 1u The reaction formula is as follows:

[0087] 2.62 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 5.5) containing 2 M substrate 1u (252 g / L) methanol (20% v / v) and 3 M glucose (540 g / L). V63L / S123T / Y207F / Y297V / W298P Wet cells, 500 mg / L GDH wet cells and 0.16 mmol / L NADP +The reaction was carried out at 25 °C under magnetic stirring. During the reaction, the pH was controlled at 5.5 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® IF, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 98%, dr Value >96:1. 1 H NMR (400 MHz, CDCl3) δ 5.04 (dd, J= 54.0, 8.4 Hz, 1H), 4.59 – 4.20 (m, 3H), 4.16 – 3.84 (m, 1H), 2.11 – 1.74(m, 1H), 1.23 (t, J = 6.4 Hz, 3H), 1.03 (dd,J = 6.7, 1.5 Hz, 3H), 0.98 (dd,J = 6.6, 1.5 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 169.08 (d, J = 22.9 Hz), 89.05 (d, J = 190.5 Hz), 75.64 (d, J = 17.4 Hz), 61.82 (d, J = 3.1 Hz), 29.94 (d, J = 6.0 Hz), 18.73 (d, J = 2.6 Hz), 14.03.

[0088] Example 15 ZPAR T26Q / V63F / R94I / Y297Q / W298L Catalytic reduction of 600 mmol / L substrate for 1w The reaction formula is as follows:

[0089] 0.3 g / L ZPAR was added to 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.0) containing 600 mM substrate 1w (96 g / L), methanol (20% v / v) and 1 M glucose (180 g / L). T26Q / V63F / R94I / Y297Q / W298L Wet cells, 50 mg / L GDH wet cells and 0.01 mmol / L NADP + The reaction was carried out at 30 °C under magnetic stirring. During the reaction, the pH was controlled at 6.0 by 0.5 mol / L potassium carbonate solution. After the reaction, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, dried overnight with anhydrous sodium sulfate, filtered and dried. The chiral HPLC (Chiracel® AS, cyclohexane / isopropanol as mobile phase) was used for analysis. ee Value ≥ 99%, dr Value >99:1. 1 H NMR (400 MHz, CDCl3) δ 5.09 (dd, J =54.0, 7.5 Hz, 1H), 4.66 (dd, J = 4.8, 3.5 Hz, 1H), 4.17 (dtd, J = 14.9, 7.4,4.8 Hz, 1H), 3.83 (s, 3H), 1.89 – 1.51 (m, 1H), 0.89 (dd, J = 10.3, 5.7 Hz, 2H), 0.64 (dd, J = 10.3, 5.7 Hz, 2H). 13 C NMR (100 MHz, CDCl3) δ 170.23 (d, J= 22.9 Hz), 89.85 (d, J = 190.5 Hz), 75.17 (d, J = 19.3 Hz), 52.56 (d, J =5.3 Hz), 17.28 (d, J = 8.3 Hz), 5.64 (d, J = 3.1 Hz).

[0090] Example 16 ZPAR W298A Catalytic reduction of 2 mol / L substrate 1a The reaction formula is as follows:

[0091] In 10 mL of potassium phosphate buffer (100 mmol / L, pH 6.5) containing 2 mol / L substrate 1a (392 g / L), methanol (10% v / v) and 4 mol / L glucose (720 g / L), 12 g / L ZPAR was added. W298A Wet cells, 1 g / L GDH wet cells and 0.1 mmol / L NADP + The reaction was carried out at 30 °C under magnetic stirring. During the reaction, the pH was controlled at 6.5 by adding 0.5 mol / L potassium carbonate solution. After the reaction was completed, an equal amount of ethyl acetate was added for extraction twice. The extracts were combined, anhydrous sodium sulfate was added for drying overnight, and the extracts were filtered and dried. The substrate conversion rate was 99% (2 R ,3 R )-2a content is 99%. 1 H NMR (400 MHz, CDCl3) δ 7.37-7.31 (m, 5H), 5.10-4.97 (m, 2H), 3.69 (s,3H), 3.36 (s, 1H).19 F NMR (376 MHz, CDCl3) δ -197.9. 13 C NMR (100 MHz, CDCl3)δ 168.3 (d, J = 23.3 Hz), 137.7 (d, J = 2.9 Hz), 128.6, 128.5, 126.8 (d, J = 1.3Hz), 91.2 (d, J = 190.5 Hz), 73.6 (d, J = 22.1 Hz), 52.4.

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that The mutant is obtained by mutating the protein having the amino acid sequence shown in SEQ ID NO: 2, and the mutant is selected from any one of the following (1) to (19): (1) replacing the threonine at position 26 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, and replacing the tyrosine at position 297 with valine; (2) the tyrosine at position 29 of the amino acid sequence as shown in SEQ ID NO: 2 is replaced by tryptophan, the asparagine at position 158 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by phenylalanine; (3) replacing the valine at position 63 of the amino acid sequence shown in SEQ ID NO: 2 with leucine, the serine at position 123 with threonine, and the tyrosine at position 207 with alanine; (4) the glutamine at position 94 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by phenylalanine, the serine at position 91 is replaced by threonine, and the tryptophan at position 298 is replaced by isoleucine; (5) In the amino acid sequence of SEQ ID NO: 2, the serine at position 91 is replaced by aspartic acid, the serine at position 123 is replaced by arginine, the glutamine at position 158 is replaced by asparagine, and the tryptophan at position 298 is replaced by alanine; (6) in the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the valine at position 63 is replaced by threonine, the arginine at position 94 is replaced by isoleucine, and the tyrosine at position 297 is replaced by valine; (7) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the glutamine at position 93 is replaced by valine, the tyrosine at position 207 is replaced by isoleucine, the tryptophan at position 298 is replaced by phenylalanine, and the tyrosine at position 246 is replaced by valine; (8) The tyrosine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is replaced by proline, the serine at position 123 is replaced by valine, the asparagine at position 158 is replaced by alanine, and the tyrosine at position 207 is replaced by threonine; (9) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by glutamine, the glutamine at position 158 is replaced by valine, the tyrosine at position 207 is replaced by alanine, and the tryptophan at position 298 is replaced by leucine; (10) The serine at position 93 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine, the serine at position 123 is replaced by aspartic acid, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by leucine; (11) The threonine at position 26 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the valine at position 63 is replaced by phenylalanine, the serine at position 123 is replaced by isoleucine, the tyrosine at position 207 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by isoleucine; (12) The serine at position 123 of the amino acid sequence shown in SEQ ID No. 2 is replaced by leucine, the asparagine at position 158 is replaced by phenylalanine, and the tyrosine at position 207 is replaced by serine; (13) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by isoleucine, the tyrosine at position 207 is replaced by alanine, and the tyrosine at position 297 is replaced by isoleucine; (14) In the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 158 is replaced by histidine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (15) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by leucine, the tyrosine at position 207 is replaced by alanine, the tyrosine at position 297 is replaced by phenylalanine, and the tryptophan at position 298 is replaced by leucine; (16) In the amino acid sequence of SEQ ID NO: 2, the valine at position 63 is replaced by leucine, the serine at position 123 is replaced by threonine, the tyrosine at position 207 is replaced by phenylalanine, the tyrosine at position 297 is replaced by valine, and the tryptophan at position 298 is replaced by proline; (17) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by phenylalanine, the valine at position 63 is replaced by valine, the arginine at position 94 is replaced by leucine, the tyrosine at position 297 is replaced by alanine, and the tryptophan at position 298 is replaced by valine; (18) In the amino acid sequence of SEQ ID NO: 2, the threonine at position 26 is replaced by glutamine, the valine at position 63 is replaced by phenylalanine, the arginine at position 94 is replaced by isoleucine, the tyrosine at position 297 is replaced by glutamine, and the tryptophan at position 298 is replaced by leucine; (19) The tryptophan at position 298 of the amino acid sequence shown in SEQ ID NO: 2 is replaced by alanine.

2. A nucleic acid molecule encoding the mutant according to claim 1.

3. A biological material containing the nucleic acid molecule according to claim 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

4. A carbonyl reductase catalyst, characterized in that The catalyst is in any of the following forms: 1) a transformant cell expressing the mutant of claim 1 or a lyophilized cell thereof; 2) A disrupted liquid of transformant cells expressing the mutant according to claim 1 or a freeze-dried enzyme powder thereof.

5. Use of the mutant according to claim 1 or the catalyst according to claim 2 in catalyzing the asymmetric reduction reaction of carbonyl compounds to prepare chiral hydroxyl compounds.

6. The use according to claim 5, characterized in that: The carbonyl compound is (2 R ,3 R )-2-fluoro-3-hydroxy ester compounds.

7. The use according to claim 5, characterized in that: The carbonyl compound is selected from any one of the following compounds 1a to 1y: 。 8. The use according to any one of claims 5 to 7, characterized in that: The asymmetric reduction reaction is carried out in a solvent in the presence of NADPH.

9. The use according to claim 8, characterized in that: The solvent is a mixed solvent of phosphate buffer and a 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 100-250 mM, pH 5.0-7.5; The co-solvent is selected from one or more of a high dielectric constant solvent, an aromatic solvent, a non-polar 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 non-polar 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.

10. The use according to claim 9, characterized in that: In the reaction system, the concentration of the carbonyl compound is 100-2000 mM, the dosage of the mutant or the catalyst is 0.01 g wet weight / L-25 g wet weight / L, the reaction temperature is 20-40°C, and the pH is 5.0-7.5.

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