Ketoreductase derived from schizosaccharomyces japonicus yfs275 and use thereof

Abstract

The application discloses a ketoreductase derived from Schizosaccharomyces japonicus yFS275 and application thereof. The ketoreductase has an amino acid sequence shown in SEQ ID NO:1, and a mutant thereof has an amino acid sequence shown in SEQ ID NO:3. The ketoreductase and the mutant thereof provided by the application can efficiently catalyze the conversion of ethyl 4-chloroacetoacetate into (R)-4-chloro-3-hydroxybutyric acid ethyl ester, and the mutant thereof has higher organic solvent resistance.

Description

Technical Field

[0001] This invention belongs to the field of functional enzyme screening technology, and relates to a ketone reductase derived from Schizosaccharomyces japonicus yFS275 and its mutants, nucleic acid molecules encoding them, vectors and recombinant cells containing these nucleic acid molecules, as well as their preparation methods and their application in the preparation of (R)-4-chloro-3-hydroxybutyrate ethyl ester. Background Technology

[0002] Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is an important chiral alcohol. The intermediate generated by the substitution of the chlorine atom with trimethylamine and subsequent alkaline hydrolysis is a crucial precursor for the preparation of L-carnitine and its derivatives (such as L-carnitine tartrates). L-carnitine is widely used in pharmaceuticals, food additives (e.g., in stage III infant formula), and sports drinks, highlighting the importance of (R)-CHBE as a precursor. The preparation of (R)-CHBE typically relies on the conversion of ethyl 4-chloroacetoacetate using a synthetic chiral catalyst. However, this process suffers from the use of catalysts synthesized from rare metals, which are relatively difficult to synthesize, and is costly. Therefore, researchers aim to achieve whole-cell production through fermentation and biotransformation.

[0003] Ketoreductase catalyzes the conversion of ethyl 4-chloroacetoacetate to ethyl (R)-4-chloro-3-hydroxybutyrate, requiring reduced nicotinamide adenine dinucleotide (NADH) as a cofactor in the catalytic process. Currently, large-scale production of ethyl (R)-4-chloro-3-hydroxybutyrate has not been achieved through fermentation and biotransformation. Furthermore, existing enzymatic reactions require the addition of a certain amount of organic solvent. However, the protein structures of biological enzymes exhibit varying and limited tolerance to organic solvents. Therefore, there is still a need to find a genetically engineered strain of ethyl (R)-4-chloro-3-hydroxybutyrate that expresses a ketone reductase with high catalytic activity and relatively high tolerance to organic solvents. Summary of the Invention

[0004] The purpose of this invention is to provide a ketone reductase and its mutants, thereby solving the problems of low enzyme activity and low tolerance to organic solvents in the existing ketone reductase-catalyzed synthesis of (R)-CHBE.

[0005] Specifically, the present invention obtained ketone reductase KREDSJ from the strain Schizosaccharomyces japonicus yFS275, the amino acid sequence of which is shown in SEQ ID NO:1 and the nucleotide sequence of which is shown in SEQ ID NO:2. Furthermore, it was mutated by error-prone PCR technology to obtain a ketone reductase mutant KREDSJ-M with significantly improved enzyme activity and organic solvent tolerance, the amino acid sequence of which is shown in SEQ ID NO:3 and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0006] In a first aspect, the present invention provides a ketone reductase having the amino acid sequence shown in SEQ ID NO:1.

[0007] The ketone reductase provided by this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, such as by using recombinant technology to express it from prokaryotes (Escherichia coli).

[0008] In some embodiments, the ketone reductase is obtained by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (e.g., E. coli BL21(DE3)), constructing a recombinant genetically engineered strain, culturing the strain, and adding an inducer to induce expression.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned ketone reductase, having the nucleotide sequence shown in SEQ ID NO:2.

[0010] The nucleic acid molecules provided by this invention can usually be obtained by amplification using a PCR instrument or by artificial synthesis.

[0011] In a third aspect, the present invention provides a mutant of the above-mentioned ketone reductase, having the amino acid sequence shown in SEQ ID NO:3, which exhibits further improvements in catalytic activity, organic solvent tolerance, and conversion rate compared to the above-mentioned ketone reductase. Compared to the amino acid sequence of the ketone reductase shown in SEQ ID NO:1, this mutant has the following mutations: L at position 28 is mutated to S, N at position 95 is mutated to S, F at position 175 is mutated to V, Y at position 240 is mutated to H, and R at position 280 is mutated to G.

[0012] The mutants provided by this invention can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically, such as by using recombination technology to express them from prokaryotes (Escherichia coli).

[0013] In some implementations, the mutant is obtained by constructing a recombinant genetically engineered bacterium by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (e.g., E.coli BL21(DE3)), then culturing the strain and adding an inducer to induce expression to obtain a ketone reductase mutant.

[0014] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned ketone reductase mutant, having the nucleotide sequence shown in SEQ ID NO:4.

[0015] The nucleic acid molecules provided by this invention can usually be obtained by amplification using a PCR instrument or by artificial synthesis.

[0016] Fifthly, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above.

[0017] In some embodiments, the recombinant vector is pET-KREDSJ or pET-ΔKREDSJ-M57, which is obtained by replacing the sequence between the BamHI and HindIII restriction sites of pET-28a(+) with the nucleic acid molecule encoding the above-mentioned ketoreductase or the nucleic acid molecule of the above-mentioned ketoreductase mutant, while keeping the rest of the sequence unchanged.

[0018] In a sixth aspect, the present invention provides a recombinant cell comprising any of the recombinant vectors described above.

[0019] In some embodiments, the recombinant cells are induced to express the aforementioned ketone reductase or a mutant of the ketone reductase.

[0020] In some implementations, the method for constructing the recombinant cells includes the following:

[0021] The recombinant vector was transformed into expression host cells, cultured, and induced to express by adding an inducer to obtain mutants expressing the above-mentioned ketone reductase or ketone reductase.

[0022] Furthermore, the recombinant vector is any of the recombinant vectors described above, and the expression host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli or yeast, with E. coli BL21(DE3) being the preferred expression host.

[0023] In some embodiments, the recombinant cells are recombinant bacteria SE and recombinant bacteria 57, and the recombinant cells can be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacteria express ketone reductase or its mutants can be any culture medium in the art that allows the recombinant genetically engineered bacteria to grow and express the ketone reductase or its mutants of the present invention, preferably TB medium.

[0024] There are no special requirements for the culture method and culture conditions. Just ensure the normal growth of the recombinant genetically engineered strain and induce the expression of ketone reductase and its mutants at 18℃.

[0025] In a seventh aspect, the present invention provides a method for preparing a ketone reductase or a mutant thereof, comprising:

[0026] The recombinant cells described above were cultured and induced with an inducing agent to obtain a culture.

[0027] Isolate the above-mentioned ketone reductase or ketone reductase mutant from the culture;

[0028] The methods for culturing and inducing recombinant cells, and for isolating ketone reductase and its mutants from the culture, are conventional methods in the field.

[0029] In an eighth aspect, the present invention provides the use of the above-described ketone reductase, any of the above-described nucleic acid molecules, mutants of the above-described ketone reductase, the above-described recombinant vector, the above-described recombinant cells, and the ketone reductase and its mutants prepared by the above-described method in the preparation of (R)-4-chloro-3-hydroxybutyrate ethyl ester.

[0030] In a ninth aspect, the present invention provides a method for preparing ethyl (R)-4-chloro-3-hydroxybutyrate, comprising: using the above-mentioned ketone reductase, the above-mentioned ketone reductase mutant, the above-mentioned recombinant cells, or the ketone reductase or its mutant prepared by the above method as a catalyst to catalyze the reaction of ethyl 4-chloroacetoacetate to convert it into ethyl (R)-4-chloro-3-hydroxybutyrate, wherein the ethyl (R)-4-chloro-3-hydroxybutyrate can be further used in the preparation of L-carnitine and its derivative salts.

[0031] In some embodiments, the temperature in the above catalytic reaction is 25-35°C, such as 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or any value or range between these values, preferably 30°C; the initial pH is 6.0-7.0, and the pH of the reaction can be adjusted using Tris or HCl (such as 500mM Tris and 1M hydrochloric acid), for example, to 6.8.

[0032] The catalytic reaction comprises ethyl 4-chloroacetoacetate, isopropanol, and NADH;

[0033] The concentration of ethyl 4-chloroacetoacetate is 100-200 g / L, for example 100 g / L, 120 g / L, 140 g / L, 160 g / L, 180 g / L, 200 g / L, or any value or range between these values, preferably 200 g / L;

[0034] The volume percentage of isopropanol is 2-3%, for example 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, or any value or range between these figures, preferably 3.0%;

[0035] The concentration of NADH is 1-10 mM, for example 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, or any value or range between these values, preferably 5 mM.

[0036] In some embodiments, the above-described catalytic reaction includes catalyzing the reaction of ethyl 4-chloroacetoacetate with any of the recombinant cells described above to generate ethyl (R)-4-chloro-3-hydroxybutyrate;

[0037] Specifically, the recombinant cells or their lyophilized powder can be used as catalysts for whole-cell catalytic production of ethyl (R)-4-chloro-3-hydroxybutyrate. If the catalyst is lyophilized powder, the dosage is 0.1-0.3 g / g ethyl 4-chloroacetoacetate, preferably 0.25 g / g ethyl 4-chloroacetoacetate. If recombinant cells are used, the dosage can be 500-700 μL of bacterial culture / ml of reaction system, wherein the OD of the bacterial culture... 600 The value is 1.8-2.2, for example, 600 μLOD. 600 The reaction system has a bacterial culture volume of 2 ml.

[0038] It should be understood that the ketone reductase KREDSJ or its mutant KREDSJ-M described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the ketone reductase KREDSJ or its mutant KREDSJ-M, or recombinant cells of this invention can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0039] The ketone reductase and its mutants of the present invention can efficiently catalyze the synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate. In particular, the mutants, under suitable conditions, with ethyl 4-chloroacetoacetate as a substrate, achieve a conversion rate of 78.4% and an enzyme activity of 181.7 U / L, which is 1.192 times that of the original enzyme. Furthermore, the mutants exhibit better tolerance in the organic solvent isopropanol compared to the original enzyme. Attached Figure Description

[0040] Figure 1 The gas chromatogram of ethyl 4-chloroacetoacetate standard.

[0041] Figure 2 Gas chromatogram of (R)-4-chloro-3-hydroxybutyrate ethyl ester standard.

[0042] Figure 3This is the gas chromatogram of the reaction solution. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0044] The Japanese fission yeast (Schizosaccharomyces japonicus) yFS275 was disclosed in the literature “Ren L, Mclean J R, Hazbun TR, et al. Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19[J]. Plos One, 2011, 6(2): e16719-e16719” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0045] pET-28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., with product catalog number B540183.

[0046] Example 1: Obtaining the gene sequence of ketone reductase

[0047] 1. Genomic DNA was extracted from Schizosaccharomyces japonicus yFS275.

[0048] 2. Using the genomic DNA obtained in step 1 as a template, PCR was performed using primer 1 (5'-ggatccatgttgaccgtctacgg-3', SEQ ID NO:5) and primer 2 (5'-aagcttttaagcagaagcagcgg-3', SEQ ID NO:6) to obtain a PCR amplification fragment containing the ketone reductase gene. The nucleotide sequence of the ketone reductase gene is shown in SEQ ID NO:2, and the amino acid sequence of the ketone reductase it encodes is shown in SEQ ID NO:1.

[0049] Example 2: Obtaining the gene sequence of a ketone reductase mutant using error-prone PCR technology

[0050] Using the PCR amplification fragment obtained in Example 1 as a template, and primers 1 and 2 as primer pairs, the following error-prone PCR was performed, resulting in 96 mutants of the ketone reductase gene.

[0051] Error-prone PCR reaction system: 5 μl of 10× amplification buffer, 4 μl each of the four dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μL of Taq DNA polymerase, Mg 2+ 7 mmol / L, add double-distilled water to 50 μl.

[0052] PCR reaction procedure: (1) Pre-denaturation: 94℃ for 3 min; (2) Denaturation: 94℃ for 30 s; Annealing: 58℃ for 30 s; Extension: 72℃ for 1.5 min; 35 cycles in total; (3) Post-extension: 72℃ for 10 min; (4) Incubate at 4℃.

[0053] Example 3: Cloning of the ketone reductase gene and construction of an expression strain

[0054] 1. The PCR amplification fragment containing the ketone reductase gene obtained in Example 1 was digested with BamHI and HindIII to obtain the gene fragment; pET-28a(+) was digested with BamHI and HindIII to obtain the vector fragment; the gene fragment and the vector fragment were ligated to obtain the recombinant expression plasmid, which was named pET-KREDSJ. The plasmid was sent for sequencing, and the results were consistent with expectations.

[0055] 2. The recombinant expression plasmid pET-KREDSJ obtained in step 1 was heat-shocked into E. coli DH5α competent cells and plated on LB solid medium containing 35 μg / mL kanamycin. The corresponding single clone strain was obtained and named SD. After amplification and plasmid extraction, the pET-KREDSJ plasmid was obtained. The obtained plasmid was chemically transformed into the expression host E. coli BL21(DE3) and plated on LB solid medium containing 35 μg / mL kanamycin for screening to obtain the recombinant strain SE expressing ketone reductase.

[0056] Example 4: Cloning of the ketone reductase mutant gene and construction of an expression strain

[0057] Following the method in Example 3, recombinant plasmids pET-ΔKREDSJ-M1 to pET-ΔKREDLH-M96 were constructed using 96 mutants of the ketone reductase gene obtained in Example 2, and recombinant bacteria 1 to recombinant bacteria 96 expressing ketone reductase mutants were obtained.

[0058] Example 5: High-throughput screening yields ketone reductase mutants with high catalytic efficiency.

[0059] The recombinant bacteria from Examples 3 and 4, which were verified by PCR, were cultured in 5 mL of TB medium, and IPTG was added at a final concentration of 0.5 mM at 18 °C to induce protein expression. After 12 h, the OD of the bacterial culture was measured. 600 The value was adjusted, and the bacterial solution was diluted with water to achieve the desired OD value. 600 The value was approximately 2. Diluted bacterial suspension was added to 96-well deep-well plates for reaction. 600 μL of bacterial suspension was added to the reaction system, bringing the total reaction volume to 1.0 ml. This mixture contained 200 mg of ethyl 4-chloroacetoacetate, 3.0% isopropanol (v / v), 5 mM NADH, and PBS to make up the volume. The initial pH was adjusted to 6.8 using hydrochloric acid. The plate reaction system was incubated at 30°C and 120 rpm for 5 hours. After the reaction was complete, 200 μL of 2 M HCl was added to terminate the reaction.

[0060] After the reaction was completed, the sample was centrifuged at 4000 rpm for 20 min, and 50 μL of the supernatant was added to 200 μL of pure water and mixed well. Then, the absorbance was measured at a wavelength of 340 nm.

[0061] The strain corresponding to the reaction solution with the lowest absorbance (recombinant strain 57) underwent plasmid extraction. The extracted plasmid was sequenced, yielding the gene sequence encoding the ketone reductase mutant, as shown in SEQ ID NO:4. The amino acid sequence of the encoded ketone reductase mutant is shown in SEQ ID NO:3. Compared to the amino acid sequence of the ketone reductase shown in SEQ ID NO:1, this ketone reductase mutant exhibits the following mutations: L at position 28 is mutated to S, N at position 95 is mutated to S, F at position 175 is mutated to V, Y at position 240 is mutated to H, and R at position 280 is mutated to G.

[0062] Ethyl 4-chloroacetoacetate and NADH react under the catalysis of ketone reductase to produce ethyl (R)-4-chloro-3-hydroxybutyrate and NAD+. + The reaction equation is shown below:

[0063]

[0064] Example 6: Enzyme preparation and enzyme activity assay

[0065] The recombinant bacteria SE obtained in Example 3 and the recombinant bacteria 57 screened in Example 5 were respectively subjected to scale-up culture. After scale-up culture, the fermentation broth was centrifuged (8000 rpm, 10 min), the cells were broken up, and the lyophilized broth was prepared to obtain ketone reductase (original enzyme) and its mutant (mutant enzyme) lyophilized powder, which was stored at -80℃.

[0066] One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 μmol of (R)-4-chloro-3-hydroxybutyrate per minute or consumes 1 μmol of the substrate ethyl 4-chloroacetoacetate per minute under the reaction conditions of Example 5 (final concentration of lyophilized powder is 0.25 g / g ethyl 4-chloroacetoacetate).

[0067] The enzyme activities of the original enzyme and the mutant enzyme were 152.43 U / L and 181.7 U / L, respectively. Compared with the original enzyme, the mutant enzyme activity was 1.192 times higher. Gas chromatography analysis of the residual ethyl 4-chloroacetoacetate substrate revealed conversion rates of 71.5% and 78.4% for the two enzymes, respectively. The specific gas chromatography detection conditions are as follows:

[0068] Centrifuge the reaction solution and collect 300 μL of the supernatant. Add 600 μL of ethyl acetate, mix well, and centrifuge again. Collect the upper layer, place it in a sample vial, and place it on a sample rack for gas chromatography detection. The gas chromatography column is an Agilent DB-5 capillary column, the carrier gas is nitrogen, and the detector is a hydrogen ion detector. The injection volume is 1 μL. The injection port temperature is 250℃, the detector temperature is 250℃, and the column oven temperature is 90℃. The column flow rate of nitrogen carrier gas is 1 mL / min.

[0069] The gas chromatograms of ethyl 4-chloroacetoacetate standard and ethyl (R)-4-chloro-3-hydroxybutyrate standard are shown below. Figure 1 and Figure 2 As shown, the peak time of ethyl 4-chloroacetoacetate was 6.598 min, and the peak time of ethyl (R)-4-chloro-3-hydroxybutyrate was 7.022 min.

[0070] The gas chromatogram of the reaction solution is as follows: Figure 3 As shown, the peak time of (R)-4-chloro-3-hydroxybutyrate ethyl ester was 7.022 min, and the peak time of 4-chloroacetoacetate ethyl ester was 6.598 min.

[0071] Example 7: Enzyme Organic Solvent Tolerance Test

[0072] Take a small amount of the lyophilized powder of the original enzyme and mutant enzyme obtained in Example 6, and react them according to the reaction conditions in Example 5, and adjust the volume percentage of isopropanol according to Table 1. The conversion rates of the two enzymes under different concentrations of isopropanol at the end of the reaction are shown in Table 1.

[0073] Table 1

[0074]

[0075] The results showed that the mutant enzyme had good tolerance to the organic solvent isopropanol. Specifically, the conversion rate was above 70% when the isopropanol content was between 0-15%, which was better than that of the original enzyme. When the isopropanol content was 15%, the conversion rate of the original enzyme decreased to 54.1%, while the conversion rate of the mutant enzyme was still above 70%.

[0076] sequence

[0077] SEQ ID NO:1

[0078] MLTVYGAKKNAWAVVTGATDGIGKEYALQLAKAGFNIVIVSRNPEKLSRVAQEITEAYRVEVQTYVIDYKIATAATFQKLAEFLKPFQVTVLVNNVGLSHNMPVSFSETTEQEMDDIMQINCFGTLHTTKAVLPSMLEQRRSNKNGPRC LILTMGSFAGLLPSPYLSTYAGSKAFLANWSASLAEEVKKEGIDVWCYQSYLVCSAMSKVRRPTATIPTPKNFVREALGSIGVQRGGNQPYISQPFPSHAALSWVLEQAASRVKGFVVAQVAAMHLSIRSRALRKQAREQQKAKETAASA

[0079] SEQ ID NO:2

[0080] atgttgaccgtctacggtgcgaagaaaaatgcttgggcagtggtaacgggtgccacggacggtattggcaaggaatacgccttacaacttgccaaggctggcttcaacatcgtgattgttagtcgtaatcccgaaaaattgagccgtgttgctcaggaaatcacggaggcctatcgtgttgaggtccaaacatatgtgatcgactacaaaattgctacggctgccactttccaaaagcttgctgagttcctgaagccattccaggtgaccgtactcgtaaacaacgtcgggttgtctcacaacatgcctgtgagtttcagtgaaaccaccgaacaagagatggatgacattatgcaaatcaactgttttggcacacttcataccactaaggccgtccttcctagcatgttggaacaacgtcgtagcaacaagaatggacctcgctgcctcattttgactatgggttctttcgctggtctgcttccctcgccttatctgtctacgtacgctggttctaaggctttcttggccaactggtctgcttccctggccgaagaggtcaagaaggagggcattgatgtttggtgctaccaatcttatcttgtctgctctgccatgtccaaggtccgtcgccctactgctactattcccactcccaagaactttgtccgcgaggcactgggcagcattggcgtccaacgcggtggtaaccagccctacatcagtcagccattcccctctcatgccgctctcagctgggttctcgaacaagctgccagtcgtgtgaaaggtttcgtcgttgctcaagtcgctgctatgcacttgagcattcgttcccgtgcccttcgcaagcaggctcgcgagcaacagaaggccaaggagaccgctgcttctgcttaa

[0081] SEQ ID NO:3

[0082] MLTVYGAKKNAWAVVTGATGIGKEYASQLAKAGFNIVIVSRNPEKLSRVAQEITEAYRWEVQTYVIDYKIATAATFQKLAEFLKPFQVTVLVNSVGLSHNMPVSFSETTEQEMDDIMQINCFGTLHTTKAVLPSMLEQRRSNKNGPRC LILTMGSFAGLLPSPYLSTYAGSKAVLANWSASLAEEVKKEGIDVWCYQSYLVCSAMSKVRRPTATIPTPKNFVREALGSIGVQRGGNQPHISQPFPSHAALSWVLEQAASRVKGFVVAQVAAMHLSIRSGALRKQAREQQKAKETAASA

[0083] SEQ ID NO:4

[0084] atgttgaccgtctacggtgcgaagaaaaatgcttgggcagtggtaacgggtgccacggacggtattggcaaggaatacgcctcacaacttgccaaggctggcttcaacatcgtgattgttagtcgtaatcccgaaaaattgagccgtgttgctcaggaaatcacggaggcctatcgtgttgaggtccaaacatatgtgatcgactacaaaattgctacggctgccactttccaaaagcttgctgagttcctgaagccattccaggtgaccgtactcgtaaacagcgtcgggttgtctcacaacatgcctgtgagtttcagtgaaaccaccgaacaagagatggatgacattatgcaaatcaactgttttggcacacttcataccactaaggccgtccttcctagcatgttggaacaacgtcgtagcaacaagaatggacctcgctgcctcattttgactatgggttctttcgctggtctgcttccctcgccttatctgtctacgtacgctggttctaaggctgtcttggccaactggtctgcttccctggccgaagaggtcaagaaggagggcattgatgtttggtgctaccaatcttatcttgtctgctctgccatgtccaaggtccgtcgccctactgctactattcccactcccaagaactttgtccgcgaggcactgggcagcattggcgtccaacgcggtggtaaccagccccacatcagtcagccattcccctctcatgccgctctcagctgggttctcgaacaagctgccagtcgtgtgaaaggtttcgtcgttgctcaagtcgctgctatgcacttgagcattcgttccggtgcccttcgcaagcaggctcgcgagcaacagaaggccaaggagaccgctgcttctgcttaa

Claims

1. A ketone reductase having the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the ketone reductase of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

2.

3. The mutant of the ketone reductase according to claim 1, the amino acid sequence of which is shown in SEQ ID NO:

3.

4. A nucleic acid molecule encoding a mutant of the ketone reductase of claim 3, the nucleotide sequence of which is shown in SEQ ID NO:

4.

5. A recombinant vector comprising the nucleic acid molecule of claim 2 or 4.

6. A recombinant cell comprising the recombinant vector of claim 5.

7. A method for preparing a ketone reductase or a mutant thereof, comprising the following steps: 1) The recombinant cells of claim 6 are cultured and ketone reductase or its mutant is induced to express; 2) Isolate the ketone reductase of claim 1 or a mutant of the ketone reductase of claim 3 from the culture obtained in 1).

8. The use of the ketone reductase of claim 1, the nucleic acid molecule of claim 2, the mutant of the ketone reductase of claim 3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant cell of claim 6, and / or the ketone reductase or its mutant prepared by the method of claim 7 in the catalytic synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate.

9. A method for preparing (R)-4-chloro-3-hydroxybutyrate ethyl ester, comprising the following steps: Using the ketone reductase of claim 1, the mutant of the ketone reductase of claim 3, the recombinant cells of claim 6, and / or the ketone reductase or its mutant prepared by the method of claim 7 as catalysts, ethyl 4-chloroacetoacetate is catalyzed to convert (R)-4-chloro-3-hydroxybutyrate ethyl ester.

10. The preparation method according to claim 9, characterized in that: The catalytic reaction temperature is 25-35℃, pH 6.0-7.0, and the reaction system contains ethyl 4-chloroacetoacetate, isopropanol, and NADH. The final concentration of ethyl 4-chloroacetoacetate is 100-200 g / L; The volume percentage of isopropanol is 2-3%; The final concentration of NADH is 1-10 mM.

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