A ketoreductase and its use in the production of (r)-4-chloro-3-hydroxybutyric acid ethyl ester

Abstract

The application discloses a ketoreductase and application of the ketoreductase in preparation of (R)-4-chloro-3-hydroxybutyric acid ethyl ester. The ketoreductase disclosed by the application has an amino acid sequence shown in SEQ ID NO:1, and a mutant of the ketoreductase has an amino acid sequence shown in SEQ ID NO:3. The ketoreductase and the mutant thereof provided by the application can realize efficient catalysis of a reaction of converting 4-chloroacetyl acetic acid ethyl ester into (R)-4-chloro-3-hydroxybutyric acid ethyl ester.

Description

Technical Field

[0001] This invention belongs to the field of functional enzyme screening technology, and relates to a ketone reductase derived from Lachnellula hyalina and its mutants, nucleic acid molecules encoding them, vectors and 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] L-Carnitine was initially discovered in extracts from red meat. In the human body, it is primarily responsible for transporting long-chain fatty acids into the mitochondria for oxidation. It is widely used in pharmaceuticals and health products. Currently, the preparation of L-carnitine mainly relies on chemical synthesis. Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is an important chiral intermediate widely used in the preparation of chiral drugs such as L-carnitine and afatinib. Through ammoniation, hydrolysis, and deionization, L-carnitine and its derivative salts can be obtained. Using ethyl 4-chloroacetoacetate as a substrate, catalysis with a chiral catalyst (a metal catalyst prepared from rare metals) is currently the main method for preparing ethyl (R)-4-chloro-3-hydroxybutyrate. While chiral catalysts are being sought as alternatives, green biocatalysis and fermentation methods are being explored, which are also currently the most suitable methods for the preparation of L-carnitine via enzymatic catalysis.

[0003] The preparation of ethyl (R)-4-chloro-3-hydroxybutyrate using enzymatic catalysis of ethyl 4-chloroacetoacetate is favored, with ketone reductase catalysis being particularly favored. This process requires the addition of a certain amount of reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme. Currently, the preparation of ethyl (R)-4-chloro-3-hydroxybutyrate using enzymatic or bio-fermentation methods has not yet reached the industrial production stage. Therefore, further research, discovery, and modification of enzymes capable of catalyzing this reaction are needed to meet the demands of industrial-scale production of ethyl (R)-4-chloro-3-hydroxybutyrate. Summary of the Invention

[0004] The purpose of this invention is to provide a ketone reductase, its mutants, its encoding gene, and its applications, thereby improving enzyme activity, reusability, and conversion rate of (R)-CHBE synthesis.

[0005] This invention obtains ketone reductase KREDLH from the strain Lachnellula hyalina, 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. By further mutagenesis, a ketone reductase mutant KREDLH-M with significantly improved enzyme activity, conversion rate and reusability is obtained, 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 expressing it from Escherichia coli using recombinant technology.

[0008] In some embodiments, the ketone reductase is obtained by transforming a recombinant vector containing its encoding gene into a recombinant genetically engineered strain of Escherichia coli expression host (e.g., E. coli BL21(DE3)), then 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, reusability stability, and conversion rate compared to the above-mentioned ketone reductase. The mutant of the above-mentioned ketone reductase provided by the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically, such as by expressing it from prokaryotes (Escherichia coli) using recombinant technology.

[0012] In some implementations, the ketone reductase mutant is obtained by constructing a recombinant genetically engineered bacterium (e.g., E. coli BL21(DE3)) by transforming a recombinant vector containing its encoding gene into an E. coli expression host, then culturing the strain and adding an inducer to induce expression.

[0013] 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.

[0014] The nucleic acid molecules provided by this invention can usually be obtained by amplification using a PCR instrument or by artificial synthesis.

[0015] Fifthly, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above.

[0016] In some embodiments, the recombinant vector is pET-KREDLH or pET-ΔKREDLH-M61, which is obtained by replacing the sequence between the EcoRI 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.

[0017] In a sixth aspect, the present invention provides a recombinant cell comprising any of the recombinant vectors described above.

[0018] In some embodiments, the recombinant cells are induced to express the aforementioned ketone reductase or a mutant of the ketone reductase.

[0019] In some implementations, the method for constructing the recombinant cells includes the following:

[0020] 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.

[0021] 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.

[0022] In some embodiments, the recombinant cells are recombinant bacteria KE and recombinant bacteria 61, 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, such as TB medium.

[0023] 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℃.

[0024] More specifically, the above-mentioned method for constructing recombinant cells includes the following steps:

[0025] (1) Amplification of the ketone reductase gene KREDLH;

[0026] (2) Obtaining the gene ΔKREDLH-M61 of the ketone reductase mutant;

[0027] (3) Construction of recombinant expression plasmids pET-KREDLH and pET-ΔKREDLH-M61;

[0028] (4) The recombinant expression plasmids pET-KREDLH and pET-ΔKREDLH-M61 were transformed into host cells;

[0029] (5) Positive clones were obtained by screening on plate resistance medium;

[0030] In a seventh aspect, the present invention provides a method for preparing a ketone reductase or a mutant thereof, comprising:

[0031] The recombinant cells described above were cultured and induced with an inducing agent to obtain a culture.

[0032] Isolate the above-mentioned ketone reductase or ketone reductase mutant from the culture;

[0033] 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.

[0034] 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 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.

[0035] 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 cell or the ketone reductase or mutant prepared by the above method as a catalyst to catalyze the reaction of ethyl 4-chloroacetoacetate to obtain ethyl (R)-4-chloro-3-hydroxybutyrate.

[0036] In some embodiments, the temperature in the above catalytic reaction is 25-35°C, for example 25°C, 30°C, 35°C, or any value or range between these values, with 30°C being preferred as the reaction temperature; 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 pH 6.8.

[0037] The catalytic reaction comprises ethyl 4-chloroacetoacetate, isopropanol, and NADH;

[0038] 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 140 g / L;

[0039] The amount of isopropanol added is 2-3% (volume percentage), preferably 3%;

[0040] 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, with 5 mM being preferred.

[0041] In some embodiments, the above-described catalytic reaction includes the catalytic reaction of any of the recombinant cells described above with ethyl 4-chloroacetoacetate to generate ethyl (R)-4-chloro-3-hydroxybutyrate;

[0042] 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 amount used is 0.1-0.25 g / g ethyl 4-chloroacetoacetate, preferably 0.2 g / g ethyl 4-chloroacetoacetate. If the catalyst is the culture medium of recombinant cells, the amount used can be 400-600 μL of bacterial culture / ml of reaction system, wherein the OD of the bacterial culture is... 600 The value is 1.8-2.2, for example, 500 μL OD. 600 The reaction system has a bacterial culture volume of 2 ml.

[0043] It should be understood that the ketone reductase KREDLH or its mutant KREDLH-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 KREDLH or its mutants can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0044] The ketone reductase and its mutant of the present invention can efficiently catalyze the synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate. In particular, the mutant, under suitable conditions, with ethyl 4-chloroacetoacetate as substrate, has a conversion rate of 82.6% and an enzyme activity of 236.8 U / L, which is 1.415 times that of the original enzyme, and its stability is also better than that of the original enzyme. Attached Figure Description

[0045] Figure 1 The gas chromatogram of ethyl 4-chloroacetoacetate standard.

[0046] Figure 2Gas chromatogram of (R)-4-chloro-3-hydroxybutyrate ethyl ester standard.

[0047] Figure 3 This is the gas chromatogram of the reaction solution. Detailed Implementation

[0048] 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.

[0049] The strain Lachnellula hyalina was published in the literature “H Park, Kim, DY, Kim, SR, & Eom, AH (2018). New records of endophytic fungi isolated from leaves of abies koreana and taxus cuspidata in korea. The Korean Journal of Mycology, 46(3), 241-248.” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0050] pET-28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., with product catalog number B540183.

[0051] Example 1: Obtaining the gene sequence of ketone reductase KREDLH

[0052] 1. Extract genomic DNA from Lachnellula hyalina.

[0053] 2. Using the genomic DNA of Lachnellula hyalina as a template, PCR was performed using primer 1 (5'-ggaattccatgaaggtctttctgagcggagg-3', SEQ ID NO:5) and primer 2 (5'-cccaagcttgggtcactccttatataatccctctttcgggtaatc-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.

[0054] MKVFLSGGSGFIAAHVLDILLEHGHTVITSVRSQEKANKIKEAHPNTPASQLEFRIVKDIAQEGAFDEAIKIDGLEAVIHTASPFHFNVTDVKKDLLDPAIIGTTGILKAIKKNAPSVKRVVITSSFASIVNPSKGNSWTEHTYSEEDWNPITEEEAVLNPSNGYRASKTFAEKAAWEFVEKEKPNFTLSTMCPPLVIGPIVHYLNSLDSLNTSNQRTANLMTGKNKSEIPDTGTYIWIDVRDLALAHVKAIELPEAANKRFFITAGYFSNKEIAEIIRKNFPALEKELPAKDVKGGDYPKEGLYKE(SEQ ID NO:1)

[0055] atgaaggtctttctgagcggaggaagtggcttcatcgccgcccacgtcctcgacatcctactcgagcatggccatactgtcatcacctcggttcgttcccaagagaaagccaacaagatcaaagaggcgcaccccaacacgcctgcctcccagctcgagttccggattgtcaaagacatagcacaggagggggcctttgacgaagccatcaagattgacggcctggaagcggtgattcacacagcctcgcctttccatttcaacgtcacagatgtcaagaaagacttgcttgaccctgccataatcggcacaacaggtatcctgaaagccatcaagaagaatgctcccagcgtgaagagagtcgtcatcacgagttcctttgccagcatcgtcaatccaagtaagggaaactcctggaccgagcacacgtacagcgaggaggactggaaccccatcacggaagaagaggcggtgctgaaccccagcaatggatacagagccagcaagacgttcgccgagaaagctgcgtgggagtttgtcgagaaggagaaaccaaactttacgttgagcactatgtgccctcctttagttataggtccaattgtccactacctcaacagcctcgatagcctcaacacctctaaccagcgaaccgccaacctcatgaccggcaagaacaagtctgaaatccccgacaccggtacctacatctggatcgatgtgcgagatctcgccctcgcccacgtcaaagccatcgagctcccagaagccgcgaacaagcgattcttcatcaccgctggttacttctccaacaaggagatcgctgagattatccgcaagaacttccccgcgctcgaaaaggaattgccggcgaaggacgtcaagggtggagattacccgaaagagggattatataaggagtga(SEQ ID NO:2)

[0056] Example 2: Obtaining the gene sequence of ketoreductase mutant KREDLH-M using error-prone PCR technology

[0057] 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 84 mutants of the ketone reductase gene.

[0058] 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.

[0059] 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; 40 cycles in total; (3) Post-extension: 72℃ for 10 min; (4) Incubate at 4℃.

[0060] Example 3: Cloning of the ketone reductase gene and construction of an expression strain

[0061] 1. The PCR amplification fragment containing the ketone reductase gene obtained in Example 1 was digested with EcoRI and HindIII to obtain the gene fragment; pET-28a(+) was digested with EcoRI 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-KREDLH. The plasmid was sent for sequencing, and the results were consistent with expectations.

[0062] 2. The recombinant expression plasmid pET-KREDLH obtained in step 1 was transformed into E. coli DH5α competent cells by heat shock. The cells were then plated on LB solid medium containing 25 μg / mL kanamycin and cultured to obtain the corresponding single clone strain, which was named K. After amplification and plasmid extraction, the pET-KREDLH plasmid was obtained. The obtained plasmid was then transformed into the expression host E. coli BL21(DE3) by chemical transformation. The cells were then plated on LB solid medium containing 25 μg / mL kanamycin for screening to obtain the recombinant strain KE expressing ketone reductase.

[0063] Example 4: Cloning of the ketone reductase mutant gene and construction of an expression strain

[0064] Following the same method as in Example 3, recombinant plasmids pET-ΔKREDLH-M1 to pET-ΔKREDLH-M84 were constructed using 84 mutants of the ketone reductase gene obtained in Example 2, and recombinant bacteria 1 to recombinant bacteria 84 expressing ketone reductase mutants were obtained.

[0065] Example 5: High-throughput screening of ketone reductase mutants

[0066] 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 0.6 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. A 1.2 ml catalytic reaction system was designed in a 96-well deep-well plate. The initial pH was adjusted to 6.8. The reaction system contained 600 μL of diluted bacterial solution, 168 mg of ethyl 4-chloroacetoacetate, 3.0% isopropanol (v / v), and 5 mM NADH, with the volume made up with PBS. The plate was incubated at 30 °C and 120 rpm for 5 h. After the reaction was completed, 200 μL of 2 M HCl was added to terminate the reaction.

[0067] After the reaction was completed, 50 μL of the supernatant was added to 200 μL of pure water and mixed well. The absorbance was then measured at a wavelength of 340 nm.

[0068] The plasmid of the strain (recombinant strain 61) corresponding to the reaction solution with the lowest absorbance value was extracted and sequenced. The gene sequence encoding the ketone reductase mutant is shown in SEQ ID NO:4, and the amino acid sequence of the encoded ketone reductase mutant is shown in SEQ ID NO:3. Compared with the amino acid sequence of wild-type ketone reductase SEQ ID NO:1, this ketone reductase mutant has undergone the following mutations: amino acid K at position 41 is mutated to T, amino acid S at position 129 is mutated to N, amino acid V at position 180 is mutated to A, and amino acid K at position 272 is mutated to Q.

[0069] MKVFLSGGSGFIAAHVLDILLEHGHTVITSVRSQEKANKITEAHPNTPASQLEFRIVKDIAQEGAFDEAIKIDGLEAVIHTASPFHFNVTDVKKDLLDPAIIGTTGILKAIKKNAPSVKRVVITSSFANIVNPSKGNSWTEHTYSEEDWNPITEE EAVLNPSNGYRASKTFAEKAAWEFAEKEKPNFTLSTMCPPLVIGPIVHYLNSLDSLNTSNQRTANLMTGKNKSEIPDTGTYIWIDVRDLALAHVKAIELPEAANKRFFITAGYFSNQEIAEIIRKNFPALEKELPAKDVKGGDYPKEGLYKE(SEQ ID NO:3)

[0070] atgaaggtctttctgagcggaggaagtggcttcatcgccgcccacgtcctcgacatcctactcgagcatggccatactgtcatcacctcggttcgttcccaagagaaagccaacaagatcacagaggcgcaccccaacacgcctgcctcccagctcgagttccggattgtcaaagacatagcacaggagggggcctttgacgaagccatcaagattgacggcctggaagcggtgattcacacagcctcgcctttccatttcaacgtcacagatgtcaagaaagacttgcttgaccctgccataatcggcacaacaggtatcctgaaagccatcaagaagaatgctcccagcgtgaagagagtcgtcatcacgagttcctttgccaacatcgtcaatccaagtaagggaaactcctggaccgagcacacgtacagcgaggaggactggaaccccatcacggaagaagaggcggtgctgaaccccagcaatggatacagagccagcaagacgttcgccgagaaagctgcgtgggagtttgccgagaaggagaaaccaaactttacgttgagcactatgtgccctcctttagttataggtccaattgtccactacctcaacagcctcgatagcctcaacacctctaaccagcgaaccgccaacctcatgaccggcaagaacaagtctgaaatccccgacaccggtacctacatctggatcgatgtgcgagatctcgccctcgcccacgtcaaagccatcgagctcccagaagccgcgaacaagcgattcttcatcaccgctggttacttctccaaccaggagatcgctgagattatccgcaagaacttccccgcgctcgaaaaggaattgccggcgaaggacgtcaagggtggagattacccgaaagagggattatataaggagtga(SEQ ID NO:4)

[0071] 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:

[0072]

[0073] Example 6: Enzyme preparation and enzyme activity assay

[0074] The recombinant bacteria KE obtained in Example 3 and the recombinant bacteria 61 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℃.

[0075] Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of (R)-4-chloro-3-hydroxybutyrate per minute or the amount of enzyme required to consume 1 μmol of the substrate ethyl 4-chloroacetoacetate per minute under the reaction conditions of Example 5 (final concentration of lyophilized powder is 0.2 g / g ethyl 4-chloroacetoacetate).

[0076] The enzyme activities of the original enzyme and the mutant enzyme were 167.35 U / L and 236.8 U / L, respectively. Compared with the original enzyme, the enzyme activity of the mutant enzyme was 1.415 times. The conversion rates of the two enzymes were 76.8% and 82.6%, respectively, by analyzing the residual amount of the substrate ethyl 4-chloroacetoacetate by gas chromatography.

[0077] The gas chromatography detection method is as follows:

[0078] After the reaction was completed, the reaction solution was centrifuged, and 400 μL of the supernatant was collected. 800 μL of ethyl acetate was added and mixed thoroughly. The mixture was then filtered through a membrane and used for gas chromatography analysis of the conversion rate. An Agilent DB-5 capillary column was used with nitrogen as the carrier gas. A hydrogen ion detector was used, with the injection port temperature at 250 °C, the detector temperature at 250 °C, the column oven temperature at 90 °C, the injection volume at 1 μL, and the column flow rate at 1 mL / min.

[0079] 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.

[0080] The gas chromatogram of the reaction solution is as follows: Figure 3As shown, the peak time of (R)-4-chloro-3-hydroxybutyrate ethyl ester was 7.018 min, and the peak time of 4-chloroacetoacetate ethyl ester was 6.612 min.

[0081] Example 7: Enzyme stability test

[0082] Take a small amount of the lyophilized powder of the original enzyme and mutant enzyme obtained in Example 6, and react it according to the reaction conditions in Example 5. After the reaction is completed, centrifuge to recover the enzyme and react it again. The conversion rates of the two enzymes in each batch are shown in Table 1.

[0083] Table 1

[0084]

[0085] The results showed that the mutant enzyme had good reusability, and the conversion rate could still reach more than 80% after 6 batches of reaction. Compared with the original enzyme, it had better stability. After 6 batches of reaction, the activity of the original enzyme decreased by 12.37%, while the activity of the recombinant enzyme only decreased by 2.67%.

Claims

1. A ketone reductase mutant, the amino acid sequence of which is shown in SEQ ID NO:

3.

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

4.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the recombinant vector of claim 3.

5. A method for preparing a ketone reductase mutant, comprising the following steps: 1) The recombinant cells of claim 4 are cultured and ketone reductase mutant expression is induced; 2) Isolate the ketone reductase mutant of claim 1 from the culture obtained in 1).

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

7. A method for preparing (R)-4-chloro-3-hydroxybutyrate ethyl ester, comprising the following steps: Using the ketone reductase mutant of claim 1, the recombinant cells of claim 4, and / or the ketone reductase mutant prepared by the preparation method of claim 5 as catalysts, ethyl 4-chloroacetoacetate is catalyzed to convert (R)-4-chloro-3-hydroxybutyrate ethyl ester.

8. The preparation method according to claim 7, characterized in that: The catalytic reaction conditions are: temperature 25-35℃, pH 6.0-7.0, and the reaction feedstock includes: 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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