CN117402839A - Ketoreductase and application thereof in preparation of (R) -4-chloro-3-hydroxybutyrate - Google Patents

Abstract

The invention discloses ketoreductase and application thereof in preparation of (R) -4-chloro-3-hydroxybutyrate ethyl ester. The ketoreductase disclosed by the invention has an amino acid sequence shown as SEQ ID NO. 1, and the mutant thereof has an amino acid sequence shown as SEQ ID NO. 3. The ketoreductase and the mutant thereof provided by the invention can realize the reaction of efficiently catalyzing the conversion of the 4-chloroacetoacetic acid ethyl ester into the (R) -4-chloro-3-hydroxybutyric acid ethyl ester.

Description

Ketoreductase and application thereof in preparation of (R) -4-chloro-3-hydroxybutyrate

Technical Field

The invention belongs to the technical field of functional enzyme screening, and relates to ketoreductase from Lachnellula hyalina, mutants thereof, nucleic acid molecules encoding the ketoreductase, vectors and cells containing the nucleic acid molecules, a preparation method of the ketoreductase, the mutants, and application of the nucleic acid molecules in preparation of (R) -4-chloro-3-hydroxybutyrate.

Background

L-carnitine is initially found in red meat extracts, is mainly responsible for transporting long-chain fatty acid into mitochondria to oxidize in human bodies, is widely applied to the fields of medicines, health-care products and the like, and is prepared by a chemical synthesis method at present. The (R) -4-chloro-3-hydroxybutyric acid ethyl ester ((R) -CHBE) is an important chiral intermediate and can be widely used for preparing chiral medicines such as L-carnitine and afatinib. The L-carnitine and derivative salts thereof can be prepared by ammonification, hydrolysis and deionized. The method takes 4-chloroacetoacetic acid ethyl ester as a substrate, is catalyzed by a chiral catalyst (a metal catalyst prepared by rare metals) which is the main means for preparing (R) -4-chloro-3-hydroxybutyric acid ethyl ester at present, the chiral catalyst is being replaced by an attempt to seek a green biocatalysis and fermentation method, and is the most suitable method for preparing L-carnitine by the catalysis of biological enzymes at present.

The biological enzyme is used for catalyzing the 4-chloroacetoacetic acid ethyl ester to prepare the (R) -4-chloro-3-hydroxybutyric acid ethyl ester, the ketoreductase is used for catalyzing the preparation, and a certain amount of reduced Nicotinamide Adenine Dinucleotide (NADH) is required to be added as coenzyme to participate in the reaction process. At present, the preparation of the (R) -4-chloro-3-hydroxy ethyl butyrate based on a biological enzyme or biological fermentation mode does not enter an industrial production stage, so that further excavation, discovery and modification of the biological enzyme capable of catalyzing the reaction are needed, and the requirement of industrially producing the (R) -4-chloro-3-hydroxy ethyl butyrate can be met.

Disclosure of Invention

The invention aims to provide ketoreductase, a mutant thereof, a coding gene and application thereof, so as to improve the enzyme activity, the repeated use stability and the conversion rate of catalytic synthesis (R) -CHBE.

According to the invention, ketoreductase KREDLH is obtained from Lachnellula hyalina bacterial strain, the amino acid sequence of the ketoreductase KREDLH is shown as SEQ ID NO. 1, the nucleotide sequence of the ketoreductase KREDLH is shown as SEQ ID NO. 2, and the ketoreductase mutant KREDLH-M which is obviously improved in enzyme activity, conversion rate and repeated use stability is obtained by further carrying out mutation treatment on the ketoreductase KREDLH, the amino acid sequence of the ketoreductase KREDLH-M is shown as SEQ ID NO. 3, and the nucleotide sequence of the ketoreductase mutant KREDLH-M is shown as SEQ ID NO. 4.

In a first aspect, the present invention provides a ketoreductase having the amino acid sequence set forth in SEQ ID NO. 1.

The ketoreductase provided by the invention can be synthesized artificially, or the encoding gene of the ketoreductase can be synthesized first and then biologically expressed, for example, the ketoreductase is obtained by expressing the ketoreductase from escherichia coli by using a recombination technology.

In some embodiments, the ketoreductase is a recombinant genetically engineered strain constructed by transforming a recombinant vector containing a gene encoding the same into an E.coli expression host (e.g., E.coli BL21 (DE 3)), then culturing the strain, and adding an inducer to induce expression to obtain the ketoreductase.

In a second aspect, the present invention provides a nucleic acid molecule encoding the ketoreductase described above, having the nucleotide sequence set forth in SEQ ID NO. 2.

The above nucleic acid molecules provided by the present invention can be obtained usually by amplification using a PCR instrument or artificial synthesis.

In a third aspect, the present invention provides a mutant of the above ketoreductase having an amino acid sequence shown in SEQ ID NO. 3, which has further improved catalytic activity, reuse stability and conversion rate as compared to the above ketoreductase. The mutant of the ketoreductase provided by the invention can be synthesized artificially, or can be obtained by synthesizing the coding gene and then biologically expressing the coding gene, for example, the coding gene is obtained by expressing the coding gene from a prokaryote (escherichia coli) by using a recombination technology.

In some embodiments, the ketoreductase mutant is obtained by transforming a recombinant vector containing the encoding gene into an escherichia coli expression host (e.g. e.coli BL21 (DE 3)) to construct a recombinant genetically engineered bacterium, culturing the strain, and adding an inducer to induce expression.

In a fourth aspect, the present invention provides a nucleic acid molecule encoding a ketoreductase mutant as described above, having the nucleotide sequence set forth in SEQ ID NO. 4.

The above nucleic acid molecules provided by the present invention can be obtained usually by amplification using a PCR instrument or artificial synthesis.

In a fifth aspect, the invention provides a recombinant vector comprising a nucleic acid molecule as described in any one of the preceding claims.

In some embodiments, the recombinant vector is pET-KREDLH or pET-delta KREDLH-M61, and the remaining sequence is obtained by replacing the EcoRI and HindIII cleavage site sequences of pET-28a (+) with the nucleic acid molecule encoding the ketoreductase or the ketoreductase mutant, respectively.

In a sixth aspect, the invention provides a recombinant cell comprising a recombinant vector as described in any one of the preceding claims.

In some embodiments, the recombinant cell expresses the ketoreductase or a mutant of the ketoreductase described above after induction.

In some embodiments, the method of constructing a recombinant cell comprises the following:

the recombinant vector is transformed into an expression host cell, and the expression is induced by culturing and adding an inducer, so that the ketoreductase or the mutant of the ketoreductase is obtained.

Further, the recombinant vector is any one of the above recombinant vectors, and the expression host cell is a prokaryotic cell or a eukaryotic cell, for example, escherichia coli, yeast, and the like, preferably escherichia coli expression host e.coli BL21 (DE 3).

In some embodiments, the recombinant cells are recombinant bacteria KE and recombinant bacteria 61, and the recombinant cells may be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacterium expresses the ketoreductase or a mutant thereof may be a culture medium, such as a TB culture medium, which is known in the art to allow the recombinant genetically engineered bacterium to grow and express the ketoreductase or a mutant thereof of the present invention.

The culture method and the culture conditions have no special requirements, the normal growth of the recombinant genetic engineering strain is ensured, and the ketoreductase and the mutant thereof are induced and expressed at 18 ℃.

More specifically, the construction method of the recombinant cell comprises the following steps:

(1) Amplifying a ketoreductase gene KREDLH;

(2) Obtaining a gene delta KREDLH-M61 of the ketoreductase mutant;

(3) Constructing recombinant expression plasmids pET-KREDLH and pET-delta KREDLH-M61;

(4) Recombinant expression plasmids pET-KREDLH and pET-delta KREDLH-M61 are transformed into host cells;

(5) Screening a plate resistance culture medium to obtain a positive clone strain;

in a seventh aspect, the present invention provides a method for preparing a ketoreductase or a mutant thereof, comprising:

culturing any recombinant cell and adding an inducer for induction to obtain a culture;

isolating the ketoreductase or ketoreductase mutant described above from the culture;

among them, the method of culturing and inducing recombinant cells, and the method of separating ketoreductase and its mutants from the culture are all conventional methods in the art.

In an eighth aspect, the present invention provides the use of a ketoreductase as described above, a nucleic acid molecule as described above, a mutant of a ketoreductase as described above, a recombinant vector as described above, a recombinant cell as described above and a ketoreductase as prepared by the method as described above, and a mutant thereof, for the preparation of ethyl (R) -4-chloro-3-hydroxybutyrate, which can be further used for the preparation of L-carnitine and its derivative salts.

In a ninth aspect, the present invention provides a process for preparing (R) -4-chloro-3-hydroxybutyric acid ethyl ester, comprising: and (3) carrying out catalytic reaction on the ethyl 4-chloroacetoacetate by taking the ketoreductase, the ketoreductase mutant, the recombinant cell or the ketoreductase or the mutant thereof prepared by the method as a catalyst to obtain the (R) -4-chloro-3-hydroxybutyrate.

In some embodiments, in the above catalytic reactions, the temperature is 25-35 ℃, e.g., 25 ℃, 30 ℃, 35 ℃, or a value or range between any two of these values, with 30 ℃ being preferred as the reaction temperature; the initial pH is 6.0-7.0, and the pH of the reaction can be adjusted, for example to pH 6.8, using Tris or HCl (e.g., 500mM Tris and 1M hydrochloric acid).

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

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

the isopropanol is added in an amount of 2-3% (volume percent), preferably 3%;

the concentration of NADH is 1-10mM, e.g., 1mM, 2mM, 4mM, 6mM, 8mM, 10mM, or a value or range between any two of these values, with 5mM being preferred.

In some embodiments, the recombinant cell comprising any of the above-described catalytic reactions catalyzes the reaction of ethyl 4-chloroacetoacetate to produce ethyl (R) -4-chloro-3-hydroxybutyrate;

specifically, the recombinant cells or the freeze-dried powder thereof can be used as a catalyst for whole-cell catalytic production of (R) -4-chloro-3-hydroxybutyric acid ethyl ester, and if the catalyst is the freeze-dried powder, the dosage of the catalyst is 0.1-0.25g/g of 4-chloroacetoacetic acid ethyl ester, preferably 0.2g/g of 4-chloroacetoacetic acid ethyl ester; if the catalyst is a culture solution of recombinant cells, the dosage of the catalyst can be 400-600 mu L of bacterial liquid/ml of reaction system, wherein the OD of the bacterial liquid ₆₀₀ 1.8-2.2, e.g. 500. Mu.L OD ₆₀₀ 2 bacteria liquid/ml reaction system.

It will be appreciated that the ketoreductase KREDLH or mutant KREDLH-M of the present invention may be used in the form of whole cells of an engineering bacterium, as a crude enzyme without purification, or as a partially purified or completely purified enzyme. The ketoreductase KREDLH or mutants thereof of the present invention may also be prepared into immobilized enzymes or immobilized cell forms of catalysts using immobilization techniques known in the art.

The ketoreductase and the mutant thereof can efficiently catalyze and synthesize (R) -4-chloro-3-hydroxy ethyl butyrate, particularly the mutant thereof, under proper conditions, 4-chloroacetoacetic acid ethyl ester is taken as a substrate, the conversion rate is 82.6%, the enzyme activity is 236.8U/L, which is 1.415 times of the original enzyme, and the stability is better than that of the original enzyme.

Drawings

FIG. 1 is a gas chromatogram of a standard of ethyl 4-chloroacetoacetate.

FIG. 2 is a gas chromatogram of (R) -4-chloro-3-hydroxybutyric acid ethyl ester standard.

FIG. 3 is a gas chromatogram of the reaction solution.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all technical means used in the examples are routine in the art or according to the experimental methods suggested by the manufacturers of the kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.

Strain Lachnellula hyalina is disclosed in the literature "H Park, kim, d.y., kim, s.r., & Eom, a.h. (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," publicly available from the vandergar chemical group inc.

pET-28a (+) is a product of the division of biological engineering (Shanghai) and has a catalog number of B540183.

Example 1: obtaining of the Gene sequence of ketoreductase KREDLH

1. Genomic DNA of Lachnellula hyalina was extracted.

2. And (3) taking the genomic DNA of Lachnellula hyalina as a template, and taking a primer 1 (5'-ggaattccatgaaggtctttctgagcggagg-3', SEQ ID NO: 5) and a primer 2 (5'-cccaagcttgggtcactccttatataatccctctttcgggtaatc-3', SEQ ID NO: 6) as primer pairs to perform PCR, so as to obtain a PCR amplified fragment containing a ketoreductase gene, wherein the nucleotide sequence of the ketoreductase gene is shown as SEQ ID NO:2, and the amino acid sequence of the coded ketoreductase is shown as SEQ ID NO: 1.

MKVFLSGGSGFIAAHVLDILLEHGHTVITSVRSQEKANKIKEAHPNTPASQLEFRIVKDIAQEGAFDEAIKIDGLEAVIHTASPFHFNVTDVKKDLLDPAIIGTTGILKAIKKNAPSVKRVVITSSFASIVNPSKGNSWTEHTYSEEDWNPITEEEAVLNPSNGYRASKTFAEKAAWEFVEKEKPNFTLSTMCPPLVIGPIVHYLNSLDSLNTSNQRTANLMTGKNKSEIPDTGTYIWIDVRDLALAHVKAIELPEAANKRFFITAGYFSNKEIAEIIRKNFPALEKELPAKDVKGGDYPKEGLYKE(SEQ ID NO:1)

atgaaggtctttctgagcggaggaagtggcttcatcgccgcccacgtcctcgacatcctactcgagcatggccatactgtcatcacctcggttcgttcccaagagaaagccaacaagatcaaagaggcgcaccccaacacgcctgcctcccagctcgagttccggattgtcaaagacatagcacaggagggggcctttgacgaagccatcaagattgacggcctggaagcggtgattcacacagcctcgcctttccatttcaacgtcacagatgtcaagaaagacttgcttgaccctgccataatcggcacaacaggtatcctgaaagccatcaagaagaatgctcccagcgtgaagagagtcgtcatcacgagttcctttgccagcatcgtcaatccaagtaagggaaactcctggaccgagcacacgtacagcgaggaggactggaaccccatcacggaagaagaggcggtgctgaaccccagcaatggatacagagccagcaagacgttcgccgagaaagctgcgtgggagtttgtcgagaaggagaaaccaaactttacgttgagcactatgtgccctcctttagttataggtccaattgtccactacctcaacagcctcgatagcctcaacacctctaaccagcgaaccgccaacctcatgaccggcaagaacaagtctgaaatccccgacaccggtacctacatctggatcgatgtgcgagatctcgccctcgcccacgtcaaagccatcgagctcccagaagccgcgaacaagcgattcttcatcaccgctggttacttctccaacaaggagatcgctgagattatccgcaagaacttccccgcgctcgaaaaggaattgccggcgaaggacgtcaagggtggagattacccgaaagagggattatataaggagtga(SEQ ID NO:2)

Example 2: obtaining the gene sequence of ketoreductase mutant KREDLH-M by error-prone PCR technique

The following error-prone PCR was performed using the PCR amplified fragment obtained in example 1 as a template and primer 1 and primer 2 as a primer pair, to obtain 84 mutants of the ketoreductase gene in total.

Error-prone PCR reaction system: 5 μl of 10 Xamplification buffer, 4 μl of each of 4 dNTP mixtures (2.5 mmol/L), 50pmol of each primer, 1.5 μg of template DNA, 0.5 μl of Taq DNA polymerase, mg ²⁺ 7mmol/L, double distilled water was added to 50. Mu.l.

PCR reaction procedure: (1) Pre-denaturation: 94 ℃ for 3min; (2) denaturation: 94 ℃ for 30s; annealing: 58 ℃ for 30s; extension: 72 ℃ for 1.5min; cycling 40 times altogether; (3) post-extension: 72 ℃ for 10min; (4) incubation at 4 ℃.

Example 3: cloning of ketoreductase Gene and construction of expression Strain

1. The PCR amplified fragment containing the ketoreductase gene obtained in example 1 was digested simultaneously with EcoRI and HindIII to obtain a gene fragment; double-enzyme cutting pET-28a (+) by EcoRI and HindIII to obtain a vector fragment; the gene fragment and the vector fragment were ligated to obtain a recombinant expression plasmid, which was designated as pET-KREDLH, and the plasmid was subjected to sequencing, and the result was consistent with that expected.

2. And (3) transferring the recombinant expression plasmid pET-KREDLH obtained in the step (1) into E.coli DH5 alpha competent cells through heat shock, coating LB solid medium containing 25 mu g/mL kanamycin, culturing to obtain corresponding monoclonal strains, naming the corresponding monoclonal strains as K, amplifying and extracting the plasmids to obtain pET-KREDLH plasmids, transferring the obtained plasmids into an expression host E.coli BL21 (DE 3) through a chemical conversion mode, coating LB solid medium containing 25 mu g/mL kanamycin, and screening to obtain recombinant bacteria KE expressing ketoreductase.

Example 4: cloning of ketoreductase mutant Gene and construction of expression Strain

Recombinant plasmids pET-. DELTA.KREDLH-M1 to pET-. DELTA.KREDLH-M84 were each constructed by the same method as in example 3 using 84 mutants of the ketoreductase gene obtained in example 2, and recombinant 1-recombinant 84 expressing the ketoreductase mutants was obtained.

Example 5: high throughput screening of ketoreductase mutants

The recombinant bacteria of examples 3 and 4, which were confirmed to be correct by PCR, were cultured in 5mL of TB medium, respectively, and protein expression was induced at 18℃by adding IPTG at a final concentration of 0.6mM, and after 12 hours, the OD of the bacterial solution was measured ₆₀₀ Value, and diluting the bacterial liquid with water to make OD ₆₀₀ The value is about 2. 1.2ml of a catalytic reaction system is designed in a deep hole 96-well plate, the initial pH is adjusted to 6.8, and the reaction system comprises 600 mu L of diluted bacterial liquid, 168mg of ethyl 4-chloroacetoacetate, 3.0% of isopropanol by volume percent and 5mM of NADH, and the volume is complemented by PBS. The well plate for the incubation reaction was incubated at 30℃for 5 hours in a 120rpm mixer, and after the reaction was completed, 200. Mu.L of HCl at a concentration of 2M was added to terminate the reaction.

After the reaction, 50. Mu.L of the supernatant was added with 200. Mu.L of pure water, and the mixture was stirred and subjected to scanning at 340nm to measure absorbance.

The plasmid of the strain (recombinant bacterium 61) corresponding to the reaction solution with the lowest absorbance value is extracted, and sequencing is carried out to obtain the gene sequence of the coded ketoreductase mutant shown as SEQ ID NO. 4, and the amino acid sequence of the coded ketoreductase mutant is shown as SEQ ID NO. 3. Compared with the amino acid sequence SEQ ID NO. 1 of the wild ketoreductase, the ketoreductase mutant has the following mutation: the 41 st amino acid K is mutated to T, the 129 th amino acid S is mutated to N, the 180 th amino acid V is mutated to A, and the 272 th amino acid K is mutated to Q.

MKVFLSGGSGFIAAHVLDILLEHGHTVITSVRSQEKANKITEAHPNTPASQLEFRIVKDIAQEGAFDEAIKIDGLEAVIHTASPFHFNVTDVKKDLLDPAIIGTTGILKAIKKNAPSVKRVVITSSFANIVNPSKGNSWTEHTYSEEDWNPITEEEAVLNPSNGYRASKTFAEKAAWEFAEKEKPNFTLSTMCPPLVIGPIVHYLNSLDSLNTSNQRTANLMTGKNKSEIPDTGTYIWIDVRDLALAHVKAIELPEAANKRFFITAGYFSNQEIAEIIRKNFPALEKELPAKDVKGGDYPKEGLYKE(SEQ ID NO:3)

atgaaggtctttctgagcggaggaagtggcttcatcgccgcccacgtcctcgacatcctactcgagcatggccatactgtcatcacctcggttcgttcccaagagaaagccaacaagatcacagaggcgcaccccaacacgcctgcctcccagctcgagttccggattgtcaaagacatagcacaggagggggcctttgacgaagccatcaagattgacggcctggaagcggtgattcacacagcctcgcctttccatttcaacgtcacagatgtcaagaaagacttgcttgaccctgccataatcggcacaacaggtatcctgaaagccatcaagaagaatgctcccagcgtgaagagagtcgtcatcacgagttcctttgccaacatcgtcaatccaagtaagggaaactcctggaccgagcacacgtacagcgaggaggactggaaccccatcacggaagaagaggcggtgctgaaccccagcaatggatacagagccagcaagacgttcgccgagaaagctgcgtgggagtttgccgagaaggagaaaccaaactttacgttgagcactatgtgccctcctttagttataggtccaattgtccactacctcaacagcctcgatagcctcaacacctctaaccagcgaaccgccaacctcatgaccggcaagaacaagtctgaaatccccgacaccggtacctacatctggatcgatgtgcgagatctcgccctcgcccacgtcaaagccatcgagctcccagaagccgcgaacaagcgattcttcatcaccgctggttacttctccaaccaggagatcgctgagattatccgcaagaacttccccgcgctcgaaaaggaattgccggcgaaggacgtcaagggtggagattacccgaaagagggattatataaggagtga(SEQ ID NO:4)

Ethyl 4-chloroacetoacetate and NADH under the catalysis of ketoreductase(R) -4-chloro-3-hydroxybutyric acid ethyl ester and NAD were produced ⁺ The reaction formula of (2) is as follows:

example 6: preparation of enzyme and measurement of enzyme Activity

Recombinant bacteria KE obtained in example 3 and recombinant bacteria 61 obtained by screening in example 5 were subjected to expansion culture, and after expansion culture, the fermentation broth was subjected to centrifugation (8000 rpm,10 min), cell disruption, and freeze-drying to obtain lyophilized powders of ketoreductase (original enzyme) and mutants thereof (mutant enzyme), and stored at-80 ℃.

The enzyme activity unit (U) is defined as: under the reaction conditions of example 5 (final concentration of lyophilized powder of 0.2g/g ethyl 4-chloroacetoacetate), the amount of enzyme required to catalyze the production of 1. Mu. Mol of ethyl (R) -4-chloro-3-hydroxybutyrate per minute or the amount of enzyme required to consume 1. Mu. Mol of substrate ethyl 4-chloroacetoacetate per minute.

The enzyme activities of the original enzyme and the mutant enzyme are 167.35U/L and 236.8U/L respectively, compared with the original enzyme, the enzyme activity of the mutant enzyme is 1.415 times that of the original enzyme, and the conversion rates of the original enzyme and the mutant enzyme are 76.8% and 82.6% respectively by analyzing the residual amount of the substrate 4-chloroacetoacetate through gas chromatography.

The gas chromatography detection method comprises the following steps:

after the reaction, taking the reaction solution, centrifuging, taking 400 mu L of supernatant, adding 800 mu L of ethyl acetate, uniformly mixing, performing film passing, and then using gas chromatography to analyze the conversion rate, wherein an Agilent DB-5 capillary column is used, carrier gas is nitrogen, a hydrogen ion detector is used, the temperature of a sample inlet is 250 ℃, the temperature of the detector is 250 ℃, the temperature of a column temperature box is 90 ℃, the sample injection amount is 1 mu L, and the column flow rate is 1mL/min.

The gas chromatograms of the ethyl 4-chloroacetoacetate standard and the ethyl (R) -4-chloro-3-hydroxybutyrate standard are respectively shown in figures 1 and 2, the peak time of the ethyl 4-chloroacetoacetate is 6.598min, and the peak time of the ethyl (R) -4-chloro-3-hydroxybutyrate is 7.022min.

The gas chromatogram of the reaction solution was shown in FIG. 3, in which the peak time of ethyl (R) -4-chloro-3-hydroxybutyrate was 7.018min and the peak time of ethyl 4-chloroacetoacetate was 6.612min.

Example 7: enzyme stability test

A small amount of the lyophilized powder of the original enzyme and the mutant enzyme obtained in example 6 was reacted under the reaction conditions of example 5, and after the reaction was completed, the reaction was repeated by centrifugation to recover the enzyme, and the conversion rates of the two enzymes in each reaction batch were shown in Table 1.

TABLE 1

The result shows that the mutant enzyme has better recycling property, the conversion rate can still reach more than 80% after the reaction is carried out for 6 batches, the mutant enzyme has better stability compared with the original enzyme, the activity of the original enzyme is reduced by 12.37% after the reaction is carried out for 6 batches, and the activity of the recombinase is reduced by only 2.67%.

Claims (10)

1. A ketoreductase having the amino acid sequence shown in SEQ ID NO. 1.

2. A nucleic acid molecule encoding the ketoreductase of claim 1 having the nucleotide sequence set forth in SEQ ID No. 2.

3. A mutant of ketoreductase as claimed in claim 1, having the amino acid sequence shown in SEQ ID NO. 3.

4. A nucleic acid molecule encoding a mutant of a ketoreductase as claimed in claim 3, having the nucleotide sequence set forth 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 ketoreductase or mutant thereof, comprising the steps of:

1) Culturing the recombinant cell of claim 6 and inducing expression of a ketoreductase or mutant thereof;

2) Isolating the ketoreductase of claim 1 or the variant of the ketoreductase process of claim 3 from the culture obtained in 1).

8. Use of a ketoreductase according to claim 1, a nucleic acid molecule according to claim 2, a mutant of a ketoreductase according to claim 3, a nucleic acid molecule according to claim 4, a recombinant vector according to claim 5, a recombinant cell according to claim 6 and/or a ketoreductase prepared by a method according to claim 7 or a mutant thereof for the preparation of ethyl (R) -4-chloro-3-hydroxybutyrate.

9. A preparation method of (R) -4-chloro-3-hydroxybutyric acid ethyl ester comprises the following steps: the ketoreductase of claim 1, the mutant of ketoreductase of claim 3, the recombinant cell of claim 6 and/or the ketoreductase or the mutant thereof prepared by the method of claim 7 are used as catalysts for catalyzing the conversion of ethyl 4-chloroacetoacetate into ethyl (R) -4-chloro-3-hydroxybutyrate.

10. The method of manufacturing according to claim 9, wherein: the catalytic reaction conditions are as follows: the temperature is 25-35 ℃, the pH is 6.0-7.0, and the reaction batch comprises: ethyl 4-chloroacetoacetate, isopropanol, and NADH;

the final concentration of the ethyl 4-chloroacetoacetate is 100-200g/L;

the volume percentage of the isopropanol is 2-3%;

the final concentration of NADH is 1-10mM.