CN112680425B - Alcohol dehydrogenase mutant and application thereof - Google Patents
Alcohol dehydrogenase mutant and application thereof Download PDFInfo
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Abstract
The invention discloses an alcohol dehydrogenase mutant and application thereof, belonging to the technical field of enzyme engineering. The alcohol dehydrogenase mutant is obtained by single point mutation of a wild type, and glutamic acid at position 197 of an amino acid sequence shown in SEQ ID NO.1 is mutated. The wild type can not catalyze 4-chloroacetoacetic acid ethyl ester, the alcohol dehydrogenase mutant can catalyze 4-chloroacetoacetic acid ethyl ester to prepare (S) -4-chloro-3-hydroxy-butyric acid ethyl ester, is a chiral intermediate for synthesizing statins, and provides a new method for producing (S) -4-chloro-3-hydroxy-butyric acid ethyl ester.
Description
Technical Field
The invention relates to an alcohol dehydrogenase mutant and application thereof, belonging to the technical field of enzyme engineering.
Background
Ethyl (S) -4-chloro-3-hydroxybutyrate ((S) -CHBE) is a chiral intermediate for the synthesis of statins, one of the most effective drugs for the treatment of cardiovascular diseases. For example, atorvastatin, which is an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, can reduce low density lipoprotein cholesterol, and is directly related to the reduction of mortality of coronary heart disease. (S) -CHBE can also be used for synthesizing Slagenins B and 1, 4-dihydropyridine type beta-receptor blockers, which are inhibitors of lymphoid surface receptor kinases.
(S) -CHBE can be obtained by asymmetric reduction catalyzed by an enzyme method, and researchers at home and abroad have found enzymes which can catalyze ethyl 4-Chloroacetoacetate (COBE) to generate the (S) -CHBE from various microorganisms. As early as 1999, Keiko Kita et al (Purification and characterization of new aldehyde products from Sporobolomyces moniolor AKU4429.journal of Molecular Catalysis B: enzymic 6:305-313(1999)) isolated from Sporobolomyces moniolor AKU4429 gave an aldehyde reductase AR II which reduced COBE to (S) -CHBE. Enzymes having the same action have been identified in various microorganisms such as yeast and Acetobacter.
The alcohol dehydrogenase (hereinafter referred to as AtQR) from Agrobacterium tumefaciens is an NADH-dependent enzyme, but the wild enzyme cannot catalyze COBE to generate (S) -CHBE, and if the wild enzyme can be modified to have catalytic performance, the alcohol dehydrogenase is greatly beneficial to the preparation and application of CHBE.
Disclosure of Invention
The inventor screens an alcohol dehydrogenase (hereinafter referred to as AtQR) from Agrobacterium tumefaciens, wherein the enzyme is NADH dependent enzyme, but the wild enzyme can not catalyze COBE to generate (S) -CHBE, and in order to enable the AtQR to catalyze COBE to generate (S) -CHBE, the inventor finds out key amino acid, and adopts a site-directed mutagenesis method to modify the AtQR to obtain the alcohol dehydrogenase mutant with COBE catalytic activity.
The invention provides an alcohol dehydrogenase mutant and application thereof, wherein the alcohol dehydrogenase mutant can catalyze 4-chloroacetoacetic acid ethyl ester to prepare (S) -4-chloro-3-hydroxy ethyl butyrate, and is a chiral intermediate for synthesizing statins.
The invention provides an alcohol dehydrogenase mutant, which is obtained by mutating 197 th site of an amino acid sequence shown in SEQ ID NO. 1.
In one embodiment, SEQ ID NO.1 is the amino acid sequence of a wild-type pure dehydrogenase derived from Agrobacterium tumefaciens.
In one embodiment, the glutamic acid at position 197 is mutated to asparagine, or leucine, or methionine, or histidine, or valine, or serine.
In one embodiment, the amino acid sequence of the mutant obtained by mutating the glutamic acid at position 197 to asparagine is shown as SEQ ID No.2, and the nucleotide sequence of the gene encoding the mutant is shown as SEQ ID No. 3.
The present invention provides a gene encoding the alcohol dehydrogenase mutant.
The invention provides an expression vector containing the gene.
The present invention provides a microbial cell expressing the alcohol dehydrogenase mutant, or containing the gene.
In one embodiment, E.coli is used as the host cell.
The invention provides a method for preparing (S) -4-chloro-3-hydroxy-ethyl butyrate, which is characterized in that an alcohol dehydrogenase mutant is used for catalyzing 4-chloroacetoacetic acid ethyl ester to obtain (S) -4-chloro-3-hydroxy-ethyl butyrate.
In one embodiment, the catalysis is carried out under conditions comprising reduced coenzyme factor.
In one embodiment, the reduced coenzyme factor is NADH.
In one embodiment, the reaction is carried out at 20-40 ℃ and pH 5.0-8.5.
The invention provides the use of said mutant, or said gene, or said microbial cell, or said method for the production of (S) -4-chloro-3-hydroxy-butyric acid ethyl ester.
The invention has the beneficial effects that:
the invention modifies alcohol dehydrogenase AtQR derived from agrobacterium tumefaciens through rational design, wherein the AtQR can not catalyze 4-chloroacetoacetic acid ethyl ester, and the modified alcohol dehydrogenase mutant which can catalyze the 4-chloroacetoacetic acid ethyl ester to generate (S) -4-chloro-3-hydroxybutyric acid ethyl ester is obtained. The specific enzyme activity of the mutant E197N was 47.28U/mg.
Drawings
FIG. 1 is a diagram showing the results of SDS-PAGE of wild-type and mutant proteins of AtQR.
Detailed Description
The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.
The invention relates to recombinant Escherichia coli with alcohol dehydrogenase gene, wherein the vector is pRSFDuet-1, and the host is Escherichia coli BL21(DE 3).
Reagents used in the downstream catalytic process: ethyl 4-chloroacetoacetate from mclin (shanghai, china); ethyl 4-chloro-3-hydroxybutyrate was purchased from alatin (shanghai, china); cofactor NADH was purchased from bio-engineering (shanghai, china); other commonly used reagents are available from the national pharmaceutical group chemical agents, ltd. As used herein, the three-letter or one-letter expression of amino acids is defined by the IUPAC code for amino acids (Eur. J. biochem.,138:9-37,1984).
Alcohol dehydrogenase enzyme activity standard detection system: appropriate amounts of enzyme solution, 5mM substrate, 0.13mM NADH, 3mL overall, and the reaction medium being 20mM phosphate buffer, pH 6.0. Reacting at 40 deg.C for 1min, and calculating enzyme activity (i.e. NADH consumption rate) by using change value of 340nm spectroscopic value within 1 min.
Example 1: construction of AtQR wild type and mutant recombinant bacteria
Entrusted also with the provision of codon optimization and gene synthesis services by biotechnology (shanghai) ltd, the wild type of AtQR (amino acid sequence shown in SEQ ID No. 1) was synthesized into the corresponding nucleotide and ligated to the pRSFDuet-1 plasmid with His tag sequence (at the N-terminus of the protein) to facilitate protein purification, and the target gene was placed between the restriction sites BamH I and EcoR I. pRSFDuet-1-AtQR-WT recombinant plasmid was obtained and transformed into E.coli BL21(DE3) competent cells.
A pair of upstream and downstream primers (E197N-F/E197N-R) containing mutation sites were designed using pRSFDuet-1-AtQR-WT recombinant plasmid as a template, and site-directed mutagenesis was performed on position 197 by PCR.
The primers E197N-F/E197N-R are shown below:
E197N-F：AACATAATCTGGGAGGCCGAACTGCGGG，SEQ ID NO.4，
E197N-R：GGCCTCCCAGATTATGTTACGTTCCTGCATAGCTGT，SEQ ID NO.5。
wherein, the reaction system of PCR is:
PCR procedure: 1) pre-denaturation at 94 ℃ for 2 min; 2) denaturation at 98 ℃ for 10 s; annealing at 58 ℃ for 10 s; extension at 72 ℃ for 10 s; circulating for 30 times; 3) extending for 5min at 72 ℃; 4) storing at 4 ℃ for 2 h.
After the PCR product was verified by agarose gel electrophoresis, the PCR product was digested with restriction enzyme Dpn I.
The digestion system is as follows: DNA 40. mu. L, Buffer 4, 4. mu. L, Dpn I1. mu.L. Placing in 37 deg.C water bath for 30 min.
Finally, the digested product is transferred into competent cells of escherichia coli BL21(DE3) by a heat shock method, and a single colony with correct sequencing is a mutant recombinant bacterium carrying pRSFDuet-1-AtQR-E197N recombinant plasmid.
Example 2: microbial culture and preparation of crude enzyme solution
1. Cultivation of microorganisms
Composition of LB liquid medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl, dissolving with deionized water, fixing the volume, and sterilizing at 121 ℃ for 20min for later use. LB solid medium: adding 20g/L agar powder on the basis of LB liquid culture medium, sterilizing at 121 ℃, cooling, pouring into a culture dish, and making into a flat plate.
Coli BL21(DE3) containing the gene of interest was inoculated into 5mL of LB medium containing 50. mu.g/mL of kanamycin and cultured overnight at 37 ℃ at 200 rpm. 1mL of the culture was transferred to 50mL of fresh LB medium containing 50. mu.g/mL of Kana and cultured at 37 ℃ and 200rpm until the OD600 was about 0.6. isopropyl-beta-D-thiogalactoside was added to a final concentration of 0.2mM to induce protein expression, and incubated at 20 ℃ at 200rpm for 24 h.
2. Preparation of crude enzyme solution
After completion of the culture, the cells were collected by centrifugation at 8000rpm for 10min at 4 ℃ and resuspended in 20mM phosphate buffer (pH 7.0), and the cells were disrupted by an ultrasonic cell disrupter on ice. Centrifuging at 4 deg.C and 8000rpm for 10min, and collecting supernatant as crude enzyme solution of corresponding enzyme.
Example 3: separation and purification of enzyme and determination of enzyme activity
1. Separation and purification of enzyme
The crude enzyme solution was purified by Ni-NTA affinity chromatography. The Ni-NTA chromatography column was first equilibrated with 3 column volumes of binding solution (20 mM phosphate buffer containing 5mM imidazole and 500mM NaCl). After equilibration, the crude enzyme was applied to a column and the protein of interest was eluted linearly with different concentrations of eluent (20 mM phosphate buffer containing 500mM imidazole and 500mM NaCl) and desalted.
2. Determination of enzyme Activity
One unit of enzyme activity is defined as the amount of enzyme required to reduce 1. mu. mol NADH per minute.
The reaction system (3mL) contained 5mM COBE, 0.13mM NADH, 20mM phosphate buffer (pH 7.0) and the appropriate amount of purified enzyme. After the mixture of COBE and 20mM phosphate buffer (pH 7.0) was left in a water bath at 40 ℃ for 10min, the enzyme and NADH were added, quickly taken out, shaken by hand, poured into a cuvette, and quickly put into an ultraviolet spectrophotometer. Measuring the change of the absorbance value of NADH at 340nm along with time, calculating the enzyme activity according to the molar absorption coefficient of NADH, and showing that the original enzyme has no catalytic activity to COBE and the V of the mutant enzyme E197Nmax，KmAnd kcat/KmValues are 47.28U/mg, 0.91mM and 24.31s, respectively-1·mM-1. E197N maintained more than 60% residual activity over a pH range of 5.0-8.5. The stability is good within the temperature range of 20-30 ℃, and the activity of more than 90 percent is maintained within 3 h.
Example 4
The 197-position glutamic acid of the wild type AtQR is mutated into other amino acids, a recombinant bacterium is constructed in the same manner as in example 2 to obtain a crude enzyme solution, the crude enzyme solution is purified, and the enzyme activity is measured, so that the results show that 6 mutants can catalyze COBE, namely E197L, E197M, E197H, E197V, E197S and E197I, and the relative enzyme activities (compared with E197N) are 84.38%, 63.42%, 61.58%, 59.53%, 48.83% and 13.74%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> alcohol dehydrogenase mutant and application thereof
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Claims (9)
1. An alcohol dehydrogenase mutant characterized in that the 197 th glutamic acid in the amino acid sequence shown in SEQ ID No.1 is mutated into asparagine, leucine, methionine, histidine, valine or serine.
2. A gene encoding the alcohol dehydrogenase mutant of claim 1.
3. An expression vector comprising the gene of claim 2.
4. A microbial cell expressing the alcohol dehydrogenase mutant of claim 1 or the gene of claim 2.
5. The microbial cell of claim 4, wherein the host cell is Escherichia coli.
6. A method for preparing (S) -4-chloro-3-hydroxy-butyric acid ethyl ester, which comprises catalyzing 4-chloroacetoacetic acid ethyl ester with the alcohol dehydrogenase mutant of claim 1 to obtain (S) -4-chloro-3-hydroxy-butyric acid ethyl ester.
7. The method of claim 6, wherein the catalysis is carried out under conditions comprising reduced coenzyme factor.
8. The method of claim 7, wherein the reaction is carried out at 20-40 ℃ and pH 5.0-8.5.
9. Use of the mutant according to claim 1, or the gene according to claim 2, or the microbial cell according to claim 4 or 5, or the method according to any one of claims 6 to 8 for the production of (S) -4-chloro-3-hydroxy-butyric acid ethyl ester.
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| GB0818328D0 (en) * | 2008-10-07 | 2008-11-12 | Isis Innovation | Novel enzyme |
| CN103555608B (en) * | 2013-09-16 | 2015-06-03 | 华东理工大学 | Quininone reductase and application thereof in asymmetric synthesis of (R)-3-quinuclidinol |
| EP3652328A1 (en) * | 2017-07-14 | 2020-05-20 | c-LEcta GmbH | Ketoreductases |
| CN108384765A (en) * | 2018-02-12 | 2018-08-10 | 江南大学 | Alcohol dehydrogenase mutant and its application in the synthesis of double aryl chiral alcohols |
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