CN112680425A - Alcohol dehydrogenase mutant and application thereof - Google Patents
Alcohol dehydrogenase mutant and application thereof Download PDFInfo
- Publication number
- CN112680425A CN112680425A CN202110039540.0A CN202110039540A CN112680425A CN 112680425 A CN112680425 A CN 112680425A CN 202110039540 A CN202110039540 A CN 202110039540A CN 112680425 A CN112680425 A CN 112680425A
- Authority
- CN
- China
- Prior art keywords
- ala
- alcohol dehydrogenase
- val
- gly
- ethyl ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
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.
Alcohol dehydrogenase (hereinafter referred to as AtQR) from Agrobacterium tumefaciens, which is an NADH-dependent enzyme, but the wild-type enzyme cannot catalyze the production of (S) -CHBE from COBE, and if the wild-type enzyme can be modified to have catalytic properties, the alcohol dehydrogenase is of great benefit for 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 in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the mutant is shown in 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 utilized to catalyze 4-chloroacetoacetic acid ethyl ester to obtain the (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 the alcohol dehydrogenase AtQR from agrobacterium tumefaciens by 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-hydroxy butyric 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 amount of enzyme solution, 5mM substrate, 0.13mM NADH, 3mL overall, reaction medium is 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.
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 to obtain 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. Placing the mixture of COBE and 20mM phosphate buffer solution (pH 7.0) in a water bath at 40 deg.C for 10min, adding enzyme and NADH, quickly taking out, shaking by hand, pouring into a cuvette, and quickly placing into an ultraviolet spectrophotometer. Measuring the change of the light absorption value of NADH at 340nm along with time, calculating the enzyme activity according to the molar light absorption coefficient of NADH, and displaying the resultThe starter enzyme had no catalytic activity on COBE, 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, and the results show that 6 mutants can catalyze COBE which are respectively E197L, E197M, E197H, E197V, E197S and E197I, and the relative enzyme activities (compared with E197N) are respectively 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
<130> BAA201673A
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 260
<212> PRT
<213> Agrobacterium tumefaciens
<400> 1
Met Ala Gly Ile Phe Asp Leu Ser Gly Arg Lys Ala Ile Val Thr Gly
1 5 10 15
Gly Ser Lys Gly Ile Gly Ala Ala Ile Ala Arg Ala Leu Asp Lys Ala
20 25 30
Gly Ala Thr Val Ala Ile Ala Asp Leu Asp Val Met Ala Ala Gln Ala
35 40 45
Val Val Ala Gly Leu Glu Asn Gly Gly Phe Ala Val Glu Val Asp Val
50 55 60
Thr Lys Arg Ala Ser Val Asp Ala Ala Met Gln Lys Ala Ile Asp Ala
65 70 75 80
Leu Gly Gly Phe Asp Leu Leu Cys Ala Asn Ala Gly Val Ser Thr Met
85 90 95
Arg Pro Ala Val Asp Ile Thr Asp Glu Glu Trp Asp Phe Asn Phe Asp
100 105 110
Val Asn Ala Arg Gly Val Phe Leu Ala Asn Gln Ile Ala Cys Arg His
115 120 125
Phe Leu Ala Ser Asn Thr Lys Gly Val Ile Val Asn Thr Ala Ser Leu
130 135 140
Ala Ala Lys Val Gly Ala Pro Leu Leu Ala His Tyr Ser Ala Ser Lys
145 150 155 160
Phe Ala Val Phe Gly Trp Thr Gln Ala Leu Ala Arg Glu Met Ala Pro
165 170 175
Lys Asn Ile Arg Val Asn Cys Val Cys Pro Gly Phe Val Lys Thr Ala
180 185 190
Met Gln Glu Arg Glu Ile Ile Trp Glu Ala Glu Leu Arg Gly Met Thr
195 200 205
Pro Glu Ala Val Arg Ala Glu Tyr Val Ser Leu Thr Pro Leu Gly Arg
210 215 220
Ile Glu Glu Pro Glu Asp Val Ala Asp Val Val Val Phe Leu Ala Ser
225 230 235 240
Asp Ala Ala Arg Phe Met Thr Gly Gln Gly Ile Asn Val Thr Gly Gly
245 250 255
Val Arg Met Asp
260
<210> 2
<211> 260
<212> PRT
<213> Artificial sequence
<400> 2
Met Ala Gly Ile Phe Asp Leu Ser Gly Arg Lys Ala Ile Val Thr Gly
1 5 10 15
Gly Ser Lys Gly Ile Gly Ala Ala Ile Ala Arg Ala Leu Asp Lys Ala
20 25 30
Gly Ala Thr Val Ala Ile Ala Asp Leu Asp Val Met Ala Ala Gln Ala
35 40 45
Val Val Ala Gly Leu Glu Asn Gly Gly Phe Ala Val Glu Val Asp Val
50 55 60
Thr Lys Arg Ala Ser Val Asp Ala Ala Met Gln Lys Ala Ile Asp Ala
65 70 75 80
Leu Gly Gly Phe Asp Leu Leu Cys Ala Asn Ala Gly Val Ser Thr Met
85 90 95
Arg Pro Ala Val Asp Ile Thr Asp Glu Glu Trp Asp Phe Asn Phe Asp
100 105 110
Val Asn Ala Arg Gly Val Phe Leu Ala Asn Gln Ile Ala Cys Arg His
115 120 125
Phe Leu Ala Ser Asn Thr Lys Gly Val Ile Val Asn Thr Ala Ser Leu
130 135 140
Ala Ala Lys Val Gly Ala Pro Leu Leu Ala His Tyr Ser Ala Ser Lys
145 150 155 160
Phe Ala Val Phe Gly Trp Thr Gln Ala Leu Ala Arg Glu Met Ala Pro
165 170 175
Lys Asn Ile Arg Val Asn Cys Val Cys Pro Gly Phe Val Lys Thr Ala
180 185 190
Met Gln Glu Arg Asn Ile Ile Trp Glu Ala Glu Leu Arg Gly Met Thr
195 200 205
Pro Glu Ala Val Arg Ala Glu Tyr Val Ser Leu Thr Pro Leu Gly Arg
210 215 220
Ile Glu Glu Pro Glu Asp Val Ala Asp Val Val Val Phe Leu Ala Ser
225 230 235 240
Asp Ala Ala Arg Phe Met Thr Gly Gln Gly Ile Asn Val Thr Gly Gly
245 250 255
Val Arg Met Asp
260
<210> 3
<211> 780
<212> DNA
<213> Artificial sequence
<400> 3
atggcgggga tttttgatct gagcggccgt aaagcgatag tcactggcgg tagtaaaggt 60
ataggcgctg cgatcgctcg cgcgctggat aaagccggcg ccacagttgc aattgccgac 120
ctggatgtga tggcagccca ggcggtagta gcgggtttgg agaatggcgg ctttgcggtt 180
gaggtggacg tcaccaagcg ggcatcggta gatgcagcaa tgcaaaaagc cattgatgca 240
ctgggtggtt tcgatctctt atgtgccaat gcgggtgtta gcaccatgag accggcggtt 300
gatatcactg acgaagaatg ggatttcaat tttgacgtaa atgcacgtgg cgttttccta 360
gccaaccaaa tcgcatgccg ccattttctg gcgtccaata caaaaggagt tattgtcaat 420
acagccagtt tggctgccaa agtaggcgcc cctttactgg cccactactc tgcaagcaaa 480
tttgctgttt tcggctggac ccaggcactt gctcgtgaaa tggcaccaaa aaacattcgg 540
gttaactgcg tttgcccagg atttgtcaaa acagctatgc aggaacgtaa cataatctgg 600
gaggccgaac tgcggggcat gacccccgaa gccgttcggg ctgagtatgt gtcactgaca 660
ccgttgggcc gaatcgaaga accagaggat gttgcggatg tggttgtatt tctggcatcg 720
gatgccgccc gctttatgac cggtcaagga attaatgtaa ccggaggggt tcgcatggac 780
<210> 4
<211> 28
<212> DNA
<213> Artificial sequence
<400> 4
aacataatct gggaggccga actgcggg 28
<210> 5
<211> 36
<212> DNA
<213> Artificial sequence
<400> 5
ggcctcccag attatgttac gttcctgcat agctgt 36
Claims (10)
1. An alcohol dehydrogenase mutant characterized by having a mutation at position 197 of the amino acid sequence shown in SEQ ID NO. 1.
2. The alcohol dehydrogenase mutant according to claim 1, wherein the glutamic acid at position 197 is mutated to asparagine, leucine, methionine, histidine, valine or serine.
3. A gene encoding the alcohol dehydrogenase mutant of claim 1 or 2.
4. An expression vector comprising the gene of claim 3.
5. A microbial cell expressing the alcohol dehydrogenase mutant of claim 1 or containing the gene of claim 3.
6. The microbial cell of claim 5, wherein the host cell is Escherichia coli.
7. 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.
8. The method of claim 7, wherein the catalysis is carried out under conditions comprising reduced coenzyme factor.
9. The method of claim 8, wherein the reaction is carried out at 20-40 ℃ and at a pH of 5.0-8.5.
10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the microbial cell according to claim 5 or 6, or the method according to any one of claims 7 to 9 for the production of (S) -4-chloro-3-hydroxy-butyric acid ethyl ester.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110039540.0A CN112680425B (en) | 2021-01-13 | 2021-01-13 | Alcohol dehydrogenase mutant and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110039540.0A CN112680425B (en) | 2021-01-13 | 2021-01-13 | Alcohol dehydrogenase mutant and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112680425A true CN112680425A (en) | 2021-04-20 |
CN112680425B CN112680425B (en) | 2022-06-10 |
Family
ID=75457626
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110039540.0A Active CN112680425B (en) | 2021-01-13 | 2021-01-13 | Alcohol dehydrogenase mutant and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112680425B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100086958A1 (en) * | 2008-10-07 | 2010-04-08 | Benjamin Guy Davis | Novel enzyme |
CN103555608A (en) * | 2013-09-16 | 2014-02-05 | 华东理工大学 | Quininone reductase and application thereof to asymmetric synthesis of (R)-3-quinuclidinol |
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 |
CN110914446A (en) * | 2017-07-14 | 2020-03-24 | C-乐克塔股份有限公司 | Ketoreductase enzymes |
-
2021
- 2021-01-13 CN CN202110039540.0A patent/CN112680425B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100086958A1 (en) * | 2008-10-07 | 2010-04-08 | Benjamin Guy Davis | Novel enzyme |
CN103555608A (en) * | 2013-09-16 | 2014-02-05 | 华东理工大学 | Quininone reductase and application thereof to asymmetric synthesis of (R)-3-quinuclidinol |
CN110914446A (en) * | 2017-07-14 | 2020-03-24 | C-乐克塔股份有限公司 | Ketoreductase enzymes |
US20200216821A1 (en) * | 2017-07-14 | 2020-07-09 | 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 |
Non-Patent Citations (4)
Title |
---|
DI LIU 等: ""Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis"", 《BIOTECHNOL APPL BIOCHEM.》 * |
FENG HOU 等: ""Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR"", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
LIU,D. 等: ""3-quinuclidinone reductase [synthetic construct],ACCESSION: QXY06225.1"", 《GENBANK》 * |
MICHIHIKO KATAOKA 等: ""Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use"", 《APPL MICROBIOL BIOTECHNOL》 * |
Also Published As
Publication number | Publication date |
---|---|
CN112680425B (en) | 2022-06-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108559735B (en) | Construction and application of leucine dehydrogenase mutant | |
CN111172124B (en) | Carbonyl reductase mutant and preparation thereofR) Application of (E) -4-chloro-3-hydroxy-butyrate | |
CN111676203B (en) | Leucine dehydrogenase mutant and application thereof | |
CN111778223B (en) | Method for modifying stereoselectivity of carbonyl reductase, carbonyl reductase mutant and application | |
CN113151230B (en) | Mutant protein of formaldehyde lyase and application thereof | |
CN113774036B (en) | Imine reductase mutant and application thereof | |
CN112877307B (en) | Amino acid dehydrogenase mutant and application thereof | |
CN113073089B (en) | Novel method for improving enzyme activity of NMN biosynthetic enzyme Nampt | |
CN115717137B (en) | Lysyl specific endonuclease mutant and preparation method and application thereof | |
CN112322606B (en) | Nitrile hydratase mutant and application thereof | |
KR20050074313A (en) | Cephalosporin c acylases | |
CN113652407B (en) | Carbonyl reductase mutant and application thereof in asymmetric synthesis of chiral compound | |
CN113201516B (en) | P-nitrobenzyl esterase mutant and application thereof | |
CN108118036A (en) | Novel grape carbohydrate oxidase mutant | |
CN112175980A (en) | Method for improving activity of polymerase large fragment by site-directed mutagenesis and application | |
CN108118038A (en) | A kind of glucose oxidase mutant | |
CN110592035B (en) | Carbonyl reductase mutant, recombinant expression vector and application of carbonyl reductase mutant in production of chiral alcohol | |
CN111057686B (en) | Alcohol dehydrogenase mutant and application thereof | |
CN116676280A (en) | Glutathione bifunctional synthetase mutant and application thereof | |
CN112680425B (en) | Alcohol dehydrogenase mutant and application thereof | |
CN113249349B (en) | Mutant alcohol dehydrogenase, recombinant vector, preparation method and application thereof | |
CN108624568B (en) | Mutant xanthine dehydrogenase and application thereof | |
CN110846289A (en) | Acinetobacter baumannii xanthine dehydrogenase mutant and application thereof | |
JP2009089649A (en) | Diaphorase gene of clostridium kluyveri and its application | |
KR101778878B1 (en) | Highly active GABA-producing glutamate decarboxylase from Bacteroides sp. and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right |
Effective date of registration: 20230224 Address after: Floor 20, Unit 2, Building 1, Jinlan West Jingyuan, No. 56, Shinan Road, Science Avenue, High-tech Industrial Development Zone, Zhengzhou City, Henan Province, 450000 Patentee after: Zhuohong Chaoyuan Biotechnology (Zhengzhou) Co.,Ltd. Address before: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee before: Jiangnan University |
|
TR01 | Transfer of patent right |