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A ketoreductase mutant for producing (r)-4-chloro-3-hydroxybutyric acid ethyl ester

A technology of ethyl hydroxybutyrate and ethyl chloroacetoacetate is applied in the directions of oxidoreductase, microorganism-based method, nucleic acid carrier, etc., and can solve the problems of high enzyme dosage, low conversion rate, and no industrial production prospects. , to achieve the effect of expanding the scope of application

Active Publication Date: 2022-07-12
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrate concentration is only 25g / L-50g / L, and the enzyme dosage is too high (5g bacteria / 25ml system), the conversion rate is low, and it does not have industrial production prospects

Method used

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  • A ketoreductase mutant for producing (r)-4-chloro-3-hydroxybutyric acid ethyl ester
  • A ketoreductase mutant for producing (r)-4-chloro-3-hydroxybutyric acid ethyl ester
  • A ketoreductase mutant for producing (r)-4-chloro-3-hydroxybutyric acid ethyl ester

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Experimental program
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Effect test

Embodiment 1

[0033] Through the method of total gene synthesis, the secondary structure and codon preference of the gene are adjusted to achieve high expression in E. coli.

[0034] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv, es / OPTIMIZER / ) for design, and ensure that the Tm difference is controlled within 3℃, and the primer length is controlled within 60base , to obtain splicing primers.

[0035] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the end-to-end primers was 0.6 μM.

[0036] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH 2 O

[0037] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplifier, and amplified according to the following procedure: 98°C for 30s, ...

Embodiment 2

[0039] Through the method of total gene synthesis, the secondary structure and codon preference of the gene are adjusted to achieve high expression in E. coli.

[0040] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) for design, and ensure that the Tm difference is controlled within 3℃, and the primer length is controlled within 60base , to obtain splicing primers.

[0041] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the end-to-end primers was 0.6 μM.

[0042] 2mM dNTP mix (2mMeach dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH 2 O

[0043] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplifier, and amplified according to the following procedure: 98°C for 30s, 55...

Embodiment 3

[0045] Through the method of total gene synthesis, the secondary structure and codon preference of the gene are adjusted to achieve high expression in E. coli.

[0046] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) for design, and ensure that the Tm difference is controlled within 3℃, and the primer length is controlled within 60base , to obtain splicing primers.

[0047] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the end-to-end primers was 0.6 μM.

[0048] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH2O Bring the total volume of the reaction system to 50 μl

[0049] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplifier, and ampli...

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Abstract

The invention discloses a ketoreductase mutant for producing (R)-4-chloro-3-hydroxybutyric acid ethyl ester, belonging to the field of biotechnology, which is derived from the wild-type ketoreductase of Meyerozyma guilliermondii and can convert Ethyl 4-chloroacetoacetate is converted to generate (R)-4-chloro-3-hydroxybutyric acid ethyl ester, which has higher alcohol dehydrogenase activity compared with the wild-type sequence, and the sequence of the ketoreductase mutant is SEQ ID NO.2, the enzyme activity of the ketoreductase mutant is at least 2-10 times higher than that of the wild-type ketoreductase. The ketoreductase mutants of the present invention and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination encoding the enzyme, mutagenesis of polynucleotides, DNA shuffling, error-prone PCR, and directed evolution methods, among others.

Description

technical field [0001] The present invention relates to a ketoreductase and its application, in particular to a ketoreductase mutant for producing (R)-4-chloro-3-hydroxybutyric acid ethyl ester, belonging to the field of biotechnology. Background technique [0002] Ketoreductases are versatile catalysts through the enantioselective reduction of aldehydes or ketones to the corresponding alcohols. (R)-specific ketoreductases have different properties than (S)-specific ketoreductases, and these catalysts are used more and more frequently in the industrial synthesis of optically active alcohols. Optical activity is a necessary condition for the selective action of many medicinal and pesticidal active compounds, and in some cases one enantiomer has beneficial pharmaceutical activity while the other enantiomer has genotoxic effects. Therefore, in the synthesis of medicinal and agricultural active compounds, it is necessary to synthesize optically active alcohols using catalysts w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N15/70C12P7/62C12Y101/01C12N2800/22Y02P20/584
Inventor 丁雪峰李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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