A ketoreductase mutant with improved enzyme activity and its application

A reductase and mutant technology, applied in the field of ketoreductase mutants, can solve the problems of high enzyme dosage, low conversion rate, lack of industrial production prospects, etc., and 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 with improved enzyme activity and its application
  • A ketoreductase mutant with improved enzyme activity and its application
  • A ketoreductase mutant with improved enzyme activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH 2 O

[0039] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and amplified according to the following procedure: ...

Embodiment 2

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH 2 O

[0045] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and amplified according to the following procedure: ...

Embodiment 3

[0047] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted,

[0048] to achieve high expression in E. coli.

[0049] 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.

[0050] 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.

[0051] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH 2 O

[0052] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and amplified according to the following proced...

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PUM

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Abstract

The invention discloses a ketoreductase mutant with improved enzymatic activity and an application thereof, belonging to the field of biotechnology. It is derived from the wild-type ketoreductase of Meyerozyma guilliermondii, and can convert ethyl 4-chloroacetoacetate to generate (R) ‑4‑chloro‑3‑hydroxybutyrate ethyl ester, the ketoreductase mutant has higher alcohol dehydrogenase activity than the wild-type sequence, and has more than 90% similarity with SEQ ID NO.8. The ketoreductase mutant of the present invention has obviously high specific enzyme activity, which is 2-10 times higher than that of the wild-type ketoreductase, the reaction conditions are mild, the equipment requirements are low, the production process does not require high temperature or cooling, and the energy consumption is low. Enzyme catalysis has high efficiency, specific selectivity, and convenient purification. In addition, most of the solvent in the reaction is water, and there is no need to add solvents such as butyl acetate to form a two-phase reaction system. The three wastes discharge is low, and the preparation process is environmentally friendly.

Description

technical field [0001] The invention relates to a ketoreductase mutant with improved enzyme activity and its application, and belongs 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 with the required stereospecificity. [0003] Chiral pure (R)-ethyl 4-c...

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
CPCC12N9/0006C12N15/70C12P7/62C12Y101/01002C12N2800/22Y02P20/584
Inventor 丁雪峰李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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