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Ketoreductase mutant with enhanced enzyme activity and applications thereof

A reductase and mutant technology, applied in the field of ketoreductase mutants, can solve the problems of high enzyme dosage, lack of industrial production prospects, low conversion rate, etc., and achieve the effect of expanding the scope of application

Active Publication Date: 2020-07-28
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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  • Ketoreductase mutant with enhanced enzyme activity and applications thereof
  • Ketoreductase mutant with enhanced enzyme activity and applications thereof
  • Ketoreductase mutant with enhanced enzyme activity and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0036] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C and the primer length is controlled within 60base , to get 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 first and last primers was 0.6 μM.

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

[0039] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according ...

Embodiment 2

[0041] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0042] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C and the primer length is controlled within 60base , to get 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 first and last primers was 0.6 μM.

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

[0045] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according ...

Embodiment 3

[0047] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0048] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C and the primer length is controlled within 60base , to get splicing primers.

[0049] 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 first and last primers was 0.6 μM.

[0050] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl wxya 2 o

[0051] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according ...

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Abstract

The invention discloses a ketoreductase mutant with enhanced enzyme activity and applications thereof, and belongs to the technical field of biology. The mutant is derived from the wild-type ketoreductase of Meyerozyma guilliermondii and can convert ethyl 4-chloroacetoacetate to generate ethyl (R)-4-chloro-3-hydroxybutyrate; and compared with wild-type sequences, the mutant has higher alcohol dehydrogenase activity and is more than 90% similar to SEQ ID NO. 8. The mutant has obvious high-specific enzyme activity which is enhanced 2-10 times than the wild-type ketoreductase; the mutant is mildin reaction condition and low in equipment requirement, and high temperature or cooling is not needed during production, so that low energy consumption can be realized; as enzyme catalysis has efficient and exclusive selection, so that convenient purification can be achieved; and in addition, the vast majority of solvents in reaction is water, and the solvents such as butyl acetate are not neededto be added to form a two-phase reaction system, so that the mutant is low in discharging of waste gas, waste water and industrial residues, green and environment-friendly in preparation process.

Description

technical field [0001] The invention relates to a ketoreductase mutant with improved enzyme activity and application thereof, belonging to the field of biotechnology. Background technique [0002] Ketoreductases are versatile catalysts for the enantioselective reduction of aldehydes or ketones to their corresponding alcohols. (R)-specific ketoreductases have different properties from (S)-specific ketoreductases, and these catalysts are being 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 pharmaceutically and agrochemically active compounds, and in some cases one enantiomer has beneficial pharmaceutical activity while the other has genotoxic effects. Therefore, in the synthesis of pharmaceutically and agriculturally active compounds, it is necessary to use catalysts with the required stereospecificity to synthesize optically active alcohols. [0003] Chirally ...

Claims

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Application Information

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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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