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Activity-enhanced ketoreductase mutant and application thereof

A technology of activity enhancement and reductase, which is applied in the direction of oxidoreductase, application, enzyme, etc., can solve the problems of increasing operation difficulty and cost, and achieve the effect of reducing production cost

Active Publication Date: 2020-03-31
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] U.S. Patent No. 8,426,178 discloses an engineered ketoreductase with improved properties, and also provides a polynucleotide encoding the engineered ketoreductase, a host cell capable of expressing the engineered ketoreductase, and a method for synthesizing various chiral compounds using the engineered ketoreductase; Engineered ketoreductase polypeptides optimized to catalyze the conversion of N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropylamine to (S)-N,N-dimethyl-3 -Hydroxy-3-(2-thienyl)-1-propanamine; this scheme uses isopropanol as the coenzyme cycle method, but the reaction requires suction filtration to form a negative pressure to remove the by-product acetone of the reaction, so that the reaction can be carried out effectively , it will increase the operation difficulty and cost when the production is enlarged

Method used

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  • Activity-enhanced ketoreductase mutant and application thereof
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 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.

[0031] Use PrimerPremier (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. 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.

[0032] 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

[0033] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the following pr...

Embodiment 2

[0037] 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.

[0038] Use PrimerPremier (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. 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.

[0039] 2mM dNTP mix (2mM eac hdNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH2O Bring the total volume of the reaction system to 50 μl

[0040] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the following pr...

Embodiment 3

[0044] 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.

[0045] Use PrimerPremier (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. 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.

[0046] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl wxya 2 o

Bring the total volume of the reaction system to 50 μl

[0047] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the follo...

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Abstract

The invention discloses an activity-enhanced ketoreductase mutant and an application thereof, which belong to the to field of biotechnology. The ketoreductase mutant is derived from wild-type ketoreductase of Lactobacillus parabuchneri, which exhibits that the ketoreductase increases the conversion rate of a substrate (e.g., 3-dimethylamino-1-(2-thienyl)-1-acetone hydrochloride) to a product (e.g., S-3-dimethylamino-1-(2-thienyl)-1-propanol). Compared with the wild-type ketoreductase shown as SEQ ID NO.7, the ketoreductase mutant shows stronger catalytic activity. The mutant disclosed by the invention can be obtained by in-vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, directed evolution methods and the like for encoding the enzyme.

Description

technical field [0001] The invention relates to an enzyme, a biocatalysis method and its application, in particular to an activity-enhanced ketoreductase mutant 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 distinct properties from (S)-specific ketoreductases, and these catalysts are Optically active alcohols are used more and more frequently in the industrial synthesis; 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 However, enantiomers have genotoxic effects; therefore, in the synthesis of pharmaceutical and agricultural active compounds, it is necessary to use catalysts with the required stereospecificity to synthesize ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/00C12R1/19
CPCC12N9/0006C12N15/70C12N2800/22C12P17/00C12Y101/01047
Inventor 丁雪峰李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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