A kind of activity enhanced ketoreductase mutant and its application

An activity-enhancing, reductase technology, applied in the directions of oxidoreductase, application, enzyme, etc., can solve the problems of increasing the difficulty of operation and cost, and achieve the effect of high cycle times of coenzymes, low three-waste emissions, and high specific enzyme activity

Active Publication Date: 2021-07-23
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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  • A kind of activity enhanced ketoreductase mutant and its application
  • A kind of activity enhanced ketoreductase mutant and its application
  • A kind of activity enhanced ketoreductase mutant and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 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.

[0030] 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 , and the obtained primers were dissolved in double-distilled water, and 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.

[0031]

[0032] 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 procedures: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0033] The DNA fragment obtained by PCR was gel-cut and purified, and cloned into the NdeI / XhoI site of pET30a by homol...

Embodiment 2

[0036] 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.

[0037] 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 , and the obtained primers were dissolved in double-distilled water, and 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]

[0039] 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 procedures: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0040] The DNA fragment obtained by PCR was gel-cut and purified, and cloned into the NdeI / XhoI site of pET30a by homol...

Embodiment 3

[0043] 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.

[0044] 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 , and the obtained primers were dissolved in double-distilled water, and 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.

[0045]

[0046] 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 procedures: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0047] The DNA fragment obtained by PCR was gel-cut and purified, and cloned into the NdeI / XhoI site of pET30a by homol...

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Abstract

The invention discloses a ketoreductase mutant with enhanced activity and its application, which belongs to the field of biotechnology. It is derived from the wild-type ketoreductase of Lactobacillus parabuchneri, which shows that the substrate (such as 3-dimethylamino-1 -(2-thienyl)-1-acetone hydrochloride) to products such as S-3-dimethylamino-1-(2-thienyl)-1-propanol) with increased conversion rates of ketoreductases. Compared with the wild-type ketoreductase of SEQ ID NO.7, the mutant ketoreductase exhibits stronger catalytic activity. The mutants of the present invention can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods that encode 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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Patent Type & Authority Patents(China)
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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