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Carbonyl reductase mutant as well as construction method and application thereof

A technology of reductase and mutant, applied in the field of biochemical industry

Active Publication Date: 2021-10-01
杭州文德阶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention aims at the problems existing in the process of asymmetrically synthesizing single-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester in the prior art to prepare (R)-α-lipoic acid, and provides a carbonyl reductase mutant , recombinant vectors, recombinant genetically engineered bacteria and their applications

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  • Carbonyl reductase mutant as well as construction method and application thereof
  • Carbonyl reductase mutant as well as construction method and application thereof
  • Carbonyl reductase mutant as well as construction method and application thereof

Examples

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Embodiment 1

[0039] This example provides a mutant of carbonyl reductase, said mutant is the 135th glycine, 136th leucine, and 143rd leucine of the amino acid sequence shown in SEQ ID NO.2. Obtained by single mutation or multi-point combination mutation; where the 135th glycine is mutated to isoleucine, the 136th leucine is mutated to alanine, and the 143rd leucine is mutated to alanine, the mutants The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3.

[0040]This embodiment provides a method for constructing a carbonyl reductase mutant, which specifically includes the following steps:

[0041] Step S1, constructing the recombinant vector:

[0042] The coding gene of carbonyl reductase and the coding gene of formate dehydrogenase are connected by fusion PCR, and the co-expression vector of carbonyl reductase and formate dehydrogenase is constructed by one-step cloning of the linked dual-enzyme gene fragment and the linearized vector fragmen...

Embodiment 2

[0101] This example provides an application of the carbonyl reductase mutant in Example 1 in catalyzing the preparation of (R)-α-lipoic acid from 6-carbonyl-8-chlorooctanoic acid ethyl ester, specifically mutating the carbonyl reductase Genetic recombinant genetically engineered bacteria catalyze the synthesis of (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester from 6-carbonyl-8-chlorooctanoic acid ethyl ester, with (S)-6-carbonyl-8-chlorooctanoic acid ethyl ester as the base The product undergoes a biotransformation reaction to produce (R)-α-lipoic acid.

[0102] The specific method of synthesizing (S)-6-hydroxyl-8-chlorooctanoic acid ethyl ester in the present embodiment is:

[0103] Step 1, preparation of recombinant bacterial cells of carbonyl reductase and its mutants

[0104] The wet thallus obtained by fermenting and culturing the recombinant genetically engineered bacteria described in Example 1 is used as a catalyst, and 6-carbonyl-8-chlorooctanoic acid ethyl ester is...

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Abstract

The invention relates to the field of biochemical engineering, and discloses a carbonyl reductase mutant as well as a construction method and application thereof.The 143rd leucine in an amino acid sequence shown in SEQ ID NO.2 is mutated into alanine, the 136th leucine is mutated into alanine, and 135th glycine is mutated into isoleucine, so that the enzyme activity of the carbonyl reductase mutant is improved by 8 times; and the e.e. Value is increased to 99%, coenzyme circulation is successfully realized by coupling with formate dehydrogenase, and finally, the conversion rate and the e.e. Value of a 450 g / L substrate within 12 hours can be over 99% when the final concentration of thalli is 20 g (dcw) / L, so that the problems of low substrate concentration, low conversion rate, low yield, low e.e. Value, relatively high production cost and the like are effectively solved.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a carbonyl reductase mutant, a gene, a recombinant vector containing the gene, and a recombinant genetically engineered bacterium prepared by transformation of the recombinant vector, and the carbonyl reductase mutant in catalyzing 6-carbonyl-8 - Application of ethyl chlorooctanoate in the synthesis of (S)-6-hydroxyl-8-ethyl chlorooctanoate. Background technique [0002] Lipoic acid (α-Lipoic acid) is a biologically active natural product that was first isolated from pig liver by Reed in 1951. Lipoic acid has been widely used in the field of clinical medicine in Europe and America, such as liver disease, senile dementia, cataract, heart disease, diabetes, AIDS, psoriasis, eczema, Parkinson's disease, rheumatism, heart disease, neurological disease, subacute necrosis It is known as "universal antioxidant" for the treatment of diseases such as encephalopathy, radiation injury, ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/02C12N15/70C12N1/21C12P17/00C12P7/62C12R1/19
CPCC12N9/0006C12N9/0008C12N15/70C12P17/00C12P7/62C12Y101/01184C12Y102/01002
Inventor 俞鑫焱胡文烨
Owner 杭州文德阶生物科技有限公司
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