Ketone reductase LEK, encoding gene, mutant and application of mutant

A technology encoding genes and reductases, which is applied in the field of ketoreductases and biocatalytic synthesis of chiral chemical intermediates, can solve the problems of reduced workload and many sense mutations, and achieve the effect of increased conversion rate

Active Publication Date: 2013-09-11
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although rational / semi-rational design requires more information and analyzes based on 3D molecular structure models, the resulting mutants produce more sense mutations and the workload is greatly reduced

Method used

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  • Ketone reductase LEK, encoding gene, mutant and application of mutant
  • Ketone reductase LEK, encoding gene, mutant and application of mutant
  • Ketone reductase LEK, encoding gene, mutant and application of mutant

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Embodiment

[0052] 6.1 Materials

[0053] 6.1.1 Strains and plasmids

[0054] The strain Lodderomyces elongisporus CGMCC2.1589 was purchased from the China General Microorganism Culture Collection; the strain Thermoplasma acidophilum DSM1728 was purchased from the German Culture Collection of Microorganisms DSMZ; the strain pET22b-gdh2-E.coli BL21 was constructed and preserved in the laboratory The genetically engineered bacteria, the specific construction method is the glucose dehydrogenase GDH2 derived from Thermoplasma acidophilum DSM1728, (Zhou L.P., Zhao Y., Wang H.F., Ding J.X., 2004. Cloning and expression of glucose dehydrogenase from Bacillus Subtilis, J. Jiangsu University (medicine edition). 14(1), 7-10.) An expression strain obtained by linking the double restriction site NdeI / BamHI with the vector pET22b. Plasmid pET28a, plasmid pET22b, plasmid pETDuet-1, E.coli DH5α, E.coli BL21(DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0055] 6.1.2 Main reagent...

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Abstract

The invention discloses a ketone reductase LEK, an encoding gene, a mutant and an application of the mutant in a preparation of ethyl (R)-4-chloro-3-hydroxybutyrate (CHBE) by biological catalysis. The ketone reductase LEK has 95% or more homology with an amino acid sequence as shown in SEQ. ID.NO. 2. The mutant of the ketone reductase LEK is a mutant W28A obtained by mutating W at position 28 in the amino acid sequence of the ketone reductase LEK into A. The R-type ketone reductase, i.e., the ketone reductase LEK, is developed, then site-directed mutagenesis and transformation of the reductase are carried out, and a coenzyme regeneration cycle reaction system is established. The conversion rate of the preparation of (R)-CHBE by catalysis reaches 100% and the e.e. value is more than 99%, and simultaneously the Km value of the reductase is relatively low and is 14.08 mM. The catalytic reaction system has no need of adding an expensive coenzyme NADPH, and thereby reducing the production cost; and at high substrate concentrations, the catalytic reaction system can be used for large-scale preparation of products with high optical purity, has relatively high production efficiency, and is in favor of industrialization production.

Description

(1) Technical field [0001] The present invention relates to a ketoreductase and a method for biocatalytically synthesizing a chiral chemical intermediate, in particular to a ketoreductase LEK derived from Lutheran yeast and its mutant strain, and the ketoreductase LEK or mutant strain capable of selectively The reduction substrate of the R-type CHBE is generated, and its e.e. value is greater than 99%. (2) Background technology [0002] β-hydroxy ester is an important part of pharmaceuticals, pesticides, materials and other fine chemical raw materials, such as 4-chloro-3-hydroxybutyric acid ethyl ester (CHBE), which is an important organic synthesis intermediate, and there are three in the molecule Functional groups (-OH, -Cl, -COOH) can be introduced into a variety of drug intermediates through chlorine displacement reactions, reduction reactions, etc. Its chiral monomer (R / S)-CHBE is a very promising chiral substance. R-CHBE can be used to synthesize L-carnitine (Eui S C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P7/62
Inventor 殷晓浦谢恬叶婷婷沈凌宏曹丹王秋岩谌容裴晓林
Owner HANGZHOU NORMAL UNIVERSITY
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