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Ketoreductase lek mutant and its application

A reductase and mutant technology, applied in the field of ketoreductase and biocatalytic synthesis of chiral chemical intermediates, can solve the problems of reduced workload and many sense mutations, and achieve the effect of improving conversion rate

Active Publication Date: 2016-02-10
HANGZHOU NORMAL UNIVERSITY
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  • 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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  • Ketoreductase lek mutant and its application
  • Ketoreductase lek mutant and its application
  • Ketoreductase lek mutant and its application

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Experimental program
Comparison scheme
Effect test

Embodiment

[0052] 6.1 Materials

[0053] 6.1.1 Strains and plasmids

[0054] The strain LodderomyceselongisporusCGMCC2.1589 was purchased from the China General Microorganism Collection Center; the strain Thermoplasmaacidophilum DSM1728 was purchased from the German Microbiology Collection Center DSMZ; the strain pET22b-gdh2-E. The specific construction method is to derive glucose dehydrogenase GDH2 from Thermoplasmaacidophilum DSM1728, (ZhouL.P., ZhaoY., WangH.F., DingJ.X., 2004.CloningandexpressionofglucosedehydrogenasefromBacillusSubtilis, J.JiangsuUniversity(medicineedition).14(1), 7-10.) The expression strain obtained by linking the double restriction site NdeI / BamHI with the vector pET22b. Plasmid pET28a, plasmid pET22b, plasmid pETDuet-1, E.coliDH5α, E.coliBL21(DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0055] 6.1.2 Main reagents

[0056] pMD-19T kit, Taq enzyme, dNTPmixture, IPTG, high-ligation, LATaqDNAPolymorase, pyrobestDNAPolymorase and restricti...

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Abstract

The invention discloses a ketoreductase LEK mutant and an application in preparing (R)-4-chloro-3-hydroxy ethyl butyrate through biological catalysis. The ketoreductase LEK mutant has an amino acid sequence shown in SEQ. ID. NO.2, and the ketoreductase LEK mutant is a mutant S209G which is obtained by mutating S on a 209th position of a ketoreductase LEK amino acid site. The R-type ketoreductase developed by the invention, namely ketoreductase LEK, is transformed through site-specific mutagenesis, and a coenzyme regeneration circular reaction system is established; a conversion rate of (R)-CHBE from catalysis reaction is 100% and an e.e. value is greater than 99%, and meanwhile, a Km value of the enzyme is relatively low, namely 6.05mM; the catalysis reaction system is free from expensive coenzyme NADPH, thus reducing production cost; and the ketoreductase LEK mutant can realize large-scale preparation of products which are high in optical purity under a relatively high substrate concentration, so that the mutant is relatively high in production efficiency and is favorable for industrial 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 as synthetic L-carnitine (EuiSC, J...

Claims

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Application Information

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