Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin

Abstract

The invention provides a clone and a pronucleus expression method of a novel B class epoxide hydrolase gene mature peptide cDNA sequence from Aspergillus usamii E001. The nucleotide sequence of the novel B class epoxide hydrolase gene mature peptide cDNA sequence is SEQ ID NO:1, the corresponding amino acid sequence is SEQ ID NO:2, and the corresponding gene is named Aus EH-B. According to the invention, good stereoselectivity is achieved for (R)-epichlorohydrin through chiral gas chromatography analysis rEH, and a produced (S)-epichlorohydrin antipode excess value achieves 99%. The pronucleus expression method provided by the invention lays the foundation for the industrialized production of epoxide hydrolase and provides the basis for preparing chiral epichlorohydrin by researching an EH enzyme kinetic resolution method for biological catalysis technology industrialization.

Description

technical field [0001] The invention relates to the cloning and prokaryotic expression of a novel B-type epoxide hydrolase (Aus EH-B) gene mature peptide cDNA sequence derived from Aspergillus usamii E001 strain, and the preparation of chiral The method for epoxystyrene belongs to the technical field of bioengineering. Background technique [0002] Chiral compounds are important chiral ligands, which are widely used in various asymmetric reactions, especially in the field of drug synthesis. Chiral epichlorohydrin is an important key intermediate in organic synthesis, used to prepare a variety of high optical purity pharmaceutical intermediates and natural products, such as aryloxypropanolamines, derivatives of cyclopentanone, antifungal Drugs (S) - Meikangling, atorvastatin, L-carnitine, etc. At present, racemic epichlorohydrin is cheap and easy to obtain, while chiral epichlorohydrin is expensive, and the quotation of the latter is basically more than 6 times that of the ...

AI Technical Summary

Problems solved by technology

The genetically engineered epoxide hydrolase has high purity, does not contain other proteases, and has stronger stereoselectivity; the Escherichia coli expression system has the advantages of high-efficiency intracellular expression of recombinant proteins, low cost, high productivity, and simple operation; and because the general substrate is Organic solvents, enzyme molecules are inhibited by the substrate concentration, and the intracellular expression host bacterial envelope forms a natural protective barrier, which is more conducive to biocatalysis at high substrate concentrations and adapts to industrial production requirements; the invention uses recombinant epoxides to hydrolyze There is no report on the preparation of chiral epoxystyrene by enzyme

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