7β-hydroxysteroid dehydrogenase mutant, its application and synthesis method

Abstract

The invention provides a mutant of 7 beta-hydroxyl steroid dehydrogenase, application of the mutant and a synthesis method. The mutant of the 7 beta-hydroxyl steroid dehydrogenase is characterized in that amino acid sequences of the mutant are Seq ID NO:4, and coded nucleotide sequences are Seq ID NO:3; or amino acid sequences of the mutant are Seq ID NO:6, and coded nucleotide sequences are Seq ID NO:5. The mutant, the application and the synthesis method have the advantages that cholic acid compounds, particularly ursodeoxycholic acid, can be catalytically synthesized by the efficient 7 beta-hydroxyl steroid dehydrogenase, mutant enzymes of the 7 beta-hydroxyl steroid dehydrogenase and coenzyme regeneration systems, accordingly, the substrate concentration can reach 100 g / L, the conversion rate is 99.2-99.5%, and the weight yield can reach 94-96%; and the enzymes can be inexpensively and easily obtained by the aid of a fermentation process, accordingly, the production cost and the product quality are superior to the production cost and the product quality of chemical synthesis methods, and the mutant and the synthesis method are applicable to industrial production.

Description

Technical field: The present invention relates to novel 7β-steroid dehydrogenase (7β-hydroxsteroid dehydrogenase, 7β-HSDH) mutants, encoding these Sequences of enzymes, methods of producing such enzymes, and their use in the enzymatic synthesis of cholic acid compounds, in particular in the synthesis of ursodeoxycholic acid (UDCA); furthermore, the invention also includes the use of mutants for the synthesis of UDCA The new method of , and the post-extraction method of UDCA. Background technique: Ursodeoxycholic acid (I, UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile, and it and its corresponding diastereoisomer chenodeoxycholic acid (II, CDCA) are clinically used to treat various Gallstone disease, various acute and chronic liver diseases, has good effect. The yield of extracting UDCA from bear bile of artificially raised bears is low, the source is limited, and it is against animal protection, so artificial synthesis ...

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Problems solved by technology

Hirano and Masuda described a NADP+-dependent 7β-HSDH from Collinsia aerogenes ATCC 25986 (Appl Environ Microbiol, 1982, 43(5):1057-1063), but no sequence information was published

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