7beta-hydroxysterol dehydrogenase mutant and application of 7beta-hydroxysterol dehydrogenase mutant in ursodeoxycholic acid synthesis

Abstract

The invention discloses a 7beta-hydroxysterol dehydrogenase mutant with increased activity and stability which is obtained through molecular evolution, recombinant expression plasmid containing the 7beta-hydroxysterol dehydrogenase mutant gene and a recombinant expression transformant and a preparation method of a recombinant mutant enzyme preparation, and the invention also provides an application of the recombinant mutant enzyme preparation in ursodeoxycholic acid synthesis. The 7beta-hydroxysterol dehydrogenase has excellent activity and heat stability, can efficiently catalyze asymmetric reduction of 7-carbonyl lithocholic acid to prepare the ursodeoxycholic acid; the 7beta-hydroxysterol dehydrogenase is subjected to immobilization and then is subjected to couple by an enzyme method with the immobilized 7beta-hydroxysterol dehydrogenase, epimerization of a substrate chenodeoxycholic acid with low cost can be directly catalyzed, ursodeoxycholic acid can be prepared through continuous conversion, and the operation is simple. Compared with the prior art reported currently, ursodeoxycholic acid prepared by hydroxysterol dehydrogenase through catalysis has the advantages of high substrate concentration, short reaction time, complete reaction, and high product purity, and has strong industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to 7β-hydroxysterol dehydrogenase (7β-hydroxysteroid dehydrogenase, 7β-HSDH) of Ruminococcus twistedensis with improved activity and stability Rt ) mutant, its recombinant expression vector and recombinant expression transformant, the preparation method of the recombinant 7β-hydroxysterol dehydrogenase catalyst, and the application of the recombinant 7β-hydroxysterol dehydrogenase catalyst in the synthesis of ursodeoxycholic acid. Background technique [0002] Bear bile products have high medicinal value and are usually used in modern medicine to treat cholestatic liver diseases such as reflux gastritis, gallstones, biliary pancreatitis, fatty liver disease, drug-induced hepatitis and viral hepatitis. Ursodeoxycholic acid (abbreviated as UDCA) is an important active ingredient in bear bile products. It has high medicinal value and can improve the damage of chronic liver disea...

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Problems solved by technology

[0007] In summary, among the existing reports on enzymatic preparation of UDCA, there are many reports on the preparation of UDCA by enzymatic catalyzed asymmetric reduction of 7-KLCA, but the activity of 7β-HSDH used is low, and Escherichia coli is used as the recombinant method. The expressed host, the expressed enzyme is an intracellular enzyme, and the recombinant bacteria need to be crushed to separate the enzyme solution. The application process is cumbersome, and 7-KLCA is not an animal bile acid raw material that can be extracted in large quantities in nature, and the price is relatively high; Although CDCA exists in a large amount in poultry bile and the price is relatively low, there are few reports on enzymatic coupling catalysis of CDCA epimerization, and the substrate concentration is not high, and in the reported two-step enzymatic reaction, the first The enzyme in the first step reaction needs to be inactivated before the enzyme in the second step reaction can be added. The steps are cumbersome, the efficiency is low, the enzyme cannot be used repeatedly, and the cost of biocatalyst application is relatively high.

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