Halohydrin dehalogenase mutant and applications thereof

Abstract

The invention discloses a halohydrin dehalogenase mutant and applications thereof, wherein the mutant is subjected to one or a plurality of the following mutations that the arginine at the site 84 isreplaced by alanine, serine, histidine or asparagine, the arginine at the site 88 is replaced by leucine, valine, threonine, cysteine, phenylalanine or isoleucine, the valine at the site 136 is replaced by threonine or serine, and the asparagine at the site 144 is replaced by cysteine, histidine or threonine. According to the present invention, the halohydrin dehalogenase HheAAM is modified by respectively using the directed evolution and the semi-rational design, wherein the results show that the sites 84, 88, 136 and 144 can affect the enzyme activity; and the four key sites are subjected tocombined mutation by using the iterative saturation mutation strategy, and are subjected to random combined mutation by using the combined active site saturation experiment strategy, such that the specific enzyme activity of the screened mutant is 801.59 U.g<-1>, and is 12 times the specific enzyme activity of the original enzyme.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a halohydrin dehalogenase mutant and application thereof. Background technique [0002] In 2017, The Lancet published the three causes of death that caused the largest number of deaths in 2016. Cardiovascular and cerebrovascular diseases have become the leading cause of human death. [0003] In the past, statins were mostly used to regulate blood lipids, and later found that they also have anti-inflammatory effects, improve arterial endothelial function, inhibit myocardial remodeling, and stabilize atherosclerotic plaques. They can be used to treat and prevent cardiovascular and cerebrovascular diseases. Among them, atorvastatin calcium is a relatively classic and effective statin drug, and (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester is the synthesis process of atorvastatin calcium The key chiral building blocks in . [0004] Halohydrin dehalo...

AI Technical Summary

Problems solved by technology

[0005] (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester can be dehalogenated by (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester Preparation, the alkaline environment in the traditional chemical synthesis process will cause the spontaneous hydrolysis of the substrate, resulting in low product yield, and the use of halohydrin dehalogenase to catalyze the reaction has mild reaction conditions and few side reactions, which has great application potential

[0006] The main problem in the existing research is that there are few types of halohydrin dehalogenases, and the enzyme activity against the substrate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester is very low

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