Enhancing Catalytic Properties Of Halohydrin Dehalogenase From Mycobacterium Sp.strain GP1(HheB_GP1) By Direction Evolution Strategy

Halohydrin dehalogenase can not only catalyze the dehalogenation of organic halides through the intramolecular nucleophilic substitution mechanism,but also accept nucleophilic reagents with negative charges to catalyze the epoxide ring-opening reaction.Therefore,the halohydrin dehalogenase can play an important role in the degradation of organic halides and the synthesis of various optically pure?-substituted alcohols and epoxides.HheB_GP1?B-type halohydrin dehalogenase from Mycobacterium sp.GP1?is significantly different from HheA and HheC in terms of substrate range and enantioselectivity.If the advantages can be complemented with other two types of enzymes and utilized,the application range of halohydrin dehalogenase can be greatly expanded.However,the catalytic properties of wild-type halohydrin dehalogenase can not meet the practical application to a certain extent.Therefore,in order to meet industrial needs,it is usually necessary to carry out reasonable transformation.At present,HheA and HheC have produced many excellent mutants through protein engineering,but the research on the catalytic activity of HheB_GP1 has not been reported.The low catalytic activity of the enzyme HheB_GP1 has become the limiting factor of its application.If HheB_GP1 with higher catalytic activity is obtained through protein engineering,it can promote its wider application in industrial production.Therefore,in this paper,the research on the catalytic properties of HheB_GP1P1 was carried out from the following three aspects.First,efficient recombinant expression of enzyme in vitro is an alternative to subsequent experiments.Therefore,this paper first optimizes the expression of HheB_GP1through four aspects:expression temperature,inducer concentration,time and the initial OD₆₀₀ of the bacterial solution.The optimal expression condition of HheB_GP1 was obtained:0.05%L-arabinose inducer was added at the same time of inoculation of bacterial solution(OD₆₀₀=0.05),and expression was induced at 30?and 120 rmp for10-12 h.Finally,the soluble protein expression level of HheB_GP1P1 is increased to about20%.Second,the structure-function information of HheB_GP1 is lacking at present.Therefore,in order to obtain HheB_GP1 with high catalytic activity,it is decided to adopt a directed evolution strategy for transformation.Among them,the method of error-prone PCR combined with large primer PCR was selected when constructing a random mutant library,and the reaction conditions were optimized.Finally,the rules of influencing factors in the reaction were summarized,and error-prone PCR reaction conditions of 0.3?M primers,100 ng template,2 mM Mg²⁺and 0.1 mM Mn²⁺,and megaprimer PCR reaction conditions of a ratio of primer to template of 5:1 were determined,and successfully constructed a high-quality random mutant library with appropriate mutation frequency?3‰-4‰?.Finally,after screening nearly 3700 monoclonals,six dominant mutants with improved catalytic activity for the substrate 1,3-DCP were obtained,of which the catalytic activity of A128T was 4.4 times that of the wild type.The most surprising is the F158Y mutant.In addition to the catalytic activity of the aliphatic substrate 1,3-DCP increased by 3.3 times that of the wild type,the catalytic activity of the aromatic substrate PNSHH also increased,and the e.e.value of the product?R?-ECH is also12.1%higher than that of the wild type.Through homology modeling and molecular docking methods,the mechanism of the mutation site influencing the catalytic properties was analyzed,which laid the foundation for further research on the structure-function relationship of HheB_GP1 and further transformation research.