Enhanced Hydrolysis And Acylation Activity Of Esterase BioH By Directed Evolution

Esterase BioH is an important enzyme in the Biotin synthesis. It contains a typical catalytic triad of Ser-His-Asp and reveals a carboxylesterase activity with a preference for short acyl chain substrates.The hydrolysis activity of BioH determined by p-nitrophenyl butyrate (p-NPB) was18U/mg and the enzyme was found as a thermostable protein, it retained77%of its hydrolysis activity after heating for40min in60℃. We found that the enzyme exhibited activities in the acylation of secondary alcohols and amines. The conversion rate of acylating was a-phenylethanol0.123mmol/(h·mg) with an eep of88%and the conversion rate of acylating a-phenylethylamine was2.1μmol/(h·mg) with an eep of40%. The purpose of this study is to explore whether the enzyme’s acylation activity can be enhanced by directed evolution of its hydrolysis activity. Because it’s hard to establish a high throughput screening method for the acylation reaction. In order to improve the hydrolysis activity, a directed evolution method was carried out. p-NPB was using as the screening substrate whose product,p-nitrophenol, showed a biggest absorption value in405nm.Error-prone PCR was used to build the mutant library by adjusting the concentration of Mn2+. And the mutation rate was kept as2-3changed bases in each gene when the concentration of Mn2+was0.1mM. In each round of directed evolution,5000-6000mutants were selected. Using wild-type BioH as the parent, three mutants with hydrolysis activity improved were selected, K.213E, Q70L/M170T and V175A. Their activities were improved for20%,30%and50%respectively. And then K213E and V175A were used as parents to undergo the next round of directed evolution. Three mutants from K213E were selected, they were K213E/M197L, K213E/L180M and K213E/C31Y/F50S. Their hydrolysis activities improved for40%,50%and95%. And one mutant from V175A was selected. It was V175A/Q129R, showed100%improvement compared with the wild type. Structure analysis showed that the mutant sites were located in loops or a-helixes that were far away from the activity sites. When choosing the father mutant for the next round of directed evolution, it was better to take the one with little influence to the protein expression and thermostability. Three single mutants Q70L, M170T, M197L and seven mutants created by combination of these four hot spots Q70L, M170T, M197L and K213E were detected for the acylation activity in order to find a further improvement. The best mutants, Q70L/M197L/K213E showed120%improvement in acylating a-phenylethylamine and Q70L/K213E showed70%improvement in acylating a-phenylethanol. The results demonstrated that the acylation activity of esterase BioH can be improved by directed evolution of its hydrolysis activity.