Modification of the substrate specificity of leucine dehydrogenase by site-directed mutagenesis based on biocomputing strategies

Abstract

Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness, high stereoselectivity, and mild reaction conditions. However, amine dehydrogenase has low catalytic activity, which greatly limits its application in the large-scale synthesis of chiral amines. In this study, a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy. Furthermore, conservation analysis, homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites, and the mutants L52S and T143C were obtained. The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg, respectively. The enzyme activity of the latter was 188% and the Tm value was 2.55 °Chigher than those of the original mutant, which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.