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

作　　者：Jiapeng Lu Zengyu Wang Yingying Jiang Zhoutong Sun Wei Luo 

机构地区：[1]The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education School of Biotechnology,Jiangnan University,Wuxi 214000,Jiangsu Province,China [2]Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China

出　　处：《Systems Microbiology and Biomanufacturing》2023年第2期384-392,共9页系统微生物学与生物制造（英文）

基　　金：supported by the Key Technology Research Plan Project of the Inner Mongolia Autonomous Region(2019GG302);the Program of the Key Laboratory of Carbohydrate Chemistry&Biotechnology,Ministry of Education,China(KLCCB-KF202103).

摘　　要：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.

关 键 词：Amine dehydrogenase L-leucine dehydrogenase Semi-rational design Protein engineering Substrate binding pocket 

分 类 号：Q814[生物学—生物工程]