定向进化提高枯草芽孢杆菌脂肪酶的活力  

Increasing Activity of Lipase from Bacillus subtilis by Directed Evolution

作　　者：赵博[1] 陶进[1] 马吉胜[1] 廉虹[1] 王岩[1] 常琳[1] 高仁钧[1] 曹淑桂[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,吉林长春130023

出　　处：《催化学报》2009年第4期291-296,共6页

基　　金：国家自然科学基金(30400081;20432010;20672045)

摘　　要：通过定向进化的方法,提高了一个新的源于枯草芽孢杆菌(Bacillus subtilis)的脂肪酶(BSL2)的活力.经过两轮易错PCR以及高通量筛选,最终获得了比活力为野生出发酶4.5倍的突变体3-1B2.基因比对结果表明,共有两个氨基酸发生了突变.突变酶的酶学性质研究发现,与野生酶相比,它的热稳定性及pH稳定性略有增加,最适温度和最适pH值则无太大的变化.同源模建了BSL2与3-1B2的结构,并与底物进行了分子对接.结果表明,突变体3-1B2的结合能比野生型BSL2低1.29 kcal/mol,活性中心Ser77残基与底物羰基的距离也由0.319 nm减小为0.278 nm,因而加快了酶反应速度,提高了酶活力.The specific activity of a Bacillus subtilis lipase was increased by directed evolution.Through two cycles of error prone PCR,coupled with a sensitive high-throughput screening method,a mutant named 3-1B2 was obtained.Its catalytic activity was 4.5-fold compared to that of wild lipase BSL2.DNA sequencing revealed that two amino acids were changed.Further experiments showed that the thermosta-bility and pH stability of 3-1B2 were slightly increased than those of BSL2,and the optimum temperature and pH of the mutant lipase were similar to those of wild lipase.The molecular structures of BSL2 and 3-1B2 were homologously modeled based on the crystal structure of BSLA and docked with the substrate.The results showed that the binding energy of 3-1B2 with substrate was 1.29 kcal/mol lower than that of BSL2,and the distance between catalytic residue Ser77 of 3-1B2 and substrate was 0.278 nm,lower than that of BSL2（0.319 nm）.This implied that small distance between Ser77 and substrate might increase reaction rate and then cause an increase in activity.

关 键 词：枯草芽孢杆菌 脂肪酶 易错聚合酶链反应 定向进化 分子对接 

分 类 号：O629.8[理学—有机化学]