多点突变提高α-L-鼠李糖苷酶热稳定性  

Enhanced thermostability of α-L-rhamnosidase by multiple-site mutation

作　　者：刘小琴 杨岩 吴喆瑜 巩建业 刘嘉男 廖辉 李文静 李利君 LIU Xiaoqin;YANG Yan;WU Zheyu;GONG Jianye;LIU Jianan;LIAO Hui;LI Wenjing;LI Lijun(College of Food and Bioengineering, Jimei University, Xiamen 361021, China;Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China;Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China)

机构地区：[1]集美大学食品与生物工程学院,福建厦门361021 [2]福建省食品微生物与酶工程重点实验室,福建厦门361021 [3]厦门市食品与生物工程技术研究中心,福建厦门361021

出　　处：《食品与发酵工业》2019年第6期23-29,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(31371751)

摘　　要：为了提高α-L-鼠李糖苷酶的热稳定性,对实验室前期构建的K406R、K440R、K573V、E631F四个不同氨基酸位点的单点突变体进行联合突变,得到了K406R-K573V、K406R-E631F、K440R-K573V、K440R-E631F四个联合突变体。结果表明,相比于野生型(wild type,WT),联合突变体K440R-K573V和K440R-E631F的热稳定性有所提高,在60℃时半衰期分别提高了1. 13倍,1. 44倍;在65℃的半衰期分别提高了1. 64倍,1. 64倍;在70℃的半衰期分别提高了1. 88倍,1. 68倍。对突变体K440R-E631F进行圆二色谱分析、分子动力学以及分析微观结构变化分析可知,K440R-E631F突变体与WT相比,内部疏水性有所提高,二级结构中α-螺旋、"-转角、无规则卷曲含量增多,可能与热稳定性的提高相关。In order to improve the thermostability of α-L-rhamnosidase,four single site mutants: K406 R,K440 R,K573 V,and E631 F,were combined and four new mutants: K406 R-K573 V,K406 R-E631 F,K440 RK573 V,and K440 R-E631 F,were obtained. In comparison to the wild type( WT),the thermostabilities of K440 RK573 V and K440 R-E631 F improved,as their half-lives at 60 ℃ increased by 1. 13-fold and 1. 44-fold,respectively.Moreover,their half-lives were both 1. 64 times higher than that of WT at 65 ℃,and 1. 88-fold( K440 R-K573 V)and 1. 68-fold( K440 R-E631 F) higher at 70 ℃. Based on circular dichroism,molecular dynamics and micro-structural analysis,it was found that mutant K440 R-E631 F in comparison to WT had increased internal hydrophobicity.Additionally,its contents of α-helix,"-turn and random coil also increased,which may be related to the increased thermostability of α-L-rhamnosidase.

关 键 词：α-L-鼠李糖苷酶 联合突变 热稳定性 

分 类 号：TS201.3[轻工技术与工程—食品科学]