食品与发酵工业2025，Vol.51Issue(15)：1-8,8.DOI:10.13995/j.cnki.11-1802/ts.040962
β-葡萄糖苷酶Bgl3热稳定性有益突变的鉴定与结构基础分析
Identification and structural analysis of beneficial mutations for thermostability of β-glucosidase Bgl3
摘要
Abstract
DNA shuffling is a wildly used strategy of directed evolution.It can quickly test the effects of multiple mutations,however,the actual beneficial mutations among them and their structural basis are often unclear.[3-Glucosidase is the rate-limiting enzyme for effi-cient degradation of cellulose,and good thermostability is a key factor affecting its practical catalytic efficiency.In this work,the beneficial mutations of a thermostable β-glucosidase mutant Bgl3-6511(containing 60 single mutations and has a T50 value 4.6℃ higher than wild-type)were identified through sequence alignment,site-directed mutagenesis,and thermostability assay.Results showed that six single point mutations,Y50F,R56K,V65I,T67A,and P143A,increased the T50 of the enzyme by 2.9,4.2,1.5,2.8,3.2,and 1.2℃,respectively.At the same time,5 single mutations decreased T50 by 1.0-3.4℃ were identified.A combination of beneficial mutations gave rise to mutant M6(Y50F/R52H/R56K/V65I/T67A/P143A)with a T50 increase of 13.4℃,indicating that the harmful mutations in 6511 indeed hampered the thermostability optimization.Structural analysis and molecular dynamic simulations showed that the beneficial mutations mainly increased/enhanced hydrogen bonds and Π-Π interactions,and made the local loops more rigid.In summary,this work identified the beneficial mutations in Bgl3-6511,analyzed their structural basis,and obtained a more thermostable mutant M6.These re-sults may be useful for the protein engineering of other enzymes.关键词
β-葡萄糖苷酶/热稳定性/DNA shuffling/有益突变/定点突变/结构分析Key words
β-glucosidase/thermostability/DNA shuffling/beneficial mutations/site-directed mutagenesis/structural analysis引用本文复制引用
许本宏,蒋奕文,罗敬时,杨向鹏,李广,袁珊,刘玉焕,曹立创..β-葡萄糖苷酶Bgl3热稳定性有益突变的鉴定与结构基础分析[J].食品与发酵工业,2025,51(15):1-8,8.基金项目
广州市科技计划项目(2024A04J3804) （2024A04J3804）
国家自然科学基金项目(32000031) （32000031）
广东省重点领域研发计划绿色生物制造重点专项(2022B1111050002) （2022B1111050002）
