食品与发酵工业2024，Vol.50Issue(24)：1-8,8.DOI:10.13995/j.cnki.11-1802/ts.038976
基于动态互相关性和折叠自由能变分析的谷氨酰胺转氨酶热稳定性改造
Thermostability modification of transglutaminase based on dynamic cross-correlation and folding free energy analysis
摘要
Abstract
Streptomyces mobaraensis transglutaminase(TGase),with its ability to catalyze protein cross-linking,is widely applied in food processing.Previously,a thermostable variant FRAPD-TGm2 of S.mobaraensis TGase was constructed to improve its application per-formance under high temperatures.This study identified a TGase variant with further improved thermostability based on dynamic cross-cor-relation analysis.Firstly,based on molecular dynamics simulation and dynamic cross-correlation analysis,this study identified 48 residues exhibiting dynamic cross-correlation with the flexible region of the FRAPD-TGm2 substrate-binding pocket.Subsequently,virtual saturation mutagenesis on these residues employing Rosetta Cartesian_ddg resulted in 20 variants with a folding free energy change of less than-1 kcal/mol.These mutants were then expressed,purified,and subjected to enzymatic property characterization.Among them,the 60℃half-life and 65 ℃ specific enzyme activity of the variant FRAPD-TGm2-Y34 W reached 100.5 min and 134.2 U/mg,respectively,which were 89.1％and 28.5％higher than that of FRAPD-TGm2.Furthermore,this study observed significant effects on enzyme activity and thermostability in FRAPD-TGm2 due to distal residues His201 and Asn32.The above results indicate that enzyme molecular redesign,based on dynamic cross-correlation and folding free energy analysis,can effectively enhance the thermostability of TGase.关键词
茂原链霉菌/谷氨酰胺转氨酶/热稳定性/动态互相关性/折叠自由能Key words
Streptomyces mobaraensis/transglutaminase/thermostability/dynamic cross-correlation/folding free energy引用本文复制引用
阳鹏辉,邱文轩,叶佳才,堵国成,刘松..基于动态互相关性和折叠自由能变分析的谷氨酰胺转氨酶热稳定性改造[J].食品与发酵工业,2024,50(24):1-8,8.基金项目
国家重点研发计划(2021YFC2101400) （2021YFC2101400）
国家自然科学基金(32071474,31771913) （32071474,31771913）
