农业生物技术学报2026，Vol.34Issue(7)：1530-1539,10.DOI:10.3969/j.issn.1674-7968.2026.07.015
近活性中心loop区非保守位点突变提高内切葡聚糖酶催化活性和热稳定性
Mutagenesis of Non-conservative Sites in Loop Region Near Active Center Improves Catalytic Activity and Thermostability of Endoglucanase
摘要
Abstract
Cellulases are key catalysts for the efficient utilization of cellulose resources,but their insufficient catalytic efficiency and thermostability have seriously restricted their widespread application.In this study,endoglucanase EG-20SJ from the glycoside hydrolase family 5(GH5),derived from Bacillus velezensis,was used as the research object.First,non-conservative sites in the loop region near the active center were screened through homology modeling and conservation analysis.Then,combined with alanine scanning and site-saturation mutagenesis,mutants were constructed,and their enzymatic properties were analyzed.Results showed that among the saturation mutants at the Asp99 and Ser264 sites,the single mutants D99R,S264R,and the double mutant D99R/S264R exhibited significantly improved activity.Specifically,the double mutant had an enzyme activity of 544.2 U/mg,which was 2.51 times that of the wild-type(217.1 U/mg).Regarding enzymatic properties,the optimal reaction temperature of the double mutant increased by 10℃(reaching 60℃),and its residual activity after incubation at 70℃for 1 h reached 61.7%.Kinetic analysis revealed a 44.0%reduction in michaelis constant(Km)and a 1.2-fold higher catalytic constant(kcat)/Km,indicating significant improvements in substrate affinity and catalytic efficiency.Molecular docking results confirmed that Arg99 and Arg264 in the double mutant enhanced substrate binding through additional hydrogen bonds and stabilized the conformation of the loop region.This study revealed the regulatory mechanism of non-conservative loop sites on enzyme function,providing a theoretical basis for the molecular modification and broad application of GH5 family enzymes.关键词
内切葡聚糖酶/loop区/定点饱和突变/催化活性/热稳定性Key words
Endoglucanase/Loop region/Site-saturation mutagenesis/Catalytic activity/Thermostability分类
农业科技引用本文复制引用
张超,贾贺雪,王婷婷,王倩,耿明瑜,宗锦楠,孙金旭..近活性中心loop区非保守位点突变提高内切葡聚糖酶催化活性和热稳定性[J].农业生物技术学报,2026,34(7):1530-1539,10.基金项目
河北省大学生创新创业训练计划(S2024101010030) （S2024101010030）
河北省高等学校科学技术研究项目(BJ2025153 （）
CXZX2025036) （）
