食品工业科技2026，Vol.47Issue(4)：59-68,10.DOI:10.13386/j.issn1002-0306.2025010032
半理性设计提升葡萄糖氧化酶活性与热稳定性
Enhancing the Activity and Thermostability of Glucose Oxidase Through Semi-rational Design
摘要
Abstract
This study aimed to simultaneously enhance the enzymatic activity and thermostability of glucose oxidase(AnGOD)derived from Aspergillus niger through a semi-rational design strategy.Mutation sites were designed using the FireProt online tool and consensus sequences analysis,and the optimal mutant was screened via site-directed mutagenesis and a two-stage combinatorial mutation approach.Molecular dynamics simulations were then conducted to elucidate the mechanism of performance enhancement.Results showed that from the initial 83 designed sites,12 single-point advantageous mutants were selected,and the optimal mutant M5(T276F/T34V/Q90R/S53F/S74T)was obtained through two-stage combinatorial mutagenesis.Compared to the wild-type(WT),M5 exhibited a 4.2-fold increase in specific activity,with a kcat value of 755.0 s-1 and a kcat/Km value of 34.52 L·mmol-1·s-1,representing 3.67-and 2.48-fold enhancements over WT,respectively.At 65℃,the half-life of M5 was 30 min,representing a 30-fold increase compared to WT(1 min).Molecular dynamics simulations revealed that the average RMSD of M5 and the RMSF in non-catalytic regions were reduced by 12.9%and 12.4%,respectively,compared to the wild-type(WT).Additionally,the number of hydrogen bonds between M5 and the substrate/coenzyme increased by 10.3%and 8.8%,respectively.Notably,the S74T mutation introduced a novel hydrogen bond network,while the Q90R mutation introduced a positive charge,enhancing surface electrostatic complementarity.These synergistic effects collectively improved both the thermostability and catalytic activity of AnGOD.Through semi-rational design and combinatorial mutagenesis,this study significantly alleviated the stability-activity trade-off in AnGOD enzyme engineering,laying a foundation for the application of GOD.关键词
葡萄糖氧化酶/活性与稳定性权衡/半理性设计/一致性分析/分子动力学模拟Key words
glucose oxidase/activity-stability trade-off/semi-rational design/consensus sequences analysis/molecular dynamics simulation分类
生物科学引用本文复制引用
李荷月,郭煜辉,裴雯雯,王倩,李娇,赵伟,秦慧民,孙媛霞,陈朋..半理性设计提升葡萄糖氧化酶活性与热稳定性[J].食品工业科技,2026,47(4):59-68,10.基金项目
国家重点研发计划(2022YFC2104900) （2022YFC2104900）
山东省重点研发计划(重大科技创新工程)项目(2023CXGC010714) （重大科技创新工程）
合成生物学海河实验室重大攻关类项目(22HHSWSS00003). （22HHSWSS00003）
