食品与发酵工业2026，Vol.52Issue(18)：21-29,9.DOI:10.13995/j.cnki.11-1802/ts.047066
蔗糖异构酶关键氨基酸残基定点突变显著提升其热稳定性与催化性能
Site-directed mutagenesis of key residues in sucrose isomerase for enhanced thermostability and catalytic performance
摘要
Abstract
Sucrose isomerase(SIase)catalyzes the conversion of sucrose to isomaltulose.Natural SIases generally exhibit low thermo-stability and catalytic efficiency.To improve SIase's characteristics,molecular structure analysis and rational design strategies were em-ployed to conduct site-directed mutagenesis of SIase from Pantoea dispersa.Based on prediction of 23 amino acid sites that potentially affect thermostability,combined with multi-ligand docking and substrate/product tunnel analysis,key residues of K1 60 and K302 were identified that may simultaneously improve thermostability and catalytic efficiency.A double mutant SI-MY(K160M/K302Y)was subsequently con-structed through molecular cloning and site-directed mutagenesis.Enzymatic characterizations revealed that the optimal temperature of SI-MY increased from 30 ℃ to 35 ℃,the half-life at 50 ℃ increased to 1.88 folds,the Km decreased from 145.5 mmol/L to 56.4 mmol/L,the Kcat increased from 4.97 s-1 to 7.57 s-1,and the catalytic efficiency(κcat/Km)increased by 2.92 folds.In isomerization of sucrose cata-lyzed by SI-MY at 50 ℃,the yield of isomaltulose increased from 84.41％to 95.63％,representing an increase of 11.22％.The results demonstrated that SIase's thermostability and catalytic efficiency were effectively improved through rational modification of key residues within the substrate/product transport tunnels.The obtained mutant represented a potential promising enzyme candidate for efficient bio-manufacturing of isomaltulose.关键词
蔗糖异构酶/底物通道/产物通道/定向进化/热稳定性/催化效率Key words
sucrose isomerase/substrate tunnel/product tunnel/site-directed mutagenesis/thermostability/catalytic efficiency引用本文复制引用
李爽,靳卓琳,张濛,Suren SINGH,牛丹丹,王正祥..蔗糖异构酶关键氨基酸残基定点突变显著提升其热稳定性与催化性能[J].食品与发酵工业,2026,52(18):21-29,9.基金项目
国家重点研发计划政府间国际科技创新合作重点专项(2025YFE0101200) （2025YFE0101200）
天津市杰出人才计划项目(JC20200309) （JC20200309）
