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OA

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.