D-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health.The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market.Some sugar isome...D-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health.The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market.Some sugar isomerases,such as D-lyxose isomerase(D-LIase),can achieve an isomerization reaction between D-mannose and D-fructose.However,the weak thermostability of D-LIase limits its efficient conversion from D-fructose to D-mannose.Nonetheless,few studies are available that have investigated the molecular modification of D-LIase to improve its thermal stability.In this study,computer-aided tools including FireProt,PROSS,and Consensus Finder were employed to jointly design D-LIase mutants with improved thermostability for the first time.Finally,the obtained five-point mutant M5(N21G/E78P/V58Y/C119Y/K170P)showed high thermal stability and cat-alytic activity.The half-life of M5 at 65◦C was 10.22 fold,and the catalytic efficiency towards 600 g/L of D-fructose was 2.6 times to that of the wild type enzyme,respectively.Molecular dynamics simulation and intramolecular forces analysis revealed a thermostability mechanism of highly rigidity conformation,newly formed hydrogen bonds andπ-cation interaction between and within protein domains,and redistributed surface electrostatic charges for the mutant M5.This research provided a promising D-LIase mutant for the industrial production of D-mannose from D-fructose.展开更多
基金supported by the National Natural Science Foundation of China(32201963)Scientific Research Foundation of Hunan Provincial Education Department(22C0137).
文摘D-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health.The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market.Some sugar isomerases,such as D-lyxose isomerase(D-LIase),can achieve an isomerization reaction between D-mannose and D-fructose.However,the weak thermostability of D-LIase limits its efficient conversion from D-fructose to D-mannose.Nonetheless,few studies are available that have investigated the molecular modification of D-LIase to improve its thermal stability.In this study,computer-aided tools including FireProt,PROSS,and Consensus Finder were employed to jointly design D-LIase mutants with improved thermostability for the first time.Finally,the obtained five-point mutant M5(N21G/E78P/V58Y/C119Y/K170P)showed high thermal stability and cat-alytic activity.The half-life of M5 at 65◦C was 10.22 fold,and the catalytic efficiency towards 600 g/L of D-fructose was 2.6 times to that of the wild type enzyme,respectively.Molecular dynamics simulation and intramolecular forces analysis revealed a thermostability mechanism of highly rigidity conformation,newly formed hydrogen bonds andπ-cation interaction between and within protein domains,and redistributed surface electrostatic charges for the mutant M5.This research provided a promising D-LIase mutant for the industrial production of D-mannose from D-fructose.
