Engineering the thermostability of d-lyxose isomerase from Caldanaerobius polysaccharolyticus via multiple computer-aided rational design for efficient synthesis of d-mannose

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 degrees 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 and pi-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.