biostudies-literatureWu HScientific Research Foundation of Hunan Provincial Education DepartmentNational Natural Science Foundation of China323-330https://www.ebi.ac.uk/biostudies/studies/S-EPMC10165151biostudies-literatureUnknown8(2)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 catalytic 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 and π-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.Synthetic and systems biotechnologyEngineering the thermostability of d-lyxose isomerase from <i>Caldanaerobius polysaccharolyticus</i> via multiple computer-aided rational design for efficient synthesis of d-mannose.PMC101651513220196322C0137Zhang WDing YPu MWen LYi MMu WCheng YWu HWu XfalseEngineering the thermostability of d-lyxose isomerase from <i>Caldanaerobius polysaccharolyticus</i> 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 catalytic 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 and π-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.2023-01-01T00:00:00Z2023 Jun2026-06-20T03:15:20.814Z2025-04-05T10:55:26.257ZS-EPMC101651513716860610.1016/j.synbio.2023.04.003