Mining and identifying a D-mannose isomerase with high fructose isomerization activity and its expression in Bacillus...

D-mannose is a functional monosaccharide with numerous positive physiological effects and holds significant commercial potential in the pharmaceutical, nutraceutical, and food industries. In this study, a hypothetical AGE family epimerase/isomerase from Stenotrophomonas maltophilia was identified and characterized as a D-mannose isomerase, named Stma-MIase, capable of efficiently converting d-fructose into D-mannose. Stma-MIase exhibited optimal activity at pH 8.5 and 60 °C, with a denaturation temperature (T_m) of 61.2 °C. The enzyme displayed a half-life of 11.1 h and 0.996 h at 50 and 55 °C, respectively. The recombinant Stma-MIase demonstrated the highest substrate affinity (K_m) and catalytic efficiency (k_cat/K_m) among other reported MIases, which was further supported by molecular dynamics simulations based on binding free energy and distance distribution. Additionally, the expression of Stma-MIase in Bacillus subtilis yielded a fermentation volume activity of 76.1 U/mL through shake-flask fermentation. Whole-cell catalysis using 500 g/L of d-fructose as substrate resulted in a conversion rate of 26.0 %. This study not only uncovers a promising Stma-MIase with high fructose isomerization efficiency but also emphasizes its potential for industrial-scale D-mannose production.