A study of targeted mutation of l-rhamnose isomerase to improve the conversion efficiency of D-allose

D -Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L -Rhamnose isomerase ( L -Rhi) is the earliest enzyme found to catalyze the production of D -allose from D -psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D -allose. In this study, L -Rhi derived from Bacillus subtilis was employed as the research subject, and D -psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure , tertiary structure , and interactions with ligands of the enzyme. The yield of D -allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D -allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively. According to modeling analysis, manganese (Mn 2+ ) had no significant effect on the production of D -psicose from D -psicose by L -Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D -psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D -psicose and facilitating its conversion to D -allose, providing the basis for the production of D -allose.