Molecular modification and properties study of Ribose-5-phosphate isomerase originating from Curtobacterium flaccumfaciens ZXL1

The enzymes used for D-allose production generally failed to meet the requirements of industrial production of D-allose in terms of enzyme activity and catalytic efficiency. In this study, 13 single mutants of CfRpiB (Ribose-5-phosphate isomerase from Curtobacterium flaccumfaciens) were constructed by combining site-specific mutations and ring-open residue mutations with rational design. It was found that the enzyme activities of all 13 single mutants were positively changed relative to the wild type. Among them, the single mutant with the highest increase in enzyme activity was C72M, which increased its enzyme activity by 30%. On this basis, three iterative mutant strains were constructed, among which C72M/S105H increased the yield of D-allose by 2.62 times. Especially, enzymatic property measurements showed that its enzyme activity increased by 50%. Molecular docking of the C72M/S105H mutant with D-allulose revealed that the increased enzymatic activity of the mutant C72M/S105H could be attributed to the smaller substrate binding site and tighter binding to the substrate. This study further reveals the relationship between mutation sites and enzymatic properties, demonstrating the feasibility of improving the efficiency of preparing rare sugars through enzyme molecular modification. Synopsis Site-directed mutagenesis of binding sites and open-open loop residues and explored the reasons for the enhanced activity of the mutated enzyme.