Enhancing Stability\nand Catalytic Activity of d‑Allulose 3‑Epimerase\nthrough Multistrategy Computational\nDesign and Cross-Regional Advantageous Mutations

d-Allulose 3-epimerase (DAEase) derived from Clostridium bolteae has excellent properties in the\ncatalytic production of d-allulose, a rare sugar with unique\nbiological functions. However, the industrial application of C. bolteae DAEase (Cb-DAEase) for d-allulose\nproduction is hindered by its low enzyme activity, poor long-term\nthermostability, and pH tolerance. In this study, we identified potential\nnoncatalytic residues in Cb-DAEase using methods such as proline substitution,\nsurface charge engineering, and surface residue prediction. The effects\nof these residues were experimentally validated, followed by structural\nanalysis, which led to the generation of multisite mutants through\ncross-regional structural combinations. The obtained mutant Cb-R2P-E6P-D137C\nshowed 155.6% of the enzyme activity of the wild type, and the <i>K</i><sub>cat</sub>/<i>K</i><sub>m</sub> increased\nby 1.3-fold, an elevated half-life of 15.7 min, and an elevated <i>T</i><sub>m</sub> value of 1.1 °C. The mutant Cb-R2P-E6P-A83D-D137C\nhad 139.7% of the enzyme activity of the wild type, the <i>K</i><sub>cat</sub>/<i>K</i><sub>m</sub> increased by 1.2-fold,\nwith an elevated half-life of 12.3 min, an elevated <i>T</i><sub>m</sub> value of 0.8 °C, and maintained 68% of the enzyme\nactivity at pH 5.0. The findings outlined a feasible approach for\nthe rational design of multiple preset functions of target enzymes\nsimultaneously.