Enhancing Stability and Catalytic Activity of <scp>d</scp>-Allulose 3-Epimerase through Multistrategy Computational Design and Cross-Regional Advantageous Mutations

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