Computer-Aided Molecular Modifications for Enhanced Activity and Thermal Stability of <scp>d</scp>-Allulose 3-Epimerase

D-Allulose 3-epimerase (DAE) can convert D-fructose into D-allulose, but its lower activity and poor thermal stability restrict its application in industrial production. This study utilized a semi-rational design and specific site mutations to enhance the enzyme activity and thermal stability of a DAE from <i>Clostridium scindens</i> ATCC 35704 (<i>Csc</i>-DAE) at the same time. Notably, the activities of six mutants (V22I, A25V, Q46I, C212N, G165A, and G267D) and the orderly superimposed double-point mutants were increased by 1.08-1.31 and 1.28-1.64 times that of the wild type (553.7 U/mg), respectively. In addition, A25V/C212N showed good thermal stability, and its optimum temperature (<i>T</i>_opt) was 65 °C and half-life (<i>t</i>_1/2) was 3.997 h at 60 °C, which increased by 5 °C and 5.76 times, respectively. The conversion ratio reached 30.62 and 31.22% at 65 and 70 °C, respectively, and the structural changes of the mutants were analyzed using molecular docking. These studies provide a beneficial mutation for industrial applications.