Combinatorial Lid-Domain Engineering and Computational Design Enable Simultaneous Enhancement of Activity and Thermostability of D-Allulose 3-Epimerase.

d-Allulose is a promising low-calorie rare sweetener; however, its industrial production is limited by the insufficient catalytic activity and thermostability of d-allulose 3-epimerases (DAEases). In this study, a combinatorial engineering strategy integrating lid-domain modulation and computational design was developed to improve enzyme performance. Lid engineering identified a key mutation (N120D) associated with enhanced catalytic turnover, while computational screening identified beneficial mutations (T69S, D71E, and S128A). The best mutant, T69S/D71E/N120D/S128A, exhibited a 1.9-fold increase in specific activity, a 2.2-fold improvement in catalytic efficiency, and a 34.1% longer half-life at 60 °C. It produced 152 g/L d-allulose from 500 g/L d-fructose within 3 h, achieving a 30.4% conversion yield under mildly acidic and high-temperature conditions. Mechanistic analyses suggested that improved performance resulted from the combinatorial effects of enhanced structural rigidity and altered lid-domain dynamics, consistent with improved catalytic turnover while maintaining thermostability. This work provides an efficient biocatalyst and a generalizable protein engineering strategy.