Loop Engineering and Whole-Cell Immobilization of d -Allulose 3-Epimerase for the Improvement of Catalytic Performance of d -Allulose Production

d-Allulose, a low-calorie, rare monosaccharide with sucrose-like sweetness, has emerged as a promising alternative to traditional sweeteners. However, its industrial-scale production remains hindered by the limited thermostability and operational longevity of the key biocatalysts d-allulose 3-epimerase (DAE). To address this bottleneck, this study engineered a DAE from Clostridium cellulolyticum H10 (CcDAE) by targeting its flexible loop 106–121 for improving thermostability. Through directed evolution, an advantageous mutant, T119K, was identified. Subsequent combinatorial mutagenesis yielded a superior triple mutant, T119K/D281G/C289R, which exhibited a significantly enhanced performance. Furthermore, to bridge the gap between laboratory-scale optimization and industrial application, a whole-cell immobilization strategy was implemented by expressing this mutant in Bacillus subtilis, the robust biocatalyst maintained remarkable activity over 64 reusable cycles. This breakthrough not only provides a highly thermostable DAE mutant but also establishes an efficient and scalable bioprocess for sustainable d-allulose production, offering critical theoretical insights and support for the food industries.