Directional immobilization of D-allulose 3-epimerase using SpyTag/SpyCatcher strategy as a robust biocatalyst for synthesizing D-allulose

D-Allulose, as low-calorie rare sugar, possessed several notable biological activities and was biosynthesized by D-allulose 3-epimerase (DAEase). Here, CcDAE from Clostridium cellulolyticum was successfully immobilization via covalent attachment (RI-CcDAE), and Resin-SpyCatcher/SpyTag-CcDAE modular (DI-CcDAE). Both immobilized CcDAEs exhibited higher thermal and pH stabilities than the free form, and they maintained 80.0 % of relative activity after 7 consecutive cycles and 25 days of storage. Predominantly, DI-CcDAE represented superior catalytic efficiency with a 2.4-fold increase of kcat/Km, compared with RI-CcDAE (0.75 s-1 mM-1 vs 0.31 s-1 mM-1). The RI-CcDAE and DI-CcDAE were then applied in mixed fruit Jiaosu to convert D-fructose into D-allulose, which exhibited the productivity of D-allulose 1.08 g/Lh-1 and 1.57 g/Lh-1, respectively. This research provided a promising directional immobilization strategy for DAEase, and robust biocatalyst for production of functional foodstuff containing D-allulose.