Tailored magnetic silica-immobilized D-allulose 3-epimerase with enhanced stability and recyclability for efficient D-allulose production.

D-allulose, a low-calorie functional sweetener, is produced by the enzymatic conversion of d-fructose via D-allulose 3-epimerase (DAE) and holds significant market potential, particularly for individuals with obesity and diabetes. However, the limited reusability and stability of DAE have restricted its industrial application. In this study, we developed functional superparamagnetic supports by integrating diatomite, a biomineralized silica-based material, with cobalt ferrite nanoparticles through a green chemical co-precipitation method. The covalent attachment of DAE enzymes to these magnetic supports resulted in enzyme-metal hybrid catalysts (DAE@mDE-NH₂) that exhibited enhanced stability and facilitated recovery and reuse via magnetic separation. These catalysts showed superior stability in acidic conditions and high temperatures, with a 24-fold increase in half-life at 60 °C compared to free DAE. They also exhibited remarkable durability, retaining 95.36 % of their activity after six months of storage at 4 °C and 70.08 % activity after 12 consecutive cycles. Utilizing this robust and recyclable biocatalyst, 147.7 g/L of D-allulose was obtained from 500 g/L of d-fructose. This study presents a sustainable strategy for advancing the production of high-value functional sweeteners like D-allulose while providing new insights into enzyme immobilization for biocatalytic processes.