Study on Immobilization of Hyperthermophilic D-Allulose 3-Epimerase and Properties of Immobilized Enzyme

D-allulose, a promising low-calorie natural sweetener and a key member of the rare sugar family, exhibits physiological benefits in mitigating multiple diseases. As the C-3 epimer of D-fructose, D-allulose was synthesized through the catalytic action of D-allulose 3-epimerase (DAE). However, industrial-scale sugar epimerization required high-temperature conditions (greater than 65 ℃), and existed DAEs could not meet this requirement. To address this bottleneck, a hyperthermophilic DAE (TI-DAE) was immobilized using diatomite as a solid core, polyethyleneimine (PEI) as a protective shell, and glutaraldehyde (GA) as a cross-linking agent, resulting in the formation of a core-shell-structured immobilized enzyme (TI-DAE@Diatomite-PEI-GA), and its structure and performance were characterized. The result revealed that immobilized enzyme TI-DAE@Diatomite-PEI-GA exhibited better pH and thermal stability than the free enzyme TI-DAE. The half-life of TI-DAE@Diatomite-PEI-GA was greater than 24 h, 11 times higher than free enzyme under 90 ℃. Furthermore, TI-DAE@Diatomite-PEI-GA retained over 70% relative activity after 20 batches of converting 500 g/L D-fructose into D-allulose. The results proved that the enzyme immobilization technology based on diatomite covalent cross-linking could greatly improve the thermostability of DAE, reduce the cost of industrial production of D-allulose, break through a technical bottleneck of current industrial production, and lay a foundation for promoting the industrialization of enzymatic synthesis of D-allulose.