Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems

D-allulose is a rare sugar with promising applications in food and health industries, owing to its low caloric value and multiple health benefits. In this study, we systematically investigated a thermostable D-allulose 3-epimerase (TcDAEase) from Thermogemmatispora carboxidivorans for food-compatible continuous production. The enzyme exhibited remarkable thermostability, with over 70% activity retained at 80 °C, and showed broad pH tolerance across the range of 8.0 to 13.0. Notably, TcDAEase exhibited high catalytic activity toward D-allulose and D-fructose even without the addition of metal ions. Moreover, food-grade Mg2+ was identified as enhancing enzyme activity by 14.3%, thus ensuring compliance with Generally Recognized as Safe (GRAS) standards for food applications. To improve industrial applicability, the enzyme was immobilized using a chitosan-diatomaceous earth (DE) matrix via three-step adsorption–crosslinking–embedding strategy. The immobilized TcDAEase achieved 48.7% ± 2.4% activity recovery and retained 90.3% ± 1.5% activity over seven reaction cycles. Furthermore, continuous production of D-allulose was realized in a packed-bed reactor, operating stably at 60 °C, pH 8.0 and 5 mM Mg2+ for 150 days, producing 756 kg of D-allulose with a conversion yield exceeding 89.7% of the theoretical maximum. Overall, this study provides a feasible strategy for the continuous and efficient production of high-value-added D-allulose in the food industry.