# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 3 Results and Discussion
### Biochemical Properties of the Immobilized TcDAEase  
The biochemical properties of immobilized TcDAEase were evaluated under optimized conditions. As shown in Figure 8a, the immobilized enzyme retained over 70% of its relative activity across the temperature range of 60-90 \({}^{\circ}\)C, with peak activity (over 90%) between 70 and 90 \({}^{\circ}\)C. Compared to the free enzyme which exhibited rapid activity loss above 80 \({}^{\circ}\)C, the immobilized form displayed enhanced thermal tolerance. Figure 8b illustrates that TcDAEase also retained high activity under strongly alkaline conditions, with a pH optimum above 10.0. However, from an industrial perspective, extremely high temperatures and pH values pose challenges such as increased energy consumption and the risk of non-enzymatic browning reactions (e.g., Maillard reaction), which can compromise product quality [34]. Combining the biochemical properties of immobilized TcDAEase derived from our experiments and the challenges of real food production, 60 \({}^{\circ}\)C and pH 8.0 were chosen as the optimal operating conditions. Under these conditions, a balance between catalytic performance, stability and process feasibility can be achieved.  
To assess operational stability, reusability tests were conducted under these conditions (60 \({}^{\circ}\)C, pH 8.0, 5 mM Mg\({}^{2+}\)). As shown in Figure 9, immobilized TcDAEase retained \(90.3\%\pm 1.5\%\) of its initial activity after seven consecutive cycles--outperforming commonly used agarose- and alginate-based systems, which typically retain only 40-70% activity under similar conditions [61]. This exceptional stability is likely attributed to the synergistic effects of the composite matrix. The glutaraldehyde-crosslinked chitosan network restricts conformational mobility and protects against thermal denaturation, while the porous diatomaceous earth framework provides a large surface area and abundant binding sites, mitigating enzyme leaching and aggregation [62]. Although structural analyses such as SEM or BET were not performed, the physical properties of the composite carrier were consistent with those of food-grade diatomite materials reported in previous literature [55]. Furthermore, the hydrophilic amino and hydroxyl groups of chitosan may create a favorable microenvironment that shields the enzyme from interfacial stresses during packed-bed operation [63].  
Figure 7: Schematic illustration of the three-step adsorption–crosslinking–embedding process for immobilizing TcDAEase onto chitosan– diatomaceous earth composite.  
Together, these results confirm that the chitosan-diatomaceous earth matrix imparts excellent thermal and operational stability to TcDAEase, supporting its application in continuous, food-compatible, and cost-effective D-allulose biomanufacturing.  
Figure 8: Biochemical characterization of immobilized TcDAEase. (**a**) Temperature-activity profile measured in 50 mM Na\({}_{2}\)HPO\({}_{4}\)-NaH\({}_{2}\)PO\({}_{4}\) buffer (pH 8.0) containing 1 mM Mg\({}^{2+}\). (**b**) pH-activity profile assessed at 60 \({}^{\circ}\)C using various buffer systems (Na\({}_{2}\)HPO\({}_{4}\)-citrate buffer (pH 4.0-5.0), Na\({}_{2}\)HPO\({}_{4}\)-NaH\({}_{2}\)PO\({}_{4}\) buffer (pH 6.0-8.0), glycine-NaOH buffer (pH 9.0–10.0)).  
Figure 9: Reusability of immobilized TcDAEase: The reaction occurred by introducing 1 g of immobilized TcDAEase into a 20 mL fructose solution comprising 50 g/L D-fructose, 1 mM Mg\({}^{2+}\), maintained at 60 \({}^{\circ}\)C and pH 8.0. Following each reaction, the immobilized TcDAEase was washed with deionized water and transferred into fresh fructose solution for successive reaction batches. After each batch, residual catalytic activity (\(\blacksquare\)) and substrate conversion (\(\blacktriangle\)) were analyzed.