# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 3 Results and Discussion
### Thermostability, pH Tolerance and Metal Ion Dependence  
The enzymatic properties of TeDAEase were evaluated under the conditions described in Section 2.4. As shown in Figure 4a, TeDAEase exhibited optimal activity at 80 \({}^{\circ}\)C and retained over 70% of its activity within the 60-90 \({}^{\circ}\)C range. Thermal inactivation analysis (Figure 4b) revealed half-lives of 720 min at 60 \({}^{\circ}\)C, 300 min at 70 \({}^{\circ}\)C, and 40 min at 80 \({}^{\circ}\)C, indicating superior thermostability compared to most previously reported DAEases. Specifically, TeDAEase offered a favorable balance between stability and activity (Table 1). The _DaeM_-derived enzyme [16] showed the highest thermal stability (t\({}_{1/2}\) = 9900 min at  
Figure 3: SDS-PAGE analysis of recombinant TcDAEase expression and purification. Lane M: Protein molecular weight marker (14.3–66.4 kDa); Lane 1: _E. coli_ BL21 (DE3) (empty pET-22b vector); Lane 2: _E. coli_ BL21 (DE3) expressing TeDAEase; Lane 3: crude cell extract; Lane 4: purified TeDAEase.  
Figure 2: Multiple sequence alignment of D-allulose 3-epimerases showing conserved residues involved in metal coordination (\(\blacksquare\)) and substrate binding (\(\bullet\)). Strictly conserved amino acids are shown in red highlights. Alignment visualization was performed using ESPript 3.0.  
60 \({}^{\circ}\)C) but low catalytic activity (1.14 U/mg). The enzyme from _Caballeronia fortuita_[33] exhibited high catalytic activity (270 U/mg) but poor thermostability (t\({}_{1/2}\) = 63 min at 60 \({}^{\circ}\)C). TcDAEase showed a moderate activity of 7.9 U/mg and a t\({}_{1/2}\) of 720 min at 60 \({}^{\circ}\)C, making it well-suited for long-term high-temperature applications.  
In terms of pH tolerance, TcDAEase displayed a broad activity profile with an optimum at pH 10.0. Notably, more than 70% of its maximal activity was retained across the pH range of 8.0-13.0, whereas activity declined sharply below pH 5.0 (Figure 4c). Long-term pH stability tests (Figure 4d) showed that the enzyme remained stable after 6 h incubation at 60 \({}^{\circ}\)C between pH 7.0 and 9.0. Although TcDAEase tolerated strongly alkaline conditions, maintaining the reaction environment below pH 9.0 is advisable to avoid non-enzymatic browning, particularly in food-related processes [34]. Therefore, we choose the pH = 8.0 for all subsequent experiments, which provides the highest enzymatic activity and maintain good enzymatic stability.  
The effects of various metal ions on enzymatic activity were assessed by supplementing 1 mM of different divalent cations (Figure 4e). Among them, Ni\({}^{2+}\), Co\({}^{2+}\), and Mg\({}^{2+}\) enhanced catalytic activity by 30.3%, 20.2%, and 14.3%, respectively. While Ni\({}^{2+}\) exhibited the strongest activation effect, its toxicity and regulatory limitations preclude its application in food-grade systems [18]. In contrast, Mg\({}^{2+}\) is FDA-GRAS approved and thus suitable for industrial use. Further investigations on Mg\({}^{2+}\) concentration (Figure 4f) revealed that enzymatic activity increased with Mg\({}^{2+}\) concentration and plateaued beyond 5 mM. And the enzyme activity increased by approximately 2.1-fold when 5 mM Mg\({}^{2+}\) was added, compared to without Mg\({}^{2+}\). Accordingly, 5 mM Mg\({}^{2+}\) was selected as the optimal cofactor concentration for continuous D-allulose production. Taken together, the combination of high thermostability, broad alkaline tolerance, and Mg\({}^{2+}\)-activation distinguishes TcDAEase from most previously characterized DAEases, reinforcing its potential for robust, food-compatible D-allulose production under industrial conditions.  
Figure 4: Effect of temperature, pH, and metal ions on TcDAEase activity. (**a**) Optimal temperature; (**b**) thermal stability at 60, 70, and 80 \({}^{\circ}\)C; (**c**) optimal pH; (**d**) pH stability; (**e**) effect of different metal ions; (**f**) effect of Mg\({}^{2+}\) concentration.