# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 3 Results and Discussion
### Immobilization Optimization and Stability Assessment of TcDAease
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Enzyme Source (Strain)** & \(k_{cat}\) (s\({}^{-1}\)) & \(K_{m}\) (mM) & \
\begin{tabular}{c} \(k_{cat}\)/\(K_{m}\) \\ (s\({}^{-1}\)mM\({}^{-1}\)) \\ \end{tabular} &
\begin{tabular}{c} **Conversion Rate** \\ **(\%)** \\ \end{tabular} & **Reference** \\ \hline _Arltrobacter_ & 2920 & 738.7 & 3.953 & 27 & [47] \\ _psycholactophilus_ & & & & \\ \hline _Blautia produca_ & 65.03 \(\pm\) 3.56 & 235.7 \(\pm\) 9.938 & 0.28 \(\pm\) 0.014 & 30 & [48] \\ \hline _Thermogemmatispora_ & 96.6 \(\pm\) 0.5 & 260.9 \(\pm\) 7.4 & 0.38 \(\pm\) 0.02 & 30 & This work \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Cont._improved chitosan precipitation and network formation. In contrast, excessively alkaline conditions may result in over-crosslinking, which can impair enzyme conformation or hinder substrate diffusion [57]. Overall, a neutral pH range (6.5-7.0) was found to provide optimal precipitation efficiency and enzymatic activity, potentially due to the favorable protonation state of chitosan for controlled gelation and Schiff base (CH=N) formation [58]. The concentration of chitosan is also one of the key factors affecting performance. Too low a concentration can result in an insufficiently intact matrix, which can weaken the particle strength. Conversely, too high a concentration will result in the formation of a dense gel, which will limit matrix diffusion and thus reduce apparent activity [59]. As shown in Figure 6c,d, the highest initial activity (9.7 \(\pm\) 0.9 U\(\cdot\)g\({}^{-1}\)) was observed at a chitosan concentration of 5 g/L. However, poor operational stability was noted under this condition, with rapid activity decay (k = 0.24 \(\pm\) 0.02 cycle\({}^{-1}\), t\({}_{1/2}\) = 2.9 cycles, R\({}^{2}\) = 0.98). In contrast, increasing the chitosan concentration to 10 g/L significantly enhanced stability (k = 0.01 \(\pm\) 0.003 cycle\({}^{-1}\), t\({}_{1/2}\) = 69.3 cycles, R\({}^{2}\) = 0.91). Under these optimized conditions, 90.3 \(\pm\) 1.5% of the initial activity was retained after seven consecutive cycles. This corresponds to a 24-fold reduction in the rate of inactivation compared to the 5 g/L condition. These improvements align with previous findings suggesting that a critical chitosan concentration (-8 g/L) is required to establish a percolation threshold for network formation on silica-based supports [60]. In summary, under optimized conditions (wet cell loading: 15 g/L; precipitation pH: 6.5-7.0; chitosan concentration: 10 g/L), the immobilized TcDAEase achieved 49 \(\pm\) 1.2% activity recovery and demonstrated favorable mechanical integrity. As shown  
Figure 6: Optimization of TcDAEase immobilization parameters. (**a**) Effect of wet cell loading (0.5-2.5 g/100 mL) on protein loading and activity recovery; (**b**) Immobilized enzyme activity at different precipitation pH values: 5.0–6.0, 6.5–7.0 and 10.0–11.0. (**c**) Initial catalytic activity versus chitosan concentration (2.5–10 g/L); (**d**) Operational stability during seven batch cycles for 5 g/L (**iii**), 10 g/L (**•**), 20 g/L (**a**), 30 g/L (**\(\blacktriangledown\)**), and 40 g/L (**\(\blacktriangledown\)**) chitosan concentrations. in Figure 7, the enzyme prepared via the three-step method exhibited excellent operational stability, with no detectable protein leaching after drying and repeated washing at 60 \({}^{\circ}\)C. These findings confirm the successful formation of a robust immobilization matrix, whose particles after filtration are shown in Figure S2.