# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 3 Results and Discussion
### Immobilization Optimization and Stability Assessment of TcDAease  
To achieve large-scale production of D-allulose, we optimized the immobilization process for TcDAease and used whole-cell immobilization techniques while circumventing the cost of enzyme purification by using the natural permeability of E. coli membranes to small molecule substrates [50; 51]. Drawing on immobilization studies of glucose isomerase [52; 53], we developed a diatomic-chitosan composite carrier that integrates the high mechanical strength of diatomaceous earth with the strong binding capacity of chitosan. The adsorption-crosslinking-embedding process was finely controlled by adjusting pH conditions. Specifically, we used glutaraldehyde pretreatment at pH 3.0 to ensure uniform dispersion of the carrier materials, adjusted the pH to 4.5 to promote adsorption and cross-linking of the cells, and finally adjusted the pH over 5.0; a gradual alkalinization promoted the precipitation of chitosan. Besides, the composite carrier was optimized for compatibility with packed-bed reactor applications. We first focus on the optimization of chitosan. Chitosan is a bio-compatible binder that enhances the mechanical robustness of the matrix and prevents the leaching of enzymes during manipulation. To improve the operational stability and reproducibility of the immobilized enzyme, three key parameters were systematically evaluated: wet cell loading, chitosan precipitation pH, and chitosan concentration. As shown in Figure 6a, protein loading increased linearly with cell concentration in the range of 0.5-1.52 g/100 mL (slope = 10.3 \(\pm\) 0.8 mg/100 mL). Beyond this range, loading efficiency declined sharply (slope = 1.8 \(\pm\) 0.3 mg/100 mL). Breakpoint analysis identified a critical saturation threshold at 1.52 \(\pm\) 0.10 g/100 mL, which is consistent with the surface saturation of the carrier material [54]. In parallel, enzyme activity recovery exhibited first-order decay kinetics (k = 0.45 \(\pm\) 0.05). The half-maximal activity was observed at 1.54 g/100 mL, which was closely aligned with the protein loading saturation point. This strong correlation suggests that excessive cell density hinders the effective utilization of the enzyme by restricting the diffusion of crosslinking agents [55]. Additionally, spatial constraints at high cell densities may induce conformational distortions that impede substrate access and ultimately reduce catalytic efficiency [56]. According to these results, 1.5 g/100 mL was selected as the optimal cell loading, yielding a protein loading of 23 \(\pm\) 0.8 mg/g and approximately 50% activity recovery. This condition offered a balanced trade-off between carrier utilization and enzyme performance. In addition, the pH for chitosan precipitation was identified as a critical factor influencing immobilization efficiency. We embedded them in three pH intervals, acidic (5.0-6.0), neutral (6.5-7.0) and basic (10.0-11.0). Although chitosan's theoretical precipitation pKa is \(\sim\)6.3, partial gelation was observed even at pH 5.0-6.0, which is likely due to localized pH fluctuations during NaOH titration under vigorous stirring. As shown in Figure 6b, immobilized TcDAease prepared at pH 5.0-6.0, 6.5-7.0 and 10.0-11.0 exhibited activities of 1.4 \(\pm\) 0.3, 6.7 \(\pm\) 0.2 and 6.1 \(\pm\) 0.2 U\(\cdot\)g\({}^{-1}\), corresponding to activity recoveries of 19 \(\pm\) 0.5%, 49 \(\pm\) 1.2% and 41 \(\pm\) 1.3%, respectively. These results indicate that neutral to mildly alkaline conditions significantly enhanced catalytic performance, likely due to