# Thermostable D-Allulose 3-Epimerase for Long-Term Food-Compatible Continuous Production Systems
## 3 Results and Discussion
### Substrate Specificity and Kinetic Parameters  
The substrate specificity of TcDAEase was evaluated using four ketoexoses: D-allulose, D-fructose, D-tagatose, and D-sorbose. As shown in Figure 5, TcDAEase exhibited the highest specific activity toward D-allulose (15.9 \(\pm\) 0.2 U-mg\({}^{-1}\)), followed by D-fructose, D-tagatose, and D-sorbose. This substrate preference supports the hypothesis of a conserved epimerization mechanism among DAEases and is consistent with previous reports on enzymes from _Arthbroacter globiformis_ M30 [13], _Clostridium scindens_ [42], _Ruminococcus_ \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Strain of Enzyme** & \multicolumn{2}{c}{**Optimal**} & \multirow{2}{*}{**Optimal pH**} & \multirow{2}{*}{**Metal Ion**} & \multirow{2}{*}{**Half-Life**} & \multirow{2}{*}{**Catalytic Activity** (U/mg)**} & \multirow{2}{*}{**Reference**} \\
**Source** & & & & & & \\ \hline _Pseudomonas_ sp. & 60 & 7.0–9.0 & NR & NR & NR & [35] \\ \hline _Pseudomonas cichorii_ & 60 & 7.5 & NR & NR & NR & [36] \\ \hline _Agrobacteriumium_ & \multirow{2}{*}{50} & \multirow{2}{*}{8.0} & \multirow{2}{*}{Mn\({}^{2+}\)} & \multirow{2}{*}{63.5 min/50 \({}^{\circ}\)C} & \multirow{2}{*}{8.89} & \multirow{2}{*}{[37]} \\ _tumeidens_ & & & & & & \\ \hline _Sinorhizobium_ sp. & 40 & 8.5 & \multirow{2}{*}{Mn\({}^{2+}\)} & \multirow{2}{*}{251 min/45 \({}^{\circ}\)C,} & \multirow{2}{*}{NR} & \multirow{2}{*}{[38]} \\  & & & & & & \\  & & & & & & \\ \hline _Rhodobacter sphaeroides_ & 40 & 9.0 & Mn\({}^{2+}\) & NR & NR & [39] \\ \hline _Clostridium_ & \multirow{2}{*}{55} & \multirow{2}{*}{8.0} & \multirow{2}{*}{Co\({}^{2+}\)} & \multirow{2}{*}{408 min/60 \({}^{\circ}\)C} & NR & [40] \\ _cellulolyticum_ & & & & & & \\ \hline _Ruminococcus_ sp. & 60 & 7.5–8.0 & Mn\({}^{2+}\) & 96 min/60 \({}^{\circ}\)C & 8.95 & [15] \\ \hline _Clostridium_ sp. & 65 & 8.0 & Co\({}^{2+}\) & 15 min/60 \({}^{\circ}\)C & NR & [41] \\ \hline _Clostridium scindens_ & 60 & 7.5 & Mn\({}^{2+}\) & 108 min/50 \({}^{\circ}\)C & NR & [42] \\ \hline _Desmospora_ sp. & 60 & 7.5 & Co\({}^{2+}\) & NR & NR & [43] \\ \hline _Clostridium botheae_ & 55 & 7.0 & Co\({}^{2+}\) & 156 min/55 \({}^{\circ}\)C & NR & [14] \\ \hline _Dora_ sp. & 70 & 6.0 & Co\({}^{2+}\) & NR & 803 & [44] \\ \hline _Treponema primitia_ & 70 & 8.0 & Co\({}^{2+}\) & 30 min/60 \({}^{\circ}\)C & NR & [31] \\ \hline _Flavorinfractor plautiti_ & 65 & 7.0 & Co\({}^{2+}\) & 40 min/65 \({}^{\circ}\)C & 20 & [32] \\ \hline _Arthbroacter globiformis_ & 70 & 7.0–8.0 & Mg\({}^{2+}\) & NR & 23.6 & [13] \\ \hline _Agrobacteriumium_ sp. & 55–60 & 7.5–8.0 & Co\({}^{2+}\) & 267 min/55 \({}^{\circ}\)C, 28.2 min/60 \({}^{\circ}\)C, 3.8 min/65 \({}^{\circ}\)C & 90.5 & [45] \\ \hline _Corynebacterium_ & \multirow{2}{*}{55} & \multirow{2}{*}{8.0} & \multirow{2}{*}{Mn\({}^{2+}\)} & \multirow{2}{*}{140 min/60 \({}^{\circ}\)C} & \multirow{2}{*}{22.7} & \multirow{2}{*}{[46]} \\ _glutamicum_ & & & & & & \\ \hline _DaeM_ & 80 & 7.0 & Co\({}^{2+}\) & 9900 min/60 \({}^{\circ}\)C, 3240 min/70 \({}^{\circ}\)C, 49 min/80 \({}^{\circ}\)C & 1.14 \(\pm\) 0.048 & [16] \\ \hline _Caballerotia fortitia_ & \multirow{2}{*}{65} & \multirow{2}{*}{7.5} & \multirow{2}{*}{Co\({}^{2+}\)} & \multirow{2}{*}{427.8 min/50 \({}^{\circ}\)C,} 307.8 min/55 \({}^{\circ}\)C, 307.8 min/55 \({}^{\circ}\)C, 63 min/60 \({}^{\circ}\)C} & \multirow{2}{*}{[270]} \\ _Arthbroacter_ & \multirow{2}{*}{70} & \multirow{2}{*}{8.5} & \multirow{2}{*}{Mg\({}^{2+}\)} & \multirow{2}{*}{128.4 min/70 \({}^{\circ}\)C} & \multirow{2}{*}{14.3} & \multirow{2}{*}{[47]} \\ _psychicotapthilus_ & & & & & & \\ \hline _Blautia produca_ & 55 & 8.0 & Mn\({}^{2+}\) & 180 min/55 \({}^{\circ}\)C & 1.76 \(\pm\) 0.099 & [48] \\ \hline _Thermogemmatispora_ & \multirow{2}{*}{80} & \multirow{2}{*}{10.0} & \multirow{2}{*}{Ni\({}^{2+}\)} & \multirow{2}{*}{720 min/60 \({}^{\circ}\)C,} 300 min/70 \({}^{\circ}\)C, 40 min/80 \({}^{\circ}\)C} & \multirow{2}{*}{7.9 \(\pm\) 0.3} & \multirow{2}{*}{This work} \\ _carboxidiorurans_ & & & & & & \\ \hline \hline \end{tabular}
\end{table}