## 3 Results and Discussion
### Biochemical Characterization of reFAEs  
The effects of temperature on the CGA hydrolysis activity of purified reFAEs and WTC FAE were shown in Figure 4A. The hydrolysis activity of the three enzymes increased with the rise in temperature within a certain range, but the enzyme activity was inhibited after each optimum temperature. The reFAE1 and reFAE2 reached their maximum activity at 60 \({}^{\circ}\)C and 50 \({}^{\circ}\)C, respectively. This was in accordance with the reported optimum reaction temperature for FAE [14]. By comparing these three enzymes, it can be found that optimal temperature of reFAE2 for CGA hydrolysis had been lowered by about 10 \({}^{\circ}\)C, indicating lower energy consumption and higher acceptability for industrial application.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Esterases** & **Total Protein (mg)** & **Total Activity (U)** & **Specific Activity (U/g)** \\ \hline reFAE1 & 21.19 \(\pm\) 0.03 \({}^{\text{b}}\) & 5.2238 \(\pm\) 0.03 \({}^{\text{a}}\) & 246.37 \(\pm\) 0.03 \({}^{\text{c}}\) \\ reFAE2 & 24.36 \(\pm\) 0.08 \({}^{\text{b}}\) & 8.3055 \(\pm\) 0.02 \({}^{\text{a}}\) & 340.95 \(\pm\) 0.04 \({}^{\text{c}}\) \\ \hline \hline \end{tabular} Different letters (a, b, c) in the same column indicate significant differences at \(p\) < 0.05.  
\end{table}
Table 1: Results of purification of recombinases.  
Figure 4: **(A)** Effect of temperature on the CGA hydrolysis activity of recombinant proteins and WTC FAE; **(B)** effect of pH on the CGA hydrolysis activity of recombinant proteins and WTC C FAE. For different pH conditions, different buffer solutions are used: sodium citrate buffer for pH 3–5, phosphate buffer for pH 6–8, and glycine-NaOH buffer for pH 8–10. **(C)** Effect of metal ions on the activity of recombinant proteins.  
When different pH values were applied to the reFAEs, the optimum pH of the purified reFAEs was equal to that from WTC at 7.0 (Figure 4B). When the pH was between 3.0 and 10.0, their enzyme activity was stable, indicating that the range for application of reFAEs was wide.  
Metal ions can chelate with the local functional groups of the enzyme protein, affecting its three-dimensional structure, and thereby changing the catalytic efficiency and stability of the enzyme. It was found that EDTA treatment had no effect on the activity of the WTC FAE and reFAEs (Figure 4C), and thus, it could be expected that there was no participation of divalent ions in the active site of the reFAEs [25]. The 10 metal ions used in the assay had different effects on the reFAE1, while Mn\({}^{2+}\) and Ca\({}^{2+}\) had a certain degree of activation effect on reFAE2, whose enzyme activity levels increased by about 8% and 12%, respectively. The monovalent metal ions had an insignificant effect on the enzyme activity of WTC FAE, while the activity was significantly inhibited by the divalent ions Zn\({}^{2+}\), Cu\({}^{2+}\) and Fe\({}^{2+}\). Among them, the activity of reFAE1 and reFAE2 decreased by 43% and 38%, respectively, under the action of Cu\({}^{2+}\). The trivalent metal ions Al\({}^{3+}\) and Fe\({}^{3+}\) had a strongly detrimental influence on enzyme activity, and the enzyme activities of the recombinases were almost completely lost after Fe\({}^{3+}\) treatment. The above findings indicated that the reFAEs could hydrolyze CGA without the participation of specific metal ions. When applied in CGA hydrolysis, trivalent metal ions should be taken away for better performance of the recombinant proteins.