## 3 Results
### Effects of Various Metal Ions, Organic Solvents and Detergents on EstDR4  
Four metal ions (Li\({}^{+}\), Na\({}^{+}\), K\({}^{+}\), and Mg\({}^{2+}\)) showed no or negligible effects on EstDR4 activity at concentrations of 1 mM and 5 mM in our assays (\(p\) > 0.05) (Figure 5a). EstDR4 activity was substantially enhanced by 1 mM Cs\({}^{+}\), Ca\({}^{2+}\), and Sr\({}^{2+}\), and 5 mM Cs\({}^{+}\). However, EstDR4 activity was significantly inhibited by 1 mM and 5 mM Co\({}^{2+}\), Cu\({}^{2+}\), and Zn\({}^{2+}\), and 5 mM Ca\({}^{2+}\), Sr\({}^{2+}\), and Mn\({}^{2+}\) (\(p\) < 0.05). Among these, 1 mM Cu\({}^{2+}\) had the greatest inhibitory effect on esterase activity; almost all enzyme activity was lost at this concentration. Interestingly, EstDR4 activity was significantly enhanced by 1 mM Sr\({}^{2+}\), increasing to 123.27% of the initial activity (\(p\) < 0.05), whereas 5 mM Sr\({}^{2+}\) significantly inhibited the activity of EstDR4.  
Figure 4: Effect of temperature and pH on enzyme activity. (**a**) Effect of temperature on esterase activity using pNPC8 as a substrate from 0–70 \({}^{\circ}\)C. (**b**) Temperature stability of EstDR4 at various temperatures (10 \({}^{\circ}\)C, 20 \({}^{\circ}\)C, 30 \({}^{\circ}\)C, 40 \({}^{\circ}\)C and 50 \({}^{\circ}\)C) for 1 to 6 h with esterase activity assayed every hour at 30 \({}^{\circ}\)C and pH 8. (**c**) Effect of pH on enzymatic activity, measured from pH 5–11. (**d**) Stability of EstDR4 at different pH values (pH 5–11) for 1 h at 30 \({}^{\circ}\)C followed by esterase activity assayed at 30 \({}^{\circ}\)C and pH 8. All-results are shown in relative activity, the maximum activity measured was taken as 100%.  
The tolerance of EstDR4 toward most organic solvents was generally low (Figure 5c); the enzyme was almost inactivated in 25% (\(v/v\)) ethanol and acetone, and had weak activity in methanol, isopropanol, 1-butanal, isoamyl alcohol, chloroform and methylbenzene (residual activity 7%-27%). However, DMSO and n-hexane only slightly diminished the activity to approximately 99.59% and 75.59% of the initial activity, respectively.  
As for detergents, EstDR4 was significantly activated by 1% of Tween 80 and Triton X-100 exhibiting 135.47% and 132.22% of original activity, respectively (\(p<0.05\)). However, EstDR4 activity was inhibited by SDS, in which it exhibited less than 10% residual activity compared with the original level. Additionally, 1% Tween 20 diminished the activity to approximately 71.74% of the original level (Figure 5b).  
Figure 5: Effect of metal ions, detergents and organic solvents on esterase activity of EstDR4. (**a**) Effect of metal ions (1 and 5 mM) on EstDR4 activity: Li\({}^{+}\), Na\({}^{+}\), K\({}^{+}\), Cs\({}^{+}\), Mg\({}^{2+}\), Ca\({}^{2+}\), Sr\({}^{2+}\), Mn\({}^{2+}\), Co\({}^{2+}\), Cu\({}^{2+}\) and Zn\({}^{2+}\). (**b**) Effect of detergents on enzyme activity: SDS, Tween 20, Tween 80 and Triton X-100. (**c**) Effect of organic solvents (25% \(v/v\)) on EstDR4 activity: dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, isopropanol, 1-butanol, isoamyl alcohol, chloroform, methylbenzene and n-hexane. The relative activity of the control without metal ions, detergents and organic solvents was regarded as 100%. The different letters between each condition indicate significant differences (\(p<0.05\)).