## 3 Results
### Properties of Est2L and Est4L
On the other hand, Est4L was not activated by the metal ions tested, but was greatly inhibited to 7.5% and 29.8% by 5 mM Cu\({}^{2+}\) and Zn\({}^{2+}\), respectively, and moderately inhibited to 63.8% by 5 mM Fe\({}^{2+}\) (Figure (b)b). No noticeable  
Figure 8: Michaelis–Menten and Lineweaver–Burk plots of Est2L (**a**) and Est4L (**b**) for C4. Experiments were performed at five different substrate concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0 mM of C4. Figure 9: Effects of organic solvents, detergents, and PMSF on enzyme activities of Est2L (**a**) and Est4L (**b**). P.I., preincubated for 30 min. effect was observed from other ions. Regarding the effect of Co\({}^{2+}\), little effect on Est2L was observed below 10 mM Co\({}^{2+}\), but inhibition was observed at 20 mM Co\({}^{2+}\). In addition, Zn\({}^{2+}\) showed little effect on Est2L activity, though it showed more significant effects at a 20 mM concentration (Figure 10c). It is concluded that Fe\({}^{2+}\), Mn\({}^{2+}\), Mg\({}^{2+}\), and Ca\({}^{2+}\) activated Est2L, and Cu\({}^{2+}\) inhibited Est2L and Est4L; however, Zn\({}^{2+}\) inhibited only Est4L but not Est2L, suggesting that metal ions in group 1 did not significantly affect Est2L and Est4L activities, but those in groups 11 or 12 inhibited the two enzymes. Ethylenediaminetetraacetic acid (EDTA) showed insignificant effects on Est2L and Est4L, which retained relative activities of 96.6% and 88.6% respectively. To observe the effects of EDTA and metal ion combinations, EDTA was preincubated and then metal ions were added, or vice versa, for Est2L and Est4L. In the experiments, little difference was observed as a result of change of preincubation order, except that inhibition by EDTA -> Cu\({}^{2+}\) was greater than that by Cu\({}^{2+}\) -> EDTA and inhibition by the combination of EDTA and Co\({}^{2+}\) was greater than those of isolated EDTA or Co\({}^{2+}\) in Est2L (Figure 11). It is possible that the bindings of Cu\({}^{2+}\) or Co\({}^{2+}\) and EDTA to Est2L were more complex than to Est4L. For the lipid hydrolysis activities, Est2L and Est4L hydrolyzed glyceryl tributyrate to 69.0% and 52.8% respectively, followed by trioctanoate to 77.9% and 71.9% respectively (Figure 12). It was confirmed that the hydrolyzing activities of fish oil, olive oil, and glyceryl trioleate by Est2L and Est4L were remarkably low. When based on glyceryl tributyrate as a substrate, Est4L showed a 25.1% higher hydrolyzing activity than Est2L. Figure 11: EDTA and metal ion effects on Est2L (**a**), and Est4L (**b**). The enzyme was preincubated with 1 mM EDTA or metal ions for 30 min, then 1 mM metal ions or EDTA were added respectively, and finally enzyme activities were measured. Figure 10: Effects of cations on the activities of Est2L (**a**) and Est4L (**b**) and of Co\({}^{2+}\) and Zn\({}^{2+}\) ions on Est2L activity (**c**). Control is the enzyme activity in the absence of metal ions. Regarding enantioselectivity, Est2L showed no preference between R- and S-forms; however, Est4L showed a preference toward S-form at a low degree of 10.6%, i.e., 89.6% and 79.3% of the residual substrate amounts in R- and S-form, respectively, under the condition (Figure 13).