## 3 Results
### Properties of Est2L and Est4L  
Est2L showed the highest activity at 60 \({}^{\circ}\)C and maintained a meaningful level (i.e., 76.2%) of its activity even at 80 \({}^{\circ}\)C (Figure 4a). Est4L showed the highest activity at 40-50 \({}^{\circ}\)C, and its activity did not noticeably increase between 30 \({}^{\circ}\)C and 60 \({}^{\circ}\)C, i.e., it remained at 86.9-93.6% of the maximum activity, as opposed to Est2L, whose activity increased significantly by 2.58 times at 60 \({}^{\circ}\)C compared to 30 \({}^{\circ}\)C (Figure 4b). Both Est2L and Est4L showed optimal activities at pH 10, indicating that they are alkaline esterases (Figure 5). In terms of thermal stability, Est2L had a half-life of 22.9 min at 50 \({}^{\circ}\)C; however, its half-life rapidly decreased to 4.3 min at 60 \({}^{\circ}\)C (Figure 6a). Est4L showed half-lives of 13.6 and 3.5 min at 40 \({}^{\circ}\)C and 50 \({}^{\circ}\)C, respectively (Figure 6b). Therefore, it was concluded that Est 2L was 6.54 times more stable than Est4L at 50 \({}^{\circ}\)C based on their respective half-lives. Regarding substrate specificities for p-NP esters, Est2L showed the highest activity with C2, followed by C4 (73.5%) and C6 (46.2%) (Figure 7a). It did not show any significant activity with p-NP ester substrates with more than 8 carbon chains, and showed the typical substrate specificity of acetylesterase. However, Est4L showed the highest reactivity with C10, followed by C2 (95.2%) and C4 (94.6%) to a similar extent (Figure 7b). Moreover, Est4L showed more than 50% of relative activities for C8 (73.6%), C12 (55.6%), and C6 (50.6%). The activities for C14 and C16 significantly decreased to 13.3% and 2.8%, respectively. On the other hand, Est2L showed BTCI and ATCI hydrolyzing activities of 13.3% and 25.4% respectively, which were higher than those of Est4L (Figure 7a,b). In kinetic studies analyzed by the Lineweaver-Burk plot, Est2L had a K\({}_{\text{m}}\) value of 0.67 mM for C4, and Est4L had a higher affinity for the substrate at 0.10 mM (Figure 8). The V\({}_{\text{max}}\) values of Est2L and Est4L were 0.072 and 0.036 U/mg, respectively. Figure 6: Thermal stabilities of Est2L (**a**) and Est4L (**b**). Enzyme activities were measured by a continuous method after preincubation of enzyme at each designated time. Figure 7: Substrate specificities of Est2L (**a**) and Est4L (**b**). Enzyme activities with C2 to C16 were measured using 1 mM \(p\)-nitrophenyl esters in 50 mM Tris-HCl (pH 8.0), and those of acetylcholinesterasease (AChE) and butyrylcholinesteraseerase (BChE) were measured using 0.5 mM ATCI and BTCI respectively, with 0.5 mM DTNB in 50 mM sodium phosphate (pH 7.5) buffer. Regarding stability with organic solvents, Est2L was stable at 5% and 30% of methanol; however, it was deactivated to 76% and 15.7% at 5% and 30% isopropanol respectively, and to 69.3% and 6.7% at 5% and 30% acetonitrile respectively (Figure (a)a). Est4L was also stable up to 30% methanol and was activated to 128.6%, and was activated to 132.4% at 5% isopropanol but inactivated to 6.9% at 30% isopropanol. Est4L was inactivated to 67% and 7.5% at 5% and 30% acetonitrile (Figure (b)b). By adding detergents, Est2L and Est4L were significantly inactivated to less than 10% at 1% SDS, but neither were inactivated at 1% Triton X-100, instead being activated to 124% and 119.8% respectively (Figure (a)a,b). In addition, phenylmethylsulfonyl fluoride (PMSF) inhibited Est2L by 73.0% at 1 mM, but had little effect on Est4L, which maintained 88.9% of its activity. However, the inhibition of Est4L by PMSF increased at a higher concentration (10 mM) and became nearly complete with preincubation of Est4L and PMSF for 30 min. Regarding the effects of metal ions on enzyme activities, Est2L was activated to 139.8%, 130.4%, 118.6%, and 117.3% by 5 mM Fe\({}^{2+}\), Mn\({}^{2+}\), Mg\({}^{2+}\), and Ca\({}^{2+}\) respectively, but inhibited to 52.4% by 5 mM Cu\({}^{2+}\) (Figure (a)a).