## 4 Discussion
Furthermore, OsmC was composed of approximately 160 residues, much shorter than CzcO with approximately 400 residues. Specific activities of Est2L (0.043 U/mg) and Est4L (0.037 U/mg) in crude extracts were lower than that of Lip3K, which had the lowest specific activity (0.25 U/mg) found in our results previously reported [29]. Thus, it was difficult to separate homogeneous proteins with two or more column steps. Although several columns were used for purification by combinational sets, recovery and purification were not successful. Therefore, we investigated the enzymatic properties with partially purified enzymes. Considering the low specific activities, actual natural substrates of Est2L and Est4L should be investigated in further research. Native Est2L was found to have dimeric forms similar to most other family IV enzymes (Table 3), although some were reported to have monomeric to tetrameric forms. EstE7 existed in various forms, including monomer, dimer, and aggregated forms with reversible oligomerization, of which the dimer form showed the highest activity [47]. Cest-2923 had dimer and tetramer forms at neutral and acidic pH, respectively [48], and REst1 had monomer and trimer forms [49]. Little information is available about the native forms of family V enzymes (Table 3). Est4L was tetramer in form, whereas Est16 [50] and OpeH [51] were monomer in form, and LacH [52] was dimer in form. Optimum temperatures of family IV enzymes were extremely diverse, ranging from 10.5 \({}^{\circ}\)C for E69 [53] to 95 \({}^{\circ}\)C for EstE1 [54], and that of Est2L (60 \({}^{\circ}\)C) was higher than that of Est3K [29] and was located in the upper range (Table 3). The range of optimum temperatures of family V enzymes was relatively narrow from 35 \({}^{\circ}\)C for H8 [55] to 78 \({}^{\circ}\)C for FClip1 [56], and that of Est4L was in the center region (Table 3). Est2L was found to be an intracellular enzyme without a signal peptide similar to most other HSL and family IV lipolytic enzymes [41], and Est4L was also an intracellular enzyme. Est2L showed a substrate preference for short pNP-esters, similar to many other family IV lipolytic enzymes, including Cest-2923 [48], Est22 [57], and EstA1 [58] (Table 3). Est4L preferred relatively long pNP-esters (especially C10), similar to FCLip1 [56], whereas most other family V enzymes prefer shorter pNP-esters (Table 3). Interestingly, Est2L hydrolyzed BTCI, which was used as a substrate for BChE, suggesting that Est2L has broad specificity to various types of esters, whereas Est4L efficiently hydrolyzed up to C12 without BChE activity, indicating that Est4L has broad substrate specificity to longer pNP esters. These properties make them highly attractive for industrial applications, especially for ester prodrugs, providing a basic strategy for masking functionalities of polar alcohol and carboxylic acid and improving permeabilities [4]. Est2L and Est4L were stable in 30% methanol but inactivated in isopropanol or acetonitrile, similar to others reported, except for PestE, which was stable in all three solvents [59], EstA1, which was stable in methanol [58], and FCIip1, which was stable in isopropanol [56]. Little information about enantioselectivity was available. The selectivity of Est2L was weak and Est4L preferred S-form, while EST2 [60], PestE [61], and LacH [52] preferred R-form. Est2L was inhibited by Cu\({}^{2+}\), similar to Est3K and Est22 [29; 57], and Est4L was inhibited by Cu\({}^{2+}\) and Zn\({}^{2+}\), similar to H8 and FCIip1 [55; 56]. On the other hand, the interactions of CzCO domain of Est2L with metal ions might be different from those of YiiP, a bacterial cation diffusion facilitator (CDF), which maintains cellular homeostasis of metal ions by transporting Zn\({}^{2+}\), Fe\({}^{2+}\), and Cd\({}^{2+}\), and contains three binding sites and tetrahedral motifs [62]. In PMSF inhibition, Est2L was more sensitive than Est4L; inhibition of Est4L by PMSF increased with higher PMSF concentration, similar to the results of other family V esterases [45; 49].