## 4 Discussion  
In this study, two esterase genes, _est2L_ and _est4L_, were isolated from a metagenomic library; they encoded a family IV Est2L and a family V Est4L esterase, respectively. Two documented genes were similar or identical to the respective genes annotated from the genome sequences; however, the amino acid sequences of Est2L and Est4L were very dissimilar compared to those of the more than 20 family IV enzymes and less than 10 family V enzymes characterized to date, respectively. The highest values were 9.8% to 27.9% and 10.33% to 21.28% (Table 3) respectively. In addition, Est2L was a unique fusion type of protein, containing two domains of a CzcO and an acetylesterase domain (a member of family IV). Due to these points, Est2L was examined in this study, along with Est4L (a member of family V), although their activities and yields were low. Bacterial family IV lipolytic enzymes show a strikingly high similarity to the mammalian hormone-sensitive lipase (HSL) [1]. Molecular masses of mammalian HSLs range from 84 to 130 kDa [38] and are larger than bacterial HSLs (bHSL), which range from 31.2 to 41.2 kDa. Human HSL (hHSL) is composed of 786 amino acid residues. In general, HSL has two domains: the N-terminal binding domain (\(\sim\)315 residues) and the C-terminal catalytic domain [38,39]; however, bHSL has a cap domain comprising 50-100 residues at the N-terminus and the C-terminal catalytic domain [39]. The N-terminal binding domain mediates protein-protein interactions, specifically with adipocyte lipid binding protein [40], whereas the cap domain plays an important role in enzyme stability and specificity [41]. Interestingly, Est2L was much larger in molecular mass, i.e., 839 residues and 92.5 kDa, than other bHSL enzymes characterized to date. It was also larger than hHSL. The size difference resulted from the large N-terminal domain (10-419 residues, CzcO superfamily)  
Figure 12: Lipid hydrolysis activities of Est2L (**a**) and Est4L (**b**), using a pH shift assay. Enzyme activities were measured at 1% substrates in 20 mM Tris-HCl (pH 8.0) by adding 0.1% phenol red. Figure 13: Enantioselectivity of Est2L (**a**) and Est4L (**b**). Enzyme activities were measured at 1% (R)-methyl-3-hydroxy-2-methyl-propionate and (S)-methyl-3-hydroxy-methyl-propionate. of Est2L. The role of CzcO has been poorly understood, with the exception of its role in enhancing the activity of CzcD, which effluxes divalent cations such as Zn\({}^{2+}\), Co\({}^{2+}\), and Cd\({}^{2+}\)[30; 31], and reduces the accumulation of these cations in _Ralstonia metallidurans_ CH34 by an unspecified mechanism [42]. Curiously, the CzcO domain was fused to the family IV bHSL domain of Est2L, notwithstanding that the two domains would not be closely related. To test the effects of divalent cations, changes in Est2L were analyzed at several concentrations of Co\({}^{2+}\) and Zn\({}^{2+}\); however, significant effects were not observed for Co\({}^{2+}\) and Zn\({}^{2+}\) except an inhibition at a high concentration of Co\({}^{2+}\), although Est2L contains the domain CzcD, which is related to efflux of Zn\({}^{2+}\), Co\({}^{2+}\), and Cd\({}^{2+}\). It could be suggested that neither Co\({}^{2+}\) or Zn\({}^{2+}\) ions bind to the CzcO domain in order to stabilize the catalytic domain of Est2L, acting unlike an N-terminal binding domain in hHSL or a cap domain in bHSL. In addition, CzcD is different from the N-terminal transmembrane domain of LipA from _Pseudomonas protegens_ Pf-5, which is short (22 amino acid residues) and contributes efficient folding, probably in order to act as an intramolecular chaperone [43]. On the other hand, an esterase EstO with OsmC (osmotically induced protein C) was identified in a marine bacterium _Pseudoalteromonas arctica_[44], and was characterized as part of a new family of carboxylesterase [45]. Recently, an esterase SLC6 with OsmC from sea sediment metagenome was characterized [46]. Interestingly, OsmC was located at the C-terminus of the proteins, unlike CzcO of Est2L.