## 3 Results
### Isolation of Est2L and Est4L  
Est2L and Est4L were not bound to HiTrap-Q under the condition of 20 mM Tris/HCI, pH 8.0, and a small number of enzymes were eluted at the gradient (Figure 2). In the control experiment using _E. coli_ DH5\(\alpha\) (pUC19), endogenous esterase activity was negligible compared to the two positive clones (Figure 2). After HiTrap-Q chromatography, the purification folds of Est2L and Est4L in the unbound fractions were 2.56 and 1.70 and had yields of 22.6% and 12.5%, respectively (Table 1). Using the pools, the two enzymes were separated further using Sephacryl S-200 HR for the purpose of purification and estimation of the molecular masses of intact enzymes. Enzyme activities in the extracts were low, and unfortunately, activities drastically decreased during the dialysis and column development stages. Thus, Est2L activity was too low to be detected, and the purification fold of Est4L decreased considerably to 0.73 (Table 1). As an alternative, Est2L was separated using Sephacryl S-200 HR as the first column, and the purification fold was 5.36 (Table 1). CHT-II, HIC, HiTrap-SP, High-S, and MMC chromatographies were not effective in the recovery of Est2L or Est4L.  
In SDS-PAGE analysis with 11.5% polyacrylamide gels, protein bands in the HiTrap-Q and Sephacryl S-200 HR pools were not homogeneous; however, predicted bands corresponding to the molecular masses of Est2L and Est4L were observed at \(\sim\)100 and 31 kDa, respectively (Figure 3). Because homogeneous and active enzymes were not obtained, these fractions were used for further study.  
The molecular masses of intact Est2L and Est4L enzymes were determined to be \(183.2\pm 23.6\) and 125.4 \(\pm\) 8.65 kDa, respectively, according to the elution volumes of Sephacryl S-200 HR chromatographies (Table 2). These results suggest that Est2L and Est4L exist in a dimer and a tetramer form respectively, by comparing their respective molecular masses of 92.5 and 30.1 kDa.  
Figure 1: A phylogenetic tree of Est2L and Est4L. The tree is drawn to scale, and branch lengths are shown in the same units as those of the evolutionary distances used to infer the phylogenetic tree, using 12 amino acid sequences. Evolutionary analyses were conducted in MEGA X using the neighbor-joining method.  
Figure 3: SDS-PAGE of Est2L and Est4L. M, size markers; C, crude extract; Q, HiTrap-Q pool; S, Sephacryl S-200 HR pool. Arrows represent the predicted protein bands corresponding to the molecular masses of Est2L and Est4L.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Enzyme** & **Preparation** & **Specific Activity (U/mg)** &
\begin{tabular}{c} **Purification** \\ **(Fold)** \\ \end{tabular} & **Yield (\%)** \\ \hline \multirow{3}{*}{Est2L} & Crude extract & 0.043 & 1.00 & 100 \\  & HiTrap-Q & 0.11 & 2.56 & 22.6 \\ \cline{2-5}  & Crude extract & 0.041 & 1.00 & 100 \\  & Sephacryl S-200 & 0.22 & 5.36 & 9.45 \\ \hline \multirow{3}{*}{Est4L} & Crude extract & 0.037 & 1.00 & 100 \\  & HiTrap-Q & 0.063 & 1.70 & 12.5 \\ \cline{1-1}  & Sephacryl S-200 & 0.027 & 0.73 & 2.25 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Specific activities and yields of Est2L and Est4L during separation.  
Figure 2: HiTrap-Q chromatograms of crude extracts of _E. coli_ DH5\(\alpha\) strains harboring a plasmid pUC19 as a control and recombinant plasmids SKE2 and SKE4 as sources for esterases Est2L and Est4L, respectively. The different volume of each fraction was appropriately measured for the esterase activity. Activity was calculated as dA/min, then divided by the volume of the fraction, and finally expressed as dA/min/mL on the Y-axis. The chromatogram of UV absorbance (blue line) at 280 nm (A.U.) of Est2L was added along with conductivity (red line) (mS/cm). The UV chromatograms of Est4L and pUC19 samples were similar to that of Est2L. Protein concentrations of the highest active fractions of Est2L, Est4L, and pUC19 samples were 1.8, 1.4, and 1.6 mg/mL, respectively.