## 2 Results and Discussion
### Operational Stability of Selected Biocatalysts  
The assessment of the operational stability of a biocatalyst is required to evaluate its implementation in industrial applications [6]. For the derivative where BTL2 was immobilized at pH 7.0 with 20 mM DTT and then basified (at pH 10.1), the activity and selectivity in sardine oil hydrolysis remained almost unchanged after ten reaction cycles (Figure 7). This was expected considering the high derivative stability observed under harsher inactivating conditions, as discussed in Section 2.2.  
In the case of EE production, the EE14 h yield (%) for the best Gx derivative (obtained when TLL was immobilized in presence of 20 mM AA at pH 7.0) decreased from 71.5 to 68, thus retaining 95% of the initial activity. For the industrial reference Novozyme(r) 435, the EE14 h yield (%) decreased from 75.1% to 70.2%, retaining 93% of the initial activity (Figure 8).  
\begin{table}
\begin{tabular}{c c c c c c} \hline  & **Derivative** & \multicolumn{4}{c}{**Ethyl Ester (EE)**} \\ \hline \multirow{2}{*}{**Enzyme**} & \multirow{2}{*}{**Immobilization Condition**} & \multicolumn{2}{c}{**Yield at 14 h (\%) a**} & \multicolumn{2}{c}{**Yield at 44 h (\%) a**} \\ \cline{3-6}  & & _Non-Dried_ & _Dried_ & _Non-Dried_ & _Dried_ \\ \hline \multirow{3}{*}{**TLL**} & Reference & 11.7 \(\pm\) 1.2 & 51.6 \(\pm\) 2.2 & 30.3 \(\pm\) 1.1 & 69.8 \(\pm\) 1.8 \\  & DTT & 5.3 \(\pm\) 0.3 & 6.3 \(\pm\) 0.5 & 5.7 \(\pm\) 0.3 & 7.4 \(\pm\) 0.3 \\  & AA & 14.3 \(\pm\) 0.8 & 71.5 \(\pm\) 2.8 & 23.2 \(\pm\) 1.8 & 74.8 \(\pm\) 2.8 \\ \hline \multirow{3}{*}{**CAL**} & Reference & 8.0 \(\pm\) 0.1 & 13.0 \(\pm\) 0.9 & 8.5 \(\pm\) 0.4 & 22.0 \(\pm\) 1.7 \\  & DTT & 5.9 \(\pm\) 0.5 & 5.3 \(\pm\) 0.7 & 6.1 \(\pm\) 0.3 & 7.2 \(\pm\) 0.5 \\  & AA & 6.4 \(\pm\) 0.4 & 5.6 \(\pm\) 0.3 & 8.5 \(\pm\) 0.6 & 7.7 \(\pm\) 0.2 \\ \hline \multirow{3}{*}{**BTL2**} & Reference & 0.9 \(\pm\) 0.2 & 8.8 \(\pm\) 0.2 & 2.0 \(\pm\) 0.5 & 16.1 \(\pm\) 0.1 \\  & DTT & 2.1 \(\pm\) 0.1 & 9.5 \(\pm\) 0.7 & 3.1 \(\pm\) 0.8 & 15.0 \(\pm\) 0.8 \\ \cline{1-1}  & AA & 1.9 \(\pm\) 0.2 & 10.3 \(\pm\) 0.6 & 2.8 \(\pm\) 0.1 & 11.2 \(\pm\) 0.5 \\ \hline \multicolumn{3}{c}{**Novozyme® 435**} & \multicolumn{2}{c}{47.2 \(\pm\) 0.2} & 75.1 \(\pm\) 2.9 & 78.0 \(\pm\) 3.5 & 79.4 \(\pm\) 1.9 \\ \multicolumn{3}{c}{(industrial reference [7; 14; 15])} & \multicolumn{2}{c}{47.2 \(\pm\) 0.2} & 75.1 \(\pm\) 2.9 & 78.0 \(\pm\) 3.5 & 79.4 \(\pm\) 1.9 \\ \hline \end{tabular}  
* Yield is defined as weight percentage (%) of ethyl esters (EE) in the oil phase after 14 h or 44 h of a reaction mixture that initially had 3:1 mol EtOH: mol palm olein and 5% (p/p) of biocatalyst at 46 \({}^{\circ}\)C and 1700 rpm.  
\end{table}
Table 5: Ethyl ester production catalyzed by novel and reference biocatalysts.  
Figure 7: Sardine oil hydrolysis activity (\(\blacklozenge\)) and selectivity (\(\blacklozenge\)) after successive reaction cycles. The Biocatalyst assayed was the most selective and was based on aminated BTL2 immobilized on Gx at pH 7.0 with 20 mM DTT and incubated at pH 10.1. Conditions according to Sections 3.4 and 3.6.  
Difficulties in the recovery of the biocatalyst particles were present especially for the agarose derivative given its smaller size when compared with Novozyme(r) 435. Aside from the above, the novel TLL biocatalyst was as stable as the commercial reference under the same EE production conditions. Such a high stability of the EE\({}_{\text{14 h}}\) yield for the novel Gx derivative is probably due to the intense interaction of the enzyme with the support within multipoint immobilizations [20; 22; 40] as expected for the proposed mechanism (Section 2.1).