## 2 Results and Discussion  
Immobilization of Enzymes at Neutral pH on Gx: Suggested Mechanism of Gx Modification and Enzyme Immobilization with Additives  
According to Table 1, in absence of additives or a previous lipase animation, the immobilization yields at pH 7 and 25 \({}^{\circ}\)C were below 40% even after 24 h; this is in concordance with previous results using DTT [31]. The selected immobilization additives present different effects on the immobilization yields (Table 1): MA was the less effective (28.0-44.3%) as determined in other imine-bond reactions [34], while DTT and AA allowed the highest immobilization yields (69.2-99.7%). DTT has been previously applied as an immobilization additive for other non-aminated enzymes [31] and functions as a reference in this work, while AA has the advantage of a relatively low toxicity and price among the related compounds assayed [37]. Therefore, these additives were used to assess other immobilization parameters. As seen in Table 2, the activity of soluble BTL2 (residual control activity) remains above 90% regardless of the additive, whereas DTT was deleterious for TLL and CAL. The latter would rely on the contrasting effects of DTT on each enzyme: it preserves key Cys residues that would otherwise be involved in BTL2 deactivation [18] and can break structurally relevant disulfide bridges present in TLL or CAL [38; 39]. This would also explain the fact that recovered activities (the activity expressed by the enzyme once immobilized [36]) were relatively low for CAL and TLL but high for BTL2 when using DTT as additive (Table 2). Hence, AA was selected as the optimal immobilization additive for TLL and CAL, while DTT was selected for BTL2. \begin{table}
\begin{tabular}{c c c} \hline
**Enzyme** & **Additive (20 mM)** & **Yield at 24 h (\%) a** \\ \hline \multirow{4}{*}{TLL} & - & 39.1 \(\pm\) 2.2 \\  & DTT & 99.7 \(\pm\) 3.6 \\  & AA & 96.8 \(\pm\) 2.7 \\  & MA & 44.3 \(\pm\) 1.1 \\  & AN & 95.6 \(\pm\) 3.0 \\ \hline \multirow{4}{*}{CAL} & - & 23.9 \(\pm\) 1.1 \\  & DTT & 88.5 \(\pm\) 2.5 \\  & AA & 83.5 \(\pm\) 2.3 \\  & MA & 32.0 \(\pm\) 1.0 \\  & AN & 90.3 \(\pm\) 3.1 \\ \hline \multirow{4}{*}{BTL2} & - & 26.1 \(\pm\) 0.9 \\  & DTT & 93.0 \(\pm\) 4.1 \\ \cline{1-1}  & AA & 69.2 \(\pm\) 2.4 \\ \cline{1-1}  & MA & 28.0 \(\pm\) 0.4 \\ \cline{1-1}  & AN & 72.0 \(\pm\) 2.3 \\ \hline \end{tabular}
\end{table} 
Table 1: Immobilization yield of aminated lipases in presence of different additives at neutral pH and 25 \({}^{\circ}\)C.  
On the other hand, the time taken to reach 50% of the 24 h immobilization yield (t50%) can be used as a measure of the immobilization rate: at pH 7.0, t50% for aminated TLL, BTL2, and CAL using additives were higher (1 h, 3 h, and 7 h, respectively, Figure 1) than those obtained under conventional immobilization conditions (0.1 h, 0.2 h, and 0.5 h, respectively, data not shown), namely, at pH 9.0 without additives [27; 28; 29]. This reflects a decreased but sufficient availability of reactive amino groups against the Gx support at neutral pH (e.g., N-terminus and surrounding amino groups, Figure 2) when compared with that at pH 9.0, where groups such as those introduced after chemical amination with EDA are more reactive.