## 2 Results and Discussion
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{2}{c}{**Gx-Derivative**} & \multicolumn{2}{c}{**Immobilization Parameters**} \\ \hline
**Enzyme** & **Additive (20 mM)** & **Residual Control Activity (\%) a** & **Recovered Activity (\%) b** \\ \hline \multirow{3}{*}{TLL} & - & 96.3 \(\pm\) 3.1 & - \\  & DTT & 5.1 \(\pm\) 0.3 & 10.5 \(\pm\) 0.4 \\  & AA & 55.2 \(\pm\) 1.5 & 58.9 \(\pm\) 1.7 \\ \hline \multirow{3}{*}{CAL} & - & 94.7 \(\pm\) 2.7 & - \\  & DTT & 64.4 \(\pm\) 1.8 & 39.7 \(\pm\) 1.1 \\  & AA & 83.2 \(\pm\) 2.3 & 50.2 \(\pm\) 2.0 \\ \hline \multirow{3}{*}{BTL2} & - & 95.0 \(\pm\) 2.0 & - \\  & DTT & 91.1 \(\pm\) 1.6 & 76.7 \(\pm\) 3.3 \\ \cline{1-1}  & AA & 95.4 \(\pm\) 3.0 & 72.1 \(\pm\) 2.8 \\ \hline \hline \end{tabular}  
* Residual control activity is defined as the percentage difference between the activity of the immobilization control (enzyme solution mixed with non-activated agarose) after 24 h and that of the control at the initial immobilization time (5 IU/g support) at 25 \({}^{\circ}\)C and pH 7.0 [27]. b Recovered activity is defined as the percentage ratio of the specific derivative hydrolytic \(p\)-NPB activity (IU/mg of immobilized protein) to that of the soluble enzyme (IU/mg free enzyme) under standard conditions [36]. Values are the mean of three different experiments where the standard deviation was never \(>\)5% of the mean value.  
\end{table} 
Table 2: Immobilization parameters of aminated lipases on Gx at neutral pH and 25 \({}^{\circ}\)C.  
Figure 1: Immobilization progress of aminated lipases on glyoxyl–agarose support at 25 \({}^{\circ}\)C and pH 7.0: TLL without additives (\(\triangle\)) and with AA 20 mM (\(\blacktriangle\)); BTL2 without additives (\(\Circinus\)) and (\(\blacktriangledown\)) with AA 20 mM; CAL without additives (\(\Diamond\)) and with DTT 20 mM (\(\blacklozenge\)). The lower rates (higher t\({}_{50\%}\)) and yields of immobilization for CAL when compared with those of TLL and BTL2 would be a consequence of a lower number of CAL potentially reactive residues as evidenced in Figure 2.  
The proposed enzyme immobilization mechanism on Gx mediated by AA at pH 7.0 is depicted in Figure 3: initially, AA molecules react reversibly with the aldehyde groups of the support through its nucleophilic amino group establishing a bifunctional and dynamic support surface with carboxylate and remaining aldehyde groups. Then, by proton transfer through its _orto_-carboxylate group [34], the immobilized additive (upper AA molecule in Figure 3) acts as a catalyst by promoting the nucleophilic attack of the most reactive enzyme amino group--probably the N-terminus at neutral pH [31]--which results in the formation of an imine enzyme-support bond and the subsequent liberation of AA, ending the catalytic cycle; an akin mechanism for DTT as additive could also take place with one sulfhydryl group acting as an anchoring point and the other as a proton donor like the _orto_-carboxylate group of AA. However, the described catalytic effect would be one component of the whole action of the additive during the enzyme immobilization since, according to the spectroscopic evidence, the interaction of the support with the immobilization solution containing 20 mM AA at pH 7.0 implies modifications on the Gx surface that are not fully reversible as expected for a mere catalytic action: even after exhaustive washings of the resulting support with an immobilization solution lacking the additive (Sections 3.2.1 and 3.7), both the UV-Vis and FTIR-ATR spectra evidence characteristic energy transitions differing from those of the plain Gx support and resembling those of AA.