## 2 Results and Discussion
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Derivative Production Conditions** & \multicolumn{2}{c}{**Imobilization Parameters**} & \multicolumn{2}{c}{**Stability Parameters \({}^{\epsilon}\)**} & \multicolumn{2}{c}{**Stabilization Factor \({}^{\dagger}\)**} \\ \hline
**Immobilization Step** & **Incubation** & **Yield (\%) \({}^{\ast}\)** & **Recovered** & **Half-Life at** & **Half-Life in 80\%** & **At 70 \({}^{\circ}\)C** & **In 80\%** \\
**(pH)** & **Step (pH)** & **Activity (\%) \({}^{\text{b}}\)** & 70 \({}^{\circ}\)C (h)** & **Dioxane (h)** & **Dioxane (h)** & **Dioxane** \\ \hline
7.0 & 7.0 & 90 & 76 & 2.1 & 10.2 & 42 & 167 \\
7.0 & 8.0 & 90 & 74 & 50.0 & 130 & 1000 & 2131 \\
7.0 & 10.1 & 90 & 72 & 54.5 & 140 & 1090 & 2295 \\
8.0 & 8.0 & 96 & 65 & 3.7 & 8.1 & 74 & 133 \\
8.0 & 10.1 & 99 & 62 & 53.7 & 195 & 1074 & 3197 \\ Reference Gx & 10.1 & 96 & 64 & 24.1 & 74.2 & 482 & 1216 \\ derivative (pH 9.0) & - & 99 & 78 & 0.050 & 0.061 & 1 & 1 \\ \hline \hline \end{tabular}  
* [FOOTNOTE:*]Footnote *: footnotetext: In immobilization yield was calculated as 100% (1 \(-\) P\({}_{\text{SI}}\)\(\times\) P\({}_{\text{CS}}\)\({}^{-1}\)), where P\({}_{\text{SI}}\) and P\({}_{\text{CS}}\) indicate the total protein content after 24 h in the supernatants that were in contact at neutral pH and 25 \({}^{\circ}\)C before the measures with Gx\(-\)agarose and with non-activated agarose, respectively. \({}^{\text{b}}\) Recovered activity is defined as the percentage ratio of the specific derivative hydrolytic \(p\)-NPB activity (II/mg of immobilized protein) to that of the soluble enzyme (IU/mg free enzyme) under standard conditions [36].\({}^{\text{c}}\) Half-life times at pH 7.0 at 70 \({}^{\circ}\)C or in 80% dioxane at 30 \({}^{\circ}\)C, according to the two-phase deactivation model [47]. \({}^{\text{d}}\) Stabilization factors were calculated as the ratio of t\({}_{1/2}\) of a given BTL2 derivative and the t\({}_{1/2}\) of the respective control CNBr derivative [22; 48].  
\end{table} 
Table 3: BTL2 immobilization on Gx in presence of 20 mM DTT (except for the references) and derivative properties. It is important to note that the proposed strategy, namely, combining the solid-phase chemical amination, the presence of the additive during the immobilization at neutral or 8.0 pH, and a subsequent basification at pH 10, produces derivatives with an overall inactivation behavior that is better under the same inactivation conditions than that previously reported for BTL2 in most cases: the higher values of E\({}_{2}\)/E and, in some cases, lower values of k\({}_{2}\) (inactivation in dioxane) make their t\({}_{1/2}\) exceed those of the reference conventional Gx derivatives (up to 2.6-fold, Table 3) and around 6.5-fold higher than the t\({}_{1/2}\) in dioxane for the most stable native BTL2 derivative obtained in the presence of DTT [31]. Therefore, it is very likely that these higher stabilizations are the result not only of aminated BTL2-support stabilizing interactions through enzyme surfaces prone to trigger inactivation by denaturants (e.g., areas comprising native Ser334 or Gln40 [22; 36]), but also as the result of the positive effects of DTT on BTL2 activity [18].