## 2 Results and Discussion
According to the above spectroscopic evidence, since not all the additive molecules attached to the Gx surface are liberated from the support, it is expected that interactions other than catalytic between the enzyme and the generated bifunctional Gx support will be present. Indeed, as also seen in Figure 5, the FTIR-ATR spectra of the derivatives of Gx and TLL obtained in the presence or absence of DTT or AA show unique features especially where protein amide bands appear [5; 46]. This suggests that the specific nature of the interactions between the enzyme and the immobilized additive (e.g., ionic for AA or disulfide exchange for DTT) influences the acquired protein structure after immobilization. Indeed, this may explain the different biocatalytic properties of the resulting lipase derivatives determined during natural oil transformations (Sections 2.3 and 2.4). Figure 4: (**a**) Comparative FTIR-ATR transmittance spectra of Gx support derivatives: plain Gx support without modifications (Gx, blue), pure anthranilic acid (AA, green), and Gx anthranilic acid derivative support (Gx–AA, red); (**b**) Comparative UV-Vis spectra: free AA spectra (brown), plain Gx support (Gx, orange), Gx after treatment with 20 mM AA (green), Gx after fuchsine staining (grey), Gx after treatment with AA 20 mM and then stained with fuchsine (blue). Enzymes immobilized following the suggested mechanism (Figure 3) would produce highly stabilized derivatives given the multipunctual nature of the enzyme-support interactions; additionally, considering that after the additive action there would be unreacted aldehyde groups on both the protein surface and the support (as suggested by the UV-Vis results after fuchsine staining, Figure 4b), the number of covalent interactions could be further augmented by increasing the pH as in other Gx immobilizations [20]. The validity of these statements and the usefulness of the obtained derivatives were assessed in experiments, the results of which will be analyzed below. Effect of the Initial and Final pH on Derivative Recovered Activity and Stability: Production of a Highly Stabilized Biocatalyst  
Given their higher recovered hydrolytic activity and immobilization yield (Table 2) and the availability of previous reports of BTL2 immobilized on Gx under different conditions as references [22; 27], the aminated BTL2 derivatives (immobilized at pH 7.0 or 8.0 with DTT as the optimal additive) were selected to study the effect of increasing the incubation pH as a post-immobilization treatment (basification) on derivative stability. Their stabilities were also compared against the classic reference BTL2-covalent CNBr and other Gx derivatives previously obtained through a similar procedure intended to increase enzyme-support bonding [22; 31]. In general, high recovered activities were observed for the novel biocatalysts (Table 3): 72-76% and 62-65% for derivatives immobilized at pH 7.0 or pH 8.0, respectively, which are values close to that of the reference Gx derivative (64%). Thus, the initial immobilization pH impacts more markedly on the recovered activity than the following basification, as previously seen in other systems [22; 31]. Figure 5: FTIR-ATR transmittance spectra within the distinctive region. Gx support without modifications (blue) and Gx biocatalysts resulting from the immobilization of TLL: at conventional pH 10 (black), in presence of 20 mM AA at pH 7.0 (green), or in presence of 20 mM DTT at pH 7.0 (red). The comparatively higher recovered activities for derivatives resulting from immobilizations at pH 7.0 may reflect that the orientation of the enzyme on the support corresponds to surfaces far from the catalytic domain (Figure 2), namely, those comprising N-termini (Section 2.1).