## 2 Results and Discussion
As seen in Figure 4a (FTIR-ATR spectra) the intensity ratio of the bands between 1650-1700 cm\({}^{-1}\) (left arrow) to the bands between 3300-3400 cm\({}^{-1}\) (O-H or N-H stretch bands) follow the order AA > Gx-AA > Gx; this could be the result of the presence of additional C=O bonds--and thus a higher probability of the respective asymmetric bond stretches [40]--from the carboxylic groups of the AA molecules that remain immobilized on the Gx support surface. However, a key distinguishing element for Gx-AA, in regard to the plain Gx support spectra, is the band between 1525-1575 cm\({}^{-1}\) (right arrow) attributable to the C=C-N stretch of the aryl-imine keto form [41] of the additive linked to the support. Figure 2: Surfaces of (left) CAL (PDB 5a71), (middle) BTL2 (PDB 2w22), and (right) TLL (PDB 1 dte) centered at the (blue) N-terminus. Other surrounding residues which are potentially reactive at neutral pH with Gx are (green) lysyl \(\varepsilon\)-NH\({}_{2}\) groups and those amino groups added upon chemical animation (purple-blue) of glutamyl and aspartyl residues. In all cases, the active center is on an enzyme surface projection different from the one shown here. Notice the lower number of reactive residues for CAL. Representations made with Pymol version 1.3. Figure 4: _Cont._  
Figure 3: Proposed one-pot mechanism of the generation of a bifunctional Gx support and enzyme immobilization. (**Left**) Bifunctional surface formed after AA reaction with the Gx support; the immobilized additive molecules could interact with the enzyme through covalent nucleophilic catalysis [34; 35] (i) and/or through cationic exchange (ii) triggering enzyme immobilization. (**Right**) The increased proximity between the immobilized enzyme reactive groups and the support promotes additional covalent bonds (iii) constituting a multipoint attachment immobilization. Representations made with Pymol version 1.3. Regarding the UV-Vis spectra (Figure 4b), bands around 300-400 nm due to \(\pi\)\(\rightarrow\)\(\pi^{\bullet}\) benzenoid transitions [42] are present for pure AA, Gx-AA and even for Gx-AA after treatment with fuchsine, the latter with an additional band between 450-650 nm in the visible region due to the \(\pi\)\(\rightarrow\)\(\pi^{\bullet}\) transitions of the quinoid fuchsine ring [43]. As discussed for the respective FTIR-ATR spectra, this strongly suggests not only that AA is immobilized on Gx but also that there are aldehyde groups able to react with the dye as free groups (Figure 3) or after the displacement of immobilized AA molecules by fuchsine; the latter removes some, but not all, AA molecules since the UV-Vis spectra of the resulting support (Gx-AA-FCS, Figure 4b) still show an intense band between 300-400 nm. This suggests an intense interaction between AA and the support that is not easily broken by the reacting amino groups of the dye which mimic those of the protein in the experiment. In Figure 5 is shown the IR region that presents the more marked differences between the Gx support and its different enzymatic derivatives. The latter present a band between 1500-1625 cm\({}^{-1}\) that is absent in Gx. Since these bands coincide with the typical protein Amide II band (right arrow), it is very likely that they account for the immobilized enzyme. Even when it is not possible to clearly distinguish other remaining protein Amide bands because of overlapping with those attributable to H-O-H, O-H, C-O-C, and C=O bonds in the agarose matrix (bound water, alcohol and glycosidic groups, etc.) and the support surface (aldehyde) [44; 45], it is very likely that the contribution of the Amide I band is responsible for the fact that in all TLL and Gx spectra derivatives, the relative size of the band near 1666 cm\({}^{-1}\) is always higher than that of the band at 1385 cm\({}^{-1}\).