## 2 Results
### Use of the GGP Biocatalyst in the Decolorization of Indigo Carmine  
Figure 3 shows that with the direct exposure of 0.05 mM IC to 0.55 mM hydrogen peroxide at 25 \({}^{\circ}\)C and pH 5, the decolorization of around 28% of the IC sample was achieved after 2 h. A concentration of 0.55 mM hydrogen peroxide was used to have enough hydrogen peroxide to fully decolor the used concentration of IC, as using only 0.1 mm, the reaction stopped before reaching a significant decolorization. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{\(\mathbf{pH}\)} & \multicolumn{2}{c}{\(\mathbf{k_{i}\) (min\({}^{-1}\))}} \\ \cline{2-3}  & **GGP** & **GGP-CLEAS** \\ \hline
1,4-dioxane & 7.72 \(\pm\) 0.41 \(\times\) 10\({}^{-1}\) & 5.05 \(\pm\) 0.43 \(\times\) 10\({}^{-1}\) \\ DMSO & 4.88 \(\pm\) 0.32 \(\times\) 10\({}^{-2}\) & 4.68 \(\pm\) 0.24 \(\times\) 10\({}^{-2}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Inactivation constant by organic solvents for GGP and GGP-CLEAS at 60 \({}^{\circ}\)C. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{\(\mathbf{pH}\)} & \multicolumn{2}{c}{\(\mathbf{t\,\%\,(min)}\)} \\ \cline{2-3}  & **GGP** & **GGP-CLEAS** \\ \hline
5 & 0.922 \(\pm\) 0.023 & 2.95 \(\pm\) 0.32 \\
7 & 0.785 \(\pm\) 0.012 & 4.09 \(\pm\) 0.23 \\
9 & Activity was 0 in the first measure & 1.78 \(\pm\) 0.12 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Half-life of GGP and optimal GGP-CLEAS at 90 \({}^{\circ}\)C. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{\(\mathbf{pH}\)} & \multicolumn{2}{c}{\(\mathbf{t\,\%\,(min)}\)} \\ \cline{2-3}  & **GGP** & **GGP-CLEAS** \\ \hline
5 & 9902 & 17,329 \\
7 & 2310 & 6931 \\
9 & 1155 & 3466 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Half-life times according to hydrogen peroxide concentration for GGP and GGP-CLEAS at 25 \({}^{\circ}\)C in the presence of 0.1 mM hydrogen peroxide. The addition of a free enzyme just marginally improved this result (to 34%). The effect of the enzyme action was clearer in the first moments. The immobilized biocatalyst allowed the IC solution to decolor by 82% in 60 min, reaching 84% after 120 min at 25 \({}^{\circ}\)C, a much better result than using the free enzyme, although in the first moments, the free enzyme seemed to present an efficiency even higher than the immobilized enzyme. These results confirmed that the GGP biocatalysts designed in this paper greatly improve GGP performance in the reaction of IC degradation, very likely as a consequence of different factors such as an increase in enzyme stability and the generation of a more adequate conformation of the enzyme. The absorption spectra of indigo carmine, untreated and treated with GGP-CLEAS under optimal parameters, are shown in Figure S1. It is interesting that the characteristic absorption peak of the chromophore group of the dye at 610 nm disappears after 120 min of reaction catalyzed by GGP-CLEAS. Probably, the C=C bond of IC is cleaved by GGP-CLEAS, decreasing the intensity of the absorption peak and producing two molecules of 2-amino-5-(sodium benzenesulfonate)-benzoic acid. Afterwards, GGP-CLEAS release a sulfonate ion for the formation of anthranilic acid. This can be degraded into benzoic acid or aniline, which can form 4-ethyl-benzaldehyde, benzaldehyde, propanoic acid, acetic acid, or formic acid. Finally, these derivatives spontaneously degrade into water, molecular nitrogen, and carbon dioxide [58]. The catalytic activity of the GGP-CLEAS was evaluated in four continuous cycles of reuse in the degradation of indigo carmine, as described in Figure 3. As shown in Figure 4, GGP crosslinked aggregates after four cycles of reaction only retain 38% of the enzymatic activity exhibited in the first cycle. Figure 4: Number of reuses of GGP-CLEAS in catalytic cycles of indigo carmine degradation. Figure 3: Indigo carmine (0.05 mM) degradation by GGP and GGP-CLEAS at 25 \({}^{\circ}\)C and pH 5 in the presence of 0.55 mM hydrogen peroxide. Reference: GGP (circles), GGP-CLEAs (triangles), and no catalyst (square).