## 3 Results and Discussion  
### Immobilization of Lacase onto Different Types of Nanoparticles  
Several strategies of lacase immobilization were evaluated on magnetic and non-magnetic nanoparticles to obtain various types of nanobiocatalysts. The enzymatic activities of both NP-lacase conjugates and supernatants were measured to determine the activity yield, washing loss, and enzyme load (Table 2). The tradeoff analysis of the different outcomes will be critical to identify the most suitable option for its further use. Covalent bonding produces stronger bonds between the enzyme and the support, allowing its reuse more easily than with other available immobilization methods [36; 37] and preventing the leaching of enzymes from the support [38; 39]. In this study, different coatings as well as single- and multi-core nanoparticles were evaluated for lacase immobilization (Table 2). The covalent bonding between the supports with carboxylic groups (polyacrylic acid) did not result in satisfactory immobilization (yields lower than 5%). This may be due to excessive crosslinking of the protein molecule (due to the presence \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Different Types of Nanoparticles** & **Washing Loss (\%)** & **Activity Yield (\%)** & **Enzyme Loading (U mg\({}^{-1}\) NPs)** \\ \hline
**Covalent immobilization** & & & \\ Fumed silica nanoparticles (fsNP) & 5.6 \(\pm\) 1.3 & 100 \(\pm\) 6.1 & 1.78 \(\pm\) 0.07 \\ Silica-coated magnetic nanoparticles & & & \\ FeO-2206W (single-core) & 16.4 \(\pm\) 2.81 & 99.7 \(\pm\) 0.35 & 2.66 \(\pm\) 0.65 \\ S-57 (multi-core) & 66.63 \(\pm\) 1.67 & 31.3 \(\pm\) 0.76 & 0.42 \(\pm\) 0.05 \\ Polyacrylic acid (PAA) magnetic nanoparticles & & & \\ FeO-2204W & 96.83 \(\pm\) 1.4 & 2.55 \(\pm\) 6.5 & 0.11 \(\pm\) 0.34 \\ FeO-36 & 99.7 \(\pm\) 2.34 & 0.12 \(\pm\) 2.3 & 0.01 \(\pm\) 0.24 \\ Polyethylenimine (PEI) magnetic nanoparticles & & & \\ VOZ-19 & 27.18 \(\pm\) 0.08 & 80.5 \(\pm\) 0.21 & 1.54 \(\pm\) 0.03 \\ \hline
**Ionic exchange immobilization** & & & \\ Non-coated magnetite & 45.3 \(\pm\) 0.9 & 58.5 \(\pm\) 1.5 & 0.69 \(\pm\) 0.05 \\ nanoparticles & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Activity yield, washing loss, and enzyme loading for the optimal doses in the immobilization processes. of both NH\({}_{2}\) and COOH groups in the enzyme and also to the instability of carbodiimide, which led to very low activity yields, as was also observed in other studies with activity yields below 14%) [40,41]. However, laccase was successfully immobilized in other nanoparticles such as PEI-mNPs, which showed an activity yield higher than 80% (Table 2). The stability of this biocatalyst was inferior to laccase immobilized onto fsNPs and FeO-2206-W smNPs (Table 2). In the specific case of PEI-mNPs, the enzyme activity after three months was 50%, which was significantly lower than those of fsNPs and FeO-2206-W (93% and 99%, respectively). Similar results were observed for a previous report with silica nanoparticles, using remarkably higher dosages of APTES and glutaraldehyde than the values considered in this research [23]. The rationale behind the high activity yields is attributed to the fact that immobilized laccases on this type of support would have high affinity for standard substrates such as ABTS. For instance, [PERSON] et al. [42] reported the hyperactivation of laccase from _T. versicolor_ after the formation of covalent bonds with silica nanoparticles; whereas [PERSON] et al. [11] described a similar behavior for laccase from _T. villosa_ (activity recovery up to 148%) after the formation of cross-linking aggregates (CLEAS\({}^{\circledgtr}\), CLEATechnologies, Delft, The Netherlands). The process of immobilization by ionic exchange is based on the interaction of the charged groups of the enzyme with the groups of opposite charges in the support. It provides a weak bond between the enzyme and the support so that the native structure of the enzyme is unaltered.