## 3 Results and Discussion
### Immobilization of Lacase onto Different Types of Nanoparticles
Moreover, the bonding is reversible and it is sensitive to changes in the pH and ionic strength, which can lead to the recovery of the support [9]. When this approach was considered for the immobilization of laccase, not only limited yield was evidenced, but also the change of basic pH led to enzyme desorption. When performing the immobilization of laccase at different pH values, the best results were observed when the immobilization process was performed at pH 5 with an activity yield higher than 50% (Table 2), possibly because the point of zero charge (PZC) of the magnetite is between pH 6.5-7.9 [43], while the isoelectric point of laccase is at pH 3 [44]. The main drawback is that laccase stability decreases with lower pH, which was evidenced by the reduction of the immobilized enzyme. Considering the best results of activity yield and enzyme load, fsNPs and FeO-2206W smNPs were selected for the following experiments of phenol biotransformation. Biotransformation of Phenol by Laccase Immobilized onto fsNPs and Silica-Coated Magnetic Nanoparticles in Batch Operation  
The capacity of free and immobilized enzymes onto fsNPs and FeO-2206W smNPs to transform phenol was assessed in batch operation. The results showed that the higher phenol transformation was achieved by free laccase (>75%), whereas phenol conversion was around 23% and 48% for fsNPs and FeO-2206W smNPs, respectively (Figure 1). Lower activity of immobilized laccase towards phenolic substrates has also been previously reported. For instance, [PERSON] et al. [42] found that bisphenol A degradation rate was much slower for immobilized laccases (from 6- to 26-fold lower than that of the free enzyme). This lower reaction rate was related to the potential aggregation of the nanoparticles which could reduce substrate accessibility. [PERSON] et al. [45] studied phenol degradation by immobilized laccase on magnetic silica nanoparticles, and similar results were observed at pH 7. The rate of phenol conversion for laccase immobilized onto FeO-2206W smNPs (2.01 uM h\({}^{-1}\)) is almost two times higher than that for fsNPs. Controls with phenol lacking laccase but with functionalized nanoparticles were performed, with no decrease in phenol concentration in all cases after 24 h.  
[PERSON] and [PERSON] [46] reported that laccase can be inactivated due to the presence of free radicals in the reaction medium generated from phenol transformation (not by the substrate). However, this effect was only evident at phenol concentrations of 2000 uM (188 mg/L), almost 20-fold higher than that used in the present work. Hence, no enzymatic activity changes occurred in any of the experiments with a noticeable enzyme deactivation when performing the experiment with free laccase. According to the results, the nanobiocatalyst with FeO-2206W as a support seems to be the most adequate, as it achieved higher phenol biotransformation yields. Moreover, the separation of the nanobiocatalyst should be much simpler under a magnetic field, while intense centrifugation should be required when considering fumed silica nanoparticles. Accordingly, the single-core silica-coated nanobiocatalyst was used to prove its potential of reuse for phenol biotransformation in sequential batches. Sequential Batch Biotransformation of Phenol by Lacase Immobilized onto Silica-Coated Magnetic Nanoparticles  
Due to the remarkable effect of pH on phenol conversion reported in previous works [45; 47], the biotransformation of phenol was assessed at different pH levels (5, 6, and 7). The conversion efficiencies and rates are shown in Table 3. An improvement of phenol conversion was observed when the pH was decreased to 6 or 5, which lead to an increase of 16%. A similar pH range was found suitable for phenol by immobilized enzyme onto silica-coated magnetic nanoparticles [45]. Furthermore, the enzymatic activity was maintained constant in all the experiments.