## 2 Results and Discussion
### Removal of 2,4-DCP  
Next, the catalytic potential of PIL (2)-NH\({}_{2}\)-GA-Lac was investigated. Many previous studies have shown that various process parameters may significantly affect the removal of harmful pollutants by enzymes [52]. Therefore, after evaluating the stability and reusability of immobilized lacase, it was crucial to determine the effects of various process conditions (e.g., pH, temperature, and substrate concentration) on its efficiency and the optimal conditions for the effective removal of phenolic contaminants. In Figure 5a-c, the bar represents the efficiency of phenol removal over time (12-36 h). The dot plot represents the removal, degradation, and adsorption of 2,4-DCP by the immobilized enzyme within a certain time range. The recycling efficiency of 2,4-DCP removal by the immobilized enzyme was also studied, as shown in Figure 5d. As shown in Figure 5a, with the increase in the substrate concentration from 1 to 20 mg/L, both the 2,4-DCP removal and degradation increased firstly and then slightly decreased. When the concentration was 15 mg/L, the maximum 2,4-DCP removal and degradation occurred (90% and 55%, respectively). However, the 2,4-DCP removal and degradation decreased slightly when the substrate concentration was 20 mg/L. This can be explained by the fact that at a higher substrate concentration, the phenolic substrate tended to aggregate through intermolecular interactions, which led to a reduction in the substrate adsorption in the immobilized system [53; 54]. In addition, it can be seen from the bar that with extension of the reaction time (12-36 h), the removal of 2,4-DCP basically showed a steady rising trend. As one item that contributed to the removal, the 2,4-DCP adsorption increased until it reached around 35%. This also can be understood as the gradual saturation of the immobilized capacity on the support [55]. The removal of 2,4-DCP was investigated over a broad pH range of 2 to 6 at 20 \({}^{\circ}\)C (Figure 5b). As can be seen from the bar, when the reaction lasted for 24 h, the 2,4-DCP removal increased substantially, reaching its maximum increment at pH 4. Furthermore, regarding their variation with the pH values, the removal and degradation showed similar trends, while the adsorption did not change significantly, remaining at about 20%. It was also found that the removal changed a little when the pH was 2-4 and reached its maximum value at pH 5 (84%, 36 h). However, when the pH exceeded 5, the degradation was negatively correlated with the pH. It can be seen that the weak acidity of the solution was conducive to the removal and degradation of 2,4-DCP. In a slightly high pH environment, the dissociation equilibrium of 2,4-DCP shifted in the direction of producing more H\({}^{+}\). According to the principle of chemical reaction equilibrium, anions (OH\({}^{-}\)) enable and promote the catalytic oxidation reaction [56]. However, in this study, this led to lower catalytic oxidation efficiency when the pH value was further increased. On the other hand, the increase in anions in the dissociation equilibrium brought about the formation of free \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Vmax (mM/(mg\(\cdot\)min))** & **K\({}_{\text{m}}\) (mmol/L)** \\ \hline Free lacase & 0.035 & 1.202 \\ PIL (1)–NH\({}_{2}\)–GA–Lac & 0.043 & 1.100 \\ PIL (2)–NH\({}_{2}\)–GA–Lac & 0.043 & 0.998 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: K\({}_{\text{m}}\) and Vmax of free lacase and immobilized lacase. phenoxy, which gave rise to C-O coupling and further led to the accumulation of 2,4-DCP dimers and a high concentration of oligomer. This may hinder the substrate molecules' ability to enter the lacase's active center, resulting in reduced 2,4-DCP removal [43]. Figure 5c demonstrates that PIL (2)-NH2-GA-Lac was characterized by 2, 4-DCP removal over a wide temperature range. As the temperature increased, the removal and degradation gradually increased, with the maximum removal recorded at 50 \({}^{\circ}\)C (24 h, 85%).