## 3 Results and Discussion
### Effect of pH on ASNase Immobilization onto MWCNTs  
The immobilization of commercial ASNase was studied through physical adsorption onto MWCNTs and f-MWCNTs. The first parameter to be evaluated was the pH of the immobilization medium. The enzyme immobilization onto support is expected to be influenced by pH since it determines the protein and the MWCNTs' superficial charges and, consequently, the affinity or repulsion between them [13]. However, it must be considered that, at specific pH values, the enzyme may exhibit low activity or even be denatured, making the results inviable. This way, the immobilization of 0.086 mg/mL of ASNase onto 2 mg of both pristine and f-MWCNTs during 60 min was tested at different pH values (4, 6, 8 and 9). A control experiment with MWCNTs without enzyme was also performed, and no reaction or interference was detected in the enzyme activity method. According to Figure 2, the highest \(RRA\) of immobilized ASNase occurred at pH 8 for almost all the MWCNTs tested. Similar results were obtained by [PERSON] et al. [42] during the immobilization of ASNase on tannic acid-modified magnetic mesoporous particles. It was also possible to verify that the ASNase immobilized onto f-MWCNTs showed higher \(RRA\) than when immobilized onto pristine MWCNTs with the same diameters and pH values. This result can be explained by the larger specific surface areas available for ASNase adsorption and the higher hydrophilicity of f-MWCNTs, which increases their dispersibility in aqueous medium and therefore the availability of adsorption sites.  
The efficacy of the immobilization should be analyzed through a balance between the enzyme \(RRA\) and the \(IY\) results. Nevertheless, similar \(IY\) values were noticed for almost all the conditions studied, attaining above 90% (Figure S1 in the Supporting Information), which corresponds to almost total ASNase adsorption onto MWCNTs.  
Considering that the \(pl\) of ASNase is 5.2 and the \(pH_{PZC}\) of pristine and f-MWCNTs is around 7 and 3, respectively, at pH \(\geq\) 8, where the highest \(RRA\) were obtained, both enzyme and support surfaces are negatively charged. Thus, it is possible to conclude that electrostatic interactions do not seem to be the main driving force for the immobilization of ASNase under the tested conditions. Other interactions such as \(\pi\)-\(\pi\) interactions, CH-\(\pi\) interactions and hydrophobic interactions can be the cause of adsorption between MWCNTs/f-MWCNTs and ASNase [17].  
Figure 1: L-asparaginase (ASNase) zeta potential values for different pH values. \(\bullet\) Experimental data; \(\blacklozenge\) isoelectric point.  
The MWCNTs that displayed the best \(RRA\) (around 80%) were the f-MWCNT-1020, revealing that they have the adequate diameter (10-20 nm) and specific surface area to maintain the ASNase activity after immobilization as much as possible. Although MWCNT have a higher specific surface area leading to \(IY\) values above 97% (Figure S1 in the Supporting Information), their diameter seems to be too small to keep all the enzyme molecules active. This correlation indicates that the surface area of the MWCNTs is an important parameter during the ASNase immobilization, but MWCNTs need to have an adequate diameter so that the adsorbed enzymes have their active sites available to react with the substrate.