## 3 Results and Discussion
### Effect of Enzyme Concentration on ASNase Immobilization onto MWCNTs  
The analysis of the enzyme concentration to be immobilized is essential since it allows to know the maximum amount of enzyme that the support can adsorb. Different tests were conducted with varying ASNase concentrations, ranging from 0.05 to 0.6 mg/mL when immobilized onto 2 mg of pristine MWCNTs at pH 8.0 during 60 min of contact time and from 0.1 to 0.7 mg/mL when immobilized onto 2 mg of f-MWCNTs, at pH 8.0 during 30 min of contact time. In Figures 4 and 5 are visible an increment in the _RRA_ with the increase in the ASNase concentration up to a particular value for all the tested MWCNTs. This behavior is due to the higher amount of ASNase molecules available to adsorb onto MWCNTs  
Figure 3: Effect of immobilization time and diameter of multi-walled carbon nanotubes (MWCNTs) on the relative recovered activity (_RRA_). Immobilization of 0.086 mg/mL of L-asparaginase (ASNase) onto 2 mg of pristine multi-walled carbon nanotubes (MWCNTs) and functionalized MWCNTs (f-MWCNTs) at pH 8. Error bars correspond to the standard deviation between replicates. surface. From that ASNase concentration value, a plateau is reached where the \(RRA\) value no longer increases with the increase in the enzyme concentration, suggesting that the MWCNTs surface attained its maximum adsorption capacity. The ASNase concentration where the \(RRA\) plateau is reached is considered the optimal concentration to be used in the immobilization process. Figures 4 and 5 also show almost total adsorption of ASNase onto all MWCNTs studied, with \(IY\) close to 100%, mainly until reaching the optimal enzyme concentration. After that concentration, there was a slight decrease in the \(IY\) value in some situations due to the high amount of non-adsorbed ASNase after the saturation of the support. [PERSON] et al. [43] reported a reduction in enzyme activity at high concentrations, suggesting that this might be attributed to the hiding of certain active sites induced by enzyme aggregation. Furthermore, [PERSON] et al. [44] demonstrated that increasing enzyme concentration might also cause mass transfer blockage owing to congested space. The best \(RRA\) and \(IY\) values were obtained with MWCNTs with diameters between 10 to 20 nm and 20 to 40 nm for both pristine and f-MWCNTs (Figures 4 and 5). However, note that the results are very similar for the different diameters studied. The slightly poor results for MWCNTs with diameters below 10 nm may be due to the very small  
Figure 4: Effect of L-asparaginase (ASNase) concentration on the immobilization yield (\(IY\)) (\(\blacksquare\)) and relative recovered activity (\(RRA\)) (\(\blacktriangle\)). ASNase immobilization onto 2 mg of pristine multi-walled carbon nanotubes (MWCNTs) at pH 8.0 for 60 min. Error bars correspond to the standard deviation between replicates. pore size, hindering the ASNase adsorption. When pristine MWCNTs with diameters of 60-100 nm were used, a certain agglomeration of the support particles was observed due to their hydrophobicity, leaving part of the support surface inaccessible, potentially affecting the attained results. One more time, the balance between the _RRA_ and _IY_ values attained was more favorable for the f-MWCNTs, making them the preferable support for the immobilization of ASNase. ASNase immobilization on MWCNTs has already been described in the literature (Table 3). [PERSON] et al. [21] studied f-MWCNTs as a carrier for an ASNase from _Aspergillus versicolor_ and found that physical adsorption yielded greater _IY_ values (54.4%) than covalent binding, with a retained ASNase activity of 100%. The immobilization of ASNase has also been reported for other different supports, for example, [PERSON] et al. [45] described an immobilized ASNase on chitosan-tripolphosphate nanoparticles with an entrapment efficiency of around 72%. [PERSON] et al. [46] showed a maximum _IY_ of 85% after optimizing the covalent ASNase immobilization on aluminum oxide pellets using response surface methodology.