## 3 Results and Discussion
### Operational Stability  
The enzyme reusability is vital from the commercial point of view since it enables process costs reduction. The immobilization of ASNase enables enzyme recovery and reuse after each reaction. Six L-asparagine hydrolysis cycles were performed to assess the reusability of the ASNase immobilized onto pristine and f-MWCNTs-1020 (as one of the best-determined diameters) at the optimal conditions, by analysis of _RRA_, where the first cycle's _RRA_ was established as 100%. After six continuous reaction cycles, the ASNase immobilized onto pristine MWCNTs retained 57 \(\pm\) 8% of its initial activity (Figure 6a). The supernatant solution was analyzed via the enzyme activity method at the end of each cycle, indicating almost no active ASNase leakage in the supernatant solutions. Therefore, the reduction in the enzymatic activity might be related to the deactivation of the immobilized enzyme. When immobilized onto f-MWCNTs, the ASNase could be reused at least six times without losing significant activity, maintaining 74 \(\pm\) 2% of its initial activity (Figure 6b). The MWCNTs functionalization provides a stronger linkage between the enzyme and the support, preventing the ASNase denaturation. The slight decrease in activity seen after repeated use could be due to active site deformation caused by frequent interactions among L-asparagine and the active site of the immobilized ASNase.  
\begin{table}
\begin{tabular}{p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt}} \hline \hline
**Organism Source** & **Imabilization Support** & **Imabilization Method** & \(\mathbf{\varGamma}\) & **RRA** & **Ref** \\ \hline _Aspergillus versicolor_ & f-MWCNTs & Physical adsorption & 54\% & 100\% & [46] \\ _E. coli_ & chitosanan-tripolphosphate nanoparticles & Entrapment & - & 72\% & [47] \\ _E. coli_ & aluminum oxide pellets & Covalent linkage & 85\% & - & [48] \\ _E. coli_ & silica nanoparticles & Cross-linkage & 95\% & - & [49] \\ _E. coli_ & f-MWCNTs & Physical adsorption & 99\% & 100\% & This work \\ \hline \hline \end{tabular}
\end{table}
Table 3: Comparison of immobilization yield (_IY_) and relative recovered activity (_RRA_) values reported in the literature on L-asparaginase (ASNase) immobilization with those obtained in this work.  
Figure 6: Operational stability of immobilized L-asparaginase (ASNase) onto (**a**) pristine and (**b**) functionalized multi-walled carbon nanotubes (f-MWCNTs-1020). Error bars correspond to the standard deviation between replicates.  
[PERSON] [48] reported similar operational stability of ASNase immobilized onto metal-organic frameworks. The enzyme retained roughly 78% of its original activity after five cycles. Furthermore, after ten cycles, approximately 45% of the original activity was preserved. These results can also be compared with the ones obtained by [PERSON] et al. [49] using poly (2-hydroxyethyl methacrylate-glycidyl methacrylate) cryogels as enzyme carriers, where ASNase was able to retain 52% after ten cycles. These results highlight the suitability of the ASNase immobilization onto f-MWCNTs employed in this study to maintain equivalent enzyme activity and avoid considerable enzyme leaching due to desorption during continuous usage.