Development and characterization of a novel l-asparaginase/MWCNT nanobioconjugate

Abstract

The enzyme l-asparaginase (ASNase) presents effective antineoplastic properties used for acute lymphoblastic leukemia treatment besides their potential use in the food sector to decrease the acrylamide formation. Considering their applications, the improvement of this enzyme's properties by efficient immobilization techniques is in high demand. Carbon nanotubes are promising enzyme immobilization supports, since these materials have increased surface area and effective capacity for enzyme loading. Accordingly, in this study, multi-walled carbon nanotubes (MWCNTs) were explored as novel supports for ASNase immobilization by a simple adsorption method. The effect of pH and contact time of immobilization, as well as the ASNase to nanoparticles mass ratio, were optimized according to the enzyme immobilization yield and relative recovered activity. The enzyme-MWCNTs bioconjugation was confirmed by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), Raman and transmission electron microscopy (TEM) studies. MWCNTs have a high ASNase loading capacity, with a maximum immobilization yield of 90%. The adsorbed ASNase retains 90% of the initial enzyme activity at the optimized conditions (pH 8.0, 60 min, and 1.5 × 10^-3 g mL^-1 of ASNase). According to these results, ASNase immobilized onto MWCNTs can find improved applications in several areas, namely biosensors, medicine and food industry.

Fig. 1. Schematic diagram summarizing the immobilization of l-asparaginase on MWCNTs and respective applications.

Fig. 2. Effect of pH on the immobilization yield (columns) and relative recovered activity (symbols, line) with the immobilization of 8.6 × 10⁻⁵ g mL⁻¹ of ASNase onto 2 mg of MWCNTs for 60 min of contact time.

Fig. 3. Effect of contact time on the immobilization yield (columns) and relative recovered activity (symbols, line) with the immobilization of 8.6 × 10⁻⁵ g mL⁻¹ of ASNase onto 2 mg of MWCNTs at pH 8.0.

Fig. 4. Effect of enzyme concentration on the immobilization yield (columns) and relative recovered activity (symbols, line) with the immobilization of ASNase onto 2 mg of MWCNTs at pH 8.0 for 45 min.

Fig. 5. Freundlich (dashed red line) and Langmuir (solid blue line) isotherm models for the adsorption of ASNase on MWCNTs. Lines correspond to the fitting (nonlinear regression) of the experimental data (symbols). Experimental conditions: 2 mg of MWCNTs, pH 8.0 and 45 min of contact time.

Fig. 6. Initial reaction rates (v₀) for different concentrations of l-asparagine with (○) free and (×) immobilized ASNase (1.5 × 10⁻³ g mL⁻¹) onto MWCNTs by physical adsorption. The solid lines represent the fit of the experimental data to the Michaelis–Menten model.

Fig. 7. TEM analysis of MWCNTs before (a) and after (b) ASNase immobilization.

Fig. 8. TG analysis of MWCNTs before (a) and after (b) ASNase immobilization.

Fig. 9. FTIR-ATR spectrum of ASNase (a) and ASNase–MWCNTs bioconjugate (b).

Fig. 10. Raman spectra of (a) multi-walled carbon nanotubes (MWCNTs) and (b) ASNase–MWCNTs bioconjugate.