Immobilization of catalase on functionalized magnetic nanoparticles: a statistical approach

Magnetic nanoparticles (MNPs) Fe₃O₄, by virtue of easily modifiable surface, high surface-to-mass ratio and super-paramagnetic properties, are one of suitable candidates for the enzyme immobilization. Optimization of five important variables viz. concentration of 3-aminopropyl-tri-ethoxy-silane (APTES), glutaraldehyde (GA) and enzyme, time and temperature of loading was carried out using central composite type of experimental design without blocks giving 50 experiments including eight replicates at the central point. Characterization, stability and reusability studies were also carried out with optimized preparation. Results established the correlation between observed and response surface method (RSM) equation envisaged value (R² 0.99, 0.97 and 0.98 for enzyme’s activity, its loading over MNPs and corresponding specific activity, respectively. The predicted values suggested by RSM equation were 64.00 mM of APTES, 10.97 µL of GA, 14.50 mg mL⁻¹ of enzyme for 67 min at 22.6 °C, resulted in activity 32.1 U mg⁻¹ MNPs, while specific activity was 97.7 U mg⁻¹. Transmission electron microscopy (TEM) showed the sizes of MNPs (10.5 ± 1.7 nm), APTES-MNPs (10.23 ± 1.74 nm), GA-APTES-MNPs (11.84 ± 1.49 nm) and Catalase-GA-APTES-MNPs (13.32 ± 2.74 nm) were statistically similar. The enzyme MNPs preparation retained 81.65% activity after 144 h at 4 °C (free enzyme retained 7.87%) and 64.34% activity after 20 reuse cycles. Statistical optimized MNPs-based catalase preparation with high activity and magnetic strength was stable and can be used for further studies related to its application as analytical recyclable enzyme or magnetically oriented delivery in the body.