Covalent Immobilization of Human Serum Catalase on Fe3O4@SiO₂–NH₂ Magnetic Nanoparticles: Stability Assessment, Apparent Kinetic Behavior, and Structural Analysis

Enzyme immobilization on nanostructured supports is frequently used to increase the stability of enzymes and make recovery from reaction medium easier. In this work, glutaraldehyde was used as a coupling agent to covalently immobilize pure human serum catalase onto amine-functionalized magnetic nanoparticles (Fe₃O₄@SiO₂–NH₂). Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and dynamic light scattering (DLS) were used to characterize the resultant support and enzyme-loaded nanocomposite. Successful surface functionalization and enzyme immobilisation were supported by the obtained characterization results. Using hydrogen peroxide as the substrate, catalytic performance was assessed and contrasted with that of the free enzyme. Following immobilization, apparent Michaelis-Menten analysis revealed a little drop in Vmax together with an increase in Km. demonstrating decreased substrate accessibility and the existence of conformational or diffusional restrictions related to covalent attachment to the solid support. Despite these modifications, compared to the free enzyme, the immobilized catalase showed better pH tolerance, heat stability, and storage stability. Furthermore, simple magnetic separation of the immobilized preparation from the reaction medium was made possible by the Fe ₃ O ₄ core. These results suggest that Fe ₃ O ₄ @SiO₂–NH₂ offers an appropriate foundation for the creation of magnetically recoverable catalase systems with improved stability.