Synergistic Stabilization of Horseradish Peroxidase by Green-Synthesized Silver-Decorated Magnetite Nanoparticles Nanoparticles: Toward Sustainable Enzyme Technology

In this study, silver-decorated magnetite nanoparticles (Ag@Fe₃O₄) were synthesized via a green method using Brachychiton populneus leaf extract and employed as an efficient support matrix for immobilization of horseradish peroxidase (HRP). The biosynthesized nanocomposite exhibited magnetic properties that facilitated easy separation and reuse, while the silver loading imparted enhanced stability and potential antimicrobial activity. Comprehensive physicochemical characterizations, including XRD, FTIR, FESEM, EDX, BET, and VSM, confirmed the successful formation of Ag@Fe₃O₄ and effective enzyme loading. The immobilization yield of HRP on Ag@Fe₃O₄ reached 93%, and the immobilized enzyme showed improved tolerance toward temperature and pH variations, with an optimal pH of 7.5 and optimal temperature of 60 °C, compared to 7.0 and 50 °C for the free enzyme. Kinetic studies revealed a moderate increase in Km but maintained or slightly increased Vmax, indicating preserved catalytic efficiency. The immobilized enzyme demonstrated excellent reusability over 15 cycles (66% residual activity) and long-term storage stability (81% activity after 60 days at 4 °C). These enhancements are attributed to the protective microenvironment provided by the Ag@Fe₃O₄ matrix, which mitigates denaturation and leaching. This work highlights the potential of Ag@Fe₃O₄ as a sustainable and reusable platform for enzyme immobilization in biocatalytic applications, particularly in environmental remediation and industrial bioprocessing.