This study investigates the synthesis and characterization of a novel magnetic mesoporous silica nanoparticle (MMS) platform, specifically Fe₃O₄@SiO₂@KCC-1, functionalized for the immobilization of the diaphorase enzyme. We developed a unique core-shell structure by integrating the superparamagnetic Fe₃O₄ core with the hierarchical, fibrous KCC-1 mesoporous silica, followed by sequential functionalization with amine groups, cyanuric chloride, and diaphorase (MMS-NH₂@CC-enz). Characterization techniques, including nitrogen adsorption-desorption isotherms, FT-IR, XRD, TGA, VSM, and FE-SEM, confirmed the successful synthesis and functionalization, preserving the mesoporous structure while reducing pore size, indicative of effective modification. The novelty of this work lies in the enhanced stability and activity of immobilized diaphorase, demonstrating improved thermal, pH, and storage stability, as well as reusability up to 5 cycles with significant activity retention. Kinetic and thermodynamic analyses revealed subtle changes in Km, Vmax, and thermodynamic parameters (Ea, ΔH, ΔG, ΔS), offering new insights into enzyme-nanoparticle interactions on this magnetic KCC-1-based support. This research introduces a multifunctional MMS platform as an innovative carrier for enzyme immobilization, with significant implications for biotechnological applications such as biosensing, biocatalysis, and bioremediation.
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