Amylases are widely used enzymes across various industries such as clinical chemistry, pharmaceuticals, food pro- cessing, detergents, textiles, and paper manufacturing. Immobilization can enhance their effectiveness. Nanostructured materials have become important in many fields, including magnetite nanoparticles (Fe3O4) for immobilizing enzymes. The study aimed to immobilize alpha-amylase extracted from Bacillus subtilis onto iron oxide nanoparticles coated with chitosan to compare its catalytic efficacy to its free form. Iron oxide nanoparticles were synthesized and coated. The substrate underwent amylase introduction with glutaraldehyde, stabilizing formation on chitosan-iron oxide nanopar- ticles. XRD, FT-IR, FE-SEM, and DLS were used to analyze immobilized amylase on chitosan + iron oxide nanoparticles. To conclude, we assessed the efficacy of immobilized enzyme versus free enzyme at different pH and temperature levels. Spectroscopy indicated a strong electrostatic bond between chitosan and Fe3O4 nanoparticles, causing chitosan to bind onto their surface during alkaline conditions. Alpha-amylase is bonded to the substrate through a Schiff base bond. Optimal temperature for free and immobilized enzymes is 40 °C, and optimal pH is 7.5. The study found that the immobilized enzyme was more active than the free enzyme at ideal pH and temperature, and examined their inactivation in the 40–60 °C range. Free enzymes showed a sharp activity decline, while immobilized enzymes had a slower decline. This study measured the Vmax and Km values of free and immobilized enzymes. The free enzyme had a Vmax of 61.52 μmol/min.mg and a Km of 0.0082 mg/ml. The immobilized enzyme had a Vmax of 48.43±0.6383 μmol/min.mg and a Km of 0.0084±0.0016 mg/ml. It also retained 47% activity after 7 cycles. This study shows a new approach to using enzyme-bound magnetic nanoparticles in various industries like paper and detergent.

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