Enhancement of Catalytic Stability and Kinetic Evaluation of Acetylcholinesterase Immobilized on Functionalized Fe₃O₄ Magnetic Nanoparticles

In this study, Fe 3 O 4 magnetic nanoparticles (MNPs) were synthesized, and the acetylcholinesterase (AChE) enzyme was immobilized on the functionalized MNPs using the cross‐linker glutaraldehyde. The immobilized enzyme (Fe 3 O 4 @SiO 2 ‐NH 2 /GA/AChE) was characterized by scanning electron microscopy‐energy scattering spectroscopy (SEM‐EDX) and FTIR. Activity for optimum pH was determined in the range of pH 5–11 for free and immobilized enzyme, and the highest activity was observed at pH 8 for free AChE and pH 10 for immobilized AChE. When the kinetic parameters were examined, the K m and V max values of immobilized AChE were 0.446 mM and 0.659 EU mL −1 , respectively, while those of free AChE were 0.163 mM and 0.965 EU mL −1 , respectively. The binding affinity of the tacrine inhibitor to the enzyme was lower for the immobilized enzyme than for the free enzyme. Storage stability tests showed that immobilized AChE stored at 4 °C for 60 days retained 95.2% of its activity, while the free enzyme lost 50.4 % of its activity. Furthermore, reuse tests performed for 20 replicates showed that immobilized AChE retained more than 95% of its initial activity. Therefore, the high stability and reusability of the enzyme as a result of the immobilization process may contribute to future studies in terms of biotechnological applications.