Acetylcholinesterase immobilization on a natural thin membrane for stable biocatalyst design

This study involved the immobilization of acetylcholinesterase (AChE) onto a natural thin membrane extracted from the inner epidermal layer of Allium cepa, with the aim of enhancing the enzyme's stability, activity, and reusability for potential biotechnological applications. The membrane, composed of biocompatible polymers and naturally occurring functional groups, was used as a sustainable support in a combined strategy involving adsorption followed by glutaraldehyde-mediated cross-linking. Immobilization conditions were optimized by evaluating key factors such as enzyme concentration, membrane quantity, cross-linker dosage, and contact time to achieve maximal catalytic efficiency. Structural and morphological changes on the membrane surface were assessed via scanning electron microscopy and infrared spectroscopy, confirming successful enzyme binding and alterations indicative of interaction. The immobilized enzyme exhibited enhanced tolerance to pH and temperature variations when compared to its free form. While both free and immobilized enzymes showed peak activity at 30 °C, the immobilized enzyme retained approximately 80% of its activity at 40 °C, whereas the free enzyme retained only around 50%. A reusability assessment demonstrated that the immobilized system preserved over 60% of its initial activity after 10 consecutive cycles. Moreover, storage stability tests revealed that the immobilized enzyme maintained nearly 40% of its catalytic function after 30 days at room temperature. These results highlight the effectiveness of the onion membrane as a green and low-cost platform for enzyme immobilization, offering considerable promise for applications in industrial biocatalysis, biosensing, and diagnostic technologies.