Biochemical Characterization of Acetylcholinesterase Immobilized on Natural and Chemically Activated Sponge Gourd

ABSTRACT In this study, Luffa cylindrica (sponge gourd, SG) fibers were investigated as sustainable lignocellulosic supports for the immobilization of acetylcholinesterase (AChE) using glutaraldehyde (GA)‐mediated covalent cross‐linking. Raw and GA‐treated SGs were comparatively evaluated to determine their effectiveness as immobilization matrices. The immobilized enzymes were characterized in terms of optimal pH and temperature, thermal and pH stability, storage stability, reusability, and kinetic properties. The GA‐treated SG exhibited significantly improved biocatalytic performance compared with the free AChE and raw SG, showing enhanced stability and operational durability. Notably, after repeated use, the AChE immobilized on raw SG lost approximately 54% of its activity after nine cycles, whereas the GA‐treated SG retained nearly 65% of its initial activity under the same conditions. Structural analyses using Fourier‐transform infrared spectroscopy (FTIR) confirmed covalent enzyme attachment, while scanning electron microscopy (SEM) observations revealed a more homogeneous enzyme distribution on GA‐treated SG. Kinetic analysis indicated slight changes in K m and increased V max following immobilization. Overall, the results demonstrate that GA‐treated SG provides a cost‐effective, biodegradable, and efficient support for AChE immobilization, highlighting their potential for applications in biosensing, environmental monitoring, and biocatalysis.