Advanced HKUST/MWCNT/Cellulose Acetate Hybrid Support for β-Glucosidase Immobilization: Enhanced Stability, Catalytic Performance, and Reusability

This study reports the fabrication of a ternary HKUST/MWCNT/CA hybrid support for the immobilization of β-glucosidase and the evaluation of its catalytic and stability properties. The support was prepared by integrating HKUST with multi-walled carbon nanotubes (MWCNTs) and cellulose acetate (CA), followed by enzyme immobilization through glutaraldehyde-mediated coupling. Structural and surface characterization by XRD, FTIR, SEM/EDX, BET, and zeta potential confirmed the successful formation of the hybrid matrix and subsequent enzyme loading. The immobilization yield reached 88%, indicating efficient enzyme attachment onto the support. Nonlinear Michaelis–Menten analysis showed an increase in apparent Km after immobilization, whereas the apparent Vmax remained comparable to that of the free enzyme, indicating preservation of substantial catalytic performance. The immobilized β-glucosidase exhibited enhanced tolerance toward non-optimal conditions, retaining higher activity over broader pH and temperature ranges than the free enzyme. Notably, the optimum temperature shifted from 50 °C for the free enzyme to 60 °C after immobilization. The immobilized preparation also demonstrated good operational stability, preserving about 74% of its initial activity after 10 cycles, and excellent storage stability, retaining nearly 79% activity after 30 days at 4 °C. These findings demonstrate that HKUST/MWCNT/CA is a promising support for developing robust and reusable β-glucosidase biocatalysts.