Immobilization and characterization of banana-derived polyphenol oxidase on multi-walled carbon nanotubes

Polyphenol oxidase (PPO) was extracted from banana (Musa spp. ‘Cavendish’), and the resulting crude enzyme extract was immobilized onto multi-walled carbon nanotubes (MWCNTs). Immobilization conditions were optimized, and the resulting PPO–MWCNT biohybrids were characterized by BET, FTIR, DTA/TG, SEM/EDX, and TEM analyses. Structural characterization supported biomolecular adsorption onto the MWCNT surface, as indicated by a decrease in BET surface area from 276.6 to 177.4 m2 g−1, an increase in total pore volume from 0.411 to 0.695 cm³ g−1, and an estimated biomolecular loading of ∼6.21 wt% based on thermogravimetric analysis. Kinetic studies showed that immobilization increased the KM value from 0.017 to 0.025 M, suggesting a reduction in apparent substrate affinity potentially associated with diffusion limitations, while catalytic activity was retained. The optimum pH shifted by approximately 0.5–1.0 units toward acidic conditions, whereas the optimum temperature remained unchanged at 30 °C. Immobilization markedly improved storage stability, with the immobilized PPO preparation retaining approximately 45% of its initial activity after 35 days, compared with only ∼5% for free PPO. These results demonstrate that MWCNTs provide a promising platform for immobilizing PPO-active crude enzyme preparations and improving their storage stability while preserving catalytic performance, highlighting their potential for developing durable enzyme-based systems for biosensing, food analysis, and environmental applications.