Immobilization, optimization, characterization and kinetic properties of polyphenol oxidase to multi-walled carbon nanotube

In this study, the kinetic properties of polyphenol oxidase (PPO) extracted from <i>Satureja cuneifolia</i> were investigated using catechol and 4-methylcatechol as substrates. Optimal pH and temperature values were determined at each purification step. Subsequently, the optimum immobilization conditions were established as 2 hours of stirring time and 0.05 g of multi-walled carbon nanotubes (MWCNTs). Characterization by BET, FTIR, DTA/TG, TEM, and SEM/EDX analyses confirmed the successful immobilization of PPO onto mesoporous MWCNTs, with notable changes in surface morphology and thermal degradation behavior. The optimum pH for the free enzyme remained constant across purification methods but varied with the substrate, while the optimum temperature was consistently found at 30 °C. Upon immobilization, the optimum temperature shifted to higher values, indicating enhanced thermal stability. Catalytic efficiency (Vmax/K_M) for catechol decreased significantly after immobilization (from 2.5 × 10⁶ to 5 × 10⁴ min^-1), whereas for 4-methylcatechol, the immobilized enzyme retained a high catalytic efficiency (Vmax/K_M =1 × 10⁶ min^-1), comparable to that of the free enzyme. This shift suggests that immobilization favored substrate specificity toward 4-methylcatechol. Overall, the MWCNT-PPO system demonstrated enhanced stability, improved reusability, and altered substrate selectivity, making it a strong candidate for industrial biocatalytic applications where operational durability and efficiency are critical.