Immobilization and characterization of celery root peroxidase on multi-walled carbon nanotubes

This study investigates the immobilization of peroxidase (POD) concentrated from celery root onto multi-walled carbon nanotubes (MWCNTs), focusing on its effects on enzymatic activity, stability, and reusability. POD was extracted using phosphate buffer, followed by ammonium sulfate precipitation and dialysis. Immobilization conditions were optimized based on contact time and support amount. The immobilized enzyme showed maximum activity after 300 minutes, whereas increasing MWCNT content led to reduced activity due to diffusion limitations. Kinetic analysis revealed that immobilized POD retained a similar V_max compared to the free enzyme, but exhibited significantly higher K_M values. Comprehensive characterization using BET, FTIR, SEM/EDX, TGA, and TEM confirmed successful immobilization and enzyme-nanotube interactions. BET analysis showed a decrease in surface area from 275 to 197 m²/g. FTIR spectra confirmed the appearance of protein-specific bands post-immobilization, and EDX data revealed increased nitrogen and oxygen levels, along with Fe as a cofactor marker. Thermal degradation profiles also changed, while SEM and TEM images demonstrated morphological alterations on the nanotube surfaces. Immobilized POD preserved activity at pH 4.0-6.0 and optimum temperature (30 °C), and retained functionality over multiple cycles and storage periods. These findings highlight the potential of MWCNT-supported POD systems in environmentally relevant and industrial biocatalytic applications.