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

Abstract

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