Functionalized carbon nanotube-based affinity chromatography for polyphenol oxidase purification from different sources and evaluation of kinetic properties

Abstract

In this study, functionalized multi-walled carbon nanotubes (MWCNT) were firstly synthesized and applied to purify polyphenol oxidase (PPO). Structural characterization of matrix was confirmed via different techniques. BET surface area decreased from 278.0 to 202.8 m2·g−1 with functionalization. Affinity chromatography achieved 16.4-fold purification for S. officinalis L., 12.9-fold purification for mushroom, 8.4-fold for S. aethiopis L., while SDS-PAGE analysis of S. officinalis L. revealed a single distinct band corresponding to an estimated molecular weight of approximately 43–45 kDa. Optimum pH and temperature values varied depending on the substrates and purification step. Kinetic analysis revealed substrate-dependent catalytic behavior, with catechol (KM = 0.0035–0.0050 mM; Vmax = 5.000–25.000 EU/mL), 4-methylcatechol (KM = 0.00129–0.00333 mM; Vmax = 10.000–14.286 EU/mL) and pyrogallol (KM = 0.00075–0.0040 mM; Vmax = 5.000–50.000 EU/mL). Pyrogallol showed the highest catalytic activity due to its high Vmax/KM value. These results highlight the potential of functionalized MWCNTs as effective affinity supports for PPO purification and position Salvia species and mushroom as a promising natural enzyme source for industrial and biotechnological applications.