Physical Adsorption of Horseradish Peroxidase on Reduced Graphene Oxide Nanosheets Functionalized by Amine: A Good System for Biodegradation of High Phenol Concentration in Wastewater

The activity and pH/thermal/storage stability have been investigated for Horseradish peroxidase (HRP) physically immobilized onto modified reduced graphene oxide (RGO·NH2) nanoparticles (NPs) and compared to the free enzyme. The NPs were synthesized with a size of about 70 nm and functionalized to have amine groups. The NPs, free and immobilized HRP were characterized using Fourier transform infrared spectra, scanning electron microscopy, zeta potential measurement, and UV/visible spectroscopy. The results obtained from activity assays showed that the catalytic constant, kcat, value became more than sixfold and the catalytic efficiency, kcat/Km, value increased about sevenfold after immobilization. The results also indicated that the catalytic activity reached 133-fold (for 2 mg mL−1 of NPs) and 120-fold (for 1.5 mg mL−1 of NPs) when HRP was immobilized. The optimum pH was also obtained at 7.0 for both free and immobilized HRP. At 50 °C, the immobilized HRP retained 75% of the initial activity, while 60% initial activity remained for the free HRP after 120 min. The excellent reusability of immobilized HRP was also observed and the activity retained about 60% of the first use after ten cycles. The results also revealed that the activities reached 50% of initial activity for the immobilized HRP when the samples were stored for 35 days. The data obtained from circular dichroism spectroscopy displayed that the α-helical content of the enzyme was decreased after immobilization. The removal efficiency for high concentration phenol (2500 mg L−1) was 100 and 53% for the immobilized HRP and free HRP, respectively.