Covalent immobilization of horseradish peroxidase on functionalized reduced graphene oxide and biodegradation of high phenol concentration

Horseradish peroxidase (HRP) (EC 1.11.1.7) is an oxidoreductase enzyme that oxidases a variety of organic and inorganic compounds. The HRP is normally applied to catalyze the oxidation of substrates such as phenols and aromatic amines by H2O2. The HRP was immobilized onto modified reduced graphene oxide (RGO) nanoparticles (NPs) through covalent immobilization. In this study, we attempted to immobilize HRP on RGO functionalized NPs to be used in removing phenol from wastewater. The residual phenol compound in supernatants was measured using a colorimetric method with potassium ferricyanide and 4-aminoantipyrine, and for colorimetric assay, the absorbance was measured at 510 nm. The calibration curve was plotted based on the standard phenol concentration with the initial reaction catalytic rate. Absorbance values were converted to the concentrations of phenol compound by using the calibration curve provided ([phenol] =Abs510+0.0952/3139.1 , R2= 0.98). The efficiency of the free/immobilized HRP and NPs were examined to remove phenol compound with the concentration 2500 mg/L in the aqueous solution. For comparison, the time for phenol degradation was set to 40 min. For free HRP, the removal efficiency lastly reached about 50 %, the removal efficiency reached 100%, when immobilized HRP was applied. This result suggested that the immobilization through covalent bonding protected effectively the HRP against inactivation during the biodegradation reaction. The graphs of removal efficiency for the alone NPs and the summation of the NPs and free HRP were also obtained separately. As a result, the immobilization through covalent bonding exhibited successfully a synergetic effect between the NPs and HRP. Additionally, it was significant to note that shorter degradation time needed, provided that HRP was immobilized on the NPs.

Keywords:

Horseradish peroxidase , covalent immobilization , reduced graphene oxide , phenol removal , synergetic effect