| The immobilizations of horseradish peroxidase (HRP) using covalent binding and cross-linking method on the modified porous ceramics have been studied. The porous ceramics are modified by using N-β-aminoethyl-γ-aminopropyl-trimethoxysilane (AEAPTS) as coupling agent. Then the immobilized HRP was used for the treatment of simulative oily wastewater.The modification of porous ceramics was researched and the conditions were discussed. The results indicated that the optimum conditions for grafting were as follows:concentration of hydrochloric acid used for activation of 2.5 mol/L, mass fraction of AEAPTS of 15.0%, pH of 4.5, temperature of 70℃, and reaction time of 3.5 h. Under these conditions,1 g of ceramics could be linked to the maximum amount of coupling agent, namely 2.13 g.The immobilization influence factors and characterization of the immobilized enzyme were studied. The optimal immobilization conditions for HRP were:silane-modified ceramics were activated in 2.5% glutaraldehyde solution for 0.75 h. And then 0.1 mg/ml HRP solution (pH 7.5) was reacted with the activated ceramics for 18 h at 4℃. The immobilized rate of protein was 46.4% and the measured activity of the immobilized HRP was about 1379.4 U/g support. The maximum HRP immobilization capacity of the ceramics was 1.16 mg/g support.Compared with free HRP, the acid-base, operational and storage stabilities of immobilized HRP were increased after the immobilization. After the tenth use the activity of immobilized HRP was still over 50% of the initial one. Compared to the enzymology property of free HRP, the one of immobilized HRP was improved largely. The immobilized HRP maintained about 90.6% of its initial activity during 30 days storage period. However the free HRP maintained only 54.7% of its initial activity. The immobilized HRP was stored more stably than the free HRP.Finally, the removal of oil from oily wastewater by immobilized HRP was studied. The results indicated that the optimum treat conditions were pH of 7.0, immobilized HRP of 0.69 g, concentration of H2O2 of 564.92 mg/L, concentration of polyethylene glycol (PEG) of 19 mg/L to 15 mL oily wastewater of 122 mg/L, temperature of 25℃and reaction time of 3 h, the oil removal efficiency was above 93.64%. Metal ions such as Fe2+, Zn2+ and Mg2+ enhanced the activity of HRP while Cu2+ and Mn2+ inhibited it. Immobilized HRP can be used for many times to catalytic oxidize the oily wastewater. After immobilized HRP was used for eight times, the oil removal efficiency was still over 35%. The reaction mechanism of oxidation was still ping-pong kinetics. There were no big differences in Km/Vmax at different concentrations of H2O2, and so were Km at different concentrations of oil. The Michaelis constant Km is 10.312 mg/L. The reaction was still inhibited by H2O2 to some extent. |
