Preparation, Characterization And Adsorption Properties Of Chitosan/ Montmorillonite Intercalation Composites

Polyphenol oxidase is a highly effective oxidase of phenolic compounds, under aerobic conditions, it can catalyze the hydroxylation of monophenols to o-diphenols and subsequent oxidation o-diphenols to quinones, eventually quinone compounds formed to the polymer precipitation insoluble in water and be removed. Chitosan is a natural polymer and has been widely used as a support material for enzyme immobilization. It possesses hydroxyl (OH) and amino (NH2) groups, which link with enzymes easily. Gold nanoparticles with excellent optical properties, catalytic activity and biocompatibility, has been successfully used enzyme sensor, and can improve the catalytic performance of enzyme molecules. MMT is a layered silicate, its layer thickness and spacing in nanometer-scale, large surface area, and has good physical adsorption properties. In this study, chitosan/montmorillonite intercalation composites (CTS/MMT) and chitosan-nanogold/montmorillonite intercalation composites (CTS-Au/MMT) have been prepared by polymer solution intercalation, and the chemical structure was characterized. Polyphenol oxidase (PPO) was immobilized onchitosan/montmorillonite intercalation composites and chitosan-nano-gold/montmorillonite intercalation composites by adsorption and glutaraldehyde cross-linking. The immobilization conditions and enzymatic properties were investigated in detail. The optimum performance immobilized enzyme was selected to catalytic oxidation phenol,4-chlorophenol and 2,4-dichlorophenol, The catalytic oxidation conditions of phenolic compounds, dynamics and reusability of immobilizated PPO were investigated in detail, provide a basis for application in phenolic wastewater treatment.The main contents and conclusions are summarized as below:(1) The CTS/MMT have been prepared by polymer solution intercalation, and then characterized with Fourier transform infrared (FTIR) and X-ray diffraction (X-RD). Polyphenol oxidase (PPO) was immobilized on the composites by adsorption and glutaraldehyde cross-link ing. The effects of glutaraldehyde concentration, pH, enzyme/support weight ratio and time on the immobilized enzyme activity and protein loading yield were investigated, L-dopa as the substrate investigated the properties of free and immobilized enzymes. The results indicated that A-PPO has a higher activity. Optimal preparation conditions of the immobilization of polyphenol oxidase by adsorption (A-PPO) were pH 5.0, enzyme/support weight ratio 20 mg/g, immobilization time 6 h. And the protein loading yield of A-PPO was 12.12 mg/g, enzyme activity of the unit support 12.76×103 U/g, and activity recovery 81.7%. A-PPO and C-PPO showed the optimum activity at pH 8.0, and 40? and 30?, respectively. Compared with free PPO, immobilization enhanced enzyme stability against changes in pH and temperature. Immobilized PPO retained more than 37.6% of its initial activity after five cycles. A-PPO and C-PPO had a higher Km values (Michaelis constant) than free enzyme, and the values were 0.56,2.11 and 0.48 mmol/L, respectively. The maximum reaction rate Vmax of A-PPO and C-PPO were decreased compared with free enzyme, and determined as 2.08,2.48 and 2.70 U/g, respectively.(2) Polyphenol oxidase (PPO) was immobilized onto chitosan/montmorillonite intercalation composites by adsorption, and then used to catalytic oxidation phenol, 4-chlorophenol and 2,4-dichlorophenol from aqueous solution. The results indicated that optimal conditions of the removal for phenolic compounds by immobilized PPO were pH 7.0 and 20? for phenol, pH 5.0 and 20? for 4-chlorophenol, pH 5.0 and 30? for 2,4-dichlorophenoL Michaelis constant indicated that 2,4-dichlorophenol possessed a highest affinity for the immobilized enzyme and a fastest catalysis rate. The ratio of Vmax/Km indicates that 2,4-dichlorophenol was optimum substrate. The efficiency of removing for phenol,4-chlorophenol and 2,4-dichlorophenol maintained 15.7%,24.2% and 27.8%, respectively, after 6 times repeated use of immobilized enzyme.(3) CTS-Au/MMT were prepared by CTS/MMT as a reducing agent react with chlorine acid solution. Optimum preparation conditions were discussed, and its structures were characterized. Polyphenol oxidase (PPO) was immobilized on CTS-Au/MMT by adsorption and glutaraldehyde cross-link ing. The immobilization conditions and enzymatic properties were investigated in detail. The results indicated that optimal preparation conditions of the CTS-Au/MMT were gold chloride acid concentration 0.05 mmol/L, pH 9.0, reaction time 4 h, temperature 80?. Under the optimal preparation conditions, the protein loading yield of A'-PPO was 17.02 mg/g, enzyme activity of the unit support 24.02×103 U/g. Compared with CTS/MMT, the activity and protein loading yield of CTS-Au/MMT immobilize enzyme has improved significantly.(4) Polyphenol oxidase (PPO) was immobilized onto CTS-Au/MMT by adsorption, and then used to remove phenol.4-chlorophenol and 2,4-dichlorophenol from aqueous solution. The results indicated that optimal conditions of the removal for phenolic compounds by immobilized PPO were pH 7.0 and 30? for phenol, pH 5.0 and 20? for 4-chlorophenol, pH 5.0 and 30? for 2,4-dichlorophenoL Michaelis constant indicated that phenol possessed a highest affinity for the immobilized enzyme, and 4-chlorophenol has the fastest catalysis rate. The ratio of Vmax/Km indicating that 4-chlorophenol was the optimum substrate. Compared with CTS/MMT, gold nanoparticles particles as an electron mediator in CTS-Au/MMT showed a good adsorption capacity and catalytic activity, and a better catalytic oxidation for phenolic compounds.