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Covalent immobilization of chloroperoxidase onto magnetic beads: Catalytic properties and stability

Bayramoglu, Guelay
Kiralp, Senem
Yilmaz, Meltem
Toppare, Levent Kamil
Arica, M. Yakup
Amino groups containing magnetic beads were used in covalent immobilization of the enzyme "chloroperoxidase (CPO)" which is one of a few enzymes that can catalyse the peroxide dependent oxidation of a wide spectrum of organic and inorganic compounds. The magnetic poly(glycidylmethacrylate-methylmethacrylate-etbyleneglycol dimethacrylate), magnetic p(GMA-MMA-EGDMA) beads were prepared via suspension polymerization in the presence of ferric ions. The magnetic beads were characterized with scanning electron microscope (SEM), Fourier transform infrared (FTIR), Mossbauer spectroscopy and vibrating sample magnetometer (VSM). The magnetic beads were derivatized sequentially with ammonia and glutaraldehyde, and CPO was covalently immobilized on the support via reaction of the amino groups of the enzyme under mild conditions. The effect of various parameters including pH, temperature and enzyme concentration on the immobilization efficiency of CPO onto glutaric dialdhyde activated magnetic beads was evaluated. Magnetic measurement revealed that the resultant CPO-immobilized magnetic beads were superparamagnetic with a saturation magnetization of 18.2 emu/g. The analysis of FTIR spectra confirmed the binding of CPO on the magnetic beads. The maximum amount of immobilized CPO on the magnetic beads was 2.94 mg/g support. The values of Michaelis constants Km for immobilized CPO was significantly larger, indicating decreased affinity by the enzyme for its substrate, whereas V-max values were smaller for the immobilized CPO. However, the CPO immobilized on the magnetic beads resulted in an increase in enzyme stability with time.