Reversible Immobilization of Catalase by Metal Chelate Affinity Interaction on Magnetic Beads

A magnetic metal-chelate adsorbent utilizing N-methacryloyl-(L)-cysteine methyl ester (MAC) as a metal-chelating ligand was prepared. MAC was synthesized using methacryloyl chloride and L-cysteine methyl ester dihydrochloride. Magnetic beads with an average size of 150-250 m were obtained by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAC carried out in a dispersion medium. Mag-poly(HEMA-MAC) beads were characterized by surface area measurements, swelling tests, electron spin resonance (ESR) spectroscopy, elemental analysis, and scanning electron microscopy (SEM). The specific surface area of the magnetic beads was found to be 92.6 m^2/g. Elemental analysis of MAC for nitrogen was estimated as 55.4 (mu)mol/g. Then, Fe3+ ions were chelated on the magnetic beads. The Fe3+ loading was 12.7 (mu)mol/g of support. Fe3+-chelated magnetic beads with a swelling ratio of 62% were used in the immobilization of catalase in a batch system. This approach to the preparation of enzyme carrier has several advantages over conventional immobilization methods. An expensive, time-consuming, and critical step in the preparation of immobilized metalaffinity carriers is the coupling of a chelating ligand to the adsorption matrix. In this procedure, comonomer MAC acts as the metalchelating ligand, and it is possible to load metal ions directly on the beads without further modification steps. The maximum catalase immobilization capacity of the mag-poly(HEMA-MAC)-Fe3+ beads was observed to be 192 mg/g at pH 5.5. The Km value for immobilized catalase [mag-poly(HEMA-MAC)-Fe3+] (32.6 mM) was higher than that for free catalase (22.8 mM). Immobilized catalase exhibits enhanced stability in reaction conditions over a wide pH range (pH 5.5-7.0) and retains an activity of 76% after 10 successive batch reactions, demonstrating the usefulness of the enzyme-loaded magnetic beads in biocatalytic applications. The optimum temperature for the immobilized preparation of mag-poly(HEMA-MAC)-Fe3+-catalase at 45 °C was 5 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with immobilization.