α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization

Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Cibacron Blue F3GA (CB) was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/CB beads (100–140 μm in diameter) carrying 68.3 μmol CB/g polymer were used in α-amylase adsorption studies to assess the effects of pH, initial protein concentration, temperature and ionic strength on enzyme activity. Maximum adsorption capacity of mPHEMA/CB beads was found to be 401 ± 11 mg/g carrier. The adsorbed amounts of α-amylase per unit mass of magnetic beads reached a plateau value at about 1.0 mg/mL at pH 5.0. The optimal pH for free and immobilized α-amylase was 7.0 and 8.0, respectively. The immobilized enzyme exhibited better thermostability than the free one. The free enzyme lost all of its activity within 35 days whereas the immobilized enzyme lost about 27% of its activity during the same period. It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the mPHEMA/CB beads.

mPHEMA beads were produced by suspension polymerization in an aqueous medium and Cibacron Blue F3GA, an anthraquinone textile dye, was covalently attached to the mPHEMA beads via a nucleophilic reaction between chloride of its triazine ring and hydroxyl groups of HEMA molecules under alkaline conditions with the elimination of NaCl, resulting in the coupling of Cibacron Blue F3GA to the mPHEMA beads. mPHEMA/Cibacron Blue F3GA beads were used for α-amylase immobilization via adsorption. As can be seen in Figure, the immobilized enzyme exhibited better thermo-stability than the free one. It means that the potential utilization of enzyme would to be extensive.Figure optionsDownload as PowerPoint slideHighlights
► Incorporation of dye ligand increased the adsorption capacity of the magnetic beads.
► Under optimal conditions, adsorption capacity of the mPHEMA/CB beads was determined as 401 ± 11 mg/g carrier.
► The pH and temperature profiles of the immobilized α-amylase were much broader than that of the free enzyme.
► The immobilized enzyme was stable on storage compared to the soluble enzyme under similar conditions.