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

WOS: 000304429700003Magnetic 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 mu m in diameter) carrying 68.3 mu mol CB/g polymer were used in a-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 a-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 a-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. (C) 2012 Elsevier B.V. All rights reserved