Poly(methyl methacrylate-acrylic acid) microspheres were prepared and the acid groups were activated by using either carbodiimide (CDI) or thionyl chloride (SOCl2). alpha-Amylase was covalently bound on these activated microspheres. The properties of the immobilized enzyme vv ere investigated and compared with those of the free enzyme. The relative activities were found to be 80.4 and 67.5% for carbodiimide and thionyl chloride bound enzymes, respectively. Maximum activities were obtained at lower pHs and higher temperatures upon immobilization compared to free enzyme. No change in V-max and approximately 12-fold increase in K-m,were observed for immobilized enzymes. The enzyme activities, after storage for 1 month, were found to be 24.5 and 52.5% of I:he initial activity values for CDI and SOCl2, activated matrices, respectively. On the other-hand the free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca2+ ions demonstrated higher stability, such as SOCl2,immobilized enzyme retained 83.7% and CDI immobilized enzyme retained 90.3% of the original activity of the enzyme. Thf immobilized enzymes that were used 20 times in 3 days in repeated batch experiments demonstrated that, in the absence of Ca2+ ions about 75% and in the presence of Ca2+ ions greater than 90% of the original enzyme activity was retained. (C) 1998 Elsevier Science B.V. All rights reserved.