Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and Nhydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360 mu g/1.2 x 10(11) spore. However, maximum activity was achieved at an enzyme concentration of approximately 60 mu g/.4 x 10(10), corresponding to an estimated activity of 8 x 10(3) IU mg(-1)/1.2 x 10(11) spore for covalent immobilization and 8.53 x 10(3) for the adsorption method. After washing the enzyme with 1 M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60 degrees C and 80 degrees C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The K-m and V-max values were determined by the GraphPad Prism software, which showed that the V-max value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface. (C) 2014 Elsevier Inc. All rights reserved.