Microbiol. Res. (1998 ) 153, 319- 325 © Gustav Fischer Ver lag Immobilization of Bacillus subtilis a-amylase and characterization of its enzymatic properties Mohamed A. Abdel-Naby, Amal M. Hashem, Mona A. Esawy, Ahmed F. Abdel-Fattah Department of Chemistry of Natural and Microbial Products , National Research Center, Dokki , Cairo, Egypt Accepted: September 9, 1998 Abstract Bacillus subtilis I a-amylase was immobilized on different carriers by different method s of immobilization. The immo- bilized enzymes were prepared by physical adsorption on aminoalkylsilane-alumina (AS-alumina), ionic binding onto DEAE-cellulose, covalent binding on chitin, and entrapment in polyacrylamide had the highest activities. The specific activity of the immobili zed enzy mes, calculated on bound protein basis, were 13.5-50% of the original specific activity exhibited by the free enzyme. The optimal pH of the immo- bilized enzymes was shifted to lower values than for the free enzyme. The optimum reaction tempe rature was determined to be 45°C for the free amylase, whereas that for the immobilized enzymes was shifted to 60- 65°C. In all cases, K; values for the hydrolysis of starch of the immobilized enzymes were higher than that of the native enzyme. Compared to the free form, the immobilized enzymes exhibited improved thermal stability and higher values of activation energy of denatura- tion. Key words: Bacillus subtilis - amylase - immobilization - properties Introduction Starch is the second-abundant renewable biopolymer present on the earth with more than 109 tons produced annually (Galliard, 1985). Traditionally, starch was, and still is, hydrol yzed to low molecul ar weight dextrins and glucose using acid, but enzymatic hydrol ysis has several advantages. First, the specificity of enzymes allows the production of suga syrups with well-defined physical and chemical properties. Second, the milder enzymatic hydrolysis results in few side reactions and less "brown- Corresponding author: M. A. Abdel-N aby ing". Indeed, for the production of glucose syrups from starch, enzymat ic hydrol ysis is essential. The hydrolysis of starch to glucose involves many enzymes, the most important of these are a-amylase (a-l,4-glucan-4-glucohydrolase, EC 3.2.1.1), lase ,4-D-glucan maltohydrolyase, EC 3.2.1.2), and glucoa mylase (EC 3.2.1.3) and the debranching en- zymes (Nigam and Singh, 1995). Immobili zation of enzymes onto water-insoluble sup- ports has become a subject of interest for many indus- tries. The advantages of this technique includ e the possibility of enzyme re-utilization, enhanced stability, easy separation of catalyst from the reaction mixture and ready application to automated continuous processes (Monsan and Combes, 1988). Since amylases have many applications in the food and fermentation industries, it is not surprising that a number of methods for immobi- lizing the enzymes have been reported (Fogarty, 1983). The present paper describes the immobilization of B. subtilis 1 a-amylase onto various carriers using physical adsorption, ionic bindin g, covalent binding and entrapment methods. The properties of the immobilized enzymes were compared with those of the free enzyme to identify the most suitable immobilization method. Materials and methods Microorganisms. Bacillus subtilis I was isolated from soil plated on agar complex medium containing gil: sol- uble starch, 2.0 ; (NH4hHP0 4' 2.0; MgS0 4 . 7H 20 , 0.5; KCl, 0.5 and agar, 30. Identification of amylolytic isolate was done according to Bergey 's Manual of Determination Bacteriology (Buchanan and Gibbons, 1984). Microbiol. Res. 153 (1998) 4 319