RESEARCH ARTICLE Immobilization of a-amylase via adsorption onto bentonite/ chitosan composite: Determination of equilibrium, kinetics and thermodynamic parameters Zu ¨ beyde Baysal, Yasemin Bulut, Murat Yavuz and C ¸ etin Aytekin Faculty of Science, Department of Chemistry, Dicle University, Diyarbakır, Turkey Immobilization of a-amylase onto bentonite/chitosan (BC) composite was studied via adsorption. The composite was characterized by FTIR, SEM, and surface area measurements. The effect of different factors such as, pH, temperature, initial enzyme concentration, and various thermodynamic parameters was determined. The maximum a-amylase adsorption capacity of the BC composite was determined as 64 mg/g at 0.8 mg/mL enzyme concentration. The activity of the immobilized enzyme was measured under varying experimental conditions. The highest enzyme activity for free and immobilized enzyme was determined at 30 and 35°C in 0.1 M phosphate buffer at pH 7.0. The effect of substrate concentration on enzyme activity of free and immobilized enzymes showed a good fit to the Lineweaver–Burk plots. Michaelis constant, K m , for the immobilized a-amylase was found to be higher than for the free enzyme. The adsorption isotherm was modeled by the Langmuir equation. Received: July 17, 2013 Revised: September 9, 2013 Accepted: September 12, 2013 Keywords: a-Amylase / Bentonite / Chitosan / Composite / Immobilization 1 Introduction Nowadays, biotechnology is considered as a useful alternative to traditional technology in industrial and analytical fields. Over the last few decades, intense research has provided many approaches that facilitate practical applications of enzymes in the enzyme technology [1]. The immobilization of enzyme allows enzyme re-utilization, facilitates separation, decreases production costs, and the generation of wastes. These properties of immobilization are of advantage for industrial processes [2–5]. The immobilization of enzymes on support matrix is among the preferred techniques developed to improve almost all enzyme properties: stability, activity, reduction of inhibition, eliminating protein contamination of the product, etc. The main advantage of enzyme immobili- zation is the possibility to reuse the enzyme for many reaction cycles in industrial applications [6]. Furthermore, compared with chemical methods, physical methods, particularly adsorption, may have a higher commercial potential because they are simpler and cheaper and can retain a high catalytic activity for the immobilization of enzymes [7, 8]. There are various methods of immobilization of enzymes, such as adsorption, entrapment, crosslinking, and covalent attach- ment [9–11]. Among these immobilization techniques, adsorption is the most general, easiest to perform and oldest protocol of physical immobilization methods. Simplicity and reversibility are the most important advantages of this method [10]. Hydrolysis of starch by a-amylase is one of the most important commercial enzymatic processes that include about 25% of universal trade of enzymes, and altered completely the chemical hydrolysis [9]. a-Amylase (1,4-a-D- glucan-glucanohydrolase) hydrolyzes a (1 ! 4) glycosidic linkages of the starch polymer in a random fashion and leads to the formation of linear and branched oligosaccharides. These products are used in bread and baking industries, textile, paper, detergent industry, and clinical and medicinal applications [12, 13]. But once used, a-amylase cannot be recovered easily from the reaction systems. Therefore, it is very important to use immobilized a-amylase rather than free a-amylase [2]. There have been many reports about the immobilization of a-amylase [1, 9, 14, 15]. Correspondence: Professor Zübeyde Baysal, Faculty of Science, Department of Chemistry, Dicle University, Diyarbakır, Turkey E-mail: zbaysal@dicle.edu.tr Fax: þ90-412-248-83-00 Abbreviations: BC, bentonite/chitosan; FTIR, fourier transform infrared spectroscopy; Km, Michaelis constant DOI 10.1002/star.201300133 484 Starch/Stärke 2014, 66, 484–490 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com