Optimization of immobilization of α-amylase in alginate gel and its comparative biochemical studies with free α-amylase

Sachin Talekar and Sandeep Chavare Department of Biotechnology Engineering, Kolhapur Institute of Technology’s College of Engineering, Kolhapur, Maharashtra, India. Abstract The α-amylase was immobilized by entrapment in calcium alginate beads. The effect of concentration of sodium alginate, calcium chloride and curing time on immobilization yield of α-amylase in calcium alginate beads were investigated and immobilized α-amylase was characterized. Three percent (w/v) sodium alginate, 1M calcium chloride and 2 h curing time were used and 90% immobilization yield of α-amylase was achieved with enhanced thermal and acidic condition stabilities. Significant changes in optimum pH and temperature values of the enzyme were recorded after immobilization. The activity of immobilized enzyme was affected by the size of the bead and bead size of 2.4mm was found to be most effective for starch hydrolysis. From the enzyme kinetic study, decrease in substrate affinity and velocity of enzyme reaction were observed after immobilization of enzyme. Immobilized α-amylase retained 35% activity after 10 reuses with 30 min of each reaction time. Keywords: calcium alginate, enzyme immobilization, entrapment, α-amylase, starch hydrolysis INTRODUCTION The α-amylase (EC 3.2.1.1) enzyme which hydrolyzes starch to maltooligosaccharide is of great importance in present day biotechnology with applications ranging from food, baking, brewing, fermentation, detergent applications, textile desizing, paper industries, etc. [1, 2]. This starch degrading enzyme has received a great deal of attention because of its perceived technological significance and economic benefits. The industrial application of enzymes is often hampered by a lack of availability, high price and limited stability under operational conditions. The use of enzymes in a free form is very uneconomical because the enzymes generally cannot be recovered at the end of the reaction. These drawbacks can be overcome by immobilization of the enzyme thereby rendering it more stable and easy to recover and recycle [3, 4]. Immobilized enzymes pave the way to industrial development of continuous enzyme reactors. This procedure prevents enzyme losses due to washout and at the same time maintains enzymes at high concentrations in order to reduce the cost of the enzymes [5]. The above features would be important in the development of an economically feasible continuous bioreactor for the starch hydrolysis industry. Thus immobilizing α- amylasewould be of great significance. Several efforts have been taken to immobilize α-amylase by binding it to solid carriers [6-16].However; these covalent binding techniques involve chemical modification of the enzyme. It is preferable that the method employed for immobilization of enzyme should cause as little disturbance to the enzyme as possible. Entrapment fulfills this criterion. Entrapment technology has been designed to entrap materials within a semi-permeable polymeric membrane and/or a gel matrix [17]. Enzyme immobilization by entrapment produces the particle structure which allows contact between the substrate and enzyme to be achieved and, additionally, it is possible to immobilize several enzymes at the same time [18]. Among the many matrices available, one of the most frequently used is entrapment within porous matrices, such as alginate often in the form of beads [19]. This sort of system is reasonably safe, simple, cheap and offering good mechanical strength, high porosity for substrate and product diffusion and above all the simple procedural requirements for immobilization [20]. Thus, in the present study, α-amylase (Diastase) was immobilized in calcium alginate gel beads. The conditions of entrapment like concentration of sodium alginate, calcium chloride and bead size were optimized for highest apparent activity. The entrapped α-amylase was characterized in terms of optimum temperature and pH, kinetic parameters and compared with those of free α-amylase. MATERIALS AND METHODS Materials Sodium alginate, calcium chloride, α-amylase (Diastase), starch and DNSA (3, 5-Dinitrosalycyclic acid) were purchased from Himedia (Mumbai). All the other chemicals used were of analytical grade. Preparation of enzyme solution Freeze-dried α-amylase (Diastase) was added to 0.1 M sodium phosphate buffer (pH 7.0) to the concentration of 1 mg/ml. This enzyme stock solution was stored at 4°C for future tests.