Australian Journal of Basic and Applied Sciences, 3(3): 2883-2887, 2009 ISSN 1991-8178 Immobilization of a Thermostable á-amylase on Calcium Alginate Beads from Bacillus Subtilis KIBGE-HAR 1 2 1 1 Aliya Riaz, Shah Ali Ul Qader, Abida Anwar and Samina Iqbal 1 Pharmaceutical Research Centre, PCSIR Laboratories complex, Karachi, Pakistan 2 The Karachi Institute of Biotechnology and Genetic Engineering 9KIBGE), University of Karachi. Abstract: Alpha-amylase from B. Subtilis KIBGE-HAR was partially purified by 40 % ammonium sulfate upto 7 folds and then immobilized by entrapment in calcium-alginate beads. The catalytic properties of the immobilized á-amylase were compared with that of the free enzyme. The optimum ph of the free enzyme was 7.0 while that of immobilized enzyme was ph 7.5. The optimum temperature for free and immobilized enzyme was 60 c and 70 c respectively. The activity yield of the immobilized enzyme was 65 %. A substrate maximum for immobilized enzyme was changed from 2 % to 3%. Incubation time for enzyme-substrate reaction was remained same i.e. 5 minutes for the free and immobilized á-amylase. Key words: amylase, Immobilization, Alginate beads, characterization INTRODUCTION A biocatalyst is termed immobilized, if its mobility has been restricted by chemical means. Immobilization of enzymes refers to techniques which represent variety of advantages over free enzyme catalysis including increased stability of enzyme, easy recovery of enzyme, easy separation of reactant and product, repeated or 1 continuous use of a single batch of enzyme (Varavinit et al., 2002) which will ultimately save the enzyme, labor and overhead costs (Gerhartz, 1990). Immobilized enzymes have been widely used for many years in different industrial processes. Usually, immobilization of enzymes is carried out by three principle means, matrix assisted entrapment of enzyme, adsorption on a solid support, ionic or covalent binding (Swaisgood, 1985; Zaborsky, 1973). Entrapment is taken as the most preferable method because it prevents excessive loss of enzyme activity after immobilization, increases enzyme stability in microenvironment of matrix, protects enzyme from microbial contamination (Cabral and Kennedy, 1993). Physical entrapment of á-amylase in calcium alginate beads has shown to a relatively easy, rapid and safe technique (Dey et al., 2003) in comparison with other immobilization methods. The method method of immobilization should be such that an enzyme faces as little conformational change as possible. The nature of the solid support or matrix plays an important role in retaining the actual confirmation and activity of enzyme in the processes that utilized immobilized biocatalysts. Thermostable á-amylase is one of the most important and widely used enzymes whose spectrum of application has widened in food, paper and detergent industries (Glazer et al., 1994, Nigam and Singh, 1995). These industries would find their boosted economy if á-amylase can be re-used which is possible by their immobilization. Therefore, the present study is attempted to immobilize á-amylase produced by Bacillus subtilis KIBGE-HAR by entrapment in calcium alginate beads. We also compared the kinetics of free and immobilized á-amylase in order to explore the benefits of immobilization of enzymes. MATERIALS AND METHODS Materials for Immobilization: Sodium alginate (4%), Calcium chloride (0.2 M) and soluble starch obtained from Merck. Corresponding Author: Dr. SHAH ALI UL QADER, Assistant Professor Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi-75270 Pakistan. Ph #: 92-321-2160109 Fax #: 92-021-2229310 Email: madar_chem@yahoo.com 2883
Media Composition: á-amylase was produced from B. Subtilis KIBGE-HAR cultivated in a medium composed of (g/l): 15.0 soluble starch, 1.0 Yeast extract, 5.0 Bacto Peptone, 0.5 MgSO4, 0.5 NaCl and 0.002 CaCl2. The ph of the medium was adjusted to 7.0 before sterilization. All conditions of growth were kept same as described previously (Aliya et al., 2007). Isolation of Crude Enzyme: After 24 hours, fermented broth was centrifuged at 10,000 r.p.m for 15 minutes at 0 C to obtain the cell free filtrate. Partial Purification of á-amylase: The cell free filtrate was then precipitated with 40 % (NH 4) 2SO 4. The precipitation was carried out at 4 c under constant stirring and then left at 4 c for 1 hour. The precipitated protein was dialyzed in 50 mm Tris- HCl buffer of ph 7.0 to remove the remaining salt. Specific activity of enzyme was estimated in the dialyzed fraction. Enzyme Immobilization: An equal volume of the dialyzed enzyme and 4 % sodium alginate solution was mixed. The mixture obtained was extruded drop wise through a burette, into a gently stirred 0.2 M CaCl 2 solution at 4 c from a height of about 2 cm. The beads were left in the CaCl2 solution for about 20 minutes and then thoroughly washed with distilled water three times and used for further studies. Immobilized Enzyme Assay: The activity of immobilized enzyme was assayed by incubating 1 ml of 3 % (w/v) starch solution (prepared in 50 mm Tris-HCl buffer of ph 7.5) with 1 g of calcium alginate beads, at 70 c for 5 minutes. The á-amylase level was determined by measuring the reducing sugar released from soluble starch (Nelson 1944, Somogyi 1945). An enzyme unit is defined as the amount of a-amylase that liberates 1 mmol of reducing sugar from the substrate per gram of beads under the assay conditions. Immobilization Calculation: Weight of the beads = 4.57 gm Volume of enzyme trapped in beads = 5.5 ml Volume of enzyme trapped = 1.2 ml / g Units of enzyme before entrapment = 800 U/ml/min Units of enzyme after immobilization = 627 U/g/min Determination of Immobilization Efficiency Units of enzyme before immobilization = 800 U/ml Units of enzyme after immobilization = 522 U/ml Immobilization efficiency = 65 % Estimation of Total Protein: Total protein was estimated by Lowry method (Lowry et al., 1951). The bovine serum albumin (250 mg/ml) was used as standard. RESULTS AND DISCUSSION Effect of Sodium Alginate Concentration: It has been reported that the degree of cross linking of the gelling agent affects the pore size of the beads (Longo et al., 1992), therefore various concentrations of sodium alginate were used to achieve the highest immobilization efficiency. The immobilization efficiency was found to be highest for 4% sodium alginate solution (figure 1). Pore size of the beads should be such that substrate and product can easily diffuse in and out of the alginate get matrix but the enzyme should retain in the micro environment of beads. Lower the concentration of sodium alginate solution, greater will be the pore size of the beads and consequently leakage of enzyme from the beads will increase. Similarly, the pore size of beads will decrease with the increase in concentration of sodium alginate solution. Increased sodium alginate concentration interfered the entry of substrate into the beads; that led to the lower immobilization efficiency (Dey et al., 2003). 2884
Fig. 1: Effect of Sodium-alginate concentration on immobilization efficiency Effect of Reaction Time for Immobilized Á-amylase Activity: Effect of reaction time on activity of immobilized and soluble á-amylase is shown in figure 2.It was found that the maximum activity of both the soluble and immobilized á-amylase was achieve at 5 minute suggesting that the reaction rate of enzyme and substrate is independent to immobilization as the diffusion limitation for substrate has not been seen after entrapment of enzyme. It may conclude that pore size of the beads is optimum for the passage of substrate into the beads. Fig. 2: Time course of immobilized á-amylase activity Effect of Substrate Concentration on Immobilized Á-amylase Activity: Optimum substrate concentration for immobilized enzyme was higher than that of the soluble enzyme (figure 3). The activity of immobilized enzyme was found to be maximum when 3% substrate was used for reaction. This increased requirement of substrate upon immobilization has been reported earlier as well (Ul Qadar et al., 2007) Diffusion of large molecule will obviously be limited by steric interactions with the matrix. As starch is high M.W. polysaccharide, its diffusional resistance from the bulk solution to the micro environment of an immobilized enzyme can limit the rate of reaction. Fig. 3: Effect of substrate concentration on soluble and immobilized á-amylase Activity Effect of Ph on Immobilized Á-amylase Activity: Figure 4 showed the ph profile of free and immobilized á-amylase activity. Optimum ph for entrapped á-amylase activity was shifted from 7.0 to 7.5 after immobilization. This result demonstrated that the immobilized enzyme has slightly higher ph stability as compare to free enzyme. This shifted ph optimum upon immobilization has been reported earlier as well (Siva Sai Kumar et al, 2006). 2885
Fig. 4: Effect of ph on soluble and immobilized á-amylase Activity Effect of Temperature on Immobilized Á-amylase Activity: In the present case, the operating temperature of immobilized enzyme was raised from 60 c to 70 c (figure 5). The higher temperature profile of immobilized á-amylase could result from a lower temperature in the gel microenvironment compared to the bulk solution (Kennedy, 1987). This property of increased stability of á- amylase at higher temperatures following immobilization (Raviyan et al., 2003) can increase its application in starch processing, brewing and sugar production (Al-Qodah et al., 2007). Fig. 5: Effect of temperature on soluble and immobilized á-amylase Activity ACKNOWLEDGMENTS The authors are very grateful to Dr. Afsheen Aman and Dr. Saeeda Bano for providing the facilities for identification study using 16s RNA technique. Conclusion: Newly isolated strain Bacillus subtilis KIBGE HAR is capable of producing a thermostable alpha amylase which can be use in industry. The immobilization of this enzyme in calcium alginate will increase its stability as well as shelf life which will help this enzyme in repeated use. REFERENCES Al-Qodah, Z., H. Daghstani, P.H. Geopel and W. Lafi, 2007. Determination of kinetic parameters of alpha-amylase producing thermophile Bacillus sphaericus. African Journal of Biotechnology, 6(6): 699-706. Aliya, R., S.A. Qader, A. Anwar, S. Iqbal, S. Bano, 2007. Effect of medium composition and time course on the production of alpha-amylase from Bacillus stearothermophilus. Pak. J. Biochem. Mol. Boil., 40(2): 51-54. Dey, G., V. Nigpal, R. Banerjee, 2002. Immobilization of Alpha-amylase produced by Bacillus circulans GRS 313 on Coconut fibre. Applied Biochemistry and Biotechnology, 103(1-3): 303-314. Gerhartz, Wolfong., 1990. Enzymes in Industry. Weinheim, FRD. A.N. Glazer and H. Nikado, 1994. Microbiol Biotechnology. W.H. Freeman and Co., New York, N.Y. 2886
Kennedy, J.F. Enzyme technology. In Biotechnology; Kennedy, J.F., Cabral, J.M.S., Eds, VCH Publ.- Verlagsgesellschaft mbh: Weinheim, Germany, 1987: 7a. Longo, M.A., I.S. Novella, L.A. Garcia and M. Diaz, 1992, Diffusion of proteases in calcium alginate beads. Enzyme Microb. Technol., 14(7): 586-590. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193(1): 265-275. Nelson, N., 1944. A photometric adaptation of Somogyi method for the determination of glucose. Journal of Biological Chemistry, 153: 375-381 Nigam, P. and D. Singh, 1995. Enzyme and microbial system involved in starch processing. Enzyme Microb. Technol., 17(9): 770-778. Qader, S.A., S. Bano, A. Aman, N. Syed and A. Azhar, 2006. Enhanced production and extracellular activity of commercially important amylolytic enzyme by a newly isolated strain of Bacillus sp. AS-1. Turk J Biochem., 31(3): 135-140. Raviyan, P., J. Tang and B.A. Rasco, 2003. Thermal stability of alpha-amylase from Aspergillus oryzae entrapped in polyacrylamide gel. J. Argic. Food Chem., 51(18): 5462-5466. Saiyavit, V., C. Narisa and S. Sujin, 2002. Immobilization of a thermostable Alpha-amylase. Science Asia, 28(3): 247-251 Swaisgood, H.E., 1985. Immobilization of enzymes and some applications in the food industry. In Enzymes and Immobilized Cells in Biotechnology. (A.I. Laskin) ed., The Benjamin/Cummings Publishing Company, Inc. London, pp: 1-24 Somogyi, M., 1945. A new reagent for the determination of sugar. Journal of Biological Chemistry., 160: 61-68. Siva Sai Kumar, R., K.S. Vishwanath, A.S. Sridevi and Appu A.G. Rao, 2006. Entrapment of á-amylase in alginate beads: Single step protocol for purification and thermal stabilization. Process Biochemistry, 41(11): 2282-2288. Zoborsky, O.R., 1973. Entrapment within cross linked polymers. In: Immobilized enzymes. CRS Press., pp: 83-91. 2887