Synthesis and characterization of alpha amylase-dextran conjugates

Amylases are extensively applied enzymes in industry which can hydrolyze the glycosidic bonds in starch. Alpha amylases (1,4-?-D-glucan glucanohydrolases; EC 3.2.1.1) are endo-enzymes responsible for the hydrolysis of internal ?-1,4 linkages of amylose and amylopectin in starch. ß-amylase can attack starch ?-1,4 bonds only on nonreducing ends of polymer. Alpha amylases are composed of about 478 amino acid residues and contained five subunits. Their molecular weights are about 50 000. Especially alpha amylase from Aspergillus oryzae (TAKA-amylase), is widely used in food, textile and detergent industries. It is known that many factors in industrial processes can influence the catalytic activity of enzymes negatively by affecting the enzyme?s structural state. Thus, chemical and genetic modification of enzymes has been used to improve their stability. In this study, ?-Amylase from Aspergillus oryzae (TAKA) is purified by gel filtration chromatography using ?Sephadex G?50?. Dextrans which molecular weights are 75 000 and 188 000, are oxidated to their aldehyde derivatives with NaIO4. and attached to the purified enzyme covalently. Conjugates are concentrated and washed by ultrafiltration system. The obtained conjugates were examined using GPC and HPLC. Activities of enzyme and conjugates are determined at different temperatures, at pH 7 and results are compared. It was observed that conjugates showed larger temperature range and higher thermal stability than purified enzyme at pH 7. Key words: Aspergillus Oryzae ?-Amylase, Dextran, ?-Amylase-Dextran conjugates, activity, enzyme stability, GPC, HPLC.