Isolation, Purification and Characterization of β-amylase from Dioscorea hispida Dennst

beta-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The enzyme is an exo-acting carbohydrolase which hydrolyzes alpha-1.4-glucosidic linkages of starch, removing maltose units from the non-reducing end of the polysaccharide chain, producing beta-maltose and beta-limit dextrin as the final product. beta-amylase is widely distributed in the higher plants such as sweet potato. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-stalling agents in baking. This enzyme was extracted from Dioscorea hispida Dennst in 0.05 M acetate buffer pH 4.8 and followed by ammonium sulfate fractionation at cold temperature (10 degrees C). Ammonium sulfate fractionation was shared into fraction of 0-60%, 60-70%, 70-80% and 80-100%. The fraction containing high of specific activity (determined by Somogyi-Nelson and Lowry methods) was futher purified by dialysis. Fraction with high enzyme activity of beta-amylase were fraction 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst were 1.32 and 1.55 mg sugar. mg protein-1. minute-1, whereas specific activity of crude extract enzyme was 0.21 mg sugar. mg protein-1. minute-1. After purified with dialysis, fraction with high enzyme activity of beta-amylase were fraction of 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst was 2.72 and 4.24 mg sugar. mg protein(-1). minute(-1). The purified Dioscorea hispida Dennst beta-amylase from dialysis showed increasing in spesific activity the crude enzyme as much as 24 folds. The characterization of enzyme showed that Dioscorea hispida Dennst derived enzyme had optimum pH of 5.5 and temperature of 70 degrees C. The kinetic parameters of purified Dioscorea hispida Dennst beta-amylase showed that the K-M(app), V-max(app) value and Hill constant were 0.0211 mg/ml, 9.63 mg sugar. minute(-1) and 1.34, respectively.