Purification and characterization of an alpha-amylase from the cotyledons of germinating lentils

Abstract

alpha-Amylase from the cotyledons of lentils germinated for 6 days (Lens culinaris, Medik) was purified by heat treatment, complexing with glycogen, Sephadex G-75 filtration and electrofocusing.Although three bands with alpha-amylase activity were separated in the purified extract from Sephadex G-75 filtration by polyacrylamide gel electrophoresis, only one alpha-amylase fraction was obtained by electrofocusing, which appeared free of contaminating proteins in the electrophoretic pattern.The purified enzyme had maximum activity at pH 5.4, an activation energy of 5.8 kcal/mol, a km for soluble starch of 3.4 X 10(-4) g/ml, an isoelectric point of 4.8 and a molecular weight of 43,000.The pH and temperature stability of the enzyme and the effect of calcium and mercuric ions on the enzyme activity and stability were also studied.