Purification and characterization of an alpha-amylase from the cotyledons of germinating lentils.
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.
D000681:Amylases / Q000302:isolation & purification*; D002122:Calcium Chloride / Q000494:pharmacology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D013696:Temperature; D000516:alpha-Amylases / Q000302:isolation & purification*
D000681:Amylases; D000516:alpha-Amylases; D002122:Calcium Chloride