Abstract
An α-amylase was purified from germinating cotyledons of Phaseolus vulgaris L. cv Toramame by precipitation with ammonium sulfate, chromatographies on β-cyclodextrin Sepharose 4B and Bio-Gel P-200 columns. This enzyme migrated as a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be about 46, 000 by SDS-disc gel electrophoresis and the optimum pH was 6.0. The rate parameters for the hydrolysis of a series of maltooligosaccharides were estimated by the Lineweaver-Burk plots. The ratios of the V values for malto-triose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrin (d.p.=18) were estimated to be 5.9: 9.2:10:14: 36:108:100, and the Km values for these substrates were 143, 88, 77, 20, 5.5, 4.8, and 3.3 mM, respectively. Based on the these rate parameters and on the action mode of this enzyme toward each maltooligosaccharide, the Subsite structure was assumed to be made up of eight subsites. Those affinities (A1 s) in the active site of the enzyme were evaluated to be 1.0, 0.15, 4.7 and 0.58 (or 0.77) kcal/mol for Subsites 3, 4, 5 and 8, respectively, and the sum of the affinities of Subsites 1 and 2 was 2.2 kcal/mol, and that of Subsites 6 and 7 was -2.8 kcal/mol.
