Purification by Affinity Chromatography of α-Amylase-A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

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In Vigna mungo cotyledons, the α-amylase activity increased markedly during germination. at 27℃ in the dark, while the activity of other amylases was very low. The α-amylase was purified from 4-day-old cotyledons by affinity chromatography on epoxy-activated Sepharose 6B substituted with β-cyclodextrin and by column chromatography on Bio-Gel P-200. Gel filtration and polyacrylamide gel electrophoresis showed that the enzyme exists mostly as a monomer (43,000 daltons), but partially aggregates to form dimer, trimer and further multimers. Ca^<2+> protected the α-amylase against heat inactivation. Incubation of the enzyme with 5 mM EDTA or dialysis against 1O mM EDTA resulted in a 50-90% loss of activity. The inactivation was partially reversed by the addition of Ca^<2+>. Other properties, such as the amino acid cornposition, K_m value, pH optimum and activation energy were similar to those of other plant α-amylases.
日本植物生理学会の論文