Purification and Some Properties of a Subtilisin Inhibitor from Adzuki Beans

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A subtilisin inhibitor was isolated from adzuki beans (Phaseolus angularly) by chromatography on CM-cellulose, Sephadex G-75, DEAE-cellulose, and SP-Sephadex C-25. The final preparation was confirmed to be homogeneous on polyacrylamide gel electrophoreses, and its pi value was 3.7. The preparation was a powerful inhibitor of microbial serine-proteinases but its activity was destroyed by trypsin and chymotrypsin. Dissociation constant of the complex of the inhibitor with subtilisin was 0.16nM. The inhibitor was heat-stable over the pH range examined in spite of a lack of intramolecular disulfide bonds: little or no subtilisin-inhibitory activity was lost at 80°C for 10min, though heating to 100°C at.pH 12 caused a decrease of about 50% in the activity. The inhibitor molecule consisted of 97 amino acid residues, containing relatively large amounts of glutamic acid and valine residues and no half-cystine residues, unlike adzuki-bean proteinase inhibitors I and II. The molecular weight of the inhibitor was determined to be 12, 300 by gelfiltration and calculated to be approximately 11, 000 on the basis of the amino acid composition.
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