ISOLATION AND CHARACTERIZATION OF A TRYPSIN-CHYMOTRYPSIN INHIBITOR FROM GLYCINE MAX SEEDS

摘要

A novel trypsin-chymotrypsin inhibitor with thermal and pH stability, designated Glycv, was isolated from Glycine max cv. seeds. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, affinity chromatography on Affigel blue gel. The 19 N-terminal amino acid sequences were determined to be EYSKPCCDLCMCTRRCPPQ, demonstrating highly homologies with the sequence of Bowman-Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by SDS-PAGE and isoelectric focusing to be 19.7 kD and 5.8, respectively. Trypsin could be completely inhibited by Glycv when the molar ratio was 8.3. The inhibitory activity of Glycv was unaffected after exposure to temperatures up to 89 degrees C, as well as pH 2-12. Moreover, the inhibitor Glycv demonstrated additional antifungal activity toward the species of Fusarium oxysporum, Alternaria alternate, Physalospora piricola, Pythium aphanidermatum, Botrytis cinerea and Fusarium solani. We report, for the first time, not only the trypsin inhibitor's purification, but also its N-terminal amino acid sequence and antifungal activity against a series of phytopathogenic fungi.