Isolation and Characterization of a Thermostable Trypsin Inhibitor from Black Soybean

A thermostable trypsin inhibitor , designated TSTI ( thermostable trypsin inhibitor ), was isolated from seeds of black soybean ( Glycine max L. merr ) . The purification steps involved ammonium sulfate precipitation , ion-exchange chromatography on CM-Sephadex C-50 , gel filtration chromatography on Sephacryl S-200HR and HPLC on POROS HS-20. Its N- terminal amino acid sequence was DEYSKPCCDLCMCTRRCPPQ , which exerted highly homologies with the sequence of Bowman-Birk type trypsin inhibitors. It was speculated that TSTI should be classified Bowman-Birk type trypsin inhibitor. The moleculr weight and isoelectric point of the inhibitor were estimated to be 23. 9 kD and 6. 2 according to SDS-PAGE and IEF , repectively. Trypsin could be completely inhibited by TSTI when the molar ratio was beyond 1. The trypsin inhibitory activity of TSTI was unaffected after exposure to temperatures up to100℃ , as well as to pH 2-12. The inhibitor showed antifungal activity toward the species of Pythium aphanidermatum , Fusarium oxysporum , Alternaria alternata ( Fr. ) Keiss , Fusarium oxysporum f. sp.