黑豆种子中一种耐热型胰蛋白酶抑制剂的分离及性质表征

Isolation and Characterization of a Thermostable Trypsin Inhibitor from Black Soybean

应用硫酸铵分级沉降、弱阳交换色谱CM-Sephadex C-50、凝胶过滤色谱Sephacryl S-200HR、强阳离子高效液相色谱POROS HS-20,从黑豆种子中分离纯化一种耐热型蛋白酶抑制剂,命名为TSTI.该蛋白的N末端序列为DEYSKPCCDLCMCTRRCPPQ,与豆科植物Bowman-Birk型胰蛋白酶抑制剂具有高度同源性,推测其属于Bowman-Birk型胰蛋白酶抑制剂.SDS-PAGE和IEF测出TSTI分子量和等电点分别为23.9 kD和6.2.TSTI对胰蛋白酶有很强的抑制作用,当二者摩尔比达到1时,胰蛋白酶活力被完全抑制.此外,该蛋白酶抑制剂具有很强的热稳定性和pH稳定性,在高达100℃温度及pH2-12范围内处理,其活性均不会受到太大影响.TSTI对植物致病菌苹果轮纹病菌、瓜果腐霉病菌、白菜黑斑病菌、甜瓜枯萎病菌和葡萄灰霉病菌具有抑制作用.

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 tol00~C, 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. nelonis and Botrytis cinerea.