摘要
目的:分离、纯化一种黑豆胰蛋白酶抑制剂,并进行酶学性质及其生物学性质研究,为其生物活性蛋白的研究提供依据。方法:采用硫酸铵分级沉降、弱阳交换色谱CM-Sephadex C-50、亲和色谱Affi-gel和SDS-PAGE等方法分离、纯化和鉴定;以BANPA为底物,检测BSTI对胰蛋白酶的抑制活性。结果:从黑豆种子中分离到一种胰蛋白酶抑制剂,命名为BSTI。SDS-PAGE和阴离子高效液相色谱鉴定表明该蛋白具有相当高的纯度。其相对分子质量为20 kD,等电点为5.6。当抑制剂与酶的物质的量比达到1时,使酶完全失活。在温度65℃以下以及经pH 2～10处理后,BSTI活性几乎不受影响。BSTI对植物致病菌苹果轮纹病菌、瓜果腐霉病菌和白菜黑斑病菌具有较强的抑制作用,而对甜瓜枯萎病菌和葡萄灰霉病菌无抑制作用。结论:本文分离、表征到一种具有抗菌活性的新型胰蛋白酶抑制剂,可以使用离子交换层析柱进行分离、纯化。
Objective：It is to purify a trypsin inhibitor from black soybean（Glycine max L.merr） seeds and study the enzymatic and biological characterizations of the protein and provide basis for the bioactive protein study.Methods：The ammonium sulfate precipitation,ion-exchange chromatography on CM-Sephadex C-50,affinity chromatography on Affi-gel blue gel were used to isolate,purify and identify the trypsin inhibitor,the inhibitory activity of BST1 to trypsin was detected using BANPA as substrate.Results：A trypsin inhibitor,designated BSTI,was isolated.BSTI showed a high purity by SDS-PAGE and HPLC.Its molecular mass and isoelectric point were estimated to be 20 kD and 5.1,respectively.Trypsin was completely inhibited by BSTI when the molar ratio reached to 1.The trypsin inhibitory activity of BSTI was unaffected after exposure to temperatures up to 65 ℃,and after treatment with pH 2-12.The inhibitor exerted antifungal activity toward the species of Physalospora piricola、 Pythium aphanidermatum and Alternaria alternata（Fr.） Keiss,but no activity to Fusarium oxysporum f.sp.nelonis and Botrytis cinerea.Conclusion：The method presented in this paper was feasible to purify a trypsin inhibitor from Glycine max L.merr seeds and the newly purified protein was proved to be a new one with novel activity.The ion-exchange chromatographic column is high available and feasible.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2010年第6期47-53,共7页
Journal of Chinese Institute Of Food Science and Technology
基金
福建省新世纪优秀人才支持计划资助(2010-2013)
关键词
黑豆
胰蛋白酶抑制剂
纯化
表征
black soybean
trypsin inhibitor
purification
characterization
