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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: To isolate and purify a black bean trypsin inhibitor and study its enzymatic properties and its biological properties to provide a basis for the study of its bioactive proteins. Methods: Separation, purification and identification were carried out by ammonium sulfate fractionation, weak cation exchange chromatography CM-Sephadex C-50, affinity chromatography Affi-gel and SDS-PAGE. The inhibitory effect of BSTI on trypsin active. Results: A trypsin inhibitor was isolated from black bean seeds and named BSTI. SDS-PAGE and anion-high-performance liquid chromatography identification showed that the protein has a relatively high purity. The relative molecular mass of 20 kD, isoelectric point of 5.6. When the amount of inhibitor to enzyme substance reaches 1, the enzyme is completely inactivated. At temperatures below 65 ° C and after pH 2 to 10 treatment, BSTI activity was almost unaffected. BSTI had a strong inhibitory effect on the pathogenic fungi of P. grisea, Pythium meliloti and Alternaria brassicae, but had no inhibitory effect on Fusarium oxysporum and Botrytis cinerea. Conclusion: A novel trypsin inhibitor with antibacterial activity was isolated and characterized, which can be separated and purified by ion exchange chromatography.