酶与抑制剂关系综述.doc

PAGE38 / NUMPAGES38 酶 与 抑 制 剂 关 系 综 述 摘 要 蛋白酶A(PrA)抑制剂因其生理特性在食品、医药、农业等领域有广泛的应用前景。马铃薯块茎汁液中含有多种蛋白类蛋白酶抑制剂，含量占所含可溶蛋白的 50 %以上，目前大多作为加工废水排放，若加以利用，可有潜在的经济效益和社会效益。本论文从马铃薯块茎中分离 PrA 抑制剂，对其基本性质做了初步鉴定，并对其在纯生啤酒及抑菌效果方面做了初步试验。 马铃薯块茎经匀浆离心后，上清液经分级盐析，取 20~30 %饱和度盐析部分，透析后经冻干即为粗抑制剂。粗抑制剂再经微孔膜过滤、Sephadex G-50 分子筛层析、CM 弱阳离子交换层析，得到进一步纯化，纯化倍数达 259.7。 PrA 抑制剂的性质研究表明：PrA 抑制剂为单亚基，分子量为 16.7 kDa在 100 ℃以下热稳定性良好；PrA抑制剂对 PrA抑制时的最佳 pH范围为 4.5～6.0，最佳反应时间为 1.5 小时以上；PrA 抑制剂对 PrA 的抑制效果随着酶抑比的增加，抑制作用迅速减弱，抑制剂对 PrA 的半抑制浓度(IC50)为 0.8mg/mL。PrA 抑制剂对几种不同来源蛋白酶：胃蛋白酶、胰蛋白酶、酸性蛋白酶、木瓜蛋白酶均有一定的抑制效果，其中对 PrA 和胰蛋白酶抑制作用最强。 PrA 抑制剂的动力学研究结果表明：PrA 抑制剂对 PrA 的抑制类型为竞争和非竞争性混合抑制作用。 作为应用研究的 PrA 抑制剂粗品的主要成分为蛋白和糖类，分别占含量的 32.8 %，22.6 %。PrA 抑制剂在纯生啤酒环境中对 PrA 有较好的抑制效果，最佳作用时间约为 2 小时以上，热处理后抑制效果稳定。对 PrA 的抑制效果随着酶抑比的增加，抑制作用迅速减弱。 抑制剂对属于革兰氏阴性菌的大肠杆菌有明显抑菌效果，而对属于革兰氏阳性菌的苏云金芽孢杆菌和枯草芽孢杆菌则没有明显抑制效果。 关键词：蛋白酶 A；抑制剂；纯化；性质；应用II  Abstract The inhibitors of proteinase A(PrA) have great prospect in food, medicine and agriculture field as it’s physiological nature. The potato tube juice contains lots of proteinase inhibitors,but it is volleyed as waste liquid nowadays, so it has potential economic returns and social effect if we utilize it. This paper’s major contents were pufification PrA inhibitor from potato tube, identifyed the characters,put it into draft beer and validated the inhibition effect to bacteriums. Potato stem tuber was homogenated then centrifuged, the supernatant was grade salted out, took the 30 % degree of saturation part as crude inhibitor after dialyzed and freeze dryed. The crude inhibitor passed microporous membranemembrane filted, Sephadex G-50 sieve chromatogram,CM cation-exchange chromatography. By these courses, the inhibitor was purified 259.7 times. The PrAI inhibitor had one subunit, it’s moleculer weight was 16.7 kDa, had good heat stability under 100 ℃; the best action condition was pH limit 4.5～6.0, action time more than 1.5 hour. The inhibition was sharped down as decreasing the ratio of enzyme to inhibitor, the IC50