马铃薯丝氨酸蛋白酶抑制剂的分离纯化、结构特征及构象稳定性研究

Potato tubers contain approximately 1.5% protein on a fresh weight basis, of which, protease inhibitors represent up to 50%. Although protease inhibitors have been long considered only as antinutritional factors, potato protease inhibitors have regained interest in recent years because of their possible anticarcinogenic and positive dietary effects with broad prospects for application in the food and pharmaceutical industries. Conventional protein recovery in the milling industry includes deleterious steps like steam pressure coagulation and acid precipitation. This results in irreversible denatured proteins of only animal feed grade.Our previous study has shown that expanded bed adsorption can be used for recovery native protein from potato fruit water, the recovered protein contained a group of proteas inhibitors, with potato serine protease inhibitor as the main fraction. Therefore, the aim of the present study is to isolate potato serine protease inhibitor from potato fruit water by gel filtration, ion exchange and hydrophobic interaction chromatography, to screen out the most abundant isoform of potato serine protease inhibitor, to study the structure of potato sereine protease inhibitor and the effect of pH and temperature on the content of α-helix,β-strand, β-turn and random coil, to understand the mechainsm of the irreversible precipitation occurring in industrial processes,thereby creating possibilities to obtain soluble potato proteins that can be used in food and pharmaceutical applications.

马铃薯块茎约含1.5%蛋白，其中约一半为蛋白酶抑制剂。过去长期把蛋白酶抑制剂当抗营养因子研究，近年来发现其具有抗癌和调节饮食的作用，在食品和制药工业中具有广阔的应用前景。马铃薯蛋白酶抑制剂重新成为马铃薯加工研究的热点。传统酸热絮凝法已导致马铃薯蛋白发生不可逆变性，回收的蛋白只能用作饲料。课题组的前期研究表明，扩张床吸附可用于从马铃薯淀粉加工分离汁水选择性回收具有天然活性的蛋白酶抑制剂，回收的蛋白含有多个蛋白酶抑制剂组分，且以丝氨酸蛋白酶抑制剂为主。本研究拟采用凝胶过滤、离子交换和疏水作用对马铃薯丝氨酸蛋白酶抑制剂进行分离纯化，筛选出相对含量和活力高的亚型，分别采用圆二色光谱、荧光光谱、FTIR和DSC对其结构和构象稳定性进行研究，明确pH和温度对蛋白α-螺旋、β-折叠、β-转角和无规则卷曲变化规律的影响，阐明马铃薯浓缩蛋白发生不可逆变性的机理，为食品级（药品级）马铃薯蛋白开发提供科学依据。

蛋白酶抑制剂大约占到马铃薯汁水中蛋白的50%。马铃薯蛋白酶抑制剂对多种蛋白酶具有广泛的抑制活性，如丝氨酸蛋白酶、半胱氨酸蛋白酶、天冬氨酸蛋白酶和金属蛋白酶。根据抑制蛋白酶的种类及其分子量大小、蛋白结构和等电点pH值，可以将马铃薯蛋白酶分成7组，抗体试验结果表明大约70%马铃薯蛋白酶抑制剂属于Kunitz型抑制剂。. 马铃薯蛋白酶抑制剂当中含量最高的是马铃薯丝氨酸蛋白酶抑制剂（Potato Serine Protease Inhibitor, PSPI），这是一种Kunitz型丝氨酸蛋白酶抑制剂，大约占到马铃薯总蛋白酶抑制剂的44%。PSPI各种同工酶占到总的胰蛋白酶和胰凝乳蛋白酶活力的80%和50%。基于结构特征，PSPI可以归类为β-II型蛋白，这种蛋白的残基含有不规则的和短的β-折叠。室温条件下，在pH3.0-7.5范围内，PSPI的二级结构和三级结构没有发生改变。而且，在这个pH范围内，PSPI的热诱导展开行为没有发生改变。PSPI的展开研究结果表明，热诱导和胍盐诱导都是在非二态机制（non-two state mechanism）下进行的，蛋白质结构当中至少有两部分是独立完成展开的。PSPI在加热时的变性聚集是通过特定的机制完成的，最终的产品是一个四聚体形式。