火龙果种子清蛋白的理化性质及其胰蛋白酶抑制活性研究

Physico-Chemical Properties and Trypsin Inhibitory Activity of Seeds Albumin From Red Pitaya (Hylocereus polyrhizus)

为阐明火龙果种子清蛋白的理化性质和生物学活性,本研究从红心火龙果中纯化获得种子清蛋白(HPA),分析其分子量、氨基酸组成、同源性及胰蛋白酶抑制活性。结果表明,HPA由两条分子量为12.6、13.2 kDa的多肽链(HPA-1,HPA-2)组成。HPA富含谷氨酸(6.297 g·100g^(-1))、精氨酸(3.992 g·100g^(-1))和天冬氨酸(2.694 g·100g^(-1))。HPA与来自甜菜和菠菜2S种子贮藏蛋白具有高度相似性。HPA-2显示出较高的胰蛋白酶抑制活性,比活力为9.19×10^(3) TIU·mg^(-1),纯化倍数为1.86。经大豆胰蛋白酶抑制剂-琼脂糖凝胶柱纯化的组分HPA-2-1是胰蛋白酶竞争性抑制剂,抑制常数(Ki)为0.62 nmol·L^(-1),具有Kunitz型抑制剂的摩尔比曲线。HPA-2-1在一定的pH值(2-10)和温度(30-70℃)范围内具有稳定的抑制活性,且抑制活性几乎不受二硫苏糖醇(DTT)的影响。本研究为挖掘火龙果种子资源,丰富胰蛋白酶抑制剂的种类提供了参考。

In order to clarify the physico-chemical properties and biological activity of seed albumin from red pitaya, this study purified seed albumin(HPA) from red pitaya. The molecular weight, amino acid composition, homology and trypsin inhibitory activity of HPA were analyzed. The results showed that HPA consisted of two polypeptide chains(HPA-1, HPA-2) with the molar mass of 12.6 and 13.2 kDa, respectively. HPA was rich in glutamic acid(6.297 g·100 g^(-1)), arginine(3.992 g·100 g^(-1)), and aspartic acid(2.694 g·100 g^(-1)) using amino acid analysis. HPA had high similarity with 2 S seed storage protein from Beta vulgaris subsp. Vulgaris and Spinacia oleracea. HPA-2 showed trypsin inhibitory activity with a specific activity of 9.19×10^(3) TIU·mg^(-1) with a 1.86-purified fold. The single fraction HPA-2-1 purified by STI-berpharose column was a trypsin competitive inhibitor with Ki value of 0.62 nmol·L^(-1), and it had molar ratio curve of Kunitz-type inhibitors. HPA-2-1 had stable inhibitory activity in a broad range of pH value(2 to 10) and temperature(30 to 70℃). Its inhibitory activity was hardly affected by dithiothreitol(DTT). This study provided a reference for exploring the seed resources of pitaya and enriching types of trypsin inhibitors.