Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra, Université de Sfax, Sfax 3038, Tunisia.
Biochemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia.
Molecules. 2020 Nov 20;25(22):5452. doi: 10.3390/molecules25225452.
The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from . Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6-7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl and sorbitol, whereas the presence of Ca and Zn ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.
本研究的主要目的是从 中提取、纯化和生化表征一种蛋白蛋白酶抑制剂。还研究了其抗菌潜力和细胞毒性作用。在 0.1 M 磷酸盐缓冲液（pH6-7）中提取蛋白酶抑制剂。然后，使用硫酸铵沉淀和 Sephadex G-50 柱过滤对蛋白酶抑制剂（命名为 PDInhibitor）进行纯化，其表观分子量为 25 kDa。PDInhibitor 的 N 端序列与 Kunitz 家族的序列具有高度的同源性。PDInhibitor 在 pH 值为 5.0-11.0 的范围内具有活性，在 pH9.0 时活性最大。它在 50°C 时完全活性，在超过 55°C 时保持 90%的稳定性。CaCl 和山梨醇明显增强了 PDInhibitor 的热稳定性，而 Ca 和 Zn 离子、Sodium taurodeoxycholate (NaTDC)、Sodium dodecyl sulfate (SDS)、Dithiothreitol (DTT) 和 β-ME 则极大地提高了其抑制活性。该蛋白酶抑制剂对现有重要治疗性蛋白酶（弹性蛋白酶和胰蛋白酶）具有显著的亲和力。PDInhibitor 还能强烈抑制 和 Proteinase K 的商业蛋白酶。该抑制剂对革兰氏阳性和革兰氏阴性细菌以及真菌菌株均具有强大的抗菌活性。有趣的是，PDInhibitor 还影响几种人类癌细胞系，即 HCT-116、MDA-MB-231 和 Lovo。因此，它可以被认为是一种有潜在效力的治疗剂。
