This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40–60%) followed by a gel filtration through Sephadex G–100 and DEAE– cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS–PAGE to be 22 kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS–PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35–40 °C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The K_m values obtained for this protease were 5.4, 13, 160 and approximately 1000 μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5 mM) or iodoacetamide (10 mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

这项研究描述了分离和分离自Fasciola hepatica的稳定蛋白酶活性的表征通过使用丙酮沉淀（40-60％），然后通过Sephadex G-100和DEAE-纤维素离子交换柱进行凝胶过滤，可以对成虫进行体外维持。通过此三步纯化，将酶纯化了11倍，比活为1893.9 U / mg，回收率为31.5％。在最后的超滤步骤之后，纯化倍数增加至13.1，总活性收率达到18.8％。通过将SDS-PAGE降低到22 kDa可以估算出纯化蛋白酶的MW，而酶解活性的检测是通过酶法对以酪蛋白为底物的非变性SDS-PAGE进行的。使用这种底物，蛋白酶在pH 5.5和35-40°C的温度下表现出极强的蛋白水解活性，并且在5.0至10.0的宽pH范围内都高度稳定。除了偏爱Z-Phe-Arg-AMC荧光底物在pH 7.0下产生最大的蛋白水解活性（99.7％）外，纯蛋白酶在pH 5.5时对血红蛋白和酪蛋白底物表现出最高的裂解活性（85.6％和82.8％） ， 分别）。K分别使用荧光底物Z-Phe-Arg-AMC，血红蛋白，酪蛋白和白蛋白，对该蛋白酶获得的_m值为5.4、13、160和约1000μM。蛋白酶活性被E-64抑制剂（5 mM）或碘乙酰胺（10 mM）完全抑制，表明其半胱氨酸性质。通过免疫印迹研究了纯化的蛋白酶作为抗原的有用性。因此，在实验中感染了肝炎链球菌的绵羊血清在感染后2周（WPI）并在7 WPI时强烈识别了蛋白酶带。早期检测到的抗肝炎链球菌抗体表明该分子可作为筋膜炎疾病血清学诊断的特异性表位。