猪胸膜肺炎放线杆菌蛋白酶的分离纯化及特性分析

Purification and characterization of the protease from Actinobacillus pleuropneumoniae serotype 7

将血清7型猪胸膜肺炎放线杆菌(APP)山东分离株接种含0.2%NAD的LB液体培养基,37℃培养48 h后,经硫酸铵盐析、DEAE-纤维素离子交换层析、葡聚糖凝胶分子筛层析,从其培养上清液中分离纯化了1种蛋白酶。SDS-聚丙烯酰胺凝胶电泳显示,APP蛋白酶含有相对分子质量约为45 000的亚单位。以酪蛋白为底物测得该酶的最适pH值为7.5,最适温度为45℃;该酶对热有一定的稳定性,80℃加热30 min仍保留部分活性;乙二胺四乙酸(EDTA)可抑制其活性,而苯甲基磺酰氟(PMSF)对其无影响。

The Actinobacillus pleuropneumoniae serotype 7 strain isolated from Shandong province had been cultivated in the LB medium(containing 0.2%NAD)for 48 hours.The extracellular protease was purified by precipitation with 65% ammonium sulfate,iron-exchange chromatography on DEAE-cellulose G-100 and chromatography on Sephadex G-200.The result of SDS-PAGE of the purified protease indicated that the molecular mass of its subunit was 45 000.The optimum pH was 7.5 and the optimum temperature was 45℃.Its activity coul...