嗜水气单胞菌胞外蛋白酶ECPase54的纯化及特性分析

PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR PROTEASE ECPASE 54 FROM AEROMONAS HYDROPHILA

嗜水气单胞菌（Ａｈ）Ｊ－１株的液体培养物上清在５６℃或与乙二胺四乙酸（ＥＤＴＡ）或丝氨酸蛋白酶抑制剂苯甲基磺酰氟（ＰＭＳＦ）作用，其胞外蛋白酶（ＥＣＰａｓｅ）的活力有不同程度的下降。将酪蛋白掺入丙烯酰胺聚合后，加ＡｈＪ－１上清作ＳＤＳ－ＰＡＧＥ，３７℃原位消化，染色，显示上清中存在至少５种分子量不同的蛋白酶。ＡｈＪ－１株的培养上清经硫酸铵沉淀、ＤＥＡＥ－纤维素离子交换层析和ＳｅｐｈａｄｅｘＧ－２００分子筛层析，获得一种纯化的蛋白酶。不经２－巯基乙醇处理后，分子量约为５４０００，而经２－巯基乙醇处理的样品，分子量为３５０００，且酶活性丧失。该酶对热稳定，ＥＤＴＡ可抑制其活性，ＰＭＳＦ对其无影响。此酶属于热稳定金属蛋白酶（ＴＳＭＰ），建议命名为ＥＣＰａｓｅ５４。它的最适ｐＨ为７．５，米氏常数（Ｋｍ）为１．７７ｍｇ／ｍｌ，对Ｖｅｒｏ细胞有毒性，腹腔注射能致死小白鼠。

At least five extracellular proteases (ECPase) in Aeromonas hydrophila strain J 1 culture supernatant were assayed by in situ proteolytic activity on SDS PAGE containing 0.1% casein. One of the ECPases from the supernatant was purified by ammonium sulfate precipitation, anion exchange chromatography on DEAE cellulose and Sephadex G 200 gel filtration chromatography. SDS PAGE of the purified protease indicated that 2 ME reduced sample migrated at 35 000, while non reduced sample migrated at 54 000. This protease was accordingly designated ECPase 54. The activity of ECPase 54 was inhibited by EDTA, but not by PMSF or by heating at 56 ℃ for 30 min. It showed that the ECPase 54 was a thermostable metalloprotease (TSMP). The ECPase 54 with optimum pH 7.5 and Km 1.45 mg/ml possessed protyolytic and cytotoxic activities. It also elicited lethal toxicity to mice.