A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl‐Sepharose CL‐4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS–PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40°C to 50°C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10‐phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N‐terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco‐2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat‐labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.
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