Purification And Characterization Of A Phospholipase C Secreted By Vibrio Harveyi

Vibrio harveyi, conditional pathogenic bacteria, is widespread in marine environment. It is the causative agent of vibrosis, one of the major bacterial disease affecting marine fishes and else cultured animals in the world. The worldwide distribution of vibrosis causes great economic loss to the aquaculture industry. Studies on vibrosis revealed that pathogenicity of V. harveyi could be attributed to the presence of extracellular virulence factors such as proteases, haemolysins, chitinases, lipases and phospholipases. This paper first described partial purification of one secreted phospholipase C from the extracellular products of a pathogenic bacteria, V. harveyi strain SF-1 isolated from diseased seaperch (Lateolabrax japonicus), and characterized the properties of the purified enzyme. A phospholipase C (PLC) was successfully purified by ammonium sulfate precipitation followed by a application of ion-exchange chromatography (DEAE-Sepharose Fast Flow) and gel filtration (Sephadex G-100 chromatography).The molecular mass of the PLC was determined as 64 kDa using SDS-PAGE, which was in agreement with the results obtained from Sephadex G-200 column chromatography (65 kDa), suggesting that the PLC was a monomeric protein. The hydrolysis products profiles of the purified enzyme and standard phospholipase C (Clostridium perfringens) by thin layer chromatography were the same, suggesting that the purified enzyme belonged to the PLC family.The physical-chemical properties of the enzyme are as follows: The optimum temperature and pH of the PLC was 50℃and pH 8.0, respectively. The enzyme activity decreased greatly when temperature was above 40℃, suggesting that the PLC was heat-labile. The enzyme was stable at pH 7.0. But the PLC activity decreased greatly, when pH was above 8.0. 1mmol/L of Co2+ and Ca2+ increased the enzyme...