Purification and characterization of hemolysin from Prevotella oris

We observed hemolytic activity in culture supernatant of Prevotella oris. Results from growth-phase experiments show that hemolysin production increased during the logarithmic growth phase and decreased during the stationary phase. The hemolysin produced by P. oris was purified from the culture supernatant by ultrafiltration, diethylaminoethyl (DEAE) and carboxymethyl (CM) ion-exchange chromatography, and gel filtration chromatography; further, we investigated the purified hemolysin characteristics, including its ability to lyse human, horse, sheep, and rabbit erythrocytes. The purified hemolysin was observed as a single, 16-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The specific activity was obtained by concentrating the purified hemolysin by 9200 fold. Although hemolysin was inactivated by heat treatment, ethylenediaminetetraacetic acid (EDTA), l-cysteine, dithiothreitol (DTT), and 2-mercaptoethanol enhanced its activity. Further, treatments using trypsin, MgCl2, CaCl2, and cholesterol did not affect its hemolytic activity. A pH of 6.0 was optimal for inducing the hemolysin activity. To the best of our knowledge, this is the first report describing the purification and characterization of hemolysin produced by P. oris.

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