A Study about the Protease Produced by Vibrio parahemolyticus bc-7

Oh, Yang Hyo; Cha, Mi Sun; Kim, Min Jung

Abstract

BACKGROUND
Bacterial extracellular protease has been implicated as an important virulence factor in several infections. In Vibrio sp., protease involvement has been demonstrated by the decreased virulence of protease-deficient mutants. We characterized the protease from V. parahemolyticus bc-7 with purification.
METHODS
100 Vibrio isolates from sea water and sea products were studied. The bacteria grown in Brain Heart Infusion broth were screened for protease production by incubating on tryptic soy agar plate containing gelatin and subsequently flooding the plate with 12.5% HgCl2-1M HCl. A clear zone indicated protease activity. Optimal conditions for protease production were determined by pH, temperature and NaCl concentration. Protease was purified from culture filtrates and its biological analysis was carried out.
RESULTS
Out of Vibrio isolates studied, V. parahemolyticus bc-7 produced protease in the highest titer. Maximal yields of the protease was obtained when the bacteria were grown in Brain Heart Infusion broth with initial pH of 8.0, 3% NaCl at 30degrees C. Production of the protease was optimal during the late exponential phase. Homogeneity of the purified protease was demonstrated by revealing a 65kDa band on SDS-PAGE. The protease was stable at 50degrees C and 70% of the activity was lost by heating at 60degrees C for 30 min. The protease was stable at a pH between 5~10, but under pH 4.0, the activity was lost completely.
CONCLUSION
General and serological studies are warranted to clarify the diffences and further characterize the protease as a virulence factor in Vibrio sp.