Identification and characterization of a protease produced by Vibrio parahaemolyticus in iron-limited medium.

Abstract

Two proteolytic proteins (about 43 and 90 kDa) were produced by clinical strains of Vibrio parahaemolyticus cultured in iron-limited medium. The 43 kDa-protease was partially purified by ammonium sulfate precipitation, ultrafiltration fractionation and DEAE-Sephacel chromatography. This protease had an optimum pH range of 7 to 8, and an optimum reaction temperature of about 40 degrees C. It was heat-labile, being partially inactivated by heat-treatment at 60 or 90 degrees C for 10 min. The protease hydrolyzed casein, gelatin, elastin, collagen and hemoglobin. As a chymotrypsin-like protease, it was inhibited only by the chymostatin among seven protease inhibitors tested. Activity of this protease was partially inhibited by 1 mM of Co2+, Cu2+, Zn2+ and Hg2+ and slightly enhanced by Ca2+ and Ba2+. It was completely inactivated by orthophenanthroline (OPA), and the OPA-inactivated sample was partially reactivated by Ca2+ and Fe2+. In conclusion, this 43-kDa protease of V. parahaemolyticus was an unstable neutral chymotrypsin-like metalloprotease; Ca2+ and/or Fe2+ was essential for its activity or stability.