Purification, virulence and characterization of an extracellular peptidase produced by Aeromonas salmonicida ssp. masoucida isolated from cultured sea cucumber Apostichopus japonicus

Strain H1, Aeromonas salmonicida ssp. masoucida isolated from an ulcer on the sea cucumber Apostichopus japonicus proved to be virulent toward this holothurian at 12°C. Extracellular products (ECP) of strain H1 were prepared, and an extracellular peptidase of 42 kDa was purified from the ECP using Sephadex G-100 gel filtration chromatography and diethylaminoethyl Sepharose ion exchange chromatography. Stepwise virulence tests on sea cucumbers indicated that the 42 kDa peptidase was the virulence factor of strain H1, and its LD₅₀ for these holothurians was 1.12 μg g^-1. A peptidase activity test using azocasein as a substrate showed that optimum pH and temperature for the peptidase are 8.0 and 40°C, respectively; the peptidase was completely inactivated above 70°C. The peptidase was inhibited strongly by 5 mM phenylmethanesulphonyl fluoride, indicating that the enzyme is a serine peptidase.