Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium, Salinivibrio sp. strain AF-2004

Among several strains of moderately halophilic bacteria that were isolated from various areas in Iran, strain AF-2004 was selected as the best producer of extracellular protease and was used for further studies. Phenotypic classification and 16S rRNA sequence analysis placed AF-2004 in the genus Salinivibrio. Maximal protease production was detected at the end of exponential growth phase in the medium containing 5% (w/v) NaCl. This protease was purified to homogeneity by a combination of acetone precipitation, Q-Sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The enzyme was a monomeric protease with a relative molecular mass of 38–43 kDa by SDS-PAGE and gel filtration chromatography. The specific activity of the purified enzyme was determined to be 171.6 μmol of tyrosine/min per mg of protein using casein as a substrate. The enzyme exhibited its optimal activity at 65 °C, pH 8.5, and 0–0.5 M NaCl with a high tolerance to salt concentrations of up to 4 M. The protease was identified as a zinc-metalloprotease, which was strongly inhibited by EDTA and 1,10-phenanthroline, and the N-terminal amino acid sequence determined showed high similarity to the zinc-metalloproteases from Vibrio species.