Purification and some properties of halophilic protease produced by a moderately halophilic marine Pseudomonas sp.

Halophilic protease in culture fluids of a moderately halophilic marine Pseudomonas sp. (A-14) was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 120 000. The optimum pH for activity was 8.0. The enzyme had maximal activity at 18% NaCl concentration. The enzyme was activated by Mg2+, Co2+, and Ca2+. Ca2+ increased the heat stability, and heavy metal ions such as Fe2+, Cu2+, and Hg2+ inactivated the enzyme. Thiol reagents and diisopropyl fluorophosphate did not affect the enzymatic activity of the protease. Metal-complexing reagents, such as ethylenediaminetetraacetic acid and o-phenanthroline, inhibited enzymatic activity, although citrate and oxalate did not affect it.