Purification and properties of a proteinase from a marine luminous bacterium, Vibrio splendidus strain FLE-2.

Abstract

A proteolytic, marine luminous bacterium was isolated from seawater and identified as Vibrio splendidus biotype I strain FLE-2. A proteinase from this strain, with a specific activity 21-fold higher than that of the culture supernatant, was purified to homogeneity. The purified enzyme had a molecular weight of 50,000. The enzyme was most active at pH 8.0 and 55°C, and stable below 35°C. The enzyme activity was completely inhibited by EDTA,orthophenanthrolin and phosphoramidon. Also metal ions such as Cu2+, Hg2+, Ni2+, Cd2+ and Co2+ inhibited the activity. These results indicate that this enzymeis a metal-chelater-sensitive, alkaline proteinase. Preparation of extracellular proteinase test medium. Extracellular proteinase activity was detected on milk seawater agar-seawater agar two layer plates. In preparing this test agar medium, the two layers were poured separately, in a similar manner to in the case of milk agarmarine agar two layer plates.21) The bottom layer consisted of 300ml of distilled water with 20g of skim milk dissolved in it, and 700ml of seawater containing 50 ml of 1 m Tris-HCl buffer (pH 7.8) and 15g of agar. The two solutions were autoclaved separately (15 min at 121°C), cooled to 55°C and then mixed aseptically just before pouring. After the milk seawater layer had solidified, the