Purification and partial characterization of two extracellular alkaline proteases from Streptomyces corchorusii ST36

Two distinct alkaline proteases were detected in the culture medium of Streptomyces corchorusii ST36 isolated from an Egyptian soil sample. These enzymes were purified by precipitation with chilled ethanol and gel filtration on carboxymethyl Sepharose and on Sephadex G-120. The enzymes were purified 6 and 6.5 fold to homogeneity. The purified enzymes had final specific activities on substrate of 3.6 and 3.9 U/mg. Protease 1 had a molecular mass of 31 kDa and protease 2 was composed of two subunits, of molecular masses 18.6 and 17.4 kDa. The optimal pH and temperature for catalytic activity of protease 1 were pH 11 and 70 °C, respectively, and for protease 2 they were pH 10 and 70 °C, respectively. Protease 1 was more thermostable than protease 2. Both enzymes showed substrate specificity to casein, serum albumin, and ovalbumin. Calcium, copper, and cobalt stimulated protease 2 but did not significantly affect protease 1. Mercury was the strongest inhibitor for protease 2. The proteolytic activities of both proteases were inhibited by 10 mM phenanthroline and 50 mM ethylenediaminetetraacetic acid (EDTA). The inhibitory effect of EDTA on both enzymes was reversed by the addition of 50 mM of copper, calcium, or cobalt. Both enzymes were more stable at −20 °C under alkaline conditions than under neutral or acidic conditions.Key words: Streptomyces corchorusii, alkaline proteases, partial characterization.