Isolation and Purification of Proteolytic Enzymes, Produced by Strains of Genus Bacillus

One hundred sixty-six bacterial strains belonging to genus Bacillus were tested for protease production. Ninety percent of the studied strains demonstrated protease activity on nutrient gelatin and milk agar. A strain Bacillus thuringiensis was selected as the most promising for enzyme production based on its initial enzyme activity of 9.2 U/ml. The nutrient medium composition and cultivating conditions were optimized aiming better yields. The highest protease activity of 15 U/ml was achieved on the following conditions: inoculation of the medium with 5% inoculum (6.0 McF), followed by 16 hours of cultivation in liquid medium containing 0.5% glucose, 0.55% Bacto Peptone, 50mM phosphate buffer and 0.2% magnesium ions. The produced enzymes were partially purified 5.6 fold by ultrafiltration and size-exclusion chromatography on Sephadex G-75 and had a specific activity of 17.7 U/mg. The approximate molecular weights were determined by SDS-PAGE to be between 45 and 66 kDa.