Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

Summary The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis PAT 05^T, was characterized. This strain, grown in a highly alkaline and saline medium, produced important levels of proteolytic activity. SDS-PAGE and zymogram analyses revealed two proteolytic active bands. Through isoelectricfocusing (IEF)-zymogram, an active band with alkaline pI and two slighter active bands with acid pI values were detected. The alkaline active enzyme in the IEF was purified and characterized. It showed a molecular mass of 29.4 kDa and its pI value was >‰10.3. Proteolytic activity of the culture supernatant showed an optimal temperature of approximately 60 °C and a plateau of maximum activity between pH 9.0 and 12.0. Such activity was not affected by H₂O₂ (10% v/v), 1,10-phenanthroline (10 mM), Triton X-100 (1% v/v) and Tween 20 (1% v/v), under the assay conditions. More than 80% of the activity was retained in 10 mM EDTA, 73% in 1 % (w/v) SDS and 63% in 2 M NaCl. The enzyme was inhibited by PMSF, indicating serine-protease activity. The proteolytic activity of the crude supernatant was thermosensitive with a half-life of 2.3 min at 70 °C, while high activity was detected at moderate temperatures. Considering PAT 05^T proteolytic activity characteristics, such as high optimum pH, high stability and residual activity in presence of oxidant, surfactant and chelating agents, this strain could be a potential source of enzymes for use as additives in detergent formulations or in the leather industry.