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

OLIVERA, NELDA; SEQUEIROS, CYNTHIA; FAUSTINO SIÑERIZ; BRECCIA, JAVIER

World Journal of Microbiology and biotechnology

Springer

Año: 2006 vol. 22 p. 737 - 743

The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis , 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 05T 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.