Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis

Abstract

An extracellular, thiol-dependent and oxidation-stable alkaline serine protease (molecular mass 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)) from Bacillus mojavensis was purified to homogeneity with 17-fold purification as unbound fractions using a single step anion exchange chromatography on fast flow Q-sepharose column pre-equilibrated with buffer of pH 10.5. The N-terminal sequence of first 15 amino acids of purified protease showed 91% similarity with other subtilisins and alkaline proteases. The enzyme exhibited pH and temperature optima of 10.5 and 60 °C, respectively, and was stable between a wide pH range of 7.0 and 11.5 for 48 h. The half-lives of protease at 60, 65, and 70 °C were 150, 15 and 7 min, respectively. Various stabilizers and additives stabilized protease for more than 4 h at 60 °C, and 45 min at 65 °C. Specific protease inhibitors, such as phenylmethyl sulfonyl fluoride (PMSF), bestatin, chymostatin, iodoacetic acid, and N-bromosuccinimide completely inhibited the enzyme activity, whereas, the enzyme activity was increased up to more than two-fold in presence of 2-mercaptoethanol, glutathione, and dithiothreitol, suggesting it to be a thiol-dependent serine protease. Among metal ions, Cu 2+ and Mn 2+ ions increased enzyme activity up to 36%. The enzyme was also stable towards several commercially available laboratory bleaches (H 2O 2, sodium perborate), surfactants (tweens, Triton X-100, sodium choleate), and other commercial detergents used in laundries. The protease hydrolyzed several native proteinacious substrates, such as gelatin, elastin, albumin, haemoglobin, and skim milk, thus making it suitable for its use as an effective detergent additive. Wash performance analysis of enzyme revealed that it could effectively remove a variety of stains, such as blood, beetle and grass most effectively at 60 °C.