Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca2+, Mg2+, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.