Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12

Abstract EN

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography.

It was a 45 kDa monomer as determined by SDS PAGE analysis.

It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60C∘, respectively.

It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and β-mercaptoethanol, respectively.

In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H2O2 and NaHClO3.

It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin.

Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides.

It also cleaved insulin B chain between Val2- Asn3, Leu6-Cys7 and His10-Leu11 residues.