Purification and partial characterization of serine-metallokeratinase from a newly isolated Bacillus pumilus NRC21.

A serine metallokeratinase enzyme (30 kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na(+), K(+), Mg(2+)), Hg(+2) had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45 °C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60 °C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(+2). These results suggest that the purified keratinase may be used in several industrial applications.