Production and characterization of thermostable metallo-keratinase from newly isolated Bacillus subtilis NRC 3

Novel keratinolytic enzyme (32 kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233 units/mg protein against 169 units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na+, K+, Mg2+, Ba2+, Ca2+, and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40 °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. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.