Purification and characterization of a novel exocellular keratinase from Kocuria rosea

A keratinolytic protease activity secreted by Kocuria rosea when cultured in bioreactors using feathers as unique carbon and nitrogen source was purified and characterized. This novel keratinase activity was purified from the bioreaction broth growing media to apparent homogeneity after single step, (24-fold purification with a high yield of 54%) using DEAE column chromatography. The native molecular mass of the enzyme determined by gel filtration chromatography was 240 kDa. K. rosea extracellular keratinase was stable in a broad range of pH (8–11) and temperature (10–60 °C) profile with optimums at pH 10 and 40 °C. Crystalline soybean trypsin inhibitor (type I-S), 4-(2-aminoethyl) benzenesulfonyl floride (AEBSF) and chymostatin, strongly inhibited the keratinolytic activity indicating that the keratinase belongs to the serine protease family. The Km for the soluble keratin degradation from feathers was 242 μM. The enzyme was resistant to denaturing or reducing agents such as dithiotreitol and 2-mercaptoethanol. All of the biochemical characteristics, raising the potential use of this enzyme in numerous industrial applications.