BACKGROUND: Keratinases are important enzymes for biotechnological processes involving keratin hydrolysis. In this work substrate specificity and kinetic properties of a keratinase from Chryseobaterium sp. were investigated. RESULTS: The optimal conditions for activity of purified keratinase with respect to pH, temperature and sodium chloride concentration were established using factorial design and surface response techniques. The optimum conditions for keratinase activity were pH from 7.4 to 9.2, temperature from 35 degrees C to 50 degrees C and NaCl concentration from 50 to 340 mmol L-1, having azocasein as substrate. Subsequently, the kinetic parameters for this substrate were determined to be K-m = 0.75 mg mL(-1) and V-max = 59.5 U min(-1). The K-i value for 1,10-phenanthroline was estimated at 0.78 mmol L-1. The enzyme specificity was evaluated over different synthetic and insoluble substrates. The protease exhibited specificity with selectivity for hydrophobic and positively charged residues. In relation to the insoluble substrates, the enzyme hydrolyzed preferably chicken nails. CONCLUSIONS: This enzyme effectively hydrolyzes insoluble keratin substrates. The knowledge of keratinase properties is an essential step in the development of biotechnological processes involving keratin hydrolysis. (C) 2008 Society of Chemical Industry