A keratinase enzyme was isolated and purified from a feather-degrading culture of Aspergillus oryzae. Fractional precipitation of the crude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at 75-85% ammonium sulfate saturation showed the highest activity and about 3.3-fold purification. This fraction was further purified by gel filtration in Sephadex G-75 followed by ion exchange chromatography on DEAE-Sephadex A-50 yielding an active major protein peak showing 11.38-fold purification. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 60 kDa. The purified enzyme was able to hydrolyze different substrates showing its highest proteolytic activity on bovine serum albumin and casein followed by keratin, chicken feathers, collagen, duck feathers and sheep wool. The purified enzyme was immobilized on various carriers. Immobilization on sintered glass beads showed the highest activity. The optimum pH of the immobilized enzyme shifted to a more neutral range (7.0-7.4) compared with the free enzyme (8.0). The optimum temperature of the reaction was determined to be 60degreesC for the immobilized enzyme and 50degreesC for the free enzyme. The free keratinase enzyme was retained 42.05% of its activity at 70 degreesC (60 min) while the immobilized keratinase preparation showed a higher thermal stability. The half-lives of the free and immobilized enzyme were 45.45 and 60.00 min, respectively. The pure enzyme was activated by calcium and barium ions while EDTA and Pb inhibited the activity. (C) 2003 Elsevier Inc. All rights reserved.