Present study deals with the covalent modification of keratinolytic protease of Chryseobacterium gleum with higher enzyme activity, improved stability, non-immunogenicity and reusability. Protease of C. gleum showing feather degradation ability was modified by covalent attachment to polyethylene glycol. This modification culminated the change in electrophoretic mobility of protease in acrylamide gel. The modified enzyme showed 1.4 times more catalytic activity with better stability than native in aqueous system containing whole feathers as keratin. It showed improved pH, thermal, storage and solvent stability with a broadened range of pH (7-9) and temperature (25-50 degrees C) than native. The differentiation between modified and native enzyme was authenticated through UV-vis spectroscopy, SEM, XRD, FTIR and DSC. This modification of protease proved to be non-immunogenic in rats. The enzyme extracted after first run could be used for several cycles which clearly demonstrated its reusability in catalytic bioprocess of keratin degradation. (C) 2013 Elsevier Ltd. All rights reserved.