Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp kr6

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic, bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967 U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64 kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1, 10-phenanthroline characterized Q I enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca2+. ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M 14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum. (c) 2006 Elsevier B.V. All rights reserved.128369370