Purification and characterization of a 43 center dot 5 kDa keratinolytic metalloprotease from Microsporum canis

A keratinolytic protease secreted by a feline clinical isolate of Microspor um canis cultivated in a broth containing feline keratin as the sole nitrog en source was purified from the culture filtrate by affinity chromatography on bacitracin-agarose and by hydrophobic chromatography on octyl-agarose. The enzyme had an apparent molecular mass of 43.5 kDa and the pI was 7.7. I t had a significant activity against keratin azure, elastin-Congo red and d enatured type I collagen (azocoll), Using the latter substrate, the optimum pH was around 8 and the apparent optimum temperature around 50 degreesC, T he protease was strongly inhibited by 1,10-phenanthroline, phosphoramidon a nd EDTA, The first 13 N-terminal amino acid sequence showed a 61% homology with that of the extracellular metalloprotease of Aspergillus fumigatus and with the neutral protease I of A. oryzae, confirming that this 43.5 kDa ke ratinase is a metalloprotease, This keratinolytic metalloprotease could be a virulence-related factor involved in pathophysiological mechanisms of M, canis dermatophytosis.