Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats

A keratinolytic protease, secreted as the major component by a feline clini cal isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin-agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pi was 11.8. The enzyme was not glycosylated and its first 15 N-ter minal amino acids showed numerous similarities with other fungal subtilisin s. The optimum pH was around 9 while inactivation of the enzyme was reversi ble at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with a n apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin i nhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The high est affinity (K-m of 0.37 mM) and physiological efficiency (k(cat)/K-m) wer e obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide . These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins cf prepared agains t the keratinase and used in an immunohistochemical test allowed the detect ion of the keratinase produced by the fungus invading hair structures in na turally infected cats. The in vitro keratinolytic activity of the enzyme an d its production in vivo suggest that it may contribute to pathogenicity.