Purification and Characterization of a 315 Kda Keratinolytic Subtilisin-Like Serine Protease from Microsporum Canis and Evidence of Its Secretion in Naturally Infected Cats

TLDR: Results indicate that the keratinase belongs to the subtilisin-like serine protease family, which is similar to other fungal subtilisins.

Abstract: A keratinolytic protease, secreted as the major component by a feline clinical 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-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 whileinactivation of the enzyme was reversible at pH4, but not at pH 11. The enzyme was stable at 37 °C with an apparent optimum temperature around 55 °C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0·37 mm) and physiological efficiency (kcat/Km) were 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 G prepar...