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Characterization of an extracellular keratinase from Microsporum canis
T Hamaguchi1, N Morishita, R Usui
1Department of Dermatology, Showa University, Fujigaoka Hospital.
Abstract:
Extracellular keratinase (Ekase) 48-, 34- and 31.5-kDa polypeptides, which were isolated from Microsporum canis and examined by immunoblotting reacted with a monoclonal antibody against Ekase of M. canis. We analyzed the amino acid and determined the first 17 amino acid NH2-terminal sequences of the 48-, 34- and 31.5-kDa polypeptides. These polypeptides had a high aspartic acid, glycine and alanine content, respectively. The first 17 amino acid residues of the 34-kDa polypeptide were homologous to those of thermomycolin. This indicated that the 34-kDa polypeptide of Ekase is homologous to the thermomycolin produced by Malbranchea pulchella. Furthermore, Ekase was very heat-stable in the presence of 50 mM CaCl2 at 55 degrees C, since 50% of the initial activity remained. In contrast, no activity was detected after heating in the absence of CaCl2. These results indicate a close relationship between dermatophytes and M. pulchella.
Insights
Extracellular keratinase (Ekase) from Microsporum canis showed heat stability with calcium chloride. Its 34-kDa form is homologous to thermomycolin, suggesting a link between dermatophytes and Malbranchea pulchella.
Area of Science:
- Mycology
- Biochemistry
- Enzymology
Background:
- Microsporum canis is a dermatophyte known to produce extracellular enzymes.
- Keratinases (Ekase) are enzymes that degrade keratin, a key structural protein.
- Understanding the properties and relationships of these enzymes is crucial for dermatophyte research.
Purpose of the Study:
- To characterize extracellular keratinase (Ekase) polypeptides from Microsporum canis.
- To determine the NH2-terminal amino acid sequences of Ekase polypeptides.
- To investigate the heat stability of Ekase and its relationship with other fungal enzymes.
Main Methods:
- Isolation and purification of 48-, 34-, and 31.5-kDa Ekase polypeptides from Microsporum canis.
- Immunoblotting using a monoclonal antibody against M. canis Ekase.
- Amino acid sequence analysis of the NH2-terminal regions of the polypeptides.
- Assessing Ekase activity and heat stability in the presence and absence of CaCl2.
Main Results:
- The 48-, 34-, and 31.5-kDa Ekase polypeptides reacted with an anti-M. canis Ekase antibody.
- The NH2-terminal sequences revealed high aspartic acid, glycine, and alanine content in the respective polypeptides.
- The 34-kDa Ekase polypeptide showed homology to thermomycolin from Malbranchea pulchella.
- Ekase exhibited significant heat stability at 55°C in the presence of 50 mM CaCl2, with 50% activity remaining, but lost activity without CaCl2.
Conclusions:
- The 34-kDa Ekase polypeptide from Microsporum canis is homologous to thermomycolin, indicating a relationship between dermatophytes and Malbranchea pulchella.
- Extracellular keratinase from M. canis is heat-stable, particularly in the presence of calcium ions.
- These findings suggest a close evolutionary or functional relationship between certain dermatophytes and Malbranchea pulchella through their keratinase enzymes.