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Preliminary characterization of extracellular proteolytic enzymes of dermatophytes by chromogenic substrates
1Department of Biology, Medical Faculty, Palacký University, Olomouc, Czechoslovakia.
Abstract:
Thirty-eight chromogenic substrates were used to study the specificity of the proteolytic enzymes of seven species of dermatophytes and three related keratinolytic soil fungi. The source of enzymes were cultivation fluids from cultures of the fungi grown on human hair. The overall specificity of the enzymes of all the keratinolytic fungi was very similar. Aminoacyl- and dipeptidyl-4-nitroanilides, substrates of aminopeptidases and dipeptidyl aminopeptidases respectively, were poor substrates compared to aminoterminally blocked oligopeptidyl derivatives. Of the latter, the best substrates were those with phenylalanine, leucine, alanine, methionine or arginine (i.e. amino acids with hydrophobic or basic side chains) in the P1 position. Of the amino acids in the P2 position, proline was the most effective at accelerating the hydrolysis of the respective substrates. Positions P3 and P4 and even the aminoterminal protecting group were also of importance. The specificity profiles of the proteolytic enzymes corresponded best to those of some well characterized serine proteinases (chymotrypsin, elastase).
Insights
Fungal proteolytic enzymes showed similar specificity across species, preferring substrates with specific amino acids like phenylalanine or leucine. These enzymes resemble serine proteinases, crucial for keratin degradation.
Area of Science:
- Mycology
- Enzymology
- Biochemistry
Background:
- Dermatophytes and related fungi are key keratin degraders.
- Understanding their proteolytic enzymes is vital for applications in dermatology and biotechnology.
- Enzyme specificity dictates substrate preference and catalytic efficiency.
Purpose of the Study:
- To investigate the substrate specificity of proteolytic enzymes from various dermatophytes and keratinolytic soil fungi.
- To compare the enzyme profiles across different fungal species.
- To identify key amino acid residues and positions influencing enzyme activity.
Main Methods:
- Cultivation of seven dermatophyte and three soil fungi species on human hair.
- Preparation of fungal culture fluid enzyme extracts.
- Assay of enzyme activity using 38 distinct chromogenic substrates, including aminoacyl-, dipeptidyl-, and oligopeptidyl derivatives.
- Analysis of substrate hydrolysis rates based on amino acid composition at P1, P2, P3, and P4 positions.
Main Results:
- Overall, the proteolytic enzymes from all tested keratinolytic fungi exhibited highly similar substrate specificity.
- Aminoterminally blocked oligopeptidyl derivatives were superior substrates compared to aminoacyl- and dipeptidyl-4-nitroanilides.
- Optimal substrate hydrolysis occurred with hydrophobic or basic amino acids (phenylalanine, leucine, alanine, methionine, arginine) at the P1 position and proline at the P2 position.
- Enzyme specificity profiles closely matched those of well-characterized serine proteinases, such as chymotrypsin and elastase.
Conclusions:
- The study elucidates the conserved substrate specificity of proteolytic enzymes in keratinolytic fungi.
- The findings suggest these fungal enzymes function similarly to serine proteinases, facilitating efficient keratin breakdown.
- This research provides insights into the enzymatic mechanisms of fungal keratinolysis, relevant for biotechnological applications.