title: Increased expression of keratinase and other peptidases by Candida parapsilosis mutants.
abstract: Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.
authors.affiliation: Laboratório Proteases de Microrganismos, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, RJ, Brasil.
authors.forename: T R
authors.initials: TR
authors.lastname: Duarte
authors.forename: S S
authors.initials: SS
authors.lastname: Oliveira
authors.forename: A
authors.initials: A
authors.lastname: Macrae
authors.forename: S M L
authors.initials: SM
authors.lastname: Cedrola
authors.forename: A M
authors.initials: AM
authors.lastname: Mazotto
authors.forename: E P
authors.initials: EP
authors.lastname: Souza
authors.forename: A C N
authors.initials: AC
authors.lastname: Melo
authors.forename: A B
authors.initials: AB
authors.lastname: Vermelho
mesh_terms: D000818:Animals; D002175:Candida / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D011200:Poultry; D013379:Substrate Specificity
chemical_list: D009153:Mutagens; D005020:Ethyl Methanesulfonate; D010447:Peptide Hydrolases; C035855:keratinase