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.
Highlights
Keratin is the major structural protein of feathers, skin and wool [1], is insoluble in water and has high mechanical stability and resistance to proteolysis [1,2]
Strains were streaked on keratin agar and single colonies screened for their ability to grow in phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.2) supplemented with 1% feather keratin for 20 days at 28°C
The keratinolytic activity of strain J5 was a serine peptidase 70% higher than that of the wild-type strain (Figure 2)
Summary
Keratin is the major structural protein of feathers, skin and wool [1], is insoluble in water and has high mechanical stability and resistance to proteolysis [1,2]. Keratin stability results from a high degree of cross-linking of disulfide bonds, hydrogen bonding and hydrophobic interactions. Despite their stability, keratins do not accumulate in nature and can be hydrolyzed by some microorganisms [3]. Since feathers are almost pure protein (keratin), they are potentially a less expensive alternative source of protein for animal feed. Conversion generally requires significant energy inputs and destroys certain amino acids [5]. It follows that degradation of feather keratin by microorganisms represents an alternative method to improve the nutritional value of feather waste and to prevent environmental contamination [6]. An aspartic peptidase keratinase from Candida albicans has been described [10], indicating that yeasts are a group with largely unexplored potential
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