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Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus Onygena corvina.

Huang Y, Busk PK, Herbst FA, Lange L - Appl. Microbiol. Biotechnol. (2015)

Bottom Line: The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3).Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro.A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Bioscience, Aalborg University Copenhagen, 2450, Copenhagen, SV, Denmark.

ABSTRACT
Poultry processing plants and slaughterhouses produce huge quantities of feathers and hair/bristle waste annually. These keratinaceous wastes are highly resistant to degradation. Onygena corvina, a non-pathogenic fungus, grows specifically on feathers, hooves, horn, and hair in nature. Hence, the proteases secreted by O. corvina are interesting in view of their potential relevance for industrial decomposition of keratinaceous wastes. We sequenced and assembled the genome of O. corvina and used a method called peptide pattern recognition to identify 73 different proteases. Comparative genome analysis of proteases in keratin-degrading and non-keratin-degrading fungi indicated that 18 putative secreted proteases from four protease families (M36, M35, M43, and S8) may be responsible for keratin decomposition. Twelve of the 18 predicted protease genes could be amplified from O. corvina grown on keratinaceous materials and were transformed into Pichia pastoris. One of the recombinant proteases belonging to the S8 family showed high keratin-degrading activity. Furthermore, 29 different proteases were identified by mass spectrometry in the culture broth of O. corvina grown on feathers and bristle. The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro. A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed.

No MeSH data available.


Related in: MedlinePlus

Degradation of keratinaceous materials in a long-term incubation. a Pig bristle treated for 4 days with culture broth supernatant and with fractions of culture broth. Culture broth on pig bristle: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with pig bristle; culture broth on chichen feathers: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with chicken feathers. b Bristles and hooves (ground into particles approx. 1–2 mm in diameter) treated for 4 days with culture broth supernatant of O. corvina grown on chicken feathers. Negative control: 2× McIlvaine buffer (pH 8). c Pig bristle treated for 24 h with culture broth on chicken feathers and commercial keratinolytic proteases (alcalase, savinase, and esperase). The dosage of enzymes was based on the same protein concentration of the keratinolytic proteases in culture broth and the commercial enzymes. The degree of degradation was calibrated to negative control
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Fig5: Degradation of keratinaceous materials in a long-term incubation. a Pig bristle treated for 4 days with culture broth supernatant and with fractions of culture broth. Culture broth on pig bristle: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with pig bristle; culture broth on chichen feathers: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with chicken feathers. b Bristles and hooves (ground into particles approx. 1–2 mm in diameter) treated for 4 days with culture broth supernatant of O. corvina grown on chicken feathers. Negative control: 2× McIlvaine buffer (pH 8). c Pig bristle treated for 24 h with culture broth on chicken feathers and commercial keratinolytic proteases (alcalase, savinase, and esperase). The dosage of enzymes was based on the same protein concentration of the keratinolytic proteases in culture broth and the commercial enzymes. The degree of degradation was calibrated to negative control

Mentions: In a further test of the ability of the proteases to degrade pig bristle, the different fractions C15, C20, and the culture broth from O. corvina grown on chicken feathers or pig bristle were incubated with pig bristle at 40 °C and degradation was followed over 4 days. After 3-day incubation, culture broth on chicken feathers, culture broth on pig bristle, fraction C20 and C15 degraded 57, 51, 46, and 43 % of the pig bristle, respectively (Fig. 5a). Moreover, as protease 8393 is an M3 family metalloprotease, the function of this protease in the C15 fraction was evaluated by adding 0.5 mM of the metalloprotease inhibitor EDTA to the C15 fraction. The results showed that addition of EDTA was associated with a strongly decreased degradation of pig bristle (Fig. 5a). Therefore, metalloprotease 8393 (M3) is important for keratin degradation in combination with endopeptidase 6877 (S8) and exopeptidase 6423 (M28) in fraction C15.Fig. 5


Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus Onygena corvina.

Huang Y, Busk PK, Herbst FA, Lange L - Appl. Microbiol. Biotechnol. (2015)

Degradation of keratinaceous materials in a long-term incubation. a Pig bristle treated for 4 days with culture broth supernatant and with fractions of culture broth. Culture broth on pig bristle: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with pig bristle; culture broth on chichen feathers: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with chicken feathers. b Bristles and hooves (ground into particles approx. 1–2 mm in diameter) treated for 4 days with culture broth supernatant of O. corvina grown on chicken feathers. Negative control: 2× McIlvaine buffer (pH 8). c Pig bristle treated for 24 h with culture broth on chicken feathers and commercial keratinolytic proteases (alcalase, savinase, and esperase). The dosage of enzymes was based on the same protein concentration of the keratinolytic proteases in culture broth and the commercial enzymes. The degree of degradation was calibrated to negative control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4628079&req=5

Fig5: Degradation of keratinaceous materials in a long-term incubation. a Pig bristle treated for 4 days with culture broth supernatant and with fractions of culture broth. Culture broth on pig bristle: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with pig bristle; culture broth on chichen feathers: culture broth supernatant from O. corvina grown for 11 days on fermentation medium with chicken feathers. b Bristles and hooves (ground into particles approx. 1–2 mm in diameter) treated for 4 days with culture broth supernatant of O. corvina grown on chicken feathers. Negative control: 2× McIlvaine buffer (pH 8). c Pig bristle treated for 24 h with culture broth on chicken feathers and commercial keratinolytic proteases (alcalase, savinase, and esperase). The dosage of enzymes was based on the same protein concentration of the keratinolytic proteases in culture broth and the commercial enzymes. The degree of degradation was calibrated to negative control
Mentions: In a further test of the ability of the proteases to degrade pig bristle, the different fractions C15, C20, and the culture broth from O. corvina grown on chicken feathers or pig bristle were incubated with pig bristle at 40 °C and degradation was followed over 4 days. After 3-day incubation, culture broth on chicken feathers, culture broth on pig bristle, fraction C20 and C15 degraded 57, 51, 46, and 43 % of the pig bristle, respectively (Fig. 5a). Moreover, as protease 8393 is an M3 family metalloprotease, the function of this protease in the C15 fraction was evaluated by adding 0.5 mM of the metalloprotease inhibitor EDTA to the C15 fraction. The results showed that addition of EDTA was associated with a strongly decreased degradation of pig bristle (Fig. 5a). Therefore, metalloprotease 8393 (M3) is important for keratin degradation in combination with endopeptidase 6877 (S8) and exopeptidase 6423 (M28) in fraction C15.Fig. 5

Bottom Line: The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3).Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro.A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Bioscience, Aalborg University Copenhagen, 2450, Copenhagen, SV, Denmark.

ABSTRACT
Poultry processing plants and slaughterhouses produce huge quantities of feathers and hair/bristle waste annually. These keratinaceous wastes are highly resistant to degradation. Onygena corvina, a non-pathogenic fungus, grows specifically on feathers, hooves, horn, and hair in nature. Hence, the proteases secreted by O. corvina are interesting in view of their potential relevance for industrial decomposition of keratinaceous wastes. We sequenced and assembled the genome of O. corvina and used a method called peptide pattern recognition to identify 73 different proteases. Comparative genome analysis of proteases in keratin-degrading and non-keratin-degrading fungi indicated that 18 putative secreted proteases from four protease families (M36, M35, M43, and S8) may be responsible for keratin decomposition. Twelve of the 18 predicted protease genes could be amplified from O. corvina grown on keratinaceous materials and were transformed into Pichia pastoris. One of the recombinant proteases belonging to the S8 family showed high keratin-degrading activity. Furthermore, 29 different proteases were identified by mass spectrometry in the culture broth of O. corvina grown on feathers and bristle. The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro. A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed.

No MeSH data available.


Related in: MedlinePlus