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

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6805-9) contains supplementary material, which is available to authorized users.

Identification of keratinases from Fervidobacterium islandicum AW‐1 using dynamic gene expression profiling

“…Overall, these results suggest that feather decomposition cannot be achieved by a protease, but it would rather require a combination of multiple enzymes, including unknown sulfitolytic components. Such a synergism for keratin degradation was also supported by several recent studies on keratin degradation by dermatophytes provided a plausible mechanism that pathogenic dermatophytes cleave disulfide bonds excreting the reducing agent sulfite through cysteine dioxygenase and sulfite efflux pump (Grumbt et al , ), and then degrade keratin peptides using a combination of several proteases, including endoproteases, exoproteases and oligopeptidases (the S8, M28 and M3 families respectively), and contribute to keratin decomposition (Huang et al , ).…”

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates

“…The disulphide bonds are cleaved first, and the keratin is denatured, giving easy access for keratinases to degrade the keratin protein even further . Many other experimental studies provided evidence that disulphide reductase and the intracellular enzyme cysteine dioxygenase can break down sulphur bridges, and lead to production and secretion of sulphite, thus giving the enzymes improved access to the keratinaceous substrate . For this reason, molecular or genetic studies in vitro are unable to examine comprehensively the extent of keratin degradation by clinical isolates of dermatophytes, and the range of animal hosts attacked by the pathogen cannot be assessed.…”

“…41,48,51 On the other hand, it is widely agreed that keratin breakdown requires more than just one enzyme. 22,41,51,53,54 This concept was developed from observations that the keratinase activity of a culture broth was not detected after purified keratinase active protein was added, which indicated at least two enzymes working synergistically together in decomposition. 22,53,55 The average standard deviation (SD) values are given in brackets.…”

A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis

“…Electrophoresis was carried out at 50 mA for ∼15 min and the gel was stained with Coomassie Brilliant Blue G250. The part of the gel that stained positive for proteins were excised into 5 fractions and subjected to in-gel digestion 83 and MS analyses, as previously described 84 . The nLC-MS setup consisted of a Ultimate3000 nano-LC (Thermo Fisher) coupled to a Q Exactive mass spectrometer (Thermo Fisher).…”