WO2014169920A2 - Utilisation d'une composition microbienne pour la dégradation de substances kératiniques - Google Patents

Utilisation d'une composition microbienne pour la dégradation de substances kératiniques Download PDF

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WO2014169920A2
WO2014169920A2 PCT/DK2014/050098 DK2014050098W WO2014169920A2 WO 2014169920 A2 WO2014169920 A2 WO 2014169920A2 DK 2014050098 W DK2014050098 W DK 2014050098W WO 2014169920 A2 WO2014169920 A2 WO 2014169920A2
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acid sequence
protease
fungal species
corvina
seq
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PCT/DK2014/050098
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WO2014169920A3 (fr
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Lene Lange
Peter Kamp Busk
Huang YUHONG
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Aalborg Universitet
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Publication of WO2014169920A3 publication Critical patent/WO2014169920A3/fr
Priority to PCT/EP2015/058083 priority Critical patent/WO2015158719A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • the present invention relates to the use of a microbial composition for the degradation of keratinaceous materials.
  • the present invention also pertains to a method for the degradation of keratinaceous materials and compositions comprising a degraded keratinaceous material.
  • the compound is very resistant to the action of weak acids, alkalis, ethanol, solution or hydrolysis of the common proteolytic enzymes such as trypsin, pepsin and papain due to a high degree of cross-linking by disulfide bonds, hydrogen-bonding and hydrophobic interaction (Riffel et al., 2011).
  • the high content of cysteine residues (3-15%) contribute to keratin stability by forming disulphide bridges between different twists in a single peptide chain and between chain in keratin. Keratin with many cysteine is highly resistant to enzymatic hydrolysis (Mercer, 1957).
  • Keratinases (EC 3.4.21/24/99.11), are robust enzymes that are mostly serine or metallo proteases (Gupta and Ramnani, 2006). Keratinases have multitude industrial application such as detergent additives, food and feed modification to upgrade the nutritional value of feather meal, dehairing of leather , medicine, cosmetics, biodegradable films and coatings and degrading prions to treat the dreaded mad cow disease. In the industrial enzyme market, the available proteases are mainly from Bacillus strains and the industrial application and commercial exploitation of keratinase is still in the stage of infancy.
  • an object of the present invention relates to the use of a microbial composition for the degradation of keratinaceous materials.
  • one aspect of the invention relates to the use of (i) one or more
  • Ascomycetous fungal species (ii) one or more Basidiomycetous fungal species and/or (iii) microbial products from (i) and/or (ii) for the degradation of keratinaceous materials.
  • Another aspect of the present invention relates to the use of one or more protein(s) from one or more Ascomycetous fungal species and/or one or more Basidiomycetous fungal species for the degradation of keratinaceous materials.
  • Yet another aspect of the present invention is to provide a method for the degradation of keratinaceous materials comprising the steps of:
  • a keratinaceous material (i) one or more Ascomycetous fungal species, (ii) one or more Basidiomycetous fungal species, (iii) microbial products from (i) and/or (ii) and/or (iv) one or more protein(s) secreted from one or more Ascomycetous fungal species and/or one or more protein(s) secreted from one or more Basidiomycetous fungal species, and
  • Still another aspect of the present invention is to provide a composition
  • one or more protein(s) comprising an amino acid sequence selected from the group consisting of:
  • Another aspect of the present invention is to provide a composition
  • a composition comprising (i) one or more Ascomycetous fungal species and/or one or more
  • Basidiomycetous fungal species and/or microbial products from (i) and (ii) a keratinaceous material Basidiomycetous fungal species and/or microbial products from (i) and (ii) a keratinaceous material.
  • Another aspect of the present invention relates a feed comprising the degraded keratinaceous material obtained by the method of the present invention.
  • a further aspect of the present invention relates to a food product comprising the degraded keratinaceous material obtained by the method of the present invention.
  • Yet another aspect of the present invention relates to a cosmetic product comprising the degraded keratinaceous material obtained by the method of the present invention.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the degraded keratinaceous material obtained by the method of the present invention.
  • the strive for unlocking the potentials of nature ' s enzymes specialized in converting keratinaceous materials into biologically accessible proteins, peptides and amino acids led to looking into the capability of the secreted enzymes from fungi specialized in growing on feather, bristles, hooves, wool and hair; and at the same time avoiding the human pathogens among such fungi.
  • the most highly specialized keratin growing fungus, Onygena corvina Alb. & Schwein. 1805 showed from the very first experiments extraordinary potentials for decomposing keratinaceaous materials. O.
  • corvina grows preferably on feather ( ⁇ -keratin, figure 1); a very closely related species O. equina grows on hooves (a-keratin).
  • O. equina grows on hooves
  • O. piligena grows on human nails; and the substrate specificity of O. apus has not been very well described.
  • the keratinase activity of O. corvina was so promising (figures 2 and 3) that it led to continued and determined studies of the enzymes involved in order to unravel the basis for such interesting (first documented ultimo 2012) keratin degrading capabilities: Genome sequencing; bioinformatic analysis; protease prediction; cloning and expression; protease characterization.
  • MS Mass spectrometry
  • FIG 1 shows feather Stalkball (Onygena corvina) as it grows in nature on feather. Lange and Hora, 1963
  • Figure 2A is showing the type of duck feather used as substrate in present work.
  • Figure 2B shows to the left an apparently totally decomposed duck feather after treatment with Onygena corvina inoculum, holding both microbial biomass and protease rich supernatant; in the middle, similarly but treated with Trichoderma asperellum -some decomposition is also taking place but much less; to the right, negative control without inoculum
  • Figure 3 shows pig bristles cut in small pieces, submerged in 2xMcIlvaine buffer, pH 8: To the left (A) with Onygena corvina culture broth supernatant added; and to the right (B) with no supernatant added. Visual inspection indicates almost total degradation of the pig bristles in A.
  • Figure 4 shows time course recordings of development in protease and keratinase activity in culture broth supernatant from Onygena corvina growing on 1.5 % duck feather. Release of soluble protein from the substrate and of thio-groups also appears from the graph.
  • Figure 5 shows the influence of different initial pH on the protease and keratinase activity in culture broth supernatant from Onygena corvina growing on 1.5 % duck feather indicates a maximum between pH 6 and pH 8.
  • Figure 6 shows the influence of different amounts of feather in the production medium on the level of protease activity.
  • Figure 7 shows the difference in keratinolytic capabilities of Onygena corvina and Trichoderma asperellum.
  • Figure 8 shows the eEffect of pH and pH stability (above) and of temperature and thermo stability (below) on the activity of protease from Onygena corvina
  • Figure 9 Zymogram showing the apparent size of the proteases from Onygena corvina: (A) molecular weight marker, (B) SDS-PAGE of the crude enzyme preparation, (C) zymogram of the crude enzyme preparation.
  • Figure 10 Spectrum of protease families in Onygena corvina genome, with indication of how many representatives from each family.
  • Figure 11 shows a comparative analysis of protease repertoire between a selection of keratin-degrading (to the left) and non-keratin-degrading fungi (to the right), used as basis for selecting keratinase genes characteristic for keratin decomposers.
  • Figure 12 shows SDS-PAGE of purified recombinant proteases.
  • were loaded on 12% and 10% (w/v) polyacrylamide gel, respectively. Gels were silver stained using the Pierce Silver Stain Kit (Thermo scientific).
  • Figure 13 shows Mass spectrometry data, identifying protease genes found in Onygena corvina secretome when grown on chicken feather and pig bristles for 11 days.
  • Figure 14 shows the elution profile of protease from the cation exchange column 5 ml HiTrap SP with 50 mM citrate buffer, pH 3.86. Culture broth supernatant (50ml) was applied to the column and a gradient from 0-lM NaCI was applied to elute the column-bound protein.
  • Figure 15 shows the elution profile of protease from anion exchange column 1 ml HiTrap Q with 20 mM Tris buffer, pH 8.6. Culture broth supernatant (50ml) was applied to the column and a gradient from 0-lM NaCI was applied to elute the column-bound protein.
  • Figure 16 shows the degree of degradation of pig bristles by treatment with different fractions.
  • A anion exchanged fractions
  • C cation exchanged fractions
  • Figure 17 shows the degree of degradation of pig bristles by treatment with a blend of different fractions (A: anion exchanged fractions; C: cation exchanged fractions). Blend of C15 and C20 indicate synergistic effect of blending the two fractions.
  • Positive controls culture broth supernatant from Onygena corvina, grown for 11 days on fermentation medium with pig bristle (P) and chicken feather (C), respectively.
  • Negative control 0.05 ml fraction was replaced by 0.05 ml 2x Mcllvaine buffer (pH 8) and incubated at 40 °C for 24 h with agitation at 1000 rpm.
  • Figure 18 shows the degree of degradation of pig bristles by treatment with purified recombinant protease and blend of fractions (A: anion exchanged fractions; C: cation exchanged fractions).
  • is the purified recombinant protease (gene SEQ ID NO: 1, protein SEQ ID NO: 2) expressed in Pichia.
  • Positive control culture broth supernatant from Onygena corvina, grown for 11 days on fermentation medium with pig bristle (P) and chicken feather (C), respectively.
  • Negative control 0.05 ml fraction was replaced by 0.05 ml 2x Mcllvaine buffer (pH 8) and incubated at 40 °C for 24 h with constant agitation at 1000 rpm.
  • Figure 19 shows the degree of degradation of pig bristles by treatment for 4 days with total supernatant culture broth and with fractions of culture broth supernatant.
  • C15 and C20 are cation exchanged fractions.
  • Fraction C15+0.5 mM EDTA: 0.05 mM metallo peptidase inhibitor Ethylene diamine tetra acetic acid (EDTA) was added when C15 fraction degrade pig bristles.
  • Culture broth supernatant treatment of pig bristles culture broth supernatant of Onygena corvina grown on pig bristles for 11 days.
  • Culture broth supernatant treatment of chicken feather culture supernatant after Onygena corvina grown on chicken feather for 11 days.
  • Negative control 0.05 ml fraction was replaced by 0.05 ml 2x Mcllvaine buffer (pH 8) and incubated at 40 °C with agitation at 1000 rpm.
  • Figure 20 shows the degree of degradation of pretreated bristles and hooves (source: slaughter house) by treatment for 4 days with culture broth
  • Negative control 0.05 ml fraction was replaced by 0.05 ml 2x Mcllvaine buffer (pH 8) and incubated at 40 °C with agitation at 1000 rpm.
  • Penicillium spp for details see Table 1 and prior art section above.
  • the human pathogens / human derpatophytes are not acceptable as production host for enzyme blends for industrial purposes.
  • human pathogens are not preferred choice as origin of genes for recombinant expression of industrially relevant enzymes as the resulting enzymes may have a strong inherent risk for workers and end user health.
  • the start of this endavour was to find a fungus which as its natural habitat was specialized to grow specifically on feather, hooves, horn, and hair incl bristles.
  • Such an invention would optimally provide basis for developing an industrially relevant enzyme composition to be used to decompose both a- and ⁇ -keratin, viz feather, hooves, horn, and hair incl bristles into bio-accessible proteins, peptides and amino acids; hereby unlocking the potentials of an, in global scale, very substantial protein resource for use as animal feed (and maybe also for essential protein nutrition and treatment for humans).
  • Genome sequencing resulted, however, in a very long and extensive list of proteases belonging to a very wide spectrum af protease families.
  • Modern industrial biotechnology have shown that commercially viable industrially applicable, (viz. fitting within the rather low priced window of opportunity) are products composed of a single recombinantly expressed (in extraordinarily high yield), as e.g. enzymes for textile, detergents and animal feed purposes.
  • the next phase contributing to the successful invention was to unlock the
  • SEQ ID NO: 1 and 2 may have, or can be engineered to have, sufficient activity to provide keratin decomposition to bioaccessible proteins, peptides and amino acids to make up a product for converting keratinaceous waste materials into valuable protein rich animal feed ingredient.
  • Trichophyton spp. The sequence identity is, however, found to be only between 72 and 84 percent. The sequence differences are interpreted to be the basis for this very strong performance of the newly discovered protease genes from the genus Onygena, more specifically O. corvina / O. equina.
  • wild type genes can be even further improved by artificial evolution of each of the genes (random and targeted mutations; linker engineering; domain and family shuffling etc). Further, hybride genes between the two selected M28 and the two S8 genes may lead to even stronger exo- and endoacting enzymes, respectively.
  • expression of such 1, 2, 3, 4, or 5 genes into one expression and production host may give basis for a production host with capability for in one fermentation to give the strongest blend of enzymes for breaking down keratin to proteins (peptides and amino acids).
  • Microorganisms are the most important sources of keratinolytic enzymes and a broad spectrum of reports of keratin growing microbes are published (see Table 1). Surprisingly as of today there is still not any widely acceptyed, efficient and commercialized process for breaking down keratinaceous, animal derived bio- waste or bio-side stream products. It is reported that a vast variety of bacteria, actinomycetes and fungi are keratin degraders. A large proportion of
  • Onygena corvina (feather stalkball) and O. equina (horn stalkball), both species of the fungal genus Onygena in the Onygenaceae family, can live as
  • the present study aimed to investigate the capability of O. corvina to degrade poultry feather and produce alkaline keratinolytic protease in liquid culture with duck feather as sole carbon and nitrogen source.
  • O. corvina readily growed on and degraded duck feather and expressed high protease and keratinase activity.
  • the protease and keratinase characterization was further analyzed with respect to pH optimum and thermal stability.
  • the findings of this study show that O. corvina (and the closely related species O. equina) have great potential of bioconverting feather waste into economically products, such as animal feed and food ingredients.
  • fungi have genes for proteases with only 80- 84 % amino acid identity to one or more of the sequences of the proteins described here from O. corvina. These fungi are the human pathogenic dermatophytes Arthroderma benhamiae, Arthroderma gypseum, Arthroderma otae (Microsporum cam ' s), Trichophyton equinum, Trichophyton rubrum, Trichophyton tonsurans and Trichophyton verrucosum. Due to the pathogenicity these fungi are not useful for direct expression of proteases or keratinases as this would pose a severe health risk.
  • proteases of these fungi have been purified or recombinantly expressed to investigate their role in infection but not for investigation of their potential use as industrial keratinases (Asahi et al., 1985; Brouta et al., 2002; Chen et al., 2010; Lee et al., 1987; Sriranganadane et al., 2011).
  • Onygena corvina was cultivated in liquid medium (initial pH 8) with 1.5 % (w/v) duck feather as sole carbon and nitrogen source. After incubated at 25 °C, 200 rpm for 8 days, the pH value of the culture filtrate increased to 8.47 and the protease activity and keratinase activity were 1435 and 72 U/ml, respectively ( Figure 4). Soluble protein and thiol formation increased from day 2 to 10. Furthermore, the amount of insoluble nondegraded feather decreased with time. The increased keratinase activity appeared to be related to an increase of soluble protein indicating that the keratinase activity depends on O. corvina growth.
  • the accumulation of soluble proteins during the cultivation may be caused by both enzyme secretion and keratin solubilization. Keratinolysis is not only accomplished by keratinase, but also by disulfide reduction mechanisms, such as through disulfide reductases, sulfite, sulfide or thiosulfate chemical mechanisms, or by a cell-bound redox system (Gupta and Ramnani, 2006). So the increase in thiol groups during cultivation may be attributed to the disruption of disulfide bridges.
  • corvina does not grow well in the medium with initial pH 9 to 11.
  • partial dissolved feather may stimulate the keratinase production resulting in complete degradation of the feather.
  • the results suggesting that the tendency towards the increase in pH of the acidic medium may be due to the keratinolysis of feather, and the decline in pH of the alkaline medium may caused by the accumulation of acidic sulfur compounds products in the medium. So, the change in pH at the end fermentation is an indicator for keratinolysis.
  • duck feather was added in the medium at pH 6 and incubated with O. corvina at 25 °C, 200 rpm for 8 days. Highest protease activity was found when the amount of duck feather was 0.5 % ( Figure 6). However, the highest yield measured as soluble protein, thiol formation and keratinase was found when the amount of duck feather was 1.5 %.
  • Trichoderma (see Table 1). Furthermore, Trichoderme species are known as extraordinarily good enzyme secreters and could be a good choice for production of such blend of enzymes for keratin decomposition. Therefore, Trichoderma asperellum was chosen as a putatively positive control for duck feather degradation. T. asperellum incubated for 8 days at 25 °C in duck feather medium lead to a slight degradation of the duck feather ( Figure 7). However, O. corvina was able to degrade most of the duck feather under the same conditions. There was no degradation of the feather in the negative control without fungi. The weight loss of the duck feather incubated with O. corvina was 75 % whereas T. asperellum only gave a weight loss of 23 %. Moreover, O.
  • corvina increased the amount of soluble protein, thiol formation and the protease and keratinase activities much more than T. asperellum did ( Figure 2).
  • the results indicate that the O. corvina has a high potential for feather recycling and bioconverting them into high value-added and economical product, such as animal meal.
  • Other kinds of nonpathogenic fungi such as Aspergillus niger, Alter n aria altanata, Curvularia lunata, Fusarium oxysporum, Myrothecium roridum and Penicillium spp., can also degrade the feather but also these species give weaker reactions and need longer time to decompose keratin as compared to O. corvina.
  • Figure 8 shows that the proteases from O. corvina were active at a broad range of pH values (pH 6 to 11) and temperature (40-60 °C). Such wide pH and temperature range might be useful for industrial application. Maximal protease activity was obtained at pH 9 and 50 °C, respectively. The protease was stable at pH 5-11 at 4 °C, and more than 71 % residual activity was conserved at these pH values. The enzyme was stable for 1 h at 30 °C, at 40 °C, the residual activity was 48 %.
  • the keratinase activity of O. corvina was partially inhibited by Mg 2+ , Cu 2+ , Zn 2+ and Mn 2+ and stimulated by Ca 2+ and Fe 2+ (Table 2). It is expected that, divalent metal ions like Ca 2+ , Mg 2+ and Mn 2+ stimulated the keratinase activity, and heavy metal ions such as Cu 2+ , Zn 2+ will inhibit the keratinase activity. But for O. corvina, Mg 2+ and Mn 2+ have negative effects on keratinase activity. Fungal keratinases mostly belong to the class of serine proteases that are inhibited by PMSF and EDTA.
  • keratinase activity was partially inhibited by 1 mM EDTA and ImM PMSF. This indicated that the keratinase activity from O. corvina may be include serine protease. Different organic solvents such as ethanol, methanol, isopropanol, tween-20, tween-80 inhibited the keratinase activity to some degree. But the enzyme from O. corvina was stable in the present of glycerol. The keratinase activity was decreased by SDS and triton X-100 detergents.
  • Reducing agents like DTT and ⁇ -mercaptoethanol generally enhance keratinase activity because the addition of reducing agents can breaking disulfide bond to help sulfitolysis but the keratinase activity of O. corvina was inhibited by DTT and ⁇ -mercaptoethanol.
  • corvina proteases were estimated to 35 kDa and 20 kDa, respectively.
  • the molecular masses of keratinases ranges from 18-200 kDa (Gupta and Ramnani, 2006), so the two proteases from for O. corvina are of medium and small size.
  • amino acid sequences or nucleic acid sequences is to be understood as referring to the organism from which it derives. Said sequence may be expressed by another organism using gene technology methods well known to a person skilled in the art. This also encompasses sequences which have been chemically synthesized. Furthermore, said sequences may comprise minor changes such as codon optimization, deletions, insertions, base substitutions or shuffling, i.e. changes in the nucleic acid sequences which do not significantly (i) affect the amino acid sequence and/or (ii) the functionality of the protein.
  • the protein(s) of interest e.g. the endo-acting protease(s), the exo-acting protease(s), the metalloprotease(s) and/ or the serine protease(s) of the present invention may in particular be produced as a recombinant protein, i.e. a nucleotide sequence encoding the polypeptide of interest may be introduced into a cell for expression of the polypeptide of interest.
  • the recombinant expression may be homologous or heterologous, i.e. the polypeptide of interest may be expressed in cell which it is naturally expressed by (homologous expression) or it may be expressed by a cell which it is not naturally expressed by (heterologous expression).
  • the recombinant polypeptide of interest may be expressed by any host cell suitable for recombinant production of the particular polypeptide of interest.
  • suitable host cells include but are not limited to prokaryotic cells, such as E.coli cells and Bacillus cells.
  • suitable eukaryotic cells include but is not limited to a fungal cell such as Ascomycete cells and
  • recombinant polypeptide or "recombinant polypeptide of interest” denotes herein a recombinant produced polypeptide.
  • the present invention pertains to the use of (i) one or more Ascomycetous fungal species, (ii) one or more Basidiomycetous fungal species and/or (iii) microbial products from (i) and/or (ii) for the degradation of keratinaceous materials.
  • the microbial products may comprise protetin(s) and be e.g. present in a culture broth supernatant from one or more Ascomycetous fungal species and/or one or more Basidiomycetous fungal species.
  • the keratinous material is selected from the group consisting of feather, hair, hoof, horn and bristles.
  • the one or more Ascomycetous fungal species belongs to Eurotiomycetes. In another embodiment the one or more
  • Ascomycetous fungal species belongs to Onygenales. In yet an embodiment the the one or more Ascomycetous fungal species belongs to Onygenaceae. In a further embodiment the one or more Ascomycetous fungal species belongs to the genus Onygena. In a preferred embodiment the one or more Ascomycetous fungal species belongs to the species Onygena equina or Onygena corvina. In a further embodiment the species Onygena equina or Onygena corvina is selected from the group consisting of strains, isolates and mutants of the species Onygena equina or Onygena corvina.
  • the invention may futher employ a consortia of bacterial species such as but not limited to Gram-positive bacteria such as Bacillus sp. and/or Gram-negative bacteria such as Pseudomonas spp.
  • a further aspect of the present invention pertains to the use of one or more protein(s) from one or more Ascomycetous fungal species and/or one or more Basidiomycetous fungal species for the degradation of keratinaceous materials.
  • the protein(s) may be membrane bound or secreted.
  • the one or more protein(s) may be selected from the group consisting of endo- acting protease(s), exo-acting protease(s), metalloprotease(s) and serine protease(s).
  • the endo-acting protease(s) may belong to the Merops family S8 or Merops family M3 whereas the exo-acting protease(s) may belong to the Merops family M28.
  • the one or more protein(s) may comprise an amino acid sequence selected from the group consisting of:
  • references to a particular protein of interest e.g. the endo-acting protease(s), exo-acting protease(s), metalloprotease(s) and serine protease(s) mentioned above , includes in the context of the present invention also functionally equivalent parts or analogues of the polypeptide of interest.
  • the polypeptide of interest is an enzyme
  • a functionally equivalent part of the enzyme could be a domain or subsequence of the enzyme which includes the necessary catalytic site to enable the domain or subsequence to exert substantially the same enzymatic activity as the full-length enzyme or alternatively a gene coding for the catalyst.
  • substantially the same enzymatic activity refers to an equivalent part or analogue having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% of the activity of the natural enzyme.
  • An example of an enzymatically equivalent analogue of the enzyme could be a fusion protein which includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme derived from another species.
  • completely synthetic molecules that mimic the specific enzymatic activity of the relevant enzyme would also constitute "enzymatic equivalent analogues".
  • the skilled person will be able to readily devise appropriate assays for the determination of enzymatic acitivity.
  • the present invention relates to one or more protein(s) with the amino acid sequence according to any one of SEQ ID NO: 1, 3, 5, 7 or 9 or an amino acid sequence that has a sequence identity of at least 84% to any one of SEQ ID NO: 1, 3, 5, 7 or 9, such as at least 85 % identity, 86 % identity, 87 % identity, 88 % identity, 89 identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, 98.1 % identity, 98.2 % identity, 98.3 % identity, 98.4 % identity, 98.5 % identity, 98.6 % identity, 98.7 % identity, 98.8 % identity, 98.9 % identity, 99 % identity, 99.1 % identity, 99.2 % identity, 99.3 % identity, 99.4 % identity, 99.5
  • the one or more protein(s) may be encoded by a nucleic acid sequence selected from the group consisting of:
  • the one or more protein(s) may be produced by native or heterologous expression of a nucleic acid sequence selected from the group consisting of:
  • the nucleic acid molecule encoding the one or more protein(s) comprises a nucleic acid sequence according to any one of SEQ ID NO: 2, 4, 6, 8 or 10 or nucleic acid sequence with a sequence identity of at least 84% to any one of SEQ ID NO: 2, 4, 6, 8 or 10, such as 85 % identity, 86 % identity, 87 % identity, 88 % identity, 89 identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, 98.1 % identity, 98.2 % identity, 98.3 % identity, 98.4 % identity, 98.5 % identity, 98.6 % identity, 98.7 % identity, 98.8 % identity
  • a further aspect of the present invention pertains to a method for the degradation of keratinaceous materials comprising the steps of:
  • a keratinaceous material i) one or more Ascomycetous fungal species, (ii) one or more Basidiomycetous fungal species, (iii) microbial products from (i) and/or (ii) and/or (iv) one or more protein(s) secreted from one or more Ascomycetous fungal species and/or one or more protein(s) secreted from one or more Basidiomycetous fungal species, and
  • the degraded keratinaceous material comprises protein(s), peptides and amino acids.
  • amino acid sequence and/or the nucleic acid sequence of the protein(s) may comprise minor changes such as codon optimization, deletions, insertions, base substitutions or shuffling, i.e. changes that may essentially improve the functionality of the proteins and lead to a keratinaceous material having improve the bioaccessibility, nutrition and/or digestability.
  • composition comprising one or more protein(s) comprising an amino acid sequence selected from the group consisting of:
  • composition may further comprising a keratinaceous material.
  • the present invention pertains to a composition
  • a composition comprising (i) one or more Ascomycetous fungal species and/or one or more Basidiomycetous fungal species and/or microbial products from one or more Ascomycetous fungal species and/or one or more Basidiomycetous fungal species, and (ii) a keratinaceous material.
  • the present invention pertains to a feed comprising the degraded keratinaceous material obtained by the method of the present invention.
  • the feed may be for non-ruminant one stomach animals.
  • the feed may be selected from the group consisting of pig, mink, chicken and fish feed.
  • the present invention pertains to a food product comprising the degraded keratinaceous material obtained by the method of the present invention.
  • the present invention pertains to a cosmetic product comprising the degraded keratinaceous material obtained by the method of the present invention.
  • the present invention pertains to a pharmaceutical composition comprising the degraded keratinaceous material obtained by the method of the present invention.
  • identity is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • the default settings with respect to e.g. "scoring matrix” and "gap penalty" may be used for alignment.
  • the BLASTN and PSI BLAST default settings may be advantageous.
  • the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • Keratinous bio-materials such as e.g. feather, hair, hooves, and horn or bone can be decomposed in domestic or industrial processes by:
  • keratinous materials such as but not limited to animal feather, hair, hooves by colonization of the material by fungal species or by consortia of fungal and/or bacterial species, which are able to colonize and grow in nature on keratinous materials such as but not limited to feather, hair, hooves, and horn or bone.
  • fungal species is an Ascomycetous fungal species able to grow on such keratinous materials such as feather, hair, hooves, and horn or bone.
  • the fungal species consists of strains, isolates or mutants of the fungal species Onygena corvina able to grow on such keratinous materials such as feather, hair, hooves and horn or bone.
  • composition but an optimized selection of 1-10 different enzymes/ proteins where one, some or all have been heterologously expressed in a suitable biological production host.
  • Keratinous bio-materials such as e.g. feather, hair (incl bristles), hooves, and horn can be decomposed in domestic or industrial processes by:
  • keratinous materials such as but not limited to animal feather, hair (incl bristles), hooves, and horn by colonization of the material by one or more fungal species or by consortia of fungal species, where the fungal species is one or more Ascomycetous or Basidiomycetous fungal species, which colonize and grow predominantly on keratinous materials such as but not limited to feather, hair (incl bristles), hooves, and horn in nature.
  • a blend composition of one or more secreted proteins produced by one or more fungal species where the fungal species is one or more Ascomycetous fungal species, which colonize and grow predominantly on such keratinous materials such as but not limited to feather, hair (incl bristles), hooves, and horn in nature.
  • the fungal species is one or more Eurotiomycetes fungal species which colonize and grow predominantly on such keratinous materials such as feather, hair (incl bristles), hooves, and horn in nature.
  • the fungal species is one or more Onygenales, which colonize and grow predominantly on such keratinous materials such as feather, hair (incl bristles), hooves, and horn in nature. 5. As claim 1 and 2 where the fungal species is one or more Onygenaceae, which colonize and grow predominantly on such keratinous materials such as feather, hair (incl bristles), hooves, and horn in nature.
  • the fungal species is one or more species of the genus Onygena, which colonize and grow predominantly on such keratinous materials such as feather, hair (incl bristles), hooves, and horn in nature.
  • the fungal species is one or more strains, isolates or mutants of a sister species of the genus Onygena, more specifically Onygena equina, known to grow on trimming clippings of hooves and horn.
  • the fungal species is one or more strains, isolates or mutants of the fungal species Onygena corvina able to grow on such keratinous materials such as feather, hair (incl bristles), hooves, and horn in nature.
  • one or more endo-acting e.g. Merops family S8 or Merops family M3 or exo-acting (e.g. Merops family M28) proteases;
  • a variant comprising the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions;
  • composition but an optimized selection of 1-10 different polypeptides, comprising but not limited to the polypeptides of claim 9, where one, some or all of the polypeptides have been heterologously expressed in a suitable biological production host; more specifically in a fungus, even more specifically in a suitable Ascomycete production host, or even more specifically in a species of Trichoderma where the homologue secreted proteins may add to the total keratinolytic capability of the resulting culture broth composition
  • Onygena corvina (accession number: CBS 281.48) was obtained from CBS- KNAW fungal Biodiversity Centre (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) and kept on potato dextrose agar plate at 4 °C. Subculturing was done once a month.
  • Onygena corvina mycellium from a PDA plate was inoculated in a minimal liquid medium containing 15 g/l duck feather/10 g/l chicken feather/lOg/l dog wool/20g/l pig bristles; 2 g/l KH 2 P0 4 , 0.15 g/l MgS0 4 -7H 2 0, 0.3 g/l CaCI 2 , 3.3 g/l Tween 80, pH 8 and cultured at 25 °C on a rotary shaker (200 rpm) for eight to eleven days.
  • Trichoderma asperellum (accession number: CBS 131938) was also obtained from CBS-KNAW fungal Biodiversity Centre (Centraalbureau voor
  • Duck feather was obtained from Valbyparkens, Denmark (2012). Chicken feather was obtained from Rose Poultry (Vinderup, Skovsgaard, Denmark) on 27. Nov.2013. Pig bristles were obtained from Danish Crown (Bragesvej, Denmark) on 12. Nov.2013. Dog wool was kindly provided by Peter Busk.
  • Pretreated bristles and hooves was obtained from Danish Crown (Bragesvej, Denmark) on 22. Mar. 2014. Feather and bristles were washed three times with tap water, distilled water and MilliQ water. Then they were cut into about 1 cm pieces and air dried. Before used as sole carbon and nitrogen source in the minimal liquid medium, they were further dried in an oven at 50 °C until the weight was constant.
  • Weight loss of the substrate in protease and keratinase production medium was estimated by determining the loss of the duck feather dry weight. Initial feather weight was determined as dry feather weight after dehydration at 50 °C. Final feather weight was measured as the dry weight of the residual feather after dehydration at 50 °C.
  • Weight loss (%) (initial feather weight-final feather weight)/initial feather weight x 100.
  • Protease activity was assayed by mixing 20 ⁇ 1.5 % Azocasein (Sigma-Aldrich. Denmark) suspension in 50 mM sodium carbonate buffer (pH 9.0) and 20 ⁇ diluted enzyme. The reactions were carried out at 50 °C for 60 min with constant agitation at 300 rpm by using a TS-100 Thermo-Shaker, SC-20 (Biosan Ltd). After incubation, the reactions were stopped by adding 100 ⁇ 0.4 M trichloroacetic acid (TCA) and incubated at 4 °C for 30 min. Then the mixture was centrifuged at 16000xg for 1 min to remove the substrate.
  • TCA trichloroacetic acid
  • One unit (U) of protease activity was defined as the amount of enzyme causing 0.01 absorbance increase between the sample and control at 405 nm under the assay conditions.
  • Keratinase activity was measured with keratin azure (Sigma-Aldrich, USA) as substrate.
  • the keratin azure was ground to a fine powder with a mortar and pestle in liquid nitrogen.
  • 0.4 g keratin azure powder was mixed with 100 ml 50 mM sodium carbonate buffer (pH 9.0).
  • the reaction mixture contained 50 ⁇ keratin azure suspension and 50 ⁇ enzyme solution.
  • Assays were carried out at 50 °C for 24 h with constant agitation at 1000 rpm in a TS-100 Thermo- Shaker, SC-20 (Biosan Ltd).
  • 0.05 ml cultural supernatant was incubated with 0.004 g pig bristles in 0.2 ml 2x Mcllvaine buffer (pH 8). Assays were carried out at 40 °C for 24 h with constant agitation at 1000 rpm. The initial and final soluble protein in supernatant was measured at 280 nm by nanodroplOOO (Thermo Scientific) before and after incubation. The increased soluble protein was calculated as the difference between the final and initial soluble protein. As a control, 0.05 ml culture supernatant was replaced by 0.05 ml 2x Mcllvaine buffer (pH 8) and incubated at 40 °C for 24 h with constant agitation at 1000 rpm.
  • the initial and final soluble protein in supernatant was measured at 280 nm before and after incubation.
  • Purified Bovine serum albumin (BSA) (10 mg/ml) was series diluted to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg/ml in 2x Mcllvaine buffer (pH 8). Standard curve was generated by measuring diluted BSA absorbance at 280 nm.
  • the soluble protein before and after pig bristles degradation were calculated according to the BSA standard curve. Pig bristles degradation degree (%) was calculated by the following equation :
  • Degradation degree (%) increased soluble protein (mg) /initial pig bristles weight (mg)*100
  • the protein content was determined by the Bradford method with the BCA Protein Assay Kit (Thermo scientific. No 23227) and bovine serum albumin (BSA) as standard.
  • Free thiol groups were analysis by mixing 100 ⁇ sample with 20 ⁇ NH 4 OH, 100 ⁇ of 0.5 g/l NaCN and 100 ⁇ MilliQ water. The mixture was incubated for 20 min at 25 °C, following by addition of 20 ⁇ of 0.5 g/l sodium nitroprusside. Absorbance at 530 nm was measured within two min.
  • enzyme reactions were carried out at different temperature (30, 40, 50, 60 °C) for 60 min in 50 mM sodium carbonate buffer (pH 9).
  • the enzyme solution was pre-incubated for 60 min at 30, 40, 50, 60 °C in 50 mM sodium carbonate buffer (pH 9) where after the residual activity was measured.
  • the optimum pH of the enzyme cultures were carried out over a pH range of 4.0-11.0 at 50 °C.
  • the enzyme cultures were incubated in buffers of different pH (McIIvaine Buffer (pH 4, 5, 6, 7 and 8) or 50 mM sodium carbonate buffer (pH 9, 10 and 11)) for 60 min at 4 °C. Residual protease activity was determined after incubation.
  • Prestained Protein Ladder (10 - 170 kDa. Thermo Scientific) was included. After the electrophoresis, the gels were silver stained following the protocol of Pierce Silver Stain Kit (Thermo Scientific).
  • protease sample was mixed with native electrophoresis sample buffer (3.1 ml 1 mol/L Tris-HCI buffer (pH 6.8), 5 ml 50% glycerol. 0.5 ml 1.0% bromophenol blue and 1.9 ml MilliQ water) in a 4: 1 ratio without heat denaturation.
  • SDS-PAGE was carried out at 4 °C with a constant voltage of 80 V in a 12 % polyacrylamide gel. The gel was washed with 2.5 % Triton X-100 (v/v) in 50 mM Tris-HCI buffer (pH 9) for 30 min.
  • Fermentation culture was harvested by centrifugation at 10.000 g for 15 min at 4 °C, and the supernatant was filtered (Sartorius, Minisart® NML Syringe Filters 16534, 0.2 ⁇ ).
  • the secreted proteins were precipitated by incubating 30 ml filtered supernatant with freshly prepared 3 g crystalline trichloroacetic acid (final concentration 10% w/v) and kept in -20°C freezer overnight.
  • the precipitate was pelleted by thawing and centrifugation at 10.000 g for 30 min at 4°C.
  • the protein pellet was washed three times with 1ml ice-cold acetone and centrifuge at 14000 g for 5 min at 4 °C. Finally, the protein pellet was air dried.
  • the protein pellet was solubilized in digestion buffer (1 % sodium deoxycholate, 50 mM triethylammonium bicarbonate, pH 8.0) and heated to 99 °C for 5-10 min.
  • the sample was kept above 37 °C and 1 pg Tris (2-carboxyethyl) phosphine was added per 25 pg sample protein and incubated for 30 min at 60 °C.
  • 1 pg iodoacetamide (IAA) was added (from a 2.5 Mg/ ⁇ iodoacetamide stock solution in water) per 10 pg sample protein and incubated for 20 min at 37 °C in the dark. Then the sample was digested by the addition of 1 pg
  • Peptides were reconstituted in 0.1% trifluoroacetic acid / 2 % acetonitrile solution. A volume of 8 ⁇ of each sample was injected by the autosampler and concentrated on a trapping column (PepmaplOO, C18, 100 m x 2 cm, 5 ⁇ , Thermo Fisher Scientific) with water containing 0.1% formic acid and 2% ACN at a flow rate of 4 ⁇ / min. After 10 min, the peptides were eluted into a separation column (PepmapRSLC, C18, 75 ⁇ x 50 cm, 2 ⁇ , Thermo Fisher Scientific).
  • PepmapRSLC C18, 75 ⁇ x 50 cm, 2 ⁇ , Thermo Fisher Scientific
  • Protein identification was done with the open-source software MaxQuant (v. 1.4.1.2). The minimum peptide length was set to seven amino acids and the maximum false discovery rate (FDR) of 0.01 was required for proteins and peptides.
  • the Onygena corvina genome/6 frame database was used as a search database. Carbamidomethyl (C) was set as a fixed modification and acetyl (Protein N-term)/Oxidation (M) modifications were included in protein quantification. Razor peptides were used for protein quantification. A standard minimum ratio count of 2 was set to quantify the analysis. The "match between runs" option was enabled with a matching time window of 1 min.
  • the Biological triplicates were performed to normalize the label-free quantification (LFQ) values, and LFQ intensities in each sample were log2-transformed. To estimate the proteome variance, comparisons were performed using T-test (two-tailed, heteroscedastic). Batch CD-search was used to search for conserved domains and annotation of identified protein
  • Onygena corvina was cultured in YPD liquid medium on a rotary shaker (130 rpm) at 25 °C for 3 days.
  • the mycelia were filtered on a nylon mesh and grinded with liquid nitrogen.
  • the genomic DNA was extracted with the DNeasy plant mini kit (Qiagen) following the manufacturer's protocol.
  • the quantity and quality of the genomic DNA was measured on a NanodroplOOO (Thermo Scientific) and by electrophoresis on a 1 % agarose gel.
  • Onygena corvina was sequenced de novo on an Illumina Hiseq 2000 in one multiplexed lane as paired-end libraries with Truseq chemistry by AROS Applied Biotechnology A/S, Denmark. Based on the estimated genome size the sequence coverage was 370 times. The raw sequences were filtered for residual adapter sequences and trimmed with AdapterRemoval vl.5.2 and Seqtk. The clean sequences were assembled with CLC Genomic Workbench. Assembly statistics were calculated with the Assemblathon script.
  • the conserved peptides covered at least ten amino acids of the ORF. (Three hexapeptides that may be overlapping can cover from 8 (maximal overlap) to 18 (no overlap) residues of an amino acid sequence).
  • Keratin-degrading fungi Arthroderma otae (Assembly: GCA_000151145.1); Arthroderma gypseum (Assembly: GCA_000150975.1); Batrachochytrium dendrobatidis (Assembly: GCA_000149865.1); Coccidioides posadasii
  • GCA_000149335.1 Onygena corvina (New assembly); Trichophyton rubrum (Assembly: GCA_000151425.1); Trichophyton tonsurans (Assembly:
  • GCA_000151455.1 Trichophyton verrucosum (Assembly: GCF_000151505.1); Non-keratin-degrading fungi : Homoloaphlyctis polyrhiza
  • the number of proteases of each Merops family found in the genome of Onygena corvina and the eight keratin-degrading fungi downloaded from GenBank were compared to the number of proteases of each Merops family found in the genomes of the four non-keratin-degrading fungi downloaded from GenBank.
  • Onygena corvina was grown on feather or pig bristles or dog wool for seven days whereafter around 100 mg of mycelium together with keratin materials (feather or pig bristles) was thoroughly disrupted in lysis buffer by 3x 20 seconds pulses in the FastPrep®-24 homogenizer (MP Bio), and total RNA was extracted with the RNeasy plant mini kit (Qiagen). Genomic DNA was removed by treatment with DNase I (RNase-free) (M0303L, New England Biolabs Inc.). The quality and quantity of the RNA was measured by NanodroplOOO (Thermo scientific) and electrophoresis on a 1% agarose gel.
  • RNAse-free H2O 200 ng total RNA was mixed with 1 ⁇ primer (oligodT: Random primer 1 : 3 (0.5 Mg/ ⁇ )), and RNAse free H2O to 5 ⁇ .
  • the sample was heated 5 min at 70 °C and chilled immediately in an ice-bath for 5 min. Then it was spun down and added to a mix made of 4 ⁇ 5x ImProm II buffer, 1 ⁇ dNTP mix (lOmM each), 2 ⁇ 25 mM MgCI 2 and ⁇ ImProm- ⁇ TM Reverse Transcriptase (Promega) and RNAse-free H2O was added to a final volume of 15 ⁇ . The reaction was heated 5 min at 25 °C and incubated 1 h at 42 °C.
  • Example 3 18 predicted keratinase genes from Onygena corvina (Example 3) were amplified from cDNA made of RNA extracted from Onygena corvina growing on feather, pig bristles or dog wool with specific primers with a His-tag-encoding sequence added at the 5'-end of the reverse primer.
  • the PCR reaction mixtures contained 1.5 ⁇ diluted cDNA, 10 ⁇ 5xPhusion® HF Buffer, 1 ⁇ 10 mM dNTP (Fermentas), 2.5 ⁇ 10 ⁇ each primer and 1 U Phusion® High-Fidelity DNA Polymerase (M0530S, New England Biolabs Inc.) in 50 ⁇ .
  • the PCR reaction was performed in Biometra Thermocyclers T3000.
  • the initial denaturation step (98 °C, 30sec) was followed by 30 cycles of denaturation (98 °C, 10 sec), annealing (30 sec), elongation (72 °C, 30 sec per kb), and a final elongation step (72 °C, 10 min) after the final cycle.
  • the PCR products were purified with the GeneJET Gel Extraction and DNA Cleanup Mini Kit (K0831, Thermo Scientific) and digested with the restriction enzymes (New England Biolabs Inc.).
  • the vector pPinka-HC (PichiaPinkTM Expression System, Invitrogen) was digested with Stul and Fsel restriction enzymes and purified. The digested PCR products were inserted into pPinka-HC vector with T4 DNA Ligase (EL0011, Thermo Scientific). The recombinant plasmids were transformed to E. coli DH5a. Positive clones were selected on LB plates with 100 ⁇ g/ml ampicillin and identified by colony PCR and sequencing.
  • a single white clone was chosen for each recombinant protease genes and cultivated in 25 ml BMGY medium at 28 °C, 260 rpm until OD600 of 2-5 (after approximately 24 h). 25 ml of the culture was transferred to 1 I BMGY medium and divided in to two 2 liter baffled flasks. The Pichia was grown at 28 °C, 260 rpm until the culture reached OD600 of 2-5 (after approximately 24 h). The cells were harvested by centrifuging in sterile centrifuge bottles at 1500 xg for 5 min at room temperature.
  • the supernatant was decanted and resuspended in 200 ml BMMY medium and incubated at 28 °C and 260 rpm. Every 24 hours 1ml of 100% methanol was added to induce enzyme production. The supernatant was harvested after 4 days incubation by centrifugation at 1500 x g for 5 minutes at room temperature and filtered through a 0.2 ⁇ filter (Minisart Syringe Filters) and store at -80 °C. 7.1.23 Purification of expressed proteases
  • the His-tagged proteases were purified by fast protein liquid chromatography (FPLC) (AKTA Purifier) by the UNICORN method on a 1 ml HisTrap FF crude affinity column (GE Healthcare).
  • FPLC fast protein liquid chromatography
  • AKTA Purifier AKTA Purifier
  • the column was equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCI, 30 mM imidazole, pH 7.4) with a flow rate of 1 ml/min.
  • 100 ml sample was loaded onto the column, followed by washing with binding buffer until the absorbance reached a steady baseline.
  • the His-tagged proteases were finally eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCI, 500 mM imidazole, pH 7.4).
  • FTC-Casein Working Reagent was prepared by diluting 5 mg/ml FTC-Casein stock solution 1 : 500 in TBS buffer (25mM Tris-HCI, 0.15 M NaCI, pH 7.2). Trypsin standard was made by diluting the stock solution (50 mg/ml) to 0.5 Mg/ml in TBS buffer, serially dilute this solution to yield 6-8 standards.
  • Protein determination for the purified recombinant protease The protein concentration of purified recombinant enzyme was calculated according to the molar extinction coefficient of the related protease protein sequence (http ://encorbio.com/protocols/Prot-MW-Abs. htm). The absorbance of the protein at the ultraviolet wavelength of 280 nm was measured with a NanodroplOOO (Thermo Scientific).
  • Culture supernatant was harvest by centrifugation at 10000 x g for 10 min at 4 °C after fermentation. The supernatant was filtered (0.45 ⁇ ). The culture was fractionated using two separate strategies: 1. Cation exchange (5 ml HiTrap SP column, 50 mM citrate buffer, pH 3.86). 2. Anion exchange (1 ml HiTrap Q column, 20 mM Tris buffer, pH 8.6). In both cases, volumes corresponding to 50 ml of culture fluid were applied to the column and a NaCI gradient from 0 - 1M NaCI was applied to elute the bound protein.
  • fractionation of the culture broth supernatant from O. corvina growing on feather led to the identification of several partially purified fractions with high keratinase activity. Some of these fractions named A10, All, C15 and C20 did not contain the full wild type composition of secreted proteins but could nevertheless degrade the keratin in pig bristles.
  • Mas spectrometry of the fractions led to the identification of sequences with SEQ ID NO: 2, 4 and 6 in fraction C15 and SEQ ID NO: 2, 4, 8 and 10 in fraction C20. From the genomic sequence the full length of SEQ ID NO: 2, 4, 6, 8 and 10 could be identified and genes encoding the polypeptides were identified as SEQ ID NO: 1, 3, 5, 7 and 9.
  • the fungus Onygena corvina can degrade feather and hair completely
  • a sample of duck feather was cut into small pieces, embedded in in a minimal liquid medium containing 2 g/1 KH 2 P0 4 , 0.15 g/1 MgS0 4 .7H 2 0, 0.3 g/1 CaCI 2 , 3.3 g/1 Tween 80, pH 8 and inoculated with a) mycelium of the ascomycetoues non-pathogenic fungus Onygena corvina, which in nature grows specifically on feather, and b) with the ascomycetous non-pathogenic fungus Trichoderma asperellum, which in nature grows saprotrophically on a range of substrates, including keratinaceous materials. Negative control : Same material without fungus. After 8 days incubation the result was scored by visual inspection (Figure 2) . Surprisingly a total break down of the keratinaceous feather was observed when O. corvina was used as inoculum.
  • Onygena corvina (accession number: CBS 281.48) was genome sequenced by Illumina Hiseq 2000.
  • the assembly sequence was divided into 8 pools. Each pool was further divided into 1445 sequences with the length of 2000 bp except the last one (eg, in fragment 1, 1
  • the 8 th pool had 1426 sequences.
  • proteases belong to S8, M35, M36 and M43 families have great potential for keratin degradation.
  • Pichia pastoris system PichiaPinkTM Expression System (Invitrogen) was chosen for expressing protease genes.
  • PichiaPinkTM Strain 4 is double knock-out for both proteinases A and B (i.e., pepA and prbl), therefore has low
  • protease genes in Table 8 were inserted to pPinka-HC vector. As expected, all 13 proteases genes (including the synthetic >830
  • the recombinant proteases were purified by fast protein liquid chromatography (FPLC) (AKTA Purifier) according to the UNICORN method on a 1 ml HisTrap FF crude affinity column (GE Healthcare). The results indicated that all of the 13 genes with His tag were successfully expressed and purified. Purified
  • FTC-Casein Recombinant proteases activity and pig bristles degradation
  • FTC-Casein is native casein that has been labeled with a large molar excess of fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • Proteases can digest fluorescein-labeled casein into smaller, labeled fragments that result in a measurable change in fluorescence properties.
  • FRET-based measurement on a Corbett Rotor Gene 6000(Corbett Life Science) to detect the change in fluorescence when FTC-Casein was degraded by the purified recombinant proteases.
  • SEQ ID NO: 1 and SEQ ID NO: 2, S8 family
  • M36 family
  • the other purified recombinant proteases had very low protease activity.
  • SDS- PAGE results show that protease >687
  • protease with highest activity >687
  • the results showed that 50 ⁇ and 25 ⁇ recombinant protease >687
  • Examples 2 and 3 The result reported in Examples 2 and 3 are based on genome sequencing, analysis and bioinformatic predictions and confirmed by activity testing of predicted candidate proteases. In order to take advantage of direct
  • Example 1 identification of the proteases present in the culture broth that was shown (Example 1) to decompose a- and ⁇ -keratin, an MS analysis was made of the Onygena corvina secretome in this culture broth.
  • Example 2 To further identify proteases involved in the keratin-degrading activity of the Onygena corvina culture broth supernatant (Example 1), the supernatant was fractionated by anionic and cationic chromatography. Subsequently activity testing of all resulting fractions was done. Based on MS data (Example 4) determination of which proteases were found in the most active fractions could be made to protein family level.
  • the buffer for ion exchange chromatography is chosen so the protein of interest is at least 1 pH unit from the isoelectric point. But since the protease of interest was of unknown composition (and isoelectric point) it was necessary to guess what would be the best pH of the buffer.
  • a citrate buffer pH 3.86 was chosen for the cation exchange fractionation and a Tris-HCI buffer (pH 8.6) was chosen for the anion exchange fractionation.
  • fungi_t_q_l_(paired)_contig_125 False: 106584, S9 Dipeptidyl-peptidase were not identified in all the fractions.
  • Anion exchanged fractions (A) have much higher abundance of proteases than the cation exchanged fractions (C).
  • Fraction A13 has 18 proteases, which followed by A10 and A14 with 15 and 14 proteases, respectively. Most of cation exchanged fractions have around 2-5 proteases.
  • C15 mainly have 3 proteases >687
  • C20 mainly have 2 proteases >642
  • Fraction A10 has 15 kinds of protease including 2 proteases >642
  • the fractions were mixed in different combinations and tested for degradation of pig bristles.
  • the blends were set up as described in the methods section.
  • fraction C15 and C20 gave the highest activity, much higher than testing the two fractions individually, when adjusted for equal enzyme load (Figure 17).
  • the enzymes found in these fractions are an endoactive protease (two S8), exoactive proteases (two M28), and a metalloprotease (M3).
  • the degradation degrees of 25 ⁇ fraction C15 and C20 were 17 % and 17%.
  • the degradation degree of blend 12.5 ⁇ fraction C15 and 12.5 ⁇ fraction C20 was 21%. Therefore, the proteases >687
  • (SEQ ID NO: 9 and 10) in fraction 20 may have synergistic effect. Fraction All and A10 have much higher degradation degree.
  • the fractions were with the mixed recombinant protease >687
  • the blends were set up as described in the methods section.
  • SEQ ID NO: 5 and 6
  • SEQ ID NO: 5 and 6
  • EDTA ethylenediaminetetraacetic acid
  • (SEQ ID NO: 5 and 6) is important for keratin (here, pig bristles) degradation in combination with proteases >687
  • Neogymnomyces virgineus a new keratinolytic species from 20 dung, and its relationships with the Onygenales. Fungal Diversity. 52, 13-34.
  • Aspergillus hair bait soil samples of five different regions like f lav us technique fertile lands, animal herds, slaughter houses, poultries and barbers'shops soil samples of five different regions like
  • protease genes amplification results from cDNA made from Onygena corvina grown on chicken feather, pig bristles and dog wool. "+” indicated the protease coding sequence can be amplified from cDNA template, the related RNA was extracted from Onygena corvina when it grown on chicken feather, pig bristles or dog wool. "-” indicated the protease coding sequence cannot be amplified from cDNA template.
  • Protease activity profile of selected fraction from anion exchange chromatography Negative control : fraction was replaced by 20 mM Tris-HCI buffer, pH 8.6.

Abstract

Cette invention concerne l'utilisation d'une composition microbienne pour la dégradation de substances kératiniques. Un procédé de dégradation de substances kératiniques et des compositions comprenant une substance kératinique dégradée sont en outre décrits.
PCT/DK2014/050098 2013-04-19 2014-04-15 Utilisation d'une composition microbienne pour la dégradation de substances kératiniques WO2014169920A2 (fr)

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EP3441475A1 (fr) * 2017-08-09 2019-02-13 Barentzymes AS Neutre thermosensible protease de serine dérivée d'o. corvine
CN111606990A (zh) * 2020-06-03 2020-09-01 江南大学 活性大分子角蛋白的制备方法及其作为生物敷料的应用
CN112795488A (zh) * 2020-12-29 2021-05-14 黄山学院 一种尖孢镰刀菌菌株及其在降解鸡毛中的用途
EP4122324A1 (fr) * 2021-07-22 2023-01-25 Symborg, S.L. Procédé de conversion de la kératine
WO2024055078A1 (fr) * 2022-09-16 2024-03-21 Veratin Ltd Produits fermentés alcooliques et non alcooliques et leur procédé de préparation

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WO2019030319A1 (fr) * 2017-08-09 2019-02-14 Barentzymes As Sérine protéase thermiquement sensible neutre dérivée d'o. corvina
CN111606990A (zh) * 2020-06-03 2020-09-01 江南大学 活性大分子角蛋白的制备方法及其作为生物敷料的应用
CN111606990B (zh) * 2020-06-03 2023-02-21 江南大学 活性大分子角蛋白的制备方法及其作为生物敷料的应用
CN112795488A (zh) * 2020-12-29 2021-05-14 黄山学院 一种尖孢镰刀菌菌株及其在降解鸡毛中的用途
EP4122324A1 (fr) * 2021-07-22 2023-01-25 Symborg, S.L. Procédé de conversion de la kératine
WO2023001946A1 (fr) * 2021-07-22 2023-01-26 Symborg, Sl Procédé de conversion de kératine
WO2024055078A1 (fr) * 2022-09-16 2024-03-21 Veratin Ltd Produits fermentés alcooliques et non alcooliques et leur procédé de préparation

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