WO2015158719A1 - Composition and method for degradation of keratinaceous materials - Google Patents

Abstract

Keratin degrading composition comprising of at least two different isolated active keratinases from at least two different MEROPS protease families, wherein at least one active serine endo-keratinase belongs to the MEROPS family S8 and use of such a composition for degrading keratinaceous materials (such as e.g. feather and pig bristles).

Description

Composition and method for degradation of keratinaceous materials Field of the invention

The present invention relates to keratin degrading composition comprising at least two different isolated active keratinases from at least two different MEROPS protease families, wherein at least one active serine endo-keratinase belongs to the MEROPS family S8 and use of such a composition for degrading keratinaceous materials (such as e.g. feather or pig bristles). Background of the invention

Keratin is a family of extremely strong fibrous structural proteins that are a major component in skin, hair, nails, hooves, horns, and teeth. The amino acids which combine to form it have several unique properties and, depending on the levels of the various amino acids, it can be inflexible and hard, like hooves, or soft, as is the case with skin, a- keratin is found in the hair (including wool and pig bristles), horns, nails, claws and hooves of mammals. The harder β-keratin is found in nails and in the scales and claws of reptiles, their shells and in the feathers, beaks, claws of birds and quills of porcupines.

Poultry-processing plants produce huge quantities of feather wastes annually. The large amounts of discarded feathers cause a serious local disposal and accumulative problem leading to environmental pollution, β-keratin is the most abundant and important structural protein found in feathers, β-keratin 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. The high content of cysteine residues in feather contribute to keratin stability by forming disulphide bridges between different twists in a single peptide chain and between chains in keratin. Keratin with many cysteine residues is highly resistant to enzymatic hydrolysis. It is reported that the content of cysteine in bird feathers reaches up to 15%, indicating that feather is very difficult to degrade. In the art is also described that pig bristles are very difficult to decompose/degrade. Applying physical and chemical methods to convert feathers into feather meal resulted in the loss of nutritionally essential amino acids and formation of non-nutritive amino acids

compositions. Thus, enzymatic processes may present a better approach for efficient keratin degradation.

Several species of bacteria including Bacillus spp., Bacillus licheniformis, B. clausii, B. subtilis (Lin et al., 1992) and Streptomyces spp. and actinomycetes; and fungi including Trichophyton spp., Microsporum spp., Aspergillus spp., Rhizomucor spp., Trichoderma spp., Onygena spp. (e.g. Onygena corvina or O. equina), and Chrysosporium spp. are well known for bioconversion of keratinaceous materials due to the elaboration of keratinolytic proteases. In the art - protease enzymes with potentials for decomposition of keratin in natural materials such as feather (predominantly β-keratin) and hair, wool, and bristles

(predominantely a-keratin) has been found and described primarily from fungi that invade animal skin and belong to the group of human dermatophytes/human pathogens.

Examples of human pathogenic fungi are the human pathogenic dermatophytes

Arthroderma benhamiae, Arthroderma gypseum, Arthroderma otae (Microsporum canis), Trichophyton equinum, Trichophyton rubrum, Trichophyton tonsurans and Trichophyton verrucosum. Due to the pathogenicity these fungi may not be industrially preferred for isolation of proteases or keratinases as this could pose a health risk. Many Onygenales are keratinophilic fungi that either behave as saprophyte on keratin substrate or are pathogens of birds, mammals and human (Doveri et al., 2012). However, relatively little is known about the keratinolytic potential of Onygena spp, more specifically O. corvina/ O. equina. Keratinases (EC 3.4.21/24/99.11), are robust enzymes that are able to hydrolyze insoluble keratins more efficiently than other proteases. Most keratinases have some common characteristics despite their different origins. They belong mainly to the extracellular serine proteases or metalloproteases (Gupta and Ramnani, 2006). Keratinases have multitude industrial application such as detergent additives, dehairing process of leather manufacture, medicine, cosmetics, biodegradable films and coatings and in degrading prions to treat the dreaded mad cow disease. In the industrial enzyme market, the available proteases are mainly from Bacillus strains/species and the industrial application and commercial exploitation of keratinases is still in the stage of infancy.

Nevertheless, the fungal keratinases have been researched increasingly for technology advantages, such as high enzyme yield, easy downstream process and medical and veterinary application.

As known in the art, the MEROPS family classification is a well-known and established classification of proteases such as e.g. keratinases (see e.g. the MEROPS database link: http://merops.sanaer.ac.uk and Rawlings, N.D., Waller, M., Barrett, A.J. & Bateman, A. (2014) MEROPS : the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 42, D503-D509). In the MEROPS family classification relates "S" (in e.g. S8) to Serine peptidase and "M" (in e.g. M28) to Metallopeptidase.

As known in the art (see e.g. the MEROPS database link: http://merops.sanqer.ac. uk/cqi- bin/famsum?family=s8), the MEROPS peptidase family S8 contains the serine

endopeptidase subtilisin and its homologues. Members of family S8 have a catalytic triad in the order Asp, His and Ser in the sequence, which is a different order to that of families SI, S9 and S10. In subfamily S8A, the active site residues frequently occurs in the motifs Asp-Thr/Ser-Gly (which is similar to the sequence motif in families of aspartic endopeptidases in clan AA (AA)), His-Gly-Thr-His and Gly-Thr-Ser-Met-Ala-Xaa-Pro. In subfamily S8B, the catalytic residues frequently occur in the motifs Asp-Asp-Gly, His-Gly- Thr-Arg and Gly-Thr-Ser-Ala/Val-Ala/Ser-Pro.

The skilled person can routinely determine if a protease of interest belongs to the MEROPS peptidase family S8.

As known in the art (see e.g. the MEROPS database link: http://merops.sanqer.ac. uk/cqi-

the MEROPS peptidase family M28 contains aminopeptidases and carboxypeptidases. In members of family M28, each zinc ion is tetrahedrally coordinated, with three amino acid ligands plus activated water; one aspartate residue binds both metal ions. The zinc ligands (with the metal ion that is bound indicated by Roman numerals) occur in the sequence in the order His (II), Asp (I and II), Glu (I), Asp or Glu (II) and His (I). In addition, two other residues, an Asp and a Glu, are believed to be important for catalysis. Four of these residues occur in the motifs His-Xaa-Asp and Glu- Glu. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate.

The skilled person can routinely determine if a protease of interest belongs to the MEROPS peptidase family M28.

As known in the art (see e.g. the MEROPS database link: http://merops.sanqer.ac. uk/cqi- bin/famsum?family= m3), the MEROPS peptidase family M3 contains metallopeptidases with varied activities. Varied peptidase reactions are catalysed by members of family M3. The commonest is a form of endopeptidase activity that is restricted to substrates of low molecule mass, and is therefore termed Oligopeptidase'. Both thimet oligopeptidase (M03.001) and neurolysin (M03.002) are oligopeptidases, acting only on substrates of less than about 19 amino acid residues, with a particular preference for cleaving near the C- terminus. The bacterial peptidyl-dipeptidase Dcp (M03.005) liberates C-terminal dipeptides, but the most unusual form of peptidase activity in the family is that of the mitochondrial intermediate peptidase (M03.006) that cleaves N-terminal octapeptides from proteins during their import into mitochondria.

The skilled person can routinely determine if a protease of interest belongs to the MEROPS peptidase family M3.

Summary of the invention

A problem to be solved by the present invention is to provide novel keratinases and keratinase compositions for enhancing the degradation of keratin - such as e.g. poultry feather wastes and pig bristles, a voluminous waste product from pig slaughter houses.

The solution is based on that the present inventors have identified that Onygena corvina was significantly better to degrade the keratin in duck feather as compared to

Trichoderma asperellum (see e.g. working examples and figures herein and figure 1 and 4).

Trichoderma species are known as extraordinarily good enzyme secretors and could prima facie be a good choice for production of a blend of enzymes for keratin decomposition.

However, as discussed above the present inventors identified that Onygena corvina was significantly better to degrade the keratin in duck feather as compared to Trichoderma asperellum.

Without being limited to theory - it is believed that no association or track record as human pathogen has been described with respect to Onygenales, preferably Onygena spp. (e.g. Onygena corvina).

Accordingly, it is believed that Onygena spp. is non-pathogenic to humans.

Based on above, the present inventors analyzed reasons for the very good properties of Onygena corvina and identified that the fungi comprises different keratinases and that the good keratin degradation effect is due to a synergistic combination of the use of at least 2 different types of keratinases and this could give a good degradation of both a- and β- keratin.

As discussed in working examples herein - the present inventors purified (via anion/cation chromatography) fractions comprising isolated samples of different keratinases from Onygena corvina. They also cloned and expressed in Pichia, the keratinase herein termed "68717 | " - (SEQ ID NO: 1 and SEQ ID NO : 2 herein) - as discussed herein this enzyme was identified to belong to the MEROPS family S8). The present inventors analyzed several of the purified fractions/blends comprising different Onygena keratinases and essentially identified that in order to get the best keratin degradation it is preferred to use a mixture comprising at least one serine endo- keratinase that belongs to the MEROPS family S8 and another different MEROPS family class keratinase (such as e.g . a exo-keratinase or e.g . a metalloprotease from M EROPS family M28 or M3).

The herein discussed positive keratin degradation synergistic effect results by using a blend comprising at least 2 different types of keratinases were demonstrated by using purified keratinases from Onygena corvina.

As discussed above - the MEROPS family classification is a well-known and established classification of proteases such as e.g . keratinases and it is known that proteases within the same MEROPS classification (e.g . S8, M28 or M3) have common structural and activity related features.

Accordingly and without being limited to theory, it is believed that the herein

demonstrated positive keratin degradation effects may be seen as a class effect - e.g . that the positive effect herein shown for e.g . a mixture/blend comprising a Onygena corvina S8 and M28 proteases makes it plausible that a similar positive effect may be obtained by using S8 and M28 proteases from other organism than Onygena.

Accordingly, a first aspect of the present invention relates to a keratin degrading composition comprising at least two different isolated active keratinases from at least two different MEROPS protease families, wherein :

(i) : at least one active serine endo-keratinase belongs to the M EROPS family S8; and (ii) at least one active keratinase does not belong to the MEROPS family S8;

and wherein :

(a) : at least 0.01 % (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii);

(b) : at least 1 % (w/w - dry matter) of the total weight of the keratinases in the composition is keratinase of item (i);

(c) : the total weight of the composition (w/w - dry matter) is from lg to 100.000 tons. As discussed above, the MEROPS family classification is a well-known and established classification of proteases such as e.g. keratinases and the skilled person can routinely determine if a protease of interest belongs to the MEROPS peptidase family S8. Accordingly, it is routine work for the skilled person to determine if a keratin degrading composition of interest comprises keratinases of item (i) and (ii) of the first aspect.

As understood by the skilled person in the present context, the term "at least one" in item

(i) and (ii) of the first aspect relates to that the composition may e.g. comprise two or more different S8 keratinases in item (i) and/or two or more different keratinases in item

(ii) which are not belonging to the S8 family.

Item (a) of the first aspect essentially relates to that a commercial relevant

product/composition must have a minimum amount of herein relevant active keratinases - it is routine work for the skilled person to determine if a composition of interest fulfills the requirement of item (a) of the first aspect. For instance, if the total weight of the composition is lOOOg then the composition must comprise at least (0.01%) 0.1 g of keratinase. Item (b) of the first aspect essentially relates to that a commercial relevant

product/composition must have a minimum ratio of herein relevant active S8 keratinases - it is routine work for the skilled person to determine if a composition of interest fulfills the requirement of item (b) of the first aspect. For instance, if the composition comprises lOOOg of keratinase then at least (1%) 10 g must belong to the S8 family.

Item (c) of the first aspect essentially relates to that a commercial relevant

product/composition must have a minimum weight in order to be useful in e.g. the poultry industry. A second aspect of the invention relates to a method for the degradation of keratinaceous materials comprising the steps of:

(I) : adding to a keratinaceous material a keratin degrading composition according to the first aspect and/or herein relevant embodiments thereof;

(II) : incubating for a suitable time under suitable conditions, wherein the keratinases of the composition degrade the keratinaceous material; and

(III) : - obtaining a degraded keratinaceous material.

Definitions All definitions of herein relevant terms are in accordance of what would be understood by the skilled person in relation to the herein relevant technical context.

The term "identity" is defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. Thus, in the present context "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. Similarly, 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.

To determine the percent identity of two nucleic acid sequences or of two amino acids, 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. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical

positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment the two sequences are the same length.

One may manually align the sequences and count the number of identical nucleic acids or amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches may be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See

http://www. ncbi.nlm.nih.gov. Alternatively, 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). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.

The term "isolated keratinase" essentially relates herein to that the keratinase is isolated from its natural environment - said in other words that the polynucleotide preparation is essentially free of other material with which it is natively associated. Herein are described keratinases isolated from Onygena corvina. Accordingly, as understood by the skilled person in the present context - the term isolated keratinase does not cover the keratinase naturally present in Onygena corvina.

The term "keratinase" should be understood as the skilled person would understand it in the present context. As discussed above, the term keratinase is a well-known term to the skilled person. Herein is described one assay of keratinase activity using keratin azure (Sigma-Aldrich, USA) as substrate. The term keratinase is herein understood as an enzyme that has activity in this keratinase assay using keratin azure (Sigma-Aldrich, USA) as substrate.

Embodiment of the present invention is described below, by way of examples only.

Drawing description

Figure 1A is showing the type of duck feather used as substrate in the present work.

Figure IB 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. Dark duck feather was chosen to be representative of chicken feather but it is even harder to decompose. Figure 2 shows pig bristles with no further pretreatment than being cut in small pieces, and hereafter 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 3 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. Furthermore, the weight loss of the substrate, the release of soluble protein from the substrate, and the release of thio-groups are shown.

Figure 4 shows the difference in keratinolytic capabilities of Onygena corvina and

Trichoderma asperellum.

Figure 5 shows a spectrum of protease families in Onygena corvina genome, with indication of the number of representatives from each family. Figure 6 shows mass spectrometry data, identifying protease genes found in Onygena corvina secretome when grown on chicken feather and pig bristles for 11 days.

Figure 7 shows the degree of degradation of pig bristles by treatment with different fractions. (A: anion exchanged fractions; C : cation exchanged fractions). Fractions C15 and C20 resulted in the strongest degradation . >687 | 7 | is a purified recombinant protease (gene SEQ ID NO: 1, protein SEQ ID NO : 2) expressed in Pichia. 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 constant agitation at 1000 rpm .

Figure 8 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 9 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). Apparently the recombinant protease >687 | 7 | did not result in additional activity when added to C15, C20, A10, or Al l . >687 | 7 | 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 10 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

metallopeptidase 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 11 shows the degree of degradation of pretreated bristles and hooves (source: slaughter house) by treatment for 4 days with culture broth supernatant of Onygena corvina grown for 11 days on chicken feather. 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.

The present invention will now be described in more detail in the following. Detailed description of the invention

As understood by the skilled person in the present context - it is understood to be most preferred to combine individual preferred embodiments described herein.

For instance, a herein described preferred S8 keratinase may in a most preferred embodiment be combined with a preferred M28 keratinase to obtain a herein most preferred keratin degrading composition.

Serine endo-keratinase that belongs to the MEROP5 family 58 - item (i) of first aspect Preferably, the keratinase of item (i) of the first aspect is a keratinase isolated from an Ascomycetous fungal specie, preferably a fungal specie that belongs to Onygenales and more preferably a fungal specie that belongs to Onygenaceae.

Even more preferably, the keratinase of item (i) is a keratinase isolated from an Onygena specie, preferably Onygena corvina or Onygena equina - most preferably Onygena corvina.

It is preferred that the fungal specie is non-pathogenic to humans. As discussed above and without being limited to theory - it is believed that no association or track record as human pathogen has been described with respect to Onygenales, preferably Onygena spp. (e.g. Onygena corvina).

Accordingly, it is believed that species of Onygenales, preferably Onygena spp. are nonpathogenic to humans.

As discussed above, Bacillus spp. (such as e.g. Bacillus licheniformis, B. clausii or B.

subtilis (Lin et al., 1992) are well known for bioconversion of feather wastes due to the elaboration of keratinolytic proteases.

Without being limited to theory - it is believed that in the prior art it is not explicitly described to use an isolated active serine endo-keratinase that belongs to the MEROPS family S8 from Bacillus in a keratin degrading composition as described herein.

Accordingly, it may be preferred that the keratinase of item (i) of the first aspect is a keratinase isolated from a Bacillus specie, preferably from Bacillus licheniformis, B. clausii or B. subtilis. Based on a herein relevant sequence identity search the present inventors found that the closest herein relevant publicly known prior art described keratinase sequences have less than 86% identity to the sequences shown as SEQ ID NO: 2 herein (MEROPS family S8).

Based on a herein relevant sequence identity search the present inventors found that the closest herein relevant publicly known prior art described keratinase sequences have less than 71% identity to the sequences shown as SEQ ID NO: 10 herein (MEROPS family S8).

Preferably, the keratinase of item (i) is at least one serine endo-keratinase that belongs to the MEROPS family S8 that comprises:

- a sequence having at least 86% (more preferably at least 90%, even more

preferably at least 95% and most preferably at least 98%) identity to SEQ ID NO. :

2; or

a sequence having at least 71% (more preferably at least 80%, even more preferably at least 90% and most preferably at least 95%) identity to SEQ ID NO. : 10.

It may be preferred that keratinase of item (i) comprises SEQ ID NO. : 2 or SEQ ID NO. : 10. As discussed herein, the present inventors cloned and expressed in Pichia the keratinase herein termed "687 | 7 | " - (SEQ ID NO: 1 and SEQ ID NO: 2 herein). Keratinase that does not belong to the MEROP5 family 58 - item (ii) of first aspect

As discussed herein - the S8 family keratinase of item (ii) is an endo-keratinase.

As known in the art - endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from their end-pieces. For this reason, endopeptidases cannot break down peptides into monomers, while exopeptidases can break down proteins into monomers. Without being limited to theory - it is believed that the herein described positive keratin degradations results may be due to a synergistic effect of using both an endo-keratinase and an exo-keratinase.

Accordingly, a herein preferred embodiment is wherein the keratinase of item (ii) is at least one exo-keratinase.

Preferably, the keratinase of item (ii) is at least one metalloprotease that belongs to the MEROPS family M28 or at least one metalloprotease that belongs to the MEROPS family M3.

Preferably, item (ii) comprises at least two different metalloproteases, which are one metalloprotease that belongs to the MEROPS family M28 and one metalloprotease that belongs to the MEROPS family M3. Preferably, the keratinase of item (ii) of the first aspect is a keratinase isolated from an Ascomycetous fungal specie, preferably a fungal specie that belongs to Onygenales and more preferably a fungal specie that belongs to Onygenaceae.

Even more preferably, the keratinase of item (ii) is a keratinase isolated from an Onygena sp., preferably Onygena corvina or Onygena equina - most preferably Onygena corvina.

It is preferred that the fungal specie is non-pathogenic to humans. As discussed above, Bacillus spp. (such as e.g. Bacillus licheniformis, B. clausii or B.

subtilis (Lin et al., 1992) are well known for byconversion of feather wastes due to the elaboration of keratinolytic proteases. Accordingly, it may be preferred that the keratinase of item (ii) of the first aspect is a keratinase isolated from a Bacillus specie, preferably from Bacillus licheniformis, B. clausii or B. subtilis.

Based on a herein relevant sequence identity search the present inventors found that the closest herein relevant publicly known prior art described keratinase sequences have less than 84% identity to the sequences shown as SEQ ID NO: 4 herein (MEROPS family M28).

Based on a herein relevant sequence identity search the present inventors found that the closest herein relevant publicly known prior art described keratinase sequences have less than 84% identity to the sequences shown as SEQ ID NO: 6 herein (MEROPS family M3).

Based on a herein relevant sequence identity search the present inventors found that the closest herein relevant publicly known prior art described keratinase sequences have less than 85% identity to the sequences shown as SEQ ID NO: 8 herein (MEROPS family M28).

Preferably, the keratinase of item (ii) is at least one is at least one metalloprotease that comprises a sequence having at least 85% (more preferably at least 90%, even more preferably at least 95% and most preferably at least 98%) identity to SEQ ID NO. : 4, 6 or 8.

Preferably, the keratinase of item (ii) is at least one metalloprotease that belongs to the MEROPS family M28 that comprises a sequence having at least 85% identity to SEQ ID NO. : 4 or 8;

or wherein the keratinase of item (ii) is at least one metalloprotease that belongs to the MEROPS family M3 that comprises a sequence having at least 85% identity to SEQ ID NO. : 6.

It is routine work for the skilled person to make a variant of an isolated keratinase as described herein - i.e. a variant, wherein e.g. one or more amino acids of e.g. SEQ ID NO: 2, 4, 6, 8 or 10 have been modified/altered.

Further - as known to the skilled person if such variant changes are not too drastic it will be plausible that the enzyme would maintain its relevant activity. Based on e.g. the sequence information disclosed herein - it is routine work for the skilled person to obtain an isolated keratinase as described herein.

This may e.g. be done by recombinant expression in a suitable recombinant host cell according to procedures known in the art.

Accordingly, it is not believed necessary to describe such standard known recombinant expression procedures in many details herein.

The term "recombinant host cell" should herein be understood according to the art. As known in the art, recombinant polynucleotide (e.g. DNA) molecules are polynucleotide (e.g. DNA) molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. As understood by the skilled person - a recombinant host cell comprises recombinant polynucleotide (e.g. DNA) molecules and a recombinant host cell will therefore not be understood as covering a natural wild type cell as such.

It may be preferred that an isolated keratinase as described herein is a recombinant produced and isolated keratinase.

Combination of keratinases from different organisms As discussed herein - the isolated active keratinases may be obtained from different organisms and it may be preferred that the keratin degrading composition as described herein comprises different keratinases isolated from different organisms.

For instance, it may be preferred that the keratinase of item (i) of the first aspect is a keratinase isolated from a Ascomycetous fungal specie (preferably from Onygena sp., preferably Onygena corvina or Onygena equina) and the keratinase of item (ii) of the first aspect is a keratinase isolated from a Bacillus specie, preferably from Bacillus

licheniformis, B. clausii or B. subtilis.

In this embodiment it is preferred that the Bacillus keratinase is a metalloprotease that belongs to the MEROPS family M28 and/or a metalloprotease that belongs to the MEROPS family M3.

Alternatively, it may be preferred that the keratinase of item (ii) of the first aspect is a keratinase isolated from a Ascomycetous fungal specie (preferably from Onygena sp., preferably Onygena corvina or Onygena equina) and the keratinase of item (i) of the first aspect is a keratinase isolated from a Bacillus specie, preferably from Bacillus

licheniformis, B. clausii or B. subtilis. In this embodiment it is preferred that the Ascomycetous keratinase is a metaiioprotease that belongs to the MEROPS family M28 and/or a metaiioprotease that belongs to the MEROPS family M3. In a keratin degrading composition as described herein, wherein the keratinase of item (i) and/or item (ii) is a keratinase isolated from a Ascomycetous fungal species (preferably a from Onygena sp., preferably Onygena corvina or Onygena equina) and wherein the keratinase of item (ii) is at least one metaiioprotease that belongs to the MEROPS family M28 or at least one metaiioprotease that belongs to the MEROPS family M3 - it may be preferred that the composition also comprises at least one (e.g. at least two) isolated active keratinase(s) obtained from a Bacillus specie, preferably from Bacillus

licheniformis, B. clausii or B. subtilis.

In this embodiment it is preferred that the Bacillus keratinase is a metaiioprotease that belongs to the MEROPS family M28 and/or a metaiioprotease that belongs to the MEROPS family M3.

In a keratin degrading composition as described herein, wherein the keratinase of item (i) is at least one serine endo-keratinase that belongs to the MEROPS family S8 that comprises:

- a sequence having at least 86% identity to SEQ ID NO. : 2; or

a sequence having at least 71% identity to SEQ ID NO. : 10

- it may be preferred that the composition also comprises at least one (e.g at least two) isolated active keratinase(s) obtained from a Bacillus specie, preferably from Bacillus licheniformis, B. clausii or B. subtilis.

In this embodiment it is preferred that the Bacillus keratinase is a metaiioprotease that belongs to the MEROPS family M28 and/or a metaiioprotease that belongs to the MEROPS family M3.

In a keratin degrading composition as described herein, wherein the wherein the keratinase of item (ii) is at least one metaiioprotease that comprises a sequence having at least 85% identity to SEQ ID NO. : 4, 6 or 8

- it may be preferred that the composition also comprises at least one (e.g. at least two) isolated active keratinase(s) obtained from a Bacillus specie, preferably from Bacillus licheniformis, B. clausii or B. subtilis.

In this embodiment it is preferred that the Bacillus keratinase is a metaiioprotease that belongs to the MEROPS family M28 and/or a metaiioprotease that belongs to the MEROPS family M3. Keratin degrading composition - amount of keratinase , weight etc

As discussed above - item (a) of the first aspect reads: "(a): at least 0.01 % (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii);".

Generally speaking it is for commercial relevant reason preferred to have a relatively concentrated keratin degrading composition.

Accordingly, in relation to item (a) it is preferred that at least 0.05 % (more preferably at least 0.5% even more preferably at least 1% and most preferably at least 5%) (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii).

Without being limited to theory - it is believed that it is preferred that the keratin degrading composition comprises a relatively high ratio of family S8 keratinase.

Accordingly in relation to item (b) of the first aspect, it is preferred that at least 5 % (more preferably at least 10%, even more preferably at least 20% and most preferably at least 50%) (w/w - dry matter) of the total weight of the keratinases in the composition is keratinase of item (i).

In relation to item (c) of the first aspect, it may be preferred that the total weight of the composition (w/w - dry matter) is from 10 g to 10.000 tons, such as from 100 g to 1.000 tons or such as from 1000 g to 100 kg. The keratin degrading composition as described herein is preferably a composition, wherein keratinase activity of the composition is from 0.1 to 1000 Kilo NOVO Protease Units (KNPU), even more preferably from 1 to 100 Kilo NOVO Protease Units (KNPU) and most preferably 10 to 25 Kilo NOVO Protease Units (KNPU). Kilo NOVO Protease Units (KNPU) is a well know unit definition for the skilled person in the present context.

As known in the art (se e.g. WO00/71683A1) - Kilo NOVO Protease Units (KNPU) is determined relatively to an enzyme standard (SAVINASE^®) , and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50°C, pH 8.3, 9 min. reaction time, 3 min. measuring time.

A method for the degradation of keratinaceous materials As discussed above - a second aspect of the invention relates to a method for the degradation of keratinaceous materials comprising the steps of:

(I) : adding to a keratinaceous material a keratin degrading composition according to any of the preceding claims;

(II) : incubating for a suitable time under suitable conditions, wherein the keratinases of the composition degrade the keratinaceous material; and

(III) : - obtaining a degraded keratinaceous material.

Before step (I), it may be preferred to perform a pretreatment step - i.e. prior to the enzymatic hydrolysis in step (I) and/or Step (II) - this pretreatment step is preferably involving pressure cooking or other chemical or physical method of the keratinaceous material.

In the present context it is routine work of the skilled person to identity preferred suitable time and conditions in step (II). As known - the skilled person will e.g. routinely try different relevant suitable time/conditions in a small scale and the up-scale preferred time/conditions to a larger scale.

Preferably, the keratinaceous material is at least one keratinaceous material selected from the group consisting of feather, hair, teeth, hoof, horn, skin, wool and bristles (such as pig bristles). More preferably the keratinaceous material is at least one keratinaceous material selected from feather, skin, hair, wool, bristles (such as pig bristles) and hoof.

Skin, hair, bristles (such as pig bristles) and/or hoofs keratinaceous material may be derived from slaughterhouse waste.

Feather keratinaceous material may be derived from poultry farm waste or slaughterhouse waste.

Hair may e.g. be pig bristles or wool.

It may be preferred that the keratinaceous material is feather or pig bristles. Generally speaking - in a herein commercial relevant context it is generally preferred to add significant amount of the keratin degrading composition to a significant amount of keratinaceous material (e.g. poultry waste).

Accordingly, a preferred method of the second aspect is wherein there in step (I) of the second aspect is added at least lg of the keratin degrading composition to at least 10 g of keratinaceous material and wherein the degraded keratinaceous material of step (III) is having a degree of degradation of at least 10%. More preferably, a preferred method of the second aspect is wherein there in step (I) of the second aspect is added at least 10 g of the keratin degrading composition to at least 100 g of keratinaceous material and wherein the degraded keratinaceous material of step (III) is having a degree of degradation of at least 30%.

It is routine work for the skilled person to determine the degree of degradation.

According to the art - degree of degradation may be determined by weight loss of the keratinaceous material (e.g. duck feather) in the following way:

Weight loss of the keratinaceous material (e.g. duck feather) in protease and keratinase production medium is estimated by determining the loss of the keratinaceous material (e.g. duck feather) dry weight. Initial keratinaceous material (e.g. feather) weight is determined as dry keratinaceous material (e.g. feather) weight after dehydration at a suitable temperature (e.g. 50 °C). Final keratinaceous material (e.g. feather) weight is measured as the dry weight of the residual keratinaceous material (e.g. feather) after dehydration at suitable temperature (e.g. 50 °C). The weight loss in each experiment is determined using the formula :

Weight loss (%) = (initial keratinaceous material weight-final keratinaceous material weight)/initial keratinaceous material weight x 100.

As discussed in working examples herein - proteases from Onygena 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.

A preferred embodiment of the method of the second aspect is wherein the temperature of step (II) of the second aspect is temperature from 15 to 70 °C and the pH is from pH 5- 11.

This embodiment is particular preferred the keratinase of item (i) and/or item (ii) of the first aspect is a keratinase isolated from a Onygena sp., preferably Onygena corvina or Onygena equina.

Examples Material and methods

Microorganism and growth conditions. Onygena corvina (accession number: CBS 281.48) and Trichoderma asperellum (accession number: CBS 131938) was obtained from CBS-KNAW fungal Biodiversity Centre

(Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) and kept on potato dextrose agar plate at 4 °C. For protease production, 4 mm² square of 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 KH₂P0₄, 0.15 g/l MgS0₄7H₂0, 0.3 g/l CaCI₂, 3.3 g/l Tween 80, pH 8 and cultured at 25 °C on a rotary shaker (200 rpm) for eight to eleven days. Keratin

Duck feather was obtained from Valbyparkens, Denmark (2012). Chicken feather was obtained from Rose Poultry (Vinderup, Skovsgaard, Denmark). Pig bristles were obtained from Danish Crown (Bragesvej, Denmark). Dog wool was kindly provided by Peter Busk. Pretreated bristles and hooves was obtained from Danish Crown (Bragesvej, Denmark).

Determination of weight loss of the substrate in protease and keratinase production medium.

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. The weight loss in each experiment was determined using the formula :

Weight loss (%) = (initial feather weight-final feather weight)/initial feather weight x 100. Determination of enzyme activities

Protease activity assay with Azocasein

Protease activity was assayed by mixing 20 μΙ 1.5 % Azocasein (Sigma-Aldrich.) suspension in 50 mM sodium carbonate buffer (pH 9.0) and 20 μΙ diluted enzyme. The reactions were carried out at 50 °C for 60 min with constant agitation at 300 rpm using a TS-100 Thermo-Shaker, SC-20 (Biosan Ltd). The reactions were stopped by adding 100 μΙ 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. 100 μΙ supernatant was transferred to a microtiter plate already containing 25 μΙ of 1.8 M NaOH . Absorbance was read at 405 nm. As a control, 20 μΙ 1.5% Azocasein suspension in the same buffer with addition of 100 μΙ 0.4 M TCA before adding 20 μΙ enzyme solution was used. 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 assay

Keratinase activity was measured with (Sigma-Aldrich, USA) as substrate. The keratin azure was ground to a fine powder with a mortar and pestle in liquid nitrogen. Next, 0.4 g keratin azure powder was mixed with 100 ml 50 mM sodium carbonate buffer (pH 9.0). The reaction mixture contained 50 μΙ keratin azure suspension and 50 μΙ 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). After incubation, the reactions were stopped by adding 100 μΙ 0.4 M TCA followed by centrifuging at 16000xg for 1 min to remove the substrate. Release of azo dye was measured as absorbance of the supernatant at 595 nm. As a control, 50 μΙ 0.4 % keratin azure suspension in the same buffer was mixed with 100 μΙ 0.4 M TCA before addition of 50 μΙ enzyme solution and incubation at 50 °C for 24 h. One unit keratinase activity was defined as the amount of enzyme that resulted in an increase of 0.01 in absorbance at 595 nm under the reaction conditions. Protease activity assay with pig bristles

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)XlOO

Protein determination

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.

Determination of thiol formation Free thiol groups were analysis by mixing 100 μΙ sample with 20 μΙ NH₄OH, 100 μΙ of 0.5 g/l NaCN and 100 μΙ Mill iQ water. The mixture was incubated for 20 min at 25 °C, following by addition of 20 μΙ of 0.5 g/l sodium nitroprusside. Absorbance at 530 nm was measured within two min.

Characterization of the Onygena corvina feather-degrading enzymes

Influence of temperature and pH on enzyme activity

To investigate the effect of temperature, enzyme reactions were carried out at different temperature (30, 40, 50, 60 °C) for 60 min in 50 mM sodium carbonate buffer (pH 9). In order to study thermo stability, 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. To study pH stability, 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.

Effects of metal ions, proteinase inhibitors, organic solvents, detergents and reducing agents

The effects of various metal ions, inhibitors, detergent and organic solvents on protease and keratinase activity were investigated by assaying the enzyme activity as described above after pre-incubation with each chemical for 15 min at room temperature. The following chemicals were used : CaCI₂, MgCI₂7H₂0, CuCI₂, ZnCI₂, FeCI₂ and MnS0₄ (1 mM), phenylmethylsulfonyl fluoride (PMSF) (1 mM), β-mercaptoethanol (1 %) and ethylene diamine tetraacetic acid (EDTA) (1 mM), SDS , DTT (5 mM), ethanol, methanol, isopropyl alcohol, glycerol, triton X-100, tween-20, tween-80 (1 %).

Supernatant precipitation for MS analysis

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 μιτι).

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. Solution Digestion and desalting of the peptides

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 μg Tris (2-carboxyethyl) phosphine was added per 25 μg sample protein and incubated for 30 min at 60 °C. Next, 1 μg iodoacetamide (IAA) was added (from a 2.5 μ9/μΙ iodoacetamide stock solution in water) per 10 μg sample protein and incubated for 20 min at 37 °C in the dark. Then the sample was digested by the addition of 1 μg Trypsin (from a 0.1 μ9/μΙ trypsin stock solution) per 50 μg sample protein and incubated overnight at 37 °C. The reaction was stopped by addition of formic acid to a final concentration of 2.0 %, mixed and incubated at room temperature for 5 min. The sample was centrifuge at 13.000 g for 20 min at 4 °C and the supernatant was dried down. Finally, the peptides were desalted by C18 microcolumn cleaning.

Analysis of proteins by LC-MS/MS

Peptides were reconstituted in 0.1% trifluoroacetic acid / 2 % acetonitrile solution. A volume of 8 μΙ of each sample was injected by the autosampler and concentrated on a trapping column (PepmaplOO, C18, 100 μπι χ 2 cm, 5 μιτι, Thermo Fisher Scientific) with water containing 0.1% formic acid and 2% ACN at a flow rate of 4 μΙ/ min. After 10 min, the peptides were eluted into a separation column (PepmapRSLC, C18, 75 μιτι x 50 cm, 2 μιτι, Thermo Fisher Scientific). Chromatography was performed with 0.1 % formic acid in solvent A (100 % water) and B (100 % acetonitrile) and the solvent B gradient was set in the first 5 min from 4 to 12 % and subsequently 30 min from 10 to 30 % solvent B. After this, solvent B was increased from 30 % to 90 % within 1 min for additional 5 min using a nano-high pressure liquid chromatography system (Ultimate3000 UHPLC, Thermo Fisher Scientific). Ionized peptides were measured and fragmented by a Q-Exactive mass spectrometer (Thermo Fisher Scientific). For an unbiased analysis continuous scanning of eluted peptide ions was carried out between 400-12000 m/ z, automatically switching to MS/MS higher energy collisional dissociation (HCD) mode and twelve MS/MS events per survey scan. For MS/MS HCD measurements a dynamic precursor exclusion of 30 s, peptide match and an apex trigger of 2 to 10 s were enabled.

MS data analysis

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 log 2 -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

(http://www. ncbi.nlm.nih.qov/5tructure/cdd/wrpsb.cqi).

Genomic DNA extraction

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.

De novo draft genome assembly

The genome of 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. Gene annotation by finding homology to peptide patterns (Hotpep)

Each genome was split into 2000 bases long fragments with 100 bases overlap between fragments. Each fragment was translated in all six reading frames, which was given a score for each subfamily-specific peptide lists for each protease family by:

1. Finding all the peptides from the list that were present in the reading frame.

2. Sum the frequency of these peptides. This gave the subfamily-specific frequency score.

A hit was considered significant if any of the open reading frames:

1. Included at least three conserved peptides from a subfamily.

2. The sum of the frequency of these peptides was higher than 1.0

3. 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). If all three conditions were met a sequence fragment was assigned to the Merops family and to the PPR-generated subfamily with the highest subfamily-specific frequency score as previously described (Busk and Lange, 2013). If two fragments were assigned to the same Merops family and the distance between them in the original genome sequence was less than 6000 bases, the fragments were considered to be part of the same gene and counted as one hit.

Although each sequence fragment could only be assigned once to each Merops family it was possible for a fragment to be assigned to two or more different families.

The predicted protein sequences of the hits were confirmed by BLAST. Full length genes of the validated proteases were obtained by assembling the related contigs with the CLC Main Workbench 6

(http://www.clcsupport.com/clcmainworkbench/current/index.php?manual=Introduction_ CLC_Main_Workbench.html), and the open reading frame (ORF) was predicted by

Augustus (http://bioinf.uni-greifswald.de/augustus/submission). The ORF were further validated by BLAST.

RNA extraction

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. cDNA synthesis

200 ng total RNA was mixed with 1 μΙ primer (oligodT: Random primer 1 : 3 (0.5 μ9/μΙ)), and RNAse free H₂0 to 5 μΙ. 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 μΙ 5x ImProm II buffer, 1 μΙ dNTP mix (lOmM each), 2 μΙ 25 mM MgCI₂ and ΙμΙ ImProm- Π™ Reverse Transcriptase (Promega) and RNAse-free H₂0 was added to a final volume of 15 μΙ. The reaction was heated 5 min at 25 °C and incubated 1 h at 42 °C.

Amplification and cloning of putative keratinase genes

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 μΙ diluted cDNA, 10 μΙ 5xPhusion® HF Buffer, 1 μΙ 10 mM dNTP (Fermentas), 2.5 μΙ 10 μΜ each primer and 1 U Phusion® High-Fidelity DNA Polymerase (M0530S, New England Biolabs Inc.) in 50 μΙ. 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 (PichiaPink™ 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 μg/ml ampicillin and identified by colony PCR and sequencing. About 5-10 μg vector with protease genes were linearized with Spel (except gene >399 | 8 | , which linearized by EcoN I) and transform in to PichiaPink™ Strain 4 (Invitrogen) by electroporation according to the manufacturer's manual. After incubated in YPDS medium 2 h at 30 °C without shaking, positive clones were selected on PAD plates (A11156, PichiaPink™ Media Kit, Invitrogen) by incubation at 30 °C for 3-7 days.

Expression of recombinant protease in PichiaPink™ Strain 4

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. To induce expression, 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 μιτι filter (Minisart Syringe Filters) and store at -80 °C. 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). First, 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. Then, 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).

Assay of purified recombinant protease activity with pig bristles

25 μΙ purified recombinant protease was incubated with 4 mg 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 as described elsewhere herein.

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).

Ion exchange chromatography

Culture supernatant was harvest by centrifugation at 10000 x g for 10 min at 4 °C after fermentation. The supernatant was filtered (0.45 μιτι). 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.

Protease identification by in-solution digestion and LC-MS/MS

200 μΙ anion exchange fractions and 400 μΙ cation exchange fractions were heated to 90 °C for 15 min and dried down. Protease identification by in-solution digestion and LC- MS/MS were performed as described elsewhere herein.

Prolonged culture broth and fraction degrading pig bristles

50 μΙ culture broth after Onygena corvina growing on fermentation medium with pig bristles or chicken feather 11 days or fraction C15 and C20 were incubated with 4 mg pig bristles in 0.2 ml 2x Mcllvaine buffer (pH 8). Assays were incubated at 40 °C for 4 days with constant agitation at 1000 rpm.

Culture broth degraded pretreated bristles and hooves 50 μΙ culture broth after Onygena corvina growing on fermentation medium with chicken feather 11 days was incubated with 4 mg pretreated bristles and hooves in 0.2 ml 2x Mcllvaine buffer (pH 8). Assays were incubated at 40 °C for 4 days with constant agitation at 1000 rpm.

Example 1

The fungus Onygena corvina can degrade feather and hair/pig bristles completely

A sample of duck feather was cut into small pieces, embedded in in a minimal liquid medium containing 2 g/l KH₂P0₄, 0.15 g/l MgS0₄.7H₂0, 0.3 g/l CaCI₂, 3.3 g/l 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). Surprisingly a total break down of the keratinaceous feather was observed when Onygena corvina was used as inoculum.

A sample of pig bristles was cut in small pieces and submerged in the supernatant from Onygena corvina culture broth. Onygena corvina had been grown for 11 days on keratinaceous materials (chicken feather) after which period the aquous supernatant was separated from the fungal biomass by centrifugation of the total culture broth. The result was scored by visual inspection (Figure 2). It was surprisingly seen that incubation in the aquous supernatant of Onygena corvina held sufficient enzyme activities for apparently almost total break down of the keratin after just 24 hrs incubation, also of pig bristles, composed of a-keratin.

Example 2

Onygena corvina genome sequencing, protease prediction and protease comparative analysis resulting in selection of list of 18 Onygena corvina keratinase candidates.

Onygena corvina (accession number: CBS 281.48) was genome sequenced by Illumina Hiseq 2000. A comprehensive list of Onygena corvina proteases, distributed on protein families, was deducted by Peptide Pattern Recognition (PPR) analysis. The proteases with the strongest propability for being capable of breaking down keratin, was identified based on a comparative analysis of proteases of non-keratin degrading fungi as compared to proteases of keratin degrading fungi. This included 18 putative secreted proteases from 4 protease families, that could be responsible for the keratin decomposition by Onygena corvina (see example 1). Genome sequencing and de novo assembly

After Illumina Hiseq 2000 sequencing, we obtained 81,538,322 PE 100 bp reads. The raw reads were cleaned, pooled together and de novo assembled with the CLC Genomic Workbenchprograme. Finally, we obtained 992 contigs with length > 198 bp. The average contig length was 22,096 bp and the maximum was 933,412 bp. N75 was 160,383 bp, N50 was 260,639 bp and N25 was 517,260 bp. The genome size of Onygena corvina is 21.92 Mb. This genome is a little smaller than other Onygenales found in GenBank.

According to the summary statistics of genome assembly, 98.92% reads were matched successfully and 91.34% reads in pairs. The GC content of of Onygena corvina genome is 48.05%.

Prediction of protease genes in Onygena corvina genome by PPR

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 | 1 | to 1444| 11 sequences with the length of 2000 bp, the last one 1445111 with the length of 1144 bp). The 8^th pool had 1426 sequences. Neighbouring sequences were overlapping with 100. The protease genes of Onygena corvina were predicted by PPR as described (Busk and Lange, 2013). After prediction, full length genes were confirmed by Blast and full length ORFs were found with Augustus. Finally, 75 kinds of proteases were identified. Clan information was based on the Merops database and the families were classified based on the conserved domains. The results show that Onygena. corvina has a high numbers of S8, S33 and M28 family proteases (Figure 5). 12 of the 13 S8 family proteases and most of M28 family have a signal peptide suggesting that they are secreted proteins, whereas, none of S33 family protease had a signal peptide.

Example 3

Predicted keratinolytic protease genes, amplification, cloning, expression, and activity testing

According to the comparative analysis of protease repertoire between keratin-degrading and non-keratin-degrading fungi (Example 2), proteases belong to S8, M35, M36 and M43 families have great potential for keratin degradation. We tried to amplify the 18 protease genes using suitable PCR primers from RNA isolated from Onygena corvina grown on chicken feather (rich in β-keratin), pig bristles or dog wool (rich in a-keratin). 12 genes could be amplified, most from the cDNA made from Onygena corvina growing on a- keratin-containing pig hair or dog wool (Table 1).

The Pichia pastoris system, PichiaPink™ Expression System (Invitrogen) was chosen for expressing protease genes. PichiaPink™ Strain 4 is double knock-out for both proteinases A and B (i.e., pepA and prbl), therefore has low background protease activity. The 13 protease genes were inserted to pPinka-HC vector. As expected, all 13 proteases genes (including the synthetic >830 | 1 | gene) were successfully cloned into pPinka-HC vector and transformed to PichiaPink™ Strain 4 which showed white color.

Recombinant proteases purification and protein determination

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 recombinant protease protein concentration was calculated according to the Molar Extinction Coefficient.

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). Proteases can digest fluorescein-labeled casein into smaller, labeled fragments that result in a measurable change in fluorescence properties. We applied a 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. Compared to the fluorescence curve, protease >687 | 7 | (SEQ ID NO: 1 and SEQ ID NO: 2, S8 family) showed highest protease activity. Also protease >399 | 8 | (M36 family) and protease >830 | 1 | (M35 family) showed high activity. The other purified recombinant proteases had very low protease activity. SDS-PAGE results show that protease >687 |7 | and >399 |8 | were very purified and only one band in the gel. The protease with highest activity, >687 | 7 | (SEQ ID NO: 1 and 2, S8 family) was further applied to degrade pig bristles. The results showed that 50 μΙ and 25 μΙ recombinant protease >687 | 7 | degrade 22% and 19%, respectively, of the pig hair in 24 hours at 40 °C. Example 4

MS analysis of protease composition of Onygena corvina secretome

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 identification of the proteases present in the culture broth that was shown (Example 1) to decompose a- and β-keratin, an MS analysis was made of the Onygena corvina secretome in this culture broth.

In all 29 different proteases were identified in the culture broth from Onygena corvina grown on chicken feather. The same composition was found in the culture broth from Onygena corvina grown on pig bristles. More M28 and S8 family proteins were found than of the other proteases families (Figure 6). Hence, these two families may play an important role for keratin degradation. Example 5

Ion exchange fractionation of supernatant of culture broth of Onygena corvina and activity testing of the resulting fractions

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 determined down to the protein family level. Usually, 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.

Both the anion and the cation exchange column were able to bind proteins with active protease activity (Azocasein used as substrate). The cation exchange experiment resulted in several fractions with strong protease activity (fractions 20-26 and possibly adjacent fractions) (Table 2). The activity for these samples was much stronger than that of the initial culture fluid. The anion experiment gave fractions with lower activity compared to the cation exchange experiment (Table 3).

In conclusion, both ion exchanges columns were able to fractionate an Onygena corvina culture supernatant. Fractionation resulted in several, distinct, fractions with high proteolytic activity. These fractions containing cation exchange fractions (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30) and anion exchange fractions (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) were further applied to protease identification and keratin materials degradation.

Protease identification of fractions with protease activity by LC-MS/MS

Fractions with protease activity were in-solution digested and LC-MS/MS analyzed to further identify the protease composition (Table 4). Compared with the 29 proteases found in culture broth supernatant from Onygena corvina grown on chicken feather and pig bristles (example 4), only 6 proteases were not identified in all the fractions. Anion exchanged fractions (A) had much higher abundance of proteases than the cation exchanged fractions (C). Fraction A13 had 18 proteases, which followed by A10 and A14 with 15 and 14 proteases, respectively. Most of cation exchanged fractions have around 2- 5 proteases. Pig bristles degradation by fractions

To further discover keratinolytic proteases, fractions with protease activity (azocasein as substrate) were applied for pig bristles degradation. The results (Figure 7) indicated that all of the fractions can degrade pig bristles at different levels. C15, C20, All and A10 have much higher keratin degradation activity compared to the other fractions. The degradation capabilities of fraction C15 and C20 are even much higher than the positive control containing the full range of proteases.

According to the protease identification results, fraction C15 mainly contains 3 proteases >687|7| (SEQ ID NO: 1 and 2), >642|3| (SEQ ID NO: 3 and 4) and >839|3| (SEQ ID NO: 5 and 6) in S8, M28 and M3 family, respectively.

C20 mainly contains 2 proteases >642|3| (SEQ ID NO: 3 and 4) and >802|5| (SEQ ID NO: 7 and 8) in M28 family and 2 proteases >687|7| (SEQ ID NO: 1 and 2) and >1165|2| (SEQ ID NO: 9 and 10) in S8 family. Both of these two fractions share one >642|3| (SEQ ID NO: 3 and 4) and one >687|7| (SEQ ID NO: 1 and 2) protease. The results indicate that the proteases in fraction C15 and C20 play an important role for keratin degradation.

Fraction All has 12 kinds of protease including the 2 proteases >642|3| and >684|4| from the M28 family, 3 proteases >1435|4|, >1181|3| and >687|7| from the S8 family, 1 protease >577|5|, >839|3|, >883|2|, >714|2|, >1339|4| and >399|8| from the Ml, M3, M20, M14, M49 and M36 family, respectively, and 1 unclassified protease >847|2|.

Fraction A10 has 15 kinds of protease including 2 proteases >642|3| and >684|4| from the M28 family, 3 proteases >1435|4|, >1181|3| and >687|7| from the S8 family, 2 proteases >294|5| and >714|2| from the M14 family, 1 protease >839|3|, >883|2|, >1339|4|, > 1029111, >900|5|, >400|5| and >399|8| from the M3, M20, M49, S28, S9, S10 and M36 family, respectively, and 1 unclassified protease >847|2|.

Example 6

Activity testing of blends, composed of mixing of most active fractions

To test for synergistic action between the different amounts and types of proteases present in the fractions from ion exchange chromatography, 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. Result

Mixing of fraction C15 and C20 gave the highest activity, much higher than testing the two fractions individually, when adjusted for equal enzyme load (Figure 8). 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 μΙ fraction C15 and C20 were 17 % and 17%. The degradation degree of blend 12.5 μΙ fraction C15 and 12.5 μΙ fraction C20 was 21%.

Therefore, the proteases >687 | 7 | (SEQ ID NO: 1 and 2), >642 | 3 | (SEQ ID NO: 3 and 4) and >839 | 3 | (SEQ ID NO: 5 and 6) in fraction C15 and >642 | 3 | (SEQ ID NO: 3 and 4), >802 | 5 | (SEQ ID NO: 7 and 8), >687 | 7 | (SEQ ID NO: 1 and 2) and > 1165 | 2 | (SEQ ID NO: 9 and 10) in fraction 20 exhibits synergistic effect on degradation of keratin in form of pig bristles.

Example 7

Comparative activity testing of best performing recombinant protease and best performing fractions

To test for synergistic action between the proteases present in the fractions and the recombinant protease >687 | 7 | (SEQ ID NO: 1 and 2), the fractions were mixed with the recombinant protease >687 | 7 | (SEQ ID NO: 1 and 2) and tested for degradation of pig bristles. The blends were set up as described in the methods section.

The results indicate that the degradation degree by the recombinant protease >687 | 7 | (SEQ ID NO: 1 and 2) was 19%. The blends with recombinant protease >687 |7 | did not show increased degradation degree (Figure 9). Thus, recombinant protease >687 | 7 | is an robust enzyme to degrade pig bristles. In combination, the results of patent examples 6 and 7 suggest that the interesting activity observed from culture broth of Onygena corvina originates from in all 5 genes belonging to 3 protein families; and that most of such activity can be achieved by using 3-4 of these protease; and most of it also by only using the protease >687 | 7 | (S8).

Example 8

To test the ability of the proteases in the culture broth from Onygena corvina and fractions from ion exchange chromatography to degrade pig bristles in a long term incubation, the different fractions C15, C20, A10, Al l and the culture broth from Onygena corvina grown on chicken feather or big brisles were incubated with pig bristles at 40 °C and the degradation was followed for four days by determining the soluble protein in the samples by measuring E280 on a Nanodrop.

Furthermore, as protease >839 |3 | (SEQ ID NO: 5 and 6) is an M3 family

metallopeptidasethe function of this protease in the C15 fraction was measured vby adding 0.5 mM metallopeptidase inhibitor ethylenediaminetetraacetic acid (EDTA) to the C15 fraction degrading pig bristles. The results showed that after addition of EDTA, the pig bristles degradation degree was strongly decreased (Figure 10). This result indicated that protease >839 | 3 | (SEQ ID NO: 5 and 6) is important for keratin (here, pig bristles) degradation in combination with proteases >687 | 7 | (SEQ ID NO: 1 and 2) and >642 |3 | (SEQ ID NO: 3 and 4) in fraction C15.

After 3 days incubation, culture broth on chicken feather, culture broth on pig bristles, fraction C20 and fraction C15 degraded 63 %, 57 %, 52 % and 49 % of the pig bristle, respectively (Figure 10). In nature, keratin degrading fungi are known to secrete sulfite to destabilize the keratin by breaking the cysteine bridges, thereby making the keratin more susceptible to proteolysis and increasing the activity of keratinases three folds (Kunert, 1992). Therefore, it is surprising that 49 - 52 % degradation can be reached with the partially purified proteases in fractions C15 and C20 without addition of sulfite to break the cysteine bridges of the keratin and is likely that even higher levels of degradation would be reached by adding sulfite as reported by Kunert (1992). It is possible that the slightly higher degradation (57 - 63 %) obtained with culture broth is due to the presence of sulfite secreted by Onygena corvina during growth. These findings therefore give additional support also to the findings of the almost complete decomposition observed by visual inspection in Example 1

To assess the degradation capacity of the Onygena corvina proteases of an industrially relevant substrate pretreated bristles and hooves obtained from a slaughter house were incubated with culture broth from chicken feather. After four days incubation, the culture broth had degraded nearly half (47 %) of the pretreated bristles and hooves (Figure 11). Hence, the proteases secreted by Onygena corvina are able to degrade this substrate to a large degree even without the addition of sulfite.

In conclusion, the results of the examples 1-8 showed that upon incubation with keratinous material such as feather, hair or pig bristles, the fungus Onygena corvina could colonize the material and degrade it completely to soluble compounds such as polypeptides or amino acids. Genome sequencing and data mining of the genome for protease-encoding genes identified 75 putative protease genes. This list was further reduced to 18 candidate genes of which 12 were amplified by PCR, cloned and

heterologously expressed in Pichia pastoris. The PCR showed that the gene with SEQ ID NO: 1 was expressed by Onygena corvina when growing on either a- or β-keratin and the product of this gene (SEQ ID NO: 2) was found in the culture broth supernatant from Onygena corvina grown on feather. Three of the heterologous expressed gene products exhibited high protease activity and one of the gene products (having SEQ ID NO: 1) was also able to degrade pig bristles.

Furthermore, fractionation of the culture broth supernatant from Onygena corvina growing on feather led to the identification of several partially purified fractions with high keratinase activity. The fractions named A10, Al l, 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.

Interestingly, combining fractions C15 and C20 lead to a higher degree of degradation of pig bristles than either of the two fractions alone. This result shows that there is a synergistic effect of this optimized blend of proteases from Onygena corvine.

In a long term incubation experiment, the culture broth supernatant from Onygena corvina grown on feather or on pig bristles was able to degrade at least 60 % of a pig bristle substrate despite the abscense of keratin-destabilizing agents, that by others, have been shown to increase the activity of keratinases. Hence, addition of such agents further enhance degradation of the material. The partially purified fractions C15 (containing SEQ ID NO: 2, 4 and 6) and C20 (containing of SEQ ID NO: 2, 4, 8, and 10) degraded 50 % of the pig bristle under the same conditions without keratin-destabilizing agents. It is very surprising that such a high degree of degradation can be achieved with the isolated proteases present in fractions C15 and C20 despite the presence of an agent that breaks the cysteine bridges of the keratin.

Finally, it was shown that the culture broth supernatant from Onygena corvina grown on feather was able to degrade almost 50 % of industrially relevant substrates such as pretreated bristles and hooves obtained from a slaughter house, despite the lack of keratin-destabilizing agents. Hence in the presence of such agents it is expected that keratinases from Onygena corvina will be able to complete degrade such material. Table 1

Predicted 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.

a The cDNA sequence of the >830 | 1 | was synthesized and cloned into pUC57 by GenScript (USA)

Table 2

Protease activity profile of selected fraction from cation exchange chromatography.

Sample prior to loading : 50 ml culture supernatant was diluted to 100 ml sample prior to load. Negative control : fraction was replaced by 50 mM citric acid buffer, pH 3.86.

Fraction start Fraction end

Fraction number (min) (min) protease activty (A405)

5 36,96 37,46 14

8 38,46 38,96 17 11 39,96 40,46 4

14 41,46 41,96 131

17 42,96 43,46 77

20 44,46 44,96 484

23 45,96 46,46 341

26 47,46 47,96 171

29 48,96 49,46 9

32 50,46 50,96 7

35 51,96 52,46 8

38 53,46 53,96 -3

41 54,96 55,46 4

44 56,46 56,96 6

47 57,96 58,46 4

50 59,46 59,96 6

53 60,96 61,46 2

56 62,46 62,96 -4

59 63,96 64,46 12

Sample prior to

loading - - 170 negative control - - -1

Table 3

Protease activity profile of selected fraction from anion exchange chromatog

Negative control : fraction was replaced by 20 mM Tris-HCI buffer, pH 8.6.

Fraction start Fraction end

Fraction number (min) (min) protease activty (A405)

7 65,14 66,14 22

9 67,14 68,14 267

13 71,14 72,14 125

15 73,14 74,14 111

17 75,14 76,14 29

19 77,14 78,14 43

21 79,14 80,14 35

23 81,14 82,14 29

25 83,14 84,14 22

27 85,14 86,14 25 29 87,14 88,14 32

31 89,14 90,14 42

33 91,14 92,14 36

35 93,14 94,14 26

37 95,14 96,14 15

39 97,14 98,14 11

41 99,14 100,14 7

43 101,14 102,14 2

45 103,14 104,14 2

Pre flow through - - 497

Load flow through - - 556

Wash flow through - - 449 negative control - - -5

Table 4

Proteases identification by LC-MS/MS for the fractions with protease activity (azocasein as substrate). A: Anion exchanged fractions; C: Cation exchanged fractions

predict

ed

gene fa A A A A A A A A A A A c c c c c c c c c c c C C C C C C C C by mi Annotatio protease ID in A A 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3

PPR iy n MS 8 9 0 1 2 3 4 5 6 7 8 9 0 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 fungi t q 1 (

>1435| serine paired) contig

S8 + + + + + + + + + + +

4| protease 114:False: 157 - - - 54

fungi t q 1 (

>577|5 M aminopep paired) contig

+

1 tidase 2 13:False:9758 - - - - - + +

0

fungi t q 1 (

>1254| SI carboxyp paired) contig

+ + +

2| 0 eptidase 17:True:1956 - +

81

fungi t q 1 (

alkaline

>1 165| paired) contig

S8 serine + + + +

2| 17:True:2553

protease

7

fungi t q 1 (

metalloca

>294|5 M paired) contig

rboxypept + + + + + +

14 19:False:7367 - + + - - - +

idases

6

fungi t q 1 (

paired) contig

22:False:2260

25

fungi t q 1 (

leucine

>642|3 M paired) contig

aminopep + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

28 22:False:2263

tidase

50

fungi t q 1 (

paired) contig

22:False:2267

73

fungi t q 1 (

>634|3 M aminopep paired) contig

+ +

18 tidase 22:True:2086 - - - - - - - - - - - + - - - + - - 33

fungi t q 1 (

paired) contig

23:True:1447

>839|3 M metallope 12

+ + +

3 ptidase fungi t q 1 ( - + + + + + + + + + + + + - - + +

paired) contig

23:True:1463

43

fungi t q 1 (

>684|4 M paired) contig

peptidase + + + +

28 24:False:2537 - + + + +

4

fungi t q 1 (

>883|2 M paired) contig

peptidase + + + + + + +

20 25:False:3917 - - - + +

3

fungi t q 1 (

glutamate

>1089| M paired) contig

carboxyp + + + + +

2| 28 25:True:4297 - - +

eptidase

18

fungi t q 1 (

Serine

>1029| S2 paired) contig

carboxyp + + + + +

1 8 29:False:9847 - - + + - - - eptidase

6

fungi t q 1 (

glutamate

>413|2 M paired) contig

carboxyp + + + + +

28 31 :True:3083 - - - - - + - - eptidase

25 fungi t q 1 (

>1 181 serine paired) contig

S8 + + + + + + + + + + + + +

3| proteinase 36:False:3449

05

fungi t q 1 (

Aspartate

>847|2 paired) contig

aminotran + + + + + + + + + +

44:False:5180 - - - sferase

86

fungi t q 1 (

>714|2 M carboxyp paired) contig

+ + + +

14 eptidase 44:True:2643 - - + + + + + +

84

Dipeptidy fungi t q 1 (

>900|5 1 paired) contig

S9 + + + +

peptidase 50:False:2026

4 51

fungi t q 1 (

leucyl

>802|5 M paired) contig

aminopep +

28 50:True:1518 - - - - - - - - - - - - - - - - + - - + + + - - + + + + + + + tidase

4

fungi t q 1 (

dipeptidyl

>1339| M paired) contig

-peptidase + + + + + + + +

4| 49 57:True:9932 - + + + +

3

8

fungi t q 1 (

paired) contig

66:False:1342

>687|7 serine 79

S8 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + protease fungi t q 1 (

paired) contig

66:False:1350

03

fungi t q 1 (

paired) contig

75:True:2233

7

fungi t q 1 (

paired) contig

75:True:2278

>399|8 M metallope 2

+ + + + + + + + + + + + + + +

36 ptidase fungi t q 1 ( - + - - - - - - - - - - + - - - - paired) contig

75:True:2321

2

fungi t q 1 (

paired) contig

75:True:2382

1

fungi t q 1 (

>400|5 SI carboxyp paired) contig

+ + + - - +

0 eptidase 92:True:1620 - - - - - - - - - - - - - - - - - - - + - - + + + - 2

fungi t q 1 (

>626|6 serine paired) contig

S8 + +

protease 99:False:2020

0

1 1 1 1 1 1 1 1 1 1

Protease number 9 9 9 6 4 5 3 5 4 5 4 4 4 2 2 5 4 5 5 5 4 3

3 3 5 2 8 4 3 4 2 0 REFERENCES

1 : Gupta, R., Ramnani, P., 2006. Microbial keratinases and their prospective applications: an overview. AppI Microbiol Biotechnol. 70, 21-33.

2 : Rawlings, N.D., Waller, M., Barrett, A. J. & Bateman, A. (2014) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 42, D503-D509. 3 : Busk, P. K., Lange, L, 2013. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs. AppI Environ Microbiol. 79, 3380-3391.

4: Lin, X., Lee, C.-G., Ellen S. Casale, Shih, J.C.H., 1992. Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain. Applied and Environmental Microbiology. 58, 3271-3275.

5 : Kunert J., 1992. Effect of reducing agents on proteolytic and keratinolytic activity of enzymes of Microsporum gypseum. Mycoses. 35(l l-12) :343-8.

6 : WO00/71683A1.

Claims

Claims

1. A keratin degrading composition comprising at least two different isolated active keratinases from at least two different MEROPS protease families, wherein :

(i) : at least one active serine endo-keratinase belongs to the MEROPS family S8; and (ii) at least one active keratinase does not belong to the MEROPS family S8;

and wherein :

(a) : at least 0.01 % (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii);

(b) : at least 1 % (w/w - dry matter) of the total weight of the keratinases in the composition is keratinase of item (i);

(c) : the total weight of the composition (w/w - dry matter) is from lg to 100.000 tons; and wherein the keratinase of item (i) and/or item (ii) is a keratinase isolated from a Onygena sp., preferably Onygena corvina or Onygena equina.

2. A keratin degrading composition comprising at least two different isolated active keratinases from at least two different MEROPS protease families, wherein :

(i) : at least one active serine endo-keratinase belongs to the MEROPS family S8; and

(ii) at least one active keratinase does not belong to the MEROPS family S8;

and wherein :

(a) : at least 0.01 % (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii);

(b) : at least 1 % (w/w - dry matter) of the total weight of the keratinases in the composition is keratinase of item (i);

(c) : the total weight of the composition (w/w - dry matter) is from lg to 100.000 tons; and wherein the keratinase of item (i) is at least one serine endo-keratinase that belongs to the MEROPS family S8 that comprises:

a sequence having at least 86% identity to SEQ ID NO. : 2; or

a sequence having at least 71% identity to SEQ ID NO. : 10.

3. The keratin degrading composition of claim 2, wherein the keratinase of item (i) is at least one serine endo-keratinase that belongs to the MEROPS family S8 that comprises: a sequence having at least 95% identity to SEQ ID NO. : 2; or

a sequence having at least 95% identity to SEQ ID NO. : 10.

4. The keratin degrading composition of any of the preceding claims, wherein the keratinase of item (ii) is at least one metaiioprotease that comprises a sequence having at least 85% identity to SEQ ID NO. : 4, 6 or 8.

5. The keratin degrading composition of claim 4, wherein the keratinase of item (ii) is at least one metalloprotease that comprises a sequence having at least 95% identity to SEQ ID NO. : 4, 6 or 8.

6. The keratin degrading composition of any claims 2 to 5, wherein the keratinase of item (i) and/or item (ii) is a keratinase isolated from a Onygena sp., preferably Onygena corvina or Onygena equina.

7. The keratin degrading composition of any of the preceding claims, wherein the keratinase of item (ii) is at least one exo-keratinase.

8. The keratin degrading composition of any of the preceding claims, wherein the keratinase of item (ii) is at least one metalloprotease that belongs to the MEROPS family M28 or the keratinase of item (ii) is at least one metalloprotease that belongs to the MEROPS family M3.

9. The keratin degrading composition of any of the preceding claims, wherein items (a) to (c) of claim 1 are:

(a) : at least 5% (w/w - dry matter) of the total weight of the composition is keratinase of item (i) and item (ii);

(b) : at least 20% (w/w - dry matter) of the total weight of the keratinases in the composition is keratinase of item (i);

(c) : the total weight of the composition (w/w - dry matter) is from 100 g to 100 kg.

10. A method for the degradation of keratinaceous materials comprising the steps of:

(I) : adding to a keratinaceous material a keratin degrading composition according to any of the preceding claims;

(II) : incubating for a suitable time under suitable conditions, wherein the keratinases of the composition degrade the keratinaceous material; and

(III) : obtaining a degraded keratinaceous material.

11. The method of claim 10, wherein the keratinaceous material is at least one

keratinaceous material selected from the group consisting feather, hair, teeth, hoof, horn, skin, wool and bristles (such as pig bristles).

12. The method of any of claims 10 to 11, wherein there in step (I) of claim 10 is added at least lg of the keratin degrading composition to at least 10 g of keratinaceous material and wherein the degraded keratinaceous material of step (III) is having a degree of degradation of at least 10%.

13. The method of any of claims 10 to 12, wherein the temperature of step (II) of cla 10 is temperature from 15 to 70 °C and the pH is from pH 5-11.