Lytic polysaccharide monooxygenases and their use in detergent

Abstract

The present invention relates to newly idenified lytic polysaccharide monooxygenases (LPMOs) and their use as enhancers of enzyme benefits in detergents as well as a detergent composition comprising said LPMOs in combination with detergency enzymes.

Description

[0001] LYTIC POLYSACCHARIDE MONOOXYGENASES AND THEIR USE IN DETERGENT

[0002] REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to newly identified lytic polysaccharide monooxygenases (LPMOs) and their use in detergent compositions, in particular as enhancers of enzyme detergency benefits in detergents, as well as detergent compositions comprising said LPMOs, optionally in combination with other enzymes. The invention also relates to a process of washing a fabric or hard surface using the LPMOs alone or in combination with one or more enzymes and / or with the detergents of the present invention. A further aspect of the invention relates to the isolated LPMO polypeptides and isolated polynucleotides encoding the polypeptides as well as to vectors and host cells comprising the polynucleotides and methods of producing the polypeptides.

[0006] BACKGROUND OF THE INVENTION

[0007] Lytic polysaccharide monooxygenases belong to a class of proteins that assist in the degradation of crystalline polymeric carbohydrate substrates (EC 1.14.99.53 / 54 / 55 / 56). They are copper-de- pendent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as chitin, cellulose, and starch in the presence of a reductant and oxygen. Interestingly, LPMOs can utilize a vast variety of electron donors (reductants), including small phenolic compounds and partner enzymes such as cellobiose dehydrogenase (Stepnov, A. A., Forsberg, Z., Sorlie, M. et al. Unraveling the roles of the reductant and free copper ions in LPMO kinetics. Biotechnol Biofuels 14, 28 (2021). https: / / doi.org / 10.1186 / s13068-021-01879-0). LPMOs act on highly crystalline substrate surfaces, rather than on isolated polysaccharide chains within amorphous regions. LPMOs are classified in the Carbohydrate-Active EnZymes database (CAZy) into eight auxiliary activity (AA) families including AA9 (formerly glycosyl hydrolase family 61 , GH61), AA10 (formerly carbohydrate-binding module family 33, CBM33), AA11 and AA13-AA17 enzymes. In insects the gene family encoding LPMO-like proteins has been identified recently and determined to belong to the AA15 family (LPMO15) (Sabbadin et al, NATURE COMMUNICATIONS (2018) 9:756).

[0008] LPMOs are known to play a pivotal role in the breakdown of polysaccharides such as cellulose and chitin, by catalyzing the oxidative, as opposed to hydrolytic, cleavage of glycosidic bonds. In this way, the initial chemical and physical recalcitrance of the polysaccharide is overcome, thereby making the substrate tractable to hydrolases.

[0009] It has been found that keratin-degrading fungi express LPMO and two hypothesis for the possible role of AA11 LPMOs in keratin degradation has been formulated (Lange et al., Appl Microbiol Biotechnol (2016) 100:2083-2096). However, no evidence for the actual involvement of LPMO on keratin-degradation has been presented.

[0010] LPMOs belonging to the AA9 familiy (formerly GH61) have been widely used in industry, e.g., in baking (WO 04 / 031378), in the conversion of cellulosic feedstock into ethanol (WO 07 / 089290) and the use in laundry detergent has also been proposed (WO 2011 / 080267).

[0011] SUMMARY OF THE INVENTION

[0012] The present invention concerns lytic polysaccaharide monooxygenase that in addition to LPMO activity also have keratinase activity, and to their use, in particular use in detergents. LPMOs of the present invention belong to the AA15 enzymes and the inventors of the present invention have surprisingly found that these new LPMOs not only act on polymeric carbohydrates, such as chitin, but also on protein making them very useful in detergents that are applied for the removal various types of stains, such as proteins, starch, mannan and (hemi)cellulose. In this context, particularly the removal of keratin is a well known problem. Keratin is a protein that helps form hair, nails and the skin's outer layer and is easily accumulate on clothes. Alpha-keratin is a fibrous structural protein made up of amino acids that form a repeating secondary structure. The secondary structure of alpha-keratin provides a tightly wound structure and makes it one of the strongest biological materials and very hard to break down. Without being bound by any theory, it is believed that the LPMOs of the present invention open the fibrious structure of the keratin making it accessible to the further breakdown of the protein to smaller polypeptides by proteases. The LPMOs of the present invention also have activity on polysaccharides, such as chitin, and are believed to work in a similar way as described for the activity on protein, i.e. making the glycosidic substrate accessible to glycoside hydrolases, such as cellulases and mannanases as well as amylases.

[0013] The LPMOs of the present invention thus have a dual activity as they act both on proteins and carbohydrates that otherwise are hard to access, allowing for the further breakdown of the substrate by hydrolytic enzymes such as proteases, amylases and glycosyl hydrolases making them beneficial in the removal of stains from objects (e.g. fabrics and hard surfaces).

[0014] In a particular embodiment the present invention relates to the mature LPMOs of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ, SEQ ID NO: 1 , ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 13 and their use in detergent.

[0015] DEFINITIONS

[0016] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0017] AEP (active enzyme protein):

[0018] Enzyme protein which has a catalytic activity. There is various way to determine AEP. For example, AEP can be calculated by dividing total activities by the enzyme’s specific activity, or by active site titration. cDNA:

[0019] The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0020] Coding sequence:

[0021] The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0022] Control sequences:

[0023] The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0024] Detergent adjunct ingredient:

[0025] The detergent adjunct ingredient is different to the LPMO of this invention. The precise nature of these additional adjunct components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the operation for which it is to be used. Suitable adjunct materials include, but are not limited to the components described below such as surfactants, builders, flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, s, s, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, anti-foaming agents, dispersants, processing aids, solvents, and / or pigments.

[0026] Detergent composition:

[0027] The term “detergent composition” refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles. The detergent composition may be used to e.g. clean textiles for both household cleaning and industrial cleaning. The terms encompass any materials / compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, bar, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and / or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; laundry boosters; and textile and laundry pre-spotters / pre-treatment). In addition to containing the enzyme of the invention, the detergent formulation may contain one or more additional enzymes (such as amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xyloglucanases, hemicellulases, xan- thanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pec- tate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, car- rageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, other endo-beta-mannanases, exo-beta-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof), and / or detergent adjunct ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers (as set forth herein), fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, bluing agents and fluorescent dyes, antioxidants, and solubilizers. The term “detergent composition” may be used interchangeably with the term “detergent”.

[0028] Detergent load

[0029] Detergent load is the amount of detergent used in a wash cycle.

[0030] Enzyme detergency benefit:

[0031] The term “enzyme detergency benefit” is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and / or cleaning.

[0032] Expression:

[0033] The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0034] Expression vector:

[0035] An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0036] Extension:

[0037] The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has lytic polysaccharide monooxygenase activity.

[0038] Fragment:

[0039] The term “fragment” means a polypeptide having one or more amino acids absent from the amino and / or carboxyl terminus of the polypeptide; wherein the fragment has lytic polysaccharide monooxygenase activity.

[0040] Fusion polypeptide:

[0041] The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and / or the C-terminus of a variant of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.

[0042] Heterologous:

[0043] The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0044] Host Strain or Host Cell:

[0045] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0046] Improved property:

[0047] The term “improved property” means a characteristic associated with a polypeptide that is improved compared to a reference enzyme / parent enzyme. Such improved properties include, but are not limited to reduced odor generation i.e. odor reduction, improved thermostabilty, improved half-life in detergent (’’detergent stability”) and improved wash performance (WP). Some aspects of the invention relate to polypeptides having an improvement factor above 1 when the polypeptide is tested for a property of interest in a relevant assay, wherein the property of the reference enzyme / parent enzyme is given a value of 1 .

[0048] Introduced:

[0049] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art. Isolated:

[0050] The term “isolated” means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell.

[0051] Laundering:

[0052] The term “laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition and optionally one or more enzymes. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand.

[0053] LPMO:

[0054] The term LPMO refers to polypeptides having lytic polysaccharide monooxygenase activity. LPMOs are listed in the carbohydrate-active enzyme (CAZy) database, where they are sequence classified into seven “Auxilliary Activity” families (AA9, AA10, AA11 , AA13, AA14, AA15 and AA16). The CAZy database describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glyco- sidic bonds, see www.cazy.org. A more detailed discussion of the maintenance of the modular classification in CAZy can be found in Drula et al: “The carbohydrate-active enzyme database: functions and literature” in Nucleic Acids Research, Volume 50, Issue D1 , 7 January 2022, Pages D571-D577, https: / / doi.org / 10.1093 / nar / gkab1045.

[0055] Mature polypeptide:

[0056] The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing and / or C-terminal processing (e.g., removal of signal peptide) as well as glycosylation and phosphorylation.

[0057] Mature polypeptide coding sequence:

[0058] The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having lytic polysaccharide monooxygenase activity.

[0059] Mutant:

[0060] The term “mutant” means a polynucleotide encoding a variant. Mutation

[0061] The term “mutation” refers to a deletion (including a truncation), insertion (including an extension) or substitution in a parent polypeptide.

[0062] Native:

[0063] The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0064] Nucleic acid:

[0065] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0066] Nucleic acid construct:

[0067] The term "nucleic acid construct" means a nucleic acid molecule, either single- or doublestranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0068] Operably linked:

[0069] The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0070] Parent or parent lytic polysaccharide monooxygenase:

[0071] The parent lytic polysaccharide monooxygenase may be a polypeptide having at least 60% sequence identity to the mature part of polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

[0072] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 1. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 1. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 1 containing at least 150 amino acid residues, e.g., at least 160 and at least 165 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0073] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0074] 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 2 containing at least 160 amino acid residues, e.g., at least 170 and at least 175 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0075] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0076] 3 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 3. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 3. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 3 containing at least 150 amino acid residues, e.g., at least 160 and at least 165 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0077] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0078] 4 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 4. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 4 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0079] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0080] 5 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 5. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 5. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 5 containing at least 150 amino acid residues, e.g., at least 160 and at least 165 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0081] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0082] 6 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 6. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 6. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 6 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0083] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0084] 7 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 7. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 7. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 7 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0085] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0086] 8 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 8. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 8. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 8 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0087] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0088] 9 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 9. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 9. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 9 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0089] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO:

[0090] 10 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 10. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 10. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 10 containing at least 170 amino acid residues, e.g., at least 180 and at least 185 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0091] In an aspect, the parent has a sequence identity to the mature part of polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lytic polysaccharide monooxygenase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 12. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 12. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 12 containing at least 150 amino acid residues, e.g., at least 160 and at least 165 amino acid residues, wherein the fragment has lytic polysaccharide monooxygenase activity.

[0092] The parent may be a fusion polypeptide or cleavable fusion polypeptide. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0093] A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.

[0094] The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

[0095] Purified:

[0096] The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0097] In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0098] Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0099] It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0100] Recombinant:

[0101] The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0102] Recover:

[0103] The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0104] Reductant:

[0105] Reductant or reducing agent is a compound that can act as electron donors in the oxidative cleavage by LPMO of a substrate.

[0106] A wide variety of reducing agents have been tested on LPMO families. These include gallic acid, cysteine and ascorbic acid for LPMO family AA10. The above compounds in addition others such as catechols, and sinapic acid were found to be active in assays of an AA9 LPMO.

[0107] Rhamnolipid:

[0108] Rhamnolipid (RL) is a glycolipid that may be used as a biodegradable surfactant. RL may be in the form of mono-rhamnolipid or di-rhamnolipid, which consist of one or two rhamnose groups respectively, wherein the length of the chain may vary: m,n being 4 to 8.

[0109]

[0110] (Appl Microbiol Biotechnol (2005) 68: 718-725).

[0111] In the context of the present invention the term “rhamnolipid” includes mono-rhamnolipid or dirhamnolipid, mixtures thereof and varying chain length as well as salts of rhamnolipid. Sequence difference:

[0112] The term "sequence difference" means the percent of amino acid differences between a polypeptide and the polypeptide of SEQ ID NO: X, where X has the value 1 , 2, 3, or 4, and is calculated as follows:

[0113] (Different Residues x 100) / (Length of SEQ ID NO: X) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the N-terminus and / or C-terminus) in the sequence.

[0114] Sequence identity:

[0115] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0116] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0117] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0118] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0119] Signal Peptide:

[0120] A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0121] Sophorolipid:

[0122] The term “sophorolipid” include sophorolipid in the lactone form and the corresponding acidic form as well as mixtures thereof. Further “sophorolipid” also includes salts of sophorolipid.

[0123] Subsequence:

[0124] The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lytic polysaccharide monooxygenase activity.

[0125] Substantially the same:

[0126] The term “substantially same” in the present invention is within the reasonable understanding of those skilled in the art, and may mean that the level of lipid removal of different detergent compositions is similar or no obvious difference, for example, the difference in the level of lipid removal is within e.g. 1 %, 2% or 3% depending on the experimental errors. Sustainability:

[0127] Sustainability and sustainable means use of renewable resources that cause little or no damage to the environment and are biodegradable.

[0128] Sustainability profile:

[0129] In the context of the present invention the term sustainability profile is used for comparing the sustainability of ingredients (e.g. in a detergent composition) where one or more ingredients can replace other less sustainable ingredients while maintaining the performance of the system (e.g. the performance of a detergent composition during wash of an item).

[0130] Textile:

[0131] The term “textile” means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling. The textile may be cellulose based such as natural cellulosics, includ-ing cotton, flax / linen, jute, ramie, sisal or coir or manmade cellulosics (e.g. originating from wood pulp) including viscose / rayon, cellulose acetate fibers (tricell), lyocell or blends thereof. The textile or fabric may also be noncellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and span- dex / elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and / or rayon / viscose with one or more companion material such as wool, synthetic fiber (e.g. polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, aramid fiber), and / or cellulose-containing fiber (e.g. rayon / viscose, ramie, flax / linen, jute, cellulose acetate fiber, lyocell). Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well. In the context of the present invention, the term “textile” also covers fabrics. In the context of the present invention, the term “textile” is used interchangeably with fabric and cloth.

[0132] TEP:

[0133] Total Enzyme Protein is measured by amino acid analyses.

[0134] Variant:

[0135] The term “variant” means a polypeptide having enzyme, e.g. lytic polysaccharide monooxygenase, activity comprising a substitution, an insertion (including extension), and / or a deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position. The lytic polysaccharide monooxygenase variants of the present invention have at least 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to mature part of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. SEQ ID NO: 12, or SEQ ID NO: 13. In the context of the present invention the terms “variant”, “lytic polysaccharide monooxygenase variant”, and “LPMO variant” may be used interchangeably unless it is clear from the context that the variant refers to another enzyme class.

[0136] The variants may further comprise changes of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0137] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0138] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like. Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for lytic polysaccharide monooxygenase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0139] Wash cycle:

[0140] The term “wash cycle” is defined herein as a washing operation wherein textiles are immersed in the wash liquor, mechanical action of some kind is applied to the textile in order to release stains and to facilitate flow of wash liquor in and out of the textile and finally the superfluous wash liquor is removed. After one or more wash cycles, the textile is generally rinsed and dried.

[0141] Wash liquor:

[0142] The term “wash liquor” refers to an aqueous solution containing a detergent composition in dilute form, such as as the wash liquor in a laundry process.

[0143] Wash performance:

[0144] The term “wash performance” is used as detergent composition’s, enzyme’s or polymer’s capability to remove stains present on the object to be cleaned or maintain color and whiteness of textile during wash. The improvement in the wash performance may be quantified by lipid removal or odor generation as described in the Experimental section.

[0145] Weight percentage:

[0146] Weight percentage is abbreviated w / w%, wt% or w%. The abbreviations are used interchangeably.

[0147] Wild-type:

[0148] The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild- type sequence). Sequence Overview

[0149] SEQ ID NO: 1 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0150] SEQ ID NO: 2 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0151] SEQ ID NO: 3 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0152] SEQ ID NO: 4 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0153] SEQ ID NO: 5 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0154] SEQ ID NO: 6 is a lytic polysaccharide monooxygenase including signal peptide from

[0155] Attagenus sminovi

[0156] SEQ ID NO: 7 is a lytic polysaccharide monooxygenase including signal peptide from

[0157] Attagenus sminovi

[0158] SEQ ID NO: 8 is a lytic polysaccharide monooxygenase including signal peptide from

[0159] Attagenus sminovi

[0160] SEQ ID NO: 9 is a lytic polysaccharide monooxygenase including signal peptide from

[0161] Attagenus sminovi

[0162] SEQ ID NO: 10 is a lytic polysaccharide monooxygenase including signal peptide from

[0163] Attagenus sminovi

[0164] SEQ ID NO: 11 is a protease from Bacillus claussi

[0165] SEQ ID NO: 12 is a lytic polysaccharide monooxygenase from Attagenus sminovi

[0166] SEQ ID NO: 13 is a lytic polysaccharide monooxygenase including signal peptide from

[0167] Attagenus sminovi

[0168] CONVENTIONS FOR DESIGNATION OF VARIANTS

[0169] For purposes of the present invention, the mature part of polypeptide disclosed in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12 may be selected as the parent LPMO enzyme and used to determine the corresponding amino acid positions in another lytic polysaccharide monooxygenase. The amino acid sequence of another lytic polysaccharide monooxygenase is aligned with the polypeptide disclosed in the parent LPMO enzyme, e.g. SEQ ID NO: 1 , and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in the parent LPMO (e.g., SEQ ID NO: 1) is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IIIPAC single letter or three letter amino acid abbreviation is employed.

[0170] Substitutions

[0171] For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”) or comma “G205R,S411 F” representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

[0172] Deletions

[0173] For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”) or commas e.g., “Gly195* + Ser411*” or “G195* + S411*”.

[0174] Insertions

[0175] For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

[0176] In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

[0177] Multiple alterations

[0178] Variants comprising multiple alterations are separated by addition marks (“+”) or by commas e.g., “Arg170Tyr+Gly195Glu”, “R170Y+G195E”, “Arg170Tyr,Gly195Glu” or “R170Y,G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Different alterations

[0179] Where different alterations can be introduced at a position, the different alterations are separated by a comma or slash e.g., “Arg170Tyr,Glu” or “Arg170Tyr / Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala + Arg170Gly,Ala” or “Tyr167Gly / Ala + Arg170Gly / Ala” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0180] Unspecified amino acid

[0181] Unless otherwise limited further, the amino acid X (or Xaa) is used herein to represent any of the 20 natural amino acids.

[0182] An “X” preceding a position means that any original amino acid at that position may be substituted. For example, X93Q means that any amino acid residue at position 93 other than Q is substituted with Q. This allows for designation of substitution to a particular amino acid in different parent mannanases, where the original amino acid may vary among different parent polypeptides.

[0183] DETAILED DESCRIPTION OF THE INVENTION

[0184] It is known that even aggressive proteases such as subtilisin proteases have difficulty in degrading keratin. The inventors of the present invention have surprisingly been able to identify a clade of AA15 lytic polysaccharide monooxygenase (LPMO) polypeptides that also are capable of breaking down the protein keratin thus allowing for the further degradation of keratin which otherwise is difficult to break down due to its compact structure. Previously characterized AA15 LPMOs are known to be active on the polysacchides chitin and cellulose. The discovery of a clade of AA15 enzyme from a dermestid that is additionally active on protein is unexpected. A selection of AA15 enzyme candidates from Attagenus smirnovi were recombinantly expressed in Aspergillus oryzae and the purified enzymes tested in wash assays where they were found to be active in removing stains both with and without supplementary enzymes such as proteases.

[0185] This makes the LPMOs of the present invention particular useful where the simultaneous breakdown of (crystalline) polysaccharide structures and (crystalline) protein structures is advantageous. This is for instance the case where laundry detergent is used for the cleaning of textile where hard-to-remove stains include stains comprising keratin.

[0186] Accordingly, the present invention relates to isolated lytic polysaccharide monooxygenase (LPMO) polypeptides that have at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to any of the mature part of any of the polypeptides of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:12 or SEQ ID NO: 13 wherein the polypeptides have lytic polysaccharide monooxygenase activity and keratin degrading activity. Lytic polysaccharide monooxygenase activity can be determined as disclosed in Example 3. The keratin degrading activity can be determined as disclosed in Example 4.

[0187] It is preferred that the LPMO polypeptide used in the present invention capable of enhancing the enzyme detergency benefit by at least 1 delta remission units compared to when the enzyme is used without the LPMO polypeptide, more preferred it as capable of enhancing the detergency benefit by 2 delta remission units, more preferably by 3 delta remission units such as by 4 delta remission units, 5 delta remission units or even6 delta remission units. Enzyme detergent benefit is determined as described in Example 5.

[0188] In a particular embodiment the present invention relates to a detergent composition comprising at least one detergent adjunct ingredient and one or more polypeptides selected from the group consisting of polypeptides having lytic polysaccharide monooxygenase activity and keratin degrading activity, characterized in that the polypeptide has at least 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to any of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:12 or SEQ ID NO:13.

[0189] The concentration of the LPMO (as AEP) in the wash liquor is typically in the range of 0.05-20 ppm (mg / L) enzyme protein, such as in the range of 0.1-15 ppm, in the range of 0.5-15 ppm, in the range of 1-15 ppm, in the range of 1-10 ppm, in the range of 2-10 ppm.

[0190] The LPMO (as formulated product) may be present in the detergent in a concentration from 0.2- 10 wt%, such as in the range of 0.5-5 wt%, such as in the range of 0.5-3 wt%, such as in the range of 0.5-2.5 wt%, or in the range of 0.5-2 wt%, or even in the range of 0.5-1 wt%.

[0191] The LPMO of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WQ92 / 19709 and WQ92 / 19708.

[0192] A polypeptide of the present invention may also be incorporated in the detergent formulations disclosed in WQ97 / 07202, which is hereby incorporated by reference.

[0193] Further aspects of the present invention are disclosed in the following paragraphs.

[0194] LPMO variants of the invention

[0195] The LPMO variants of the present invention have at least 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to mature part of any of SEQ I D NO: 1 , SEQ I D NO: 2, SEQ I D NO: 3, SEQ I D NO: 4, or SEQ ID NO: 5. The variants may further comprise changes of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0196] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0197] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0198] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for LPMO activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et a / ., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / pro- teins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589. In the following, unless otherwise explicitly stated, the numbering of amino acids of the variants refers to the numbering obtained when aligned with SEQ ID:4.

[0199] Preparation of variants

[0200] The present invention also relates to methods for obtaining a variant having mannase activity with the substitutions and deletions disclosed herein. In one embedment the method comprises: (a) introducing into a parent LPMO a deletion or substitution at one or more positions of the polypeptide, wherein the variant has LPMO activity; and (b) recovering the variant.

[0201] In particular the invention relates to methods for obtaining a variant having mannase activity disclosed in the paragraph Variants above.

[0202] The variants can be prepared using any mutagenesis procedure known in the art, such as site- directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0203] Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

[0204] Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 7Q: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

[0205] Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004 / 0171154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

[0206] Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

[0207] Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

[0208] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204) and region- directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a / ., 1988, DNA 7: 127).

[0209] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0210] Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

[0211] Polynucleotides

[0212] The present invention also relates to polynucleotides encoding a LPMO of the present invention. The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. In an aspect, the polynucleotide is isolated. In another aspect, the polynucleotide is purified.

[0213] Nucleic Acid Constructs

[0214] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0215] The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0216] Promoters

[0217] The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0218] Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier, and Song et al., 2016, PLOS One 11 (7): e0158447.

[0219] Terminators

[0220] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

[0221] Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB). mRNA Stabilizers

[0222] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0223] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94 / 25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacterid. 177: 3465- 3471).

[0224] Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce / / 5(11): 1838-1846.

[0225] Leader Sequences

[0226] The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0227] Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

[0228] Polyadenylation Sequences

[0229] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0230] Signal Peptides

[0231] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

[0232] Propeptides

[0233] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0234] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

[0235] Regulatory Sequences

[0236] It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. Transcription Factors

[0237] The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, i.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7- 23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.

[0238] Expression vectors

[0239] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0240] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0241] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0242] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0243] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non-homologous recombination, such as non-homologous end-joining (NHEJ).

[0244] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0245] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0246] Host cells

[0247] The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.

[0248] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention. The host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.

[0249] The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

[0250] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.

[0251] For purposes of this invention, Bacillus classes / genera / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

[0252] The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0253] The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0254] Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.

[0255] In an aspect, the host cell is isolated, preferably the host cell is purified. Methods of production

[0256] The present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.

[0257] The host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small- scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0258] The variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant.

[0259] The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, a cell- free fermentation broth comprising the polypeptide is recovered.

[0260] The variant may be purified by a variety of procedures known in the art to obtain substantially pure variants and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0261] In an alternative aspect, the variant is not recovered.

[0262] Detergent Compositions

[0263] In one embodiment, the invention is directed to detergent compositions comprising a LPMO in combination with one or more additional cleaning composition components. In one embodiment, the detergent composition comprises a polypeptide having LPMO activity with an amino acid sequence having at least 60% identity, such as 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identity to the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:12 or SEQ ID NO:13. In one embodiment the detergent composition is in powder form. In another embodiment, the detergent composition is in a liquid or gel form. In another embodiment a bar form. In one embodiment the detergent may be wrapped in water soluble PVOH film. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

[0264] Formulation of Detergent Products

[0265] The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a sheet, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and / or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009 / 0011970 A1.

[0266] Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

[0267] A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous. Laundry Soap Bars

[0268] The LPMO of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and / or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and / or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.

[0269] The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and / or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na+, K+or NH4+and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.

[0270] The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and / or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkox- ylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and / or other compounds known in the art.

[0271] The laundry soap bar may be processed in conventional laundry soap bar making equipment such as, but not limited to, mixers, plodders, e.g. a two-stage vacuum plodder, extruders, cutters, logostampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, LPMO, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared, and the mixture is then plodded. The LPMO and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and / or wrapping. LPMO Granules

[0272] The present invention also relates to enzyme granules / particles comprising a polypeptide of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.

[0273] The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core diameter, measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO13320 (2020).

[0274] In an embodiment, the core comprises a polypeptide of the present invention.

[0275] The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

[0276] The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.

[0277] The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and / or an acidic buffer component, typically as a homogenous blend.

[0278] The core may include an inert particle with the polypeptide absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

[0279] The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.

[0280] The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

[0281] The coating may be applied in an amount of at least 0.1 % by weight of the core, e.g., at least 0.5%, at least 1 %, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.

[0282] The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.

[0283] The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.

[0284] The coating can further contain other materials as known in the art, e.g. , fillers, antisticking agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc. A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

[0285] To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.

[0286] The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.

[0287] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water. The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

[0288] The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0289] Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO3 (CH2o°c=92%), NaNO3(CH2O"C=73%), Na2HPO4(CH2o"c=95%), Na3PO4(CH25°c=92%), NH4CI (CH2o"c = 79.5%), (NH4)2HPO4(CH2O"C = 93,0%), NH4H2PO4(CH20°c = 93.1%), (NH4)2SO4(CH2o°c=81 .1%), KOI (CH2O"C=85%), K2HPO4(CH2O"C=92%), KH2PO4(CH2O°C=96.5%), KNO3(CH2O"C=93.5%), Na2SO4(CH2O"C=93%), K2SO4(CH2O"C=98%), KHSO4(CH2O"C=86%), MgSO4(CH2o"c=9O%), ZnSO4(CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH3PO4, (NH4)H3PO4, CuSO4, Mg(NO3)2 and magnesium acetate.

[0290] The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na3SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO47H3O), zinc sulfate heptahydrate (ZnSO47H3O), sodium phosphate dibasic heptahydrate (Na2HPO4 ?H2O), magnesium nitrate hexahydrate (Mg(NC>3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0291] Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

[0292] The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.

[0293] The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0294] The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0295] Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,

[0296] (a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker).

[0297] (b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97 / 23606.

[0298] (c) Absorbed core particles, wherein rather than coating the polypeptide as a layer around the core, the enzyme is absorbed onto and / or into the surface of the core. Such a process is described in WO 97 / 39116.

[0299] (d) Extrusion or pelletized products, wherein a polypeptide-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140- 142; Marcel Dekker).

[0300] (e) Prilled products, wherein a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). The product obtained is one wherein the polypeptide is uniformly distributed throughout an inert material instead of being concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

[0301] (f) Mixer granulation products, wherein a polypeptide-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , WO 90 / 09440 and WO 90 / 09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. Granulates consisting of polypeptide, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles, are more robust, and release less enzymatic dust.

[0302] (g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0303] (h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.

[0304] (i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the polypeptide contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w / w water.

[0305] Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.

[0306] The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D. Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013 / 188331.

[0307] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.

[0308] In an embodiment, the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes (such as amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xyloglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, other endo-beta-mannanases, exo-beta- mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof).

[0309] Liquid Formulations

[0310] The present invention also relates to liquid compositions comprising a polypeptide of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).

[0311] In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials. In an aspect, the liquid formulation comprises 20-80% w / w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative.

[0312] In another embodiment, the invention relates to liquid formulations comprising:

[0313] (A) 0.001-25% w / w of a polypeptide of the present invention;

[0314] (B) 20-80% w / w of polyol;

[0315] (C) optionally 0.001-2% w / w preservative; and

[0316] (D) water.

[0317] In another embodiment, the invention relates to liquid formulations comprising:

[0318] (A) 0.001-25% w / w of a polypeptide of the present invention;

[0319] (B) 0.001-2% w / w preservative;

[0320] (C) optionally 20-80% w / w of polyol; and

[0321] (D) water.

[0322] In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0323] In another embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2- propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

[0324] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w / w preservative, e.g., 0.05-1 % w / w preservative or 0.1 -0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative ( / .e., total amount of preservative), e.g., 0.02-1.5% w / w preservative, 0.05-1% w / w preservative, or 0.1 -0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0325] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xyloglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, endo-beta-mannanases, exo-beta- mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof.

[0326] Detergent Ingredients

[0327] The choice of detergent components may include, for textile care, the consideration of the type of textile to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

[0328] Any detergent components known in the art for use in detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegra- tion agents, dyes, enzyme stabilizers (including boric acid, borates, and / or polyols such as propylene glycol), fabric conditioners including clays, fillers / processing aids, fluorescent whitening agents / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in detergents may be utilized. The choice of such ingredients is well within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

[0329] Surfactants

[0330] The cleaning composition may comprise one or more surfactants, which may be anionic and / or cationic and / or non-ionic and / or semi-polar and / or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a surfactant system (comprising more than one surfactant) e.g. a mixture of one or more nonionic surfactants and one or more anionic surfactants. In one embodiment the detergent comprises at least one anionic surfactant and at least one nonionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 20:1 to 1 :20. In one embodiment the amount of anionic surfactant is higher than the amount of non-ionic surfactant e.g. the weight ratio of anionic to non-ionic surfactant may be from 10: 1 to 1.1 : 1 or from 5: 1 to 1.5: 1. The amount of anionic to non-ionic surfactant may also be equal and the weight ratios 1 :1. In one embodiment the amount of non-ionic surfactant is higher than the amount of anionic surfactant and the weight ratio may be 1 : 10 to 1 : 1.1. Preferably the weight ratio of anionic to non-ionic surfactant is from 10: 1 to 1 : 10, such as from 5: 1 to 1 :5, or from 5: 1 to 1 : 1.2. Preferably, the weight fraction of non-ionic surfactant to anionic surfactant is from 0 to 0.5 or 0 to 0.2 thus non-ionic surfactant can be present or absent if the weight fraction is 0, but if non-ionic surfactant is present, then the weight fraction of the nonionic surfactant is preferably at most 50% or at most 20% of the total weight of anionic surfactant and non-ionic surfactant. Light duty detergent usually comprises more nonionic than anionic surfactant and there the fraction of non-ionic surfactant to anionic surfactant is preferably from 0.5 to 0.9. The total weight of surfactant(s) is typically present at a level of from about 0.1% to about 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and may include any conventional surfactant(s) known in the art. When included therein the detergent will usually contain from about 1% to about 40% by weight of an anionic surfactant, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 15% to about 20%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, typically available as sodium or potassium salts or salts of monoethanolamine (MEA, 2- aminoethan-1-ol) or triethanolamine (TEA, 2,2',2"-nitrilotriethan-1-ol); in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS such as branched alkylbenzenesulfonates (BABS) and phenylalkanesulfonates; olefin sulfonates, in particular alpha-olefinsulfonates (AOS); alkyl sulfates (AS), in particular fatty alcohol sulfates (FAS), i.e., primary alcohol sulfates (PAS) such as dodecyl sulfate (SLS); alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates); paraffin sulfonates (PS) including alkane-1 -sulfonates and secondary alkanesulfonates (SAS); ester sulfonates, including sulfonated fatty acid glycerol esters and alphasulfo fatty acid methyl esters (alpha-SFMe or SES or MES); alkyl- or alkenylsuccinic acids such as dodecenyl / tetradecenyl succinic acid (DTSA); diesters and monoesters of sulfosuccinic acid; fatty acid derivatives of amino acids. Anionic surfactants may be added as acids, as salts or as ethanolamine derivatives.

[0331] When included therein the detergent will usually contain from about 0, 1 % to about 40% by weight of a cationic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12% or from about 10% to about 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyl- distearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.

[0332] When included therein the detergent will usually contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO) e.g. the AEO-series such as AEO-7, alcohol propoxylates, in particular propoxylated fatty alcohols (PFA), ethoxylated and propoxylated alcohols, alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters (in particular methyl ester ethoxylates, MEE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0333] When included therein the detergent will usually contain from about 0.01 to about 10 % by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxides, in particular N-(coco alkyl)-N,N-dimethylamine oxide and N-(tal- low-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, and combinations thereof.

[0334] When included therein the detergent will usually contain from about 0.01 % to about 10 % by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0335] Additional bio-based surfactants may be used e.g. wherein the surfactant is a sugar-based non-ionic surfactant which may be a hexyl-p-D-maltopyranoside, thiomaltopyranoside or a cyclic-maltopyra- noside, such as described in EP2516606 B1. Other biosurfactants may include rhamnolipids and sophorolipids.

[0336] Hydrotropes

[0337] A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however, the molecular structure of hydrotropes generally do not favor spontaneous selfaggregation, see e.g. review by Hodgdon and Kaier (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

[0338] The detergent may contain 0-10% by weight, for example 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0339] Builders and Co-Builders

[0340] The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof. The builder and / or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and / or co- builder known in the art for use in cleaning detergents may be utilized.

[0341] Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Clariant), ethanolamines such as 2- aminoethan-1-ol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethan-1-ol), triethanolamine (TEA, also known as 2,2',2"-nitrilotriethan-1-ol), and (carboxymethyl)inulin (CMI), and combinations thereof.

[0342] The detergent composition may also contain from about 0-50% by weight, such as about 5% to about 30%, of a detergent co-builder. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). According to the present invention, these components can be included in lower levels than in currently available detergent compositions. Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2, 2’, 2”-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N’-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1 ,1- diylbis(phosphonic acid (HEDP),ethylenediaminetetramethylenetetrakis(phosphonic acid) (EDTMPA),diethylenetriaminepentamethylenepentakis(phosphonic acid) (DTMPA or DTPMPA), N- (2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid- N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glu- tamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), a-al- anine-N,N-diacetic acid (a-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N -di acetic acid (ANDA), sulfan- ilic acid-N,N -di acetic acid (SLDA) , taurine-N,N-diacetic acid (TLIDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)ethylenediamine-N,N’,N”-triacetic acid (HEDTA), diethanolglycine (DEG), aminotrimethylenetris(phosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described in, e.g., WO 09 / 102854 and US 5977053.

[0343] Polymers and Dispersants

[0344] Generally, detergent compositions may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and / or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the below-mentioned motifs. Exemplary polymers include poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of polyethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine- / V-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include polyethylene oxide and polypropylene oxide (PEO-PPO), diquaternium ethoxy sulfate, styrene / acrylic copolymer and perfume capsules Other exemplary polymers are disclosed in, e.g., WO 2006 / 130575. Salts of the above-mentioned polymers are also contemplated.

[0345] The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.

[0346] Fabric Hueinq Agents

[0347] The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in W02005 / 03274, W02005 / 03275, W02005 / 03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007 / 087257 and W02007 / 087243.

[0348] Dye Transfer Inhibiting Agents

[0349] The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine / V-oxide polymers, copolymers of / V-vinylpyrrolidone and / V-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01 % to about 5% or even from about 0.1% to about 3% by weight of the composition.

[0350] Fluorescent Whitening Agent

[0351] The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulfonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanola- mino-4-anilino-s-triazin-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-yla- mino) stilbene-2.2'-disulfonate, 4,4'-bis-(2-anilino-4-( / V-methyl- / \ / -2-hydroxy-ethylamino)-s-triazin- 6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1 ,2,3-triazol-2-yl)stilbene-2,2'-disulfonate and sodium 5-(2 / 7-naphtho[1 ,2-c(][1 ,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s- triazin-6-ylamino) stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl- styryl)-disulfonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is a 4.4'-bis-(sulfostyryl)-biphenyl disodium salt also known as Disodium Distyrylbiphenyl Disulfonate. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins.

[0352] Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt%.

[0353] Soil Release Polymers

[0354] The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009 / 087523 (hereby incorporated by reference). Furthermore, random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (hereby incorporated by reference).

[0355] Anti-redeposition Agents

[0356] The detergent compositions of the present invention may also include one or more anti-redepo- sition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyoxyethylene and / or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid. The cellulose-based polymers described under soil release polymers above may also function as anti-redeposition agents.

[0357] Rheology Modifiers

[0358] The detergent compositions of the present invention may also include one or more rheology modifiers, structurants or thickeners, as distinct from viscosity reducing agents. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the agueous liguid matrix of a liguid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040. Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and / or structure elasticizing agents.

[0359] Additional Enzymes

[0360] The detergent additive as well as the detergent composition may contain one or more additional enzymes (such as amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xy- loglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, endo-beta-mannanases, exo-beta-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof) In general, the properties of the selected enzyme(s) should be compatible with the selected detergent, ( / .e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

[0361] Cellulases

[0362] The term “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. The two terms polypeptide having cellulase activity and cellulase are used interchangeably. Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172. Such enzymes include endoglu- canase(s) (e.g. EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. The cellulase may for example be a mono-component or a mixture of mono-component endo-1 ,4-beta-glu- canase also referred to as endoglucanase.

[0363] DNases (deoxyribonuclease)

[0364] The term “DNase” means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.

[0365] Mannanases

[0366] Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999 / 064619. A commercially available mannanase include Mannaway (Novozymes A / S) and Mannaway 200L (Novozymes A / S). Preferably, the mannanase is a GH5 mannanase (WO2018 / 206300, WO2018 / 206302, WO18185367, WO18184767, WO18220273, WO1 8220274, WO 2020207882, WO2021058452, WO17079751) or a GH26 mannanase (WO2019 / 068713, WO2019 / 068715, WO 2023 / 247348).

[0367] Proteases

[0368] Suitable proteases may be of any origin, but are preferably of bacterial or fungal origin, optionally in the form of protein engineered or chemically modified mutants. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as a subtilisin. A metalloprotease may for example be a thermolysin, e.g. from the M4 family, or another metalloprotease such as those from the M5, M7 or M8 families.

[0369] The term "subtilases" refers to a sub-group of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into six subdivisions, the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

[0370] Although proteases suitable for detergent use may be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases have generally been obtained from bacteria and in particular tromBacillus. Examples of Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and e.g. protease PD138 (described in WO 93 / 18140). Other useful proteases are e.g. those described in WO 01 / 16285 and WO 02 / 16547.

[0371] Examples of trypsin-like proteases include the Fusarium protease described in WO 94 / 25583 and WO 2005 / 040372, and the chymotrypsin proteases derived from Cellumonas described in WO 2005 / 052161 and WO 2005 / 052146.

[0372] Examples of metalloproteases include the neutral metalloproteases described in WO 2007 / 044993 such as those derived from Bacillus amyloliquefaciens, as well as e.g. the metalloproteases described in WO 2015 / 158723 and WO 2016 / 075078.

[0373] Examples of useful proteases are the protease variants described in WO 89 / 06279 WO 92 / 19729, WO 96 / 34946, WO 98 / 20115, WO 98 / 20116, WO 99 / 11768, WO 01 / 44452, WO 03 / 006602, WO 2004 / 003186, WO 2004 / 041979, WO 2007 / 006305, WO 2011 / 036263, WO 2014 / 207227, WO 2016 / 087617 and WO 2016 / 174234.

[0374] Suitable commercially available protease enzymes include those sold under the trade names Al- calase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Pri- mase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Co- ronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In and Progress® Excel (Novozymes A / S), those sold under the tradename Maxatase™, Maxacai™, Maxapem®, Pura- fect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, FN4ex™, Excellase®, Excellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz® P100, Purafect Prime, Preferenz P110™, Effectenz P1000™, Purafect®, Effectenz P1050™, Purafect® Ox, Effectenz ™ P2000, Purafast™, Properase®, Opticlean™ and Optimase® (Danisco / DuPont), BLAP (sequence shown in Figure 29 of US 5352604) and variants hereof (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0375] Lipases and Cutinases

[0376] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96 / 13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcali- genes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (W095 / 06720 & W096 / 27002), P. wisconsinensis (WO96 / 12012), GDSL-type Streptomyces lipases (W010 / 065455), cutinase from Magnaporthe grisea (WO10 / 107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W011 / 084412), Geobacillus stearothermophilus lipase (W011 / 084417), lipase from Bacillus subtilis (W011 / 084599), and lipase from Streptomyces griseus (WO11 / 150157) and S. pristinaespiralis (WO12 / 137147).

[0377] Other examples are lipase variants such as those described in EP407225, WO92 / 05249, WO94 / 01541 , WO94 / 25578, WO95 / 14783, WO95 / 30744, WO95 / 35381 , WO95 / 22615,

[0378] W096 / 00292, W097 / 04079, W097 / 07202, WO00 / 34450, WO00 / 60063, W001 / 92502,

[0379] W007 / 87508 and WO09 / 109500.

[0380] Preferred commercial lipase products include include Lipolase 100T / L, Lipex 100T / L, Lipex 105T, Lipex Evity 100L, Lipex Evity 200L (all Novozymes A / S), Preferenz® L 100 (DuPont).

[0381] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10 / 111143), acyltransferase from Mycobacterium smegmatis (WO05 / 56782), perhydrolases from the CE 7 family (WO09 / 67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10 / 100028).

[0382] Amylases

[0383] Suitable amylases include an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.

[0384] Suitable amylases include amylases having SEQ ID NO: 2 in WO 95 / 10603 or variants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO: 4 of WO 99 / 019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.

[0385] Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02 / 010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

[0386] Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594 or variants having 90% sequence identity thereof.

[0387] Other examples are amylase variants such as those described in WO2011 / 098531 , WO2013 / 001078 and WO2013 / 001087.

[0388] Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, StainzymeTM, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ Amplify; Amplify Prime; (from Novo- zymes A / S), and Rapidase™ , Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont).

[0389] Peroxidases / Oxidases

[0390] Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guar- dzyme™ (Novozymes A / S).

[0391] A suitable peroxidase is preferably a peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7, as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom, exhibiting peroxidase activity. Suitable peroxidases also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions. The haloperoxidase may be a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a van- adate-containing haloperoxidase. In a preferred method the vanadate-containing haloperoxidase is combined with a source of chloride ion.

[0392] Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

[0393] Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.

[0394] The haloperoxidase may be derivable from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or from Drechslera hartlebii as described in WO 01 / 79459, Dendryphiella salina as described in WO 01 / 79458, Phaeotrichoconis crotalarie as described in WO 01 / 79461 , or Geniculosporium sp. as described in WO 01 / 79460.

[0395] Suitable oxidases include, in particular, any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), or a bilirubin oxidase (EC 1.3.3.5).

[0396] Preferred laccase enzymes are enzymes of microbial origin. The enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).

[0397] Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Pomes, Lentinus, Pleurotus, Trametes, e.g., T. vil- losa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, My- celiophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radiata (\NO 92 / 01046), or Coriolus, e.g., C. hirsutus (JP 2238885).

[0398] Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.

[0399] A laccase derived from Coprinopsis or Myceliophthora is preferred; in particular a laccase derived from Coprinopsis cinerea, as disclosed in WO 97 / 08325; or from Myceliophthora thermophila, as disclosed in WO 95 / 33836. Licheninases

[0400] Licheninases (or lichenases) {e.g. EC 3.2.1.73) hydrolyse (1 ,4)-beta-D-glucosidic linkages in beta-D-glucans containing (1 ,3)- and (1 ,4)-bonds and can act on lichenin and cereal beta-D-glu- cans, but not on beta-D-glucans containing only 1 ,3- or 1 ,4-bonds.

[0401] Pectate Lyases

[0402] Pectate lyases catalyze the cleavage of a-1 ,4-D-galacturonan (i.e., homogalacturonan or polyga- lacturonic acid) by an eliminative pathway leaving a double bond between C4 and C5 at the +1 subsite and a reducing sugar at the -1 subsite. Pectate lyases may also have pectin lyase activity.

[0403] Formulation of Detergent Products

[0404] The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

[0405] Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and / or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009 / 0011970 A1.

[0406] Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

[0407] A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

[0408] Embodiments of the Invention

[0409] The invention is further defined in the following embodiments:

[0410] E1 : An isolated lytic polysaccharide monooxygenase, characterized in that the lytic polysaccharide monooxygenase has lytic polysaccharide monooxygenase activity and keratin degrading activity, and optionally belongs to the family AA15.

[0411] E2: The lytic polysaccharide monooxygenase according to E1 , characterized in that lytic polysaccharide monooxygenase has at least 60% sequence identity to the mature part of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 13.

[0412] E3: The lytic polysaccharide monooxygenase according to E1 or E2, characterized in that lytic polysaccharide monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to the mature part of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 13.

[0413] E4: A detergent composition comprising at least one detergent adjunct ingredient, one or more of the polypeptides having lytic polysaccharide monooxygenase activity and keratin degrading activity of any of E1 , E2 or E3 and optionally one or more additional enzymes.

[0414] E5: The detergent composition according to E4, wherein the one or more additional enzymes is selected from the group consistling of amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xyloglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alphaamylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, endo-beta-mannanases, exo-beta- mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof.

[0415] E6: The detergent composition according to E4 or E5, wherein the detergent composition comprises between 1 uM to 800 uM ascorbic acid, sue as between 1.4 to 500 uM ascorbic acid. E7: The detergent composition according to E4, E5 or E6, wherein the detergent composition does not comprise chelator.

[0416] E8: Use of the polypeptide according to any of E1 , E2 or E3 or the detergent composition of E4 to E7 in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.

[0417] E9: A method of cleaning an item, comprising exposing the item to a wash liquor comprising the lytic polysaccharide monooxygenase of any of E1 , E2 or E3 or to the detergent composition of E4 to E7, e.g. wherein the item is a textile or a hard surface.

[0418] E10: The use accordin to E8 or the method according to E9, wherein the wash liquour comprises from about 0.2 g detergent composition of E4 to E7 per liter wash liquour to about about 5 g detergent composition of E4 to E7 per liter wash liquour.

[0419] E11 : A polynucleotide encoding the polypeptide of any of E1 , E2 or E3.

[0420] E12: A nucleic acid construct or expression vector comprising the polynucleotide of E11.

[0421] E13: A recombinant host cell transformed with the polynucleotide of E12.

[0422] E14: A method of producing a lytic polysaccharide monooxygenase, comprising a. Cultivating the host cell of E13 under conditions suitable for expression of the lytic polysaccharide monooxygenase; and b. Recovering the lytic polysaccharide monooxygenase.

[0423] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0424] EXAMPLES

[0425] Materials and Methods

[0426] Strains

[0427] Escherichia coli Top-10 strain purchased from Invitrogen (Thermofisher Inc.) was used to propagate the expression vector

[0428] Aspergillus oryzae strain MT3568 (described in WO2015040159) was used for heterologous expression of the polypeptides of the invention as described in Table 1. The sequence of the mature polypeptide (the column ‘Sequence’), the associated signal peptide is listed in the column ‘Signal peptide’.

[0429] Table 1 : Polypeptides

[0430] Media

[0431] DAP4C medium is composed of 11 g MgSC r W, 1 g KH2 O4, 2.2 g Citric acid FLO, 20 g glucose, 10 g maltose, 5.2 g K3PO4 H2O, 0.5 g yeast extract, 1.25 g CaCCh, 0.5 ml AMG Trace element solution and deionized water to 1 liter. After autoclaving, 3.3 ml of 20% Lactic Acid (autoclaved) and 9.3 ml of 50% (NF^HPCL (sterile filtered) was added to every 400 ml of the above medium.

[0432] AMG Trace element solution is composed of 6.8 g ZnCh, 2.5 g CUSO4.5H2O, 0.24 g NiCh SFLO, 13.9 g FeSO4.7H2O, 13.6 g MnSO4.5H2O, 3 g Citric acid-FhO, and deionised water to 1000 ml.

[0433] LB plates are composed of 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15g of Bacto-agar, and deionised water to 1000 ml.

[0434] LB medium is composed of 1g of Bacto-tryptone, 5 g of yeast extract, and 10 g of sodium chloride, and deionised water to 1000 ml.

[0435] COVE sucrose plates are composed of 342 g of sucrose, 20 g of agar powder, 20 ml of COVE salt solution, and deionized water to 1 liter. The medium was sterilized by autoclaving. For the transformation of MT3568, 10 mM acetamide was added, when the medium was cooled to 60°C.

[0436] COVE-2 plate / tube for isolation if single transformants: 30 g / L sucrose, 20 ml / L COVE salt solution, 10 mM acetamide, 30 g / L noble agar (Difco, Cat#214220).

[0437] COVE salt solution is composed of 26 g of MgSO4'7H2O, 26g of KCL, 26g of KH2PO4, 50 ml of COVE trace metal solution, and deionised water to 1000 ml.

[0438] COVE trace metal solution is composed of 0.04g of Na2B40y 10H20, 0.4g of CUSO4 5H2O, 1.2g of FeSO4'7H2O, 0.7g of MnSO4'H2O, 0.8g of Na2MoO4'2H2O, 10g of ZnSO4'7H2O, and deionised water to 1000 ml. Wash performance assay

[0439] The following materials were used in wash performance assay:

[0440] • Ascorbic acid, Sigma # A92902

[0441] • Medley Brilliant 100L is a commercially available produdct from Novozymes A / S compris- ing mannanase, cellulase, pectate lyase, protease (subtilisin), lipase, alpha-amylase enzymes

[0442] • Model detergent:

[0443] Insert table w composition

[0444] • Test swatches: o D-ConsumerTec Dingy-TN / CP / EU with protein mix including animal keratin and collagen (CFT, Vlaardingen, The Netherlands) o C-H152 and C-H156 from CFT (CFT, Vlaardingen, The Netherlands )

[0445] Detergent 1 :

[0446] Table A: Detergent 1 composition (liquid)

[0447] Detergent 1 was produced by mixing water, propylene glycol, triethanolamine and sodium hydroxide. Then topped palm kernel fatty acid, Na-LAS, SLES and AEO were added in portions and stirred for few hours. Trisodium citrate dihydrate, DTPMP Na7 and 2-phenoxyethanol were added and pH adjusted within target (pH 8.2 - 8.6) after overnight stirring at room temperature. (Commercial raw materials were used and dosage of raw material was adjusted with the purity of the individual ingredients to achieve the listed active content)

[0448] Example 1 : Cloning, expression and fermentation of LPMOs of the invention.

[0449] Live Attgenus smirnovi larvae were obtained from the Institute for Plant Protection and Pests, University of Aarhus, Forsogsvej 1 , 4200 Slagelse, Denmark. The larvae were flash frozen in liquid nitrogen. The material was treated with TRIzol RNA Isolation Reagent (Thermofisher Inc.) using the following modified protocol:

[0450] 200 mgs of the flash frozen material were ground with baked quartz sand in liquid nitrogen and placed in an RNAse free 2 ml Eppendorf tube in which 800 uls TRIzol reagent was added. The tube was mixed rapidly to dissolve the frozen material and incubated in a 50°C water bath for 5 mins. 200uls of chloroform were added and the tube vortexed for 20 seconds. The tube was allowed to settle for 3 minutes whereupon it was vortexed again and centrifuged at room temperature for 10 min. The top liquid phase from the tube was transferred to a new tube and an equal volume of phenol chloroform was added. The phenol used was acid phenokchloroform pH 4.5. This mixture was again vortexed and centrifuged under the same conditions for 10 minutes. The top phase was again transferred to an RNAase free fresh tube and an equal volume of chloroform isoamyl alcohol was added. This was then vortexed and centrifuged for 10 minutes. The top aqueous phase was transferred to a new tube and precipitated in 1 / 10 volume 3M NaOAc and 2 volumes 96% ethanol. The tube was mixed by inversion and incubated at room temperature for 15 minutes. RNA was recovered by centrifuging the tube at 20,000g for 15 minutes at 4°C. Supernatant was carefully removed and 700 uls of 70% ethanol was added then the solution centrifuged for 5 minutes, 4°C at 20,000g. The supernatant was carefully removed, and the pellet allowed to air dry. The dry pellet was resuspended in 100uls DEPC treated water. Heating at 65°C for 10 minutes aided in resuspension. The resulting total RNA was stored- 80°C until use.

[0451] 20uls total RNA at 5.8ugs / ml was sent to FASTERIS (Rue du Champ-Blanchod 4, 1228 Plan-les- Ouates, Switzerland). Illumina mRNASeq was performed with 100bp paired end reads.

[0452] The Illumina reads were assembled using Trinity version r2012-05-18. The assemblies were translated using ESTScan-2.0b. The program hmmscan3 on a Pfam-A replicate was used to annotate protein function (from May 16-2012 (1 ,121 ,061 ,052 bytes). The assembled gene models were then searched by BLAST using a HMM for AA15 (Cazy.org) to identify candidates. The Atagenus smirinovi expressed sequence tag (EST) project was found to be unusually rich in AA15 enzymes. Some of these enzymes appear related to AA15s in the Red Flour Beetle (TriboHum castaneum) while others appear to be in a unique clade distant from this vegetarian. It is therefore hypothesized that the divergent AA15s in Atagenus are active on their proteinaceous food sources which typically are rich in keratin and collagen. The AA15 translated cDNA was used to create an Aspergillus oryzae codon optimized gene compatible with the A. oryzae expression plasmid pDau109. The plasmid pDAu109 and its use are described in (WO 2005 / 042735). The open reading frames from each cDNA were codon optimized for Aspergillus oryzae expression and synthetic genes ordered from TWIST Bioscience (South San Francisco, USA). The synthetic genes were received as intact plasmid constructs with the AA15 synthetic genes inserted into pDau109 in the BamHI-Xhol restriction sites. These plasmids were used to transform the Aspergillus oryzae strain MT3568 (WO 11 / 057140) by the methods described in Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO 04 / 032648. Transformants were selected during regeneration from protoplasts based on the ability, conferred by a selectable marker in the expression vector, to utilize acetamide as a nitrogen source, and were subsequently re-isolated under selection.

[0453] Production of the recombinant AA15 peptides was evaluated by culturing the transformants in 96- well deep-well microtiter plates for 4 days at 30°C in either a 0.25ml or 0.75ml volume of either or both YPG medium (WO 05 / 066338) or DAP-4C-1 medium (WO 12 / 103350) and monitoring peptide expression by SDS-PAGE. A single Aspergillus transformant was selected for each gene based on expression yields as evaluated in microtiter plate fermentation.

[0454] Spores of the best expressed transformant were spread on COVE-2 plates for re-isolation in order to isolate single colonies. Then a single colony was spread on a COVE-2 tube until sporulation.

[0455] For larger-scale production of the recombinant enzymes, and the Aspergillus transformants were cultured in 500ml baffled flasks containing 150 ml of fermentation medium. Transformants expressing the SOD peptides were fermented in DAP-4C-1 medium (WO 12 / 103350). The cultures were shaken on a rotary table at 150 RPM at for 4 days, and the broth was subsequently separated from cellular material by passage through a 0.22 urn filtration unit.

[0456] Example 2: Purification of LPMOs of the invention

[0457] Approximately 100 mis of the samples filtered through a 0.22nM vacuum filter to remove microorganisms and debris. The samples were desalted through 53 mis G25 Desalting columns (GE Healthcare Cat. No. 17-5087-01) according to the manufacturer’s instructions and eluted into 50mM HEPES + 100mM NaCI, pH 7. The samples were then loaded onto 5 ml Phenyl Sepharose FF columns. After loading the column was washed with equilibration buffer and the protein sample eluted in a 50mM HEPES pH7, NaCI salt gradient according to the manufacturer’s instructions (Cytiva, Cat. No. 17108203).

[0458] Example 3: Determination of LPMO activity

[0459] LPMO activity can be measured as disclosed in Brander, S., Lausten, S., Ipsen, J.0. et al. Colorimetric LPMO assay with direct implication for cellulolytic activity. Biotechnol Biofuels 14, 51 (2021). https: / / do i . org / 10.1186 / s 13068-021 -01902-4. Example 4: Determination of keratinase activity

[0460] In order to determine if the AA15 enzymes of the present invention were active on keratin, a test on Keratin Azure (Sigma-Aldrich K8500) was conducted.

[0461] Assay procedure: 50ul of 1 % Keratin azure solution was added to 1.5 ml Eppendorf tubes. 50 ul of assay solution containing 5mM Cysteine, 500nM CuSO4, 1 mMCaCI2 and 50mM HEPES buffer pH7 was added to the tubes. 20ppm of each LPMO enzyme sample were then added to each tube. The tubes were incubated for 4 hours at 37°C after which the tubes were spun and

[0462] 100 ul of assay mix was taken out for each sample to 96 well NunclonTM Delta surface plate.

[0463] 2 ul of the protease having SEQ ID NO: 11 was added to each tube and the tubes were then incubated overnight. Samples were centrifuged at 14000rpm for 2 min. 200ul of sample was transferred to 96 well NunclonTM Delta surface plate and absorbance was measured at 595nm.

[0464] 4h-without protease 23h+protease

[0465] SEQ ID NO: 1 0.0792 0.0951

[0466] SEQ ID NO: 2 0.0861 0.2178

[0467] SEQ ID NO: 3 0.0623 0.0845

[0468] SEQ ID NO: 4 0.0635 0.1045

[0469] SEQ ID NO: 5 0.0617 0.0904 control no LPMO 0.0637 0.0721 (SEQ ID NO: 11 added after 4h).

[0470] The results confirmed that the LPMO of the present invention are active in keratin decomposition. The present invention relates to the use of isolated LPMO polypeptides in general. Preferably, the LPMO polypeptide used in the present invention has a cysteine activity of at least 0.01 absorbance units after 23 hours incubation with SEQ ID NO: 11 compared to SEQ ID NO: 11 alone (control).

[0471] Example 5: Enzymatic effects on sebum removal during wash

[0472] 25 mL wash Liquid (deionized water, Model 1 3.33 g / L detergent and water hardness ions giving 15°dH at the ratio of 4:1 :7.5 (Ca;Mg:HCO3),) was added to 60 mL Nalgene bottles with each 10 metal beads. Each bottle also contained two quatered pieces each final measure of 2.5 x 2.5 cm of D-ConsumerTec Dingy-TN / CP / EU (real item body soil-like soiling on cotton swatches) and 4 other quarters of equally sized soiled ballast cotton swatches (C-H152 and C-H156 from CFT).

[0473] Enzymes were added to each bottle according to Table 2 below. Wash was started by closing the bottle lids and placing closed bottles within Wascator FOM71 CLS (Electrolux) which used 25 L water to exercise the closed bottles during the wash time.

[0474] After a 30 minutes wash the swatches were removed from each bottle, rinsed in a container under running tap water and added to a European Miele Frontloader W1935 WTL full scale wash machine to be centrifugated according to the centrifugation program. Wash performance is expressed as a delta remission measured value (ARem). After washing and rinsing the swatches are spread out flat and allowed to air dry at room temperature overnight. Light reflectance evaluations of the swatches are done using a Datacolor 800v reflectance spectrophotometer with large aperture. Delta remission values for individual swatches are calculated by subtracting the remission value at 460 nm of the control swatch from the remission value at 460 nm of the washed swatch. Calculating the enzyme effect is done by taking the measurements from washed swatches with enzymes and subtract with the measurements from washed without enzyme for each stain. The total enzyme performance is calculated as the average of individual ARem.

[0475] Table 2: Wash effects of LPMO on Consumertec sebum swatches. Delta values are based on subtracting the wash effects of the detergent alone.

[0476] From the data above (Delta Remission 460, Table 2), it is evident that the LPMOs capable of catalyzing oxidative cleavage of polysaccharides (lytic polysaccharide monooxygenase activity) and keratin degrading activity have the requested detergent enzyme benefit on stains with protein mix including animal keratin and collagen.

[0477] Example 6: Enzymatic effects on sebum removal during wash in the presence of ascorbic acid and enzyme blend

[0478] The same test conditions as in Example 5 above was applied, except that ascorbic acid was added as electron donor and the effect of combination of LPMO and commercially available enzyme blend Medley Brilliant 100L was added.

[0479] Table 3: Wash effects of LPMO on Consumertec sebum swatches. Delta values are based on subtracting the wash effects of the detergent alone. Ascorbic acid is added as a reductant. Commercially available enzyme blend Medley Brilliant 100L (available from Novozymes A / S) is added to assist removal of sebum.

[0480] From the data above (Delta Remission 460, Table 3), it is evident that the LPMOs capable of catalyzing oxidative cleavage of polysaccharides (lytic polysaccharide monooxygenase activity) and keratin degrading activity have the requested detergent enzyme benefit on stains with protein mix including animal keratin and collagen when combined with an electron donor (in this case ascorbic acid) and other detergent enzymes. Medley Brilliant 100L is a commercially available enzyme blend of protease (subtilisin), alpha-amylase, pectate lyase, mannanase (mannan endo- beta-1 , 4-mannosidase), cellulase and lipase enzymes from Novozymes A / S.

Claims

CLAIMS1 . An isolated lytic polysaccharide monooxygenase, characterized in that the lytic polysaccharide monooxygenase has lytic polysaccharide monooxygenase activity and keratin degrading activity.

2. The lytic polysaccharide monooxygenase according to claim 1 , characterized in that the lytic polysaccharide monooxygenase belongs to the family AA153. The lytic polysaccharide monooxygenase according to claim 1 or 2, characterized in that the lytic polysaccharide monooxygenase has at least 60% sequence identity to the mature part of any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

4. The lytic polysaccharide monooxygenase according to claim 1 to 3, characterized in that lytic polysaccharide monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to the mature part of any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

5. A detergent composition comprising at least one detergent adjunct ingredient, one or more of the lytic polysaccharide monooxygenases of any of claims 1 to 4 and optionally one or more additional enzymes.

6. The detergent composition according to claim 5, wherein the one or more additional enzymes is selected from the group consistling of amylases, proteases, proteases, peroxidases, cellulases, betaglucanases, xyloglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyl transferases, phospholipases, esterases, laccases, catalases, aryl esterases, amylases, alphaamylases, glucoamylases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, endo-beta-mannanases, exo-beta- mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, and combinations thereof, or any mixture thereof.

7. Use of the lytic polysaccharide monooxygenase according to any of claims 1 to 4 or the detergent composition of claim 5 and 6 in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.

8. A method of cleaning an item, comprising exposing the item to a wash liquor comprising the lytic polysaccharide monooxygenase of any of claims 1 to 4 or to the detergent composition of claim 5 or 6, e.g. wherein the item is a textile or a hard surface.

9. The use according to claim 7 or the method of claim 8, wherein the wash liquour comprises from about 0.2 g detergent composition of claim 5 or 6 per liter wash liquour to about about 5 g detergent composition of claim 5 or 6 per liter wash liquour.

10. A polynucleotide encoding the lytic polysaccharide monooxygenase of any of claims 1 to 4.

11. A nucleic acid construct or expression vector comprising the polynucleotide of claim 10.

12. A recombinant host cell transformed with the polynucleotide of claim 10.

13. A method of producing a lytic polysaccharide monooxygenase, comprising a. Cultivating the host cell of claim 12 under conditions suitable for expression of the lytic polysaccharide monooxygenase; and b. Recovering the lytic polysaccharide monooxygenase.

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