GH26 mannanases and their use in detergent

Abstract

The present invention relates to newly idenified GH26 mannanases and their use as enhancers of enzyme benefits in detergents as well as a detergent composition comprising said mannanases.

Description

[0001] GH26 MANNANASES 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] BRIEF DESCRIPTION OF THE FIGURES

[0005] Figure 1 is an alignment of the polypeptides of SEQ ID NOs: 1 , 2, 3,4, and 5.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to newly identified GH26 mannanases and their use in particular in detergent compositions, in particular as enhancers of enzyme detergency benefits in detergents, as well as detergent compositions comprising said mannanases, optionally in combination with other enzymes. The invention also relates to a process of washing a fabric or hard surface using the mannanases 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 mannanase polypeptides and isolated polynucleotides encoding the polypeptides as well as to vectors and host cells comprising the polynucleotides and methods of producing the polypeptides.

[0008] BACKGROUND OF THE INVENTION

[0009] Mannans are polysaccharides with a backbone of (3-1 ,4-linked D-mannopyranosyl residues, which can contain galactose or acetyl substitutions and may have glucose residues in the backbone. The main enzyme type participating in the degradation of mannans are endo-1 ,4-p- mannanases (EC 3.2.1.78), which hydrolyze the internal glycoside bonds in the mannan backbone.

[0010] Mannans are a type of hemicellulose representing up to 25% of wood dry weight in softwoods, but are also found in other plant material, especially in a variety of seeds. The mannan containing guar gum is used as a stabilizer in many food products.

[0011] According to CAZy (cazy.org), endo-1 ,4-p-mannanases have been found in glycoside hydrolyase families 5, 26, 113 and 134. The glycoside hydrolyase families have different substrate specificity. Within the household care industry, it has been known to use mannanases in e.g. laundry detergents. In WO 1999 / 064619 an alkaline mannanase, which exhibits mannanase activity also in the alkaline pH range when applied in cleaning compositions, is disclosed.

[0012] In WO2019 / 068713, WO2019 / 068715, W02021 / 152120 and WO2021 / 152123 mannanases of family GH 26 exhibiting beta-mannanase activity are disclosed. WO 2023 / 247348 discloses variants of a parent GH26 mannanase. SEQ ID NO: 6 of the present disclosure is a GH26 mannanase wild-type disclosed in WO2019 / 068715. SUMMARY OF THE INVENTION

[0013] Mannanases are widely used in a variety of industrial and home care applications, such as laundering. All commercially availble mannanases for use in laundry detergents are GH5 mannanases and in spite of having a very good wash performance, the substrate specificity of GH5 mannanases leaves a gap in terms of mannan-containing products that can be broken down, e.g. fenugreek gum, often found in sauces and seasoning, and mofied guar gum found in e.g. shampoo. This gap can be closed by the use of GH26 mannanases as they have a much higher specificity for e.g. fenugreek gum and modified guar gum than GH5 mannanases have.

[0014] Accordingly, the present invention concerns mannanases belonging to the GH26 family, and to their use, in particular use in detergents. In a particular embodiment the present invention relates to the mature mannanases of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, variants thereof such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22, and their use in detergent.

[0015] The present invention also relates to a composition comprising a mannanase as herein disclosed, use of such a composition in a domestic or industrial cleaning process; in particular laundry detergent compositions comprising GH26 mannanase and GH5 mannanase are considered relevant embodiements of the invention. Further, methods of washing using such composition are part of the present invention.

[0016] Other uses of the mannanases of the invention are in the production of bioethanol from softwood and palm kernel press cake, for the improvement of animal feed and in the hydrolysis of coffee. Furthermore, guar gum is used in many food products and in the oil and gas industry, so the mannanases of the invention could be used in detergents to remove mannan containing stains, for hydraulic fracturing to create subterranean fractures that extend from the borehole into rock formation in order to increase the rate at which fluids can be produced by the formation or for cleaning borehole filtercake. The use aspect is discussed in more detail in the section “Uses”.

[0017] In a further aspect, the present invention concerns an isolated polynucleotide encoding the mannanase of the invention; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants herein disclosed.

[0018] DEFINITIONS

[0019] 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. AEP (active enzyme protein)

[0020] 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.

[0021] AlphaFold structure calculation

[0022] AlphaFold version 2 (AlphaFold2, AF2) is a computational method for calculating the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., 2021 , Nature 596: 583-589). Three-dimensional structures of millions of polypeptides deposited in the UniProt database have been calculated and deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 algorithm (Varadi et al., 2021 , Nucleic Acids Res. 50(D1):D439- D444). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

[0023] In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and calculating structures of new polypeptides at source code repositories such as Github.com under deepmind / alphafold / , using notebooks / AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokryp- ton / ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

[0024] AlphaFold 2 produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction. cDNA:

[0025] 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. Coding sequence:

[0026] 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.

[0027] Control sequences:

[0028] 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.

[0029] Detergent adjunct ingredient:

[0030] The detergent adjunct ingredient is different to the mannanase 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, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-build- ers, fabric hueing agents, anti-foaming agents, dispersants, processing aids, solvents, and / or pigments.

[0031] Detergent composition:

[0032] 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 amylase, beta-glu- canase, cellulase, xyloglucanase, DNase, hexosaminidase, mannanase, protesase, lipase, cu- tinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations 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”.

[0033] Detergent load

[0034] Detergent load is the amount of detergent used in a wash cycle.

[0035] Enzyme detergency benefit:

[0036] 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.

[0037] Expression:

[0038] 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.

[0039] Expression vector:

[0040] 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.

[0041] Extension:

[0042] 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 mannanase activity. Fragment:

[0043] The term “fragment” means a polypeptide having one or more amino acids absent from the amino and / or carboxyl terminus of the polypeptide.

[0044] Heterologous:

[0045] 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.

[0046] Host Strain or Host Cell:

[0047] 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.

[0048] Improved property:

[0049] 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 .

[0050] Introduced:

[0051] 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.

[0052] Isolated:

[0053] The term “isolated” means a polypeptide, 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 polypeptide expressed in a host cell. Laundering:

[0054] 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.

[0055] Mannanase:

[0056] The term mannanase refers to polypeptides having mannanase activity. Mannanases are listed in the carbohydrate-active enzyme (CAZy) database. The CAZy database describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic 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.

[0057] Mature polypeptide:

[0058] 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.

[0059] Mature polypeptide coding sequence:

[0060] The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having mannanase activity.

[0061] Mutant:

[0062] The term “mutant” means a polynucleotide encoding a variant.

[0063] Mutation

[0064] The term “mutation” refers to a deletion (including a truncation), insertion (including an extension) or substitution in a parent polypeptide.

[0065] Native:

[0066] The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0067] Nucleic acid:

[0068] 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.

[0069] Nucleic acid construct:

[0070] 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.

[0071] Operably linked:

[0072] 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.

[0073] Parent or parent mannanase:

[0074] The parent mannanase may be a polypeptide having at least 60% sequence identity to the mature part of polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0075] In an aspect, the parent mannanase 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%, and has mannanase 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 370 amino acid residues, e.g., at least 380 amino acid residues, e.g. at least 390 amino acid residues, e.g. , at least 400 amino acid residues, e.g. at least 410 amino acid residues, e.g., at least 420 amino acid residues, e.g. at least 430 amino acid residues, e.g., at least 440 amino acid residues, such as at least 480 amino acid residues, wherein the fragment has mannanase activity.

[0076] In an aspect, the parent mannanase has a sequence identity to the mature part of polypeptide of SEQ ID NO: 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%, and has mannanase 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 390 amino acid residues, e.g., at least 400 amino acid residues, e.g. at least 410 amino acid residues, e.g. , at least 420 amino acid residues, e.g. at least 430 amino acid residues, e.g., at least 440 amino acid residues, e.g. at least 450 amino acid residues, e.g., at least 460 amino acid residues, e.g. at least 470 amino acid residues, such as at least 480 amino acid residues, wherein the fragment has mannanase activity. In an aspect, the parent mannanase has a sequence identity to the mature part of polypeptide of SEQ ID NO: 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%, and has mannanase 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 390 amino acid residues, e.g., at least 400 amino acid residues, e.g. at least 410 amino acid residues, e.g. , at least 420 amino acid residues, e.g. at least 430 amino acid residues, e.g., at least 440 amino acid residues, e.g. at least 450 amino acid residues, e.g., at least 460 amino acid residues, e.g. at least 470 amino acid residues, such as at least 480 amino acid residues, wherein the fragment has mannanase activity. In an aspect, the parent mannanase has a sequence identity to the mature part of polypeptide of SEQ ID NO: 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%, and has mannanase 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 370 amino acid residues, e.g., at least 380 amino acid residues, e.g. at least 390 amino acid residues, e.g. , at least 400 amino acid residues, e.g. at least 410 amino acid residues, e.g., at least 420 amino acid residues, e.g. at least 430 amino acid residues, e.g., at least 440 amino acid residues, such as at least 480 amino acid residues, wherein the fragment has mannanase activity.

[0077] In an aspect, the parent mannanase has a sequence identity to the mature part of polypeptide of SEQ ID NO: 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%, and has mannanase 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 370 amino acid residues, e.g., at least 380 amino acid residues, e.g. at least 390 amino acid residues, e.g. , at least 400 amino acid residues, e.g. at least 410 amino acid residues, e.g., at least 420 amino acid residues, e.g. at least 430 amino acid residues, e.g., at least 440 amino acid residues, such as at least 480 amino acid residues, wherein the fragment has mannanase activity.

[0078] The parent mannanase may be a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1 .0, to the three-dimensional structure of the polypeptide of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the threedimensional structure is calculated by Alphafold.

[0079] 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.

[0080] Purified:

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] Recombinant:

[0086] 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”. Recover:

[0087] 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.

[0088] Rhamnolipid:

[0089] 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.

[0090] (Appl Microbiol Biotechnol (2005) 68: 718-725).

[0091] In the context of the present invention the term “rhamnolipid” includes mono-rhamnolipid or di- rhamnolipid, mixtures thereof and varying chain length as well as salts of rhamnolipid. Sequence difference:

[0092] The term "sequence difference" means the percent of amino acid differences between a polypeptide and the polypeptide of SEQ ID NO: X, where X represents the sequence number (i.e. 1 , 2, 3 etc.), and is calculated as follows:

[0093] (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.

[0094] Sequence identity:

[0095] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0096] 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:

[0097] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0098] 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:

[0099] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0100] Signal Peptide:

[0101] 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. Sophorolipid:

[0102] 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.

[0103] Subsequence:

[0104] 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 mannanase activity.

[0105] Substantially the same:

[0106] 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.

[0107] Sustainability:

[0108] Sustainability and sustainable means use of renewable resources that cause little or no damage to the environment and are biodegradable.

[0109] Sustainability profile:

[0110] 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).

[0111] Textile:

[0112] 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-contaming 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.

[0113] TEP:

[0114] Total Enzyme Protein is measured by amino acid analyses.

[0115] Variant:

[0116] The term “variant” means a polypeptide having enzyme, e.g. mannanase, 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. In the context of the present invention the terms “variant”, “mannanase variant” may be used interchangeably unless it is clear from the context that the variant refers to another enzyme class.

[0117] Wash cycle:

[0118] 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.

[0119] Wash liquor:

[0120] 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.

[0121] Wash performance:

[0122] 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.

[0123] Weight percentage:

[0124] Weight percentage is abbreviated w / w%, wt% or w%. The abbreviations are used interchangeably. Wild-type:

[0125] 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).

[0126] Sequence Overview

[0127] SEQ ID NO: 1 is a GH26 mannanase from Preussia minima

[0128] SEQ ID NO: 2 is a GH26 mannanase from Didymella glomerata

[0129] SEQ ID NO: 3 is a GH26 mannanase from Ascophyta fabae

[0130] SEQ ID NO: 4 is a GH26 mannanase from Paraphoma radiciana

[0131] SEQ ID NO: 5 is a GH26 mannanase from Paraphoma sp.

[0132] SEQ ID NO: 6 is a GH26 mannanase from Paenibacillus illinoisensis

[0133] SEQ ID NO: 7 is a GH5 mannanase from Alkalihalobacterium bogoriense

[0134] SEQ ID NO: 8 is a GH5 mannanase from Alkalihalobacterium bogoriense

[0135] SEQ ID NO: 9 is a GH5 mannanase from Paenibacillus sp.

[0136] SEQ ID NO: 10 is a GH5 mannanase from Bacillus sporothermodurans

[0137] SEQ ID NO: 11 is a GH5 mannanase from Bacillus clausii

[0138] SEQ ID NO: 12 is a GH5 mannanase from unidentified organism

[0139] SEQ ID NO: 13 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0140] SEQ ID NO: 14 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0141] SEQ ID NO: 15 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0142] SEQ ID NO: 16 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0143] SEQ ID NO: 17 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0144] SEQ ID NO: 18 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0145] SEQ ID NO: 19 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0146] SEQ ID NO: 20 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0147] SEQ ID NO: 21 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima

[0148] SEQ ID NO: 22 is a variant of the mannanase of SEQ ID NO: 1 from Preussia minima CONVENTIONS FOR DESIGNATION OF VARIANTS

[0149] For purposes of the present invention, the mature part of polypeptide disclosed in SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 may be selected as the parent mannanase enzyme and used to determine the corresponding amino acid positions in another mannanase. In a preferred embodiment SEQ ID NO: 1 is selected as the parent mannanase enzyme and used to determine the corresponding amino acid positions in another mannanase. The amino acid sequence of another mannanase is aligned with the polypeptide disclosed in the parent mannanase 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 mannanase (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.

[0150] 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.

[0151] Substitutions

[0152] 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 (“,”), e.g., “Gly205Arg + Ser411 Phe” or “G205R + “S411 F”, Gly205Arg,Ser411 Phe” or “G205R,S411 F” representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

[0153] Deletions

[0154] 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*”.

[0155] Insertions

[0156] 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 ammo acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

[0157] 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:

[0158] Multiple alterations

[0159] 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.

[0160] Different alterations

[0161] 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”.

[0162] Unspecified amino acid

[0163] Unless otherwise limited further, the amino acid X (or Xaa) is used herein to represent any of the 20 natural amino acids.

[0164] 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.

[0165] DETAILED DESCRIPTION OF THE INVENTION

[0166] The present invention relates to isolated mannanase 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, SEQ ID NO:5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22 wherein the polypeptides have mannanase activity. Mannanase activity can be determined as disclosed in the Experimantal section.

[0167] In an embodiment the mannanase polypeptide has a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22, wherein the three dimensional structure is calculated by Alphafold.

[0168] 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 mannanase 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, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22.

[0169] The detergent composition above preferably also comprises a GH5 mannanase in addition to the GH26 mannanase, wherein the GH5 mannanase has at least 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to a GH5 mannanase selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and SEQ ID NO: 12.

[0170] When used in laundry detergents, it is preferred that the mannanase polypeptide of the present invention is capable of enhancing the enzyme detergency benefit by at least 1 delta remission units compared to when the detergent is used without the mannanase 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 the Experimental section.

[0171] Further aspects of the present invention are disclosed in the following paragraphs.

[0172] Mannanases and mannanase variants of the invention

[0173] The mannanases and mannanase 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: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22.

[0174] In an embodiment the mannanase variants may be variants of SEQ ID NO: 1 , comprising one or more of the following substitutions V17I, S49T, I57R, K65R, L711 , D143E, S168T, V272L, K278R, P308V, S356T, P362A, M389P, or E390D wherein numbering is obtained when aligned with SEQ ID NO: 1 , wherein the variants have at east 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity to mature part of any of SEQ I D NO: 1 .

[0175] In a more particular embodiment the mannanase variants may be variants of SEQ ID NO: 1 , comprising one or more of the following substitutions S49T, K65R, L711, K278R, P308V, S356T, P362A, or M389P, wherein numbering is obtained when aligned with SEQ ID NO: 1 , wherein the variants have at east 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity to mature part of any of SEQ ID NO: 1.

[0176] In an even more particular embodiment the mannanase variants may be variants of SEQ ID NO: 1 , comprising one or more of the following substitutions K65R, K278R, or S356T, such as K65R+K278R or K278R+S356T, wherein numbering is obtained when aligned with SEQ ID NO: 1 , wherein the variants have at east 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity to mature part of any of SEQ ID NO: 1.

[0177] In a particular embodiment the mannanase variants comprise substitutions selected from the group consisting of S356T+P362A+M389P, K65R+L71 I+K278R, K278R+P308V+S356T, and S49T+K65R+K278R, wherein numbering is obtained when aligned with SEQ ID NO: 1 , wherein the variants have at east 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity to mature part of any of SEQ ID NO: 1.

[0178] 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.

[0179] 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). Ammo 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, AlaA / al, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, LeuA / al, Ala / Glu, and Asp / Gly.

[0180] 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.

[0181] 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 mannanase 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 polypep- tides / 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.

[0182] 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 NO:1.

[0183] Preparation of variants

[0184] The present invention also relates to methods for obtaining a variant having mannase activity with the substitutions and deletions disclosed herein. In one embodiment the method comprises: (a) introducing into a parent mannanase a deletion or substitution at one or more positions of the polypeptide, wherein the variant has mannanase activity; and (b) recovering the variant.

[0185] In particular the invention relates to methods for obtaining a variant having mannase activity disclosed in the paragraph Variants above. 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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).

[0192] 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. 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.

[0193] Polynucleotides

[0194] The present invention also relates to polynucleotides encoding a mannanase 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.

[0195] Nucleic Acid Constructs

[0196] 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.

[0197] 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.

[0198] Promoters

[0199] 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.

[0200] 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.

[0201] Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Tnchoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0202] Terminators

[0203] 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.

[0204] 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).

[0205] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology. mRNA Stabilizers

[0206] 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.

[0207] 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. Bacteriol. 177: 3465- 3471).

[0208] 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.

[0209] Leader Sequences

[0210] 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.

[0211] 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.

[0212] Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. 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.

[0213] Signal Peptides

[0214] 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.

[0215] 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.

[0216] In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. Factors

[0217] 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.

[0218] Expression vectors

[0219] 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.

[0220] 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.

[0221] 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. 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.

[0222] 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).

[0223] 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.

[0224] 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.

[0225] Host cells

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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. 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0235] Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave- mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470- 1474, Christensen etal., 1988, Bio / TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

[0236] The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Fili basidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0237] For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0238] In an aspect, the host cell is isolated, preferably the host cell is purified.

[0239] Methods of production

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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).

[0245] In an alternative aspect, the variant is not recovered.

[0246] Detergent Compositions

[0247] In one embodiment, the invention is directed to detergent compositions comprising a mannanse in combination with one or more additional cleaning composition components. In one embodiment, the detergent composition comprises a polypeptide having mannanase activity with an amino acid sequence having 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: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22. The detergent composition preferably also comprises a GH5 mannanase, wherein the GH5 mannanase has at least 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to a GH5 mannanase selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , and SEQ ID NO: 12.

[0248] 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. Formulation of Detergent Products

[0249] 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.

[0250] 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.

[0251] 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.

[0252] Laundry Soap Bars

[0253] The mannanase 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.

[0254] 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.

[0255] 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.

[0256] 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, mannanase, 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 mannanase 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.

[0257] Mannanse Granules

[0258] 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.

[0259] 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).

[0260] In an embodiment, the core comprises a polypeptide of the present invention.

[0261] 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.

[0262] The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.

[0263] 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.

[0264] The core may include an inert particle with the polypeptide absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

[0265] The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.

[0266] 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).

[0267] 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%.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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. 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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 (Na2HPO47H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0277] Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.,

[0282] (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).

[0283] (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.

[0284] (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.

[0285] (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). (e) Prilled products, wherein a polypeptide-contaming 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.

[0286] (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.

[0287] (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.

[0288] (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.

[0289] (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.

[0290] 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.

[0291] 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.

[0292] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.

[0293] 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 amylase, beta-glucanase, cellulase, xyloglucanase, DNase, hexosaminidase, mannanase, protesase, lipase, cutinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations thereof).

[0294] Liquid Formulations

[0295] 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).

[0296] 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.

[0297] 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.

[0298] In another embodiment, the invention relates to liquid formulations comprising:

[0299] (A) 0.001-25% w / w of a polypeptide of the present invention;

[0300] (B) 20-80% w / w of polyol;

[0301] (C) optionally 0.001-2% w / w preservative; and

[0302] (D) water.

[0303] In another embodiment, the invention relates to liquid formulations comprising:

[0304] (A) 0.001-25% w / w of a polypeptide of the present invention;

[0305] (B) 0.001-2% w / w preservative;

[0306] (C) optionally 20-80% w / w of polyol; and

[0307] (D) water. 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.

[0308] 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).

[0309] 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.

[0310] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as such as amylase, beta-glucanase, cellulase, xyloglucanase, DNase, Hexosaminidase, mannanase, protesase, lipase, cutinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations thereof. Detergent Ingredients

[0311] 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.

[0312] 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.

[0313] Surfactants

[0314] 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 non-ionic 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.

[0315] 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.

[0316] 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. 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.

[0317] 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.

[0318] 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.

[0319] Hydrotropes

[0320] 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.

[0321] 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. Builders and Co-Builders

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] Fabric Hueinq Agents

[0328] 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.

[0329] Dye Transfer Inhibiting Agents

[0330] 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.

[0331] Fluorescent Whitening Agent

[0332] 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-cf][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.

[0333] 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%.

[0334] Soil Release Polymers

[0335] 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).

[0336] Anti-redeposition Agents

[0337] 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.

[0338] Rheology Modifiers

[0339] 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.

[0340] 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 liguid detergents and / or structure elasticizing agents.

[0341] Additional Enzymes

[0342] The detergent additive as well as the detergent composition may contain one or more additional enzymes (such as amylase, beta-glucanase, cellulase, xyloglucanase, DNase, hexosaminidase, mannanase, protesase, lipase, cutinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations thereof).

[0343] 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. Beta-qlucanases

[0344] Licheninases (or lichenases) (EC 3.2.1.73) hydrolyze (1 ,4)-beta-D-glucosidic linkages in 1 ,3-1 , 4- beta-D-glucan 4-glucanohydrolase. Licheninase may also be named beta-glucanase and belongs to GH16 family. Licheninase from Bacillus agaradhaerens is disclosed in WO 2017 / 097866, and the combined use of licheninase and DNase is described in WO 2021 / 148364. Commercially available licheninase is Bonito® and Lift Intent® (both from Novozymes A / S).

[0345] Another subgroup of beta-glucanases is laminarinases, which can be classified as endo-1 ,3-beta- glucanases (EC 3.2.1.6 and EC 3.2.1.39) or exo-1 ,3-beta-glucanases (EC 3.2.1.58). Laminarinases can be used to catalyze the hydrolysis of the beta-1 , 3-glucosidic bonds, or beta- 1 ,4-glucosidic bonds when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is substituted at C3 to release glucose or oligosaccharides. These enzymes can act on laminarin, lichenin and cereal beta-D-glucans. WO 2020 / 201403 discloses polypeptides having laminarinase activity and the use thereof in cleaning and detergent compositions. The laminarinase may be obtained from Bacillus, e.g., Bacillus sp., Paenibacillus, e.g., Paenibacillus elgii or Paenibacillus sp., Thermobacillus, e.g., Thermobacillus sp. or from Cohnella, e.g., Cohnella sp.

[0346] Cellulases and xyloqlucanases

[0347] The terms “cellulase” and “xyloglucanase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material or xyloglucan, respectively. Some glycoside hydrolases may be capable of breaking down cellulose as well as xyloglucan and can accordingly be characterized as either cellulase or xyloglucanase. Structurally, xyloglucans consist of a cellulose-like beta-1 , 4- linked glucose backbone which is frequently substituted with various side chains.

[0348] Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH6, GH7, GH8, G9, GH12, GH44, and GH45. The cellulase may for example be an endoglucanase which cleaves internal beta-1 , 4-glycosidic bonds within the cellulose chain, an exoglucanase (cellobiohydrolase) which acts on the non-reducing ends of cellulose chains, releasing cellobiose units from the chain, and a beta-glucosidase which hydrolyzes cellobiose and other short cello- oligosaccharides into glucose monomers.

[0349] Suitable cellulases and xyloglucanases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. Suitable cellulases or xyloglucanases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., T. reesei or T. viride. Other suitable cellulases are from Thielavia, e.g., Thielavia terrestris as described in WO 96 / 29397 or the fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89 / 09259 and WO 91 / 17244. Further suitable cellulases are disclosed in WO 02 / 099091 , WO 2004 / 053039, in particular SEQ ID NO: 2, and WO 2020 / 074498, in particular SEQ ID NO: 13.

[0350] Exemplary xyloglucanases include xyloglucanases obtained from Paenibacillus, in particular Paenibacillus polymyxa as disclosed in WO 01 / 62903, and WO 2022 / 043321.

[0351] Cellulases with improved stability against protease degradation in detergents are disclosed in WO 2019 / 201783, WO 2019 / 201785, WO 2020 / 208056, WO 2021 / 204838 and WO 2023 / 061928.

[0352] DNases (deoxyribonucleases)

[0353] The term “DNase” means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. DNase polypeptides have been found to be useful for deep cleaning of microbial biofilm that may be present on surfaces such as textiles or dishware or other hard surfaces, and which consists of a matrix of extracellular polymeric substance (EPS) composed of extracellular DNA, proteins, and polysaccharides. The DNase polypeptide is typically a microbial enzyme, preferably of fungal or bacterial origin, or a genetically engineered variant of a microbial DNase.

[0354] Suitable bacterial DNases may, for example, be obtained from species of Bacillus and related genera (cf. Patel and Gupta, int. J. Syst. Evol. Microbiol. 2020; 70:406-438, who proposed six new Bacillaceae genera from species formerly classified as belonging to the genus Bacillus), e.g. from Bacillus, Cytobacillus, Metabacillus, Alkalihalobacillus, Rossellomorea or Mesobacillus. Examples of species from which DNases may be obtained include Bacillus licheniformis, Bacillus subtilis, Bacillus horikoshii, Cytobacillus horneckiae, Metabacillus indicus, Alkalihalobacillus algicola, Rossellomorea vietnamensis, Alkalihalobacillus hwajinpoensis, Metabacillus indicus, Mesobacillus campisalis, Bacillus idriensis, Bacillus algicola, Bacillus marisflavi and Bacillus luciferensis. Preferred bacterial DNases include those obtained from Metabacillus indicus (previously known as Bacillus cibi) and variants thereof.

[0355] DNases may also be obtained from a fungal species. Examples of preferred fungal DNases are those obtained from Aspergillus, for example from Aspergillus oryzae, from Trichoderma, for example from Trichoderma harzianum, from Vibressa, for example from Vibressea flavovirens, from Morchella, for example from Morchella costata, and from Rhizoctonia, for example from Rhizoctonia solani, as well as variants thereof. Preferred fungal DNases include those obtained from Aspergillus oryzae and variants thereof.

[0356] Suitable DNases, DNase variants, and use thereof in detergent compositions are disclosed, for example, in WO 2014 / 087011 , WO 2015 / 155350, WO 2015 / 155351 , WO 2017 / 060475, WO 2017 / 060493, WO 2017 / 060505, WO 2017 / 064269, WO 2018 / 011277, WO 2018 / 177203, WO 2018 / 177936, WO 2018 / 177938, WO 2019 / 081724, WO 2019 / 081721 , WO 2021 / 130167, WO 2022 / 194668, WO 2022 / 194673 and WO 2023 / 165950.

[0357] Hexosaminidases (dispersins)

[0358] Detergent compositions comprising a DNase of the invention may also include one or more hexosaminidases. The term hexosaminidase includes “dispersin’’ and the abbreviation “Dsp”, which means a polypeptide having hexosaminidase activity, EC 3.2.1.-, that catalyzes the hydrolysis of P-1 ,6-glycosidic linkages of N-acetyl-glucosamine polymers found e.g. in biofilm. The term hexosaminidase includes polypeptides having N-acetylglucosaminidase activity and p-N-acetylglu- cosaminidase activity.

[0359] A polypeptide having hexosaminidase activity may be obtained from microorganisms of any genus, in particular from bacteria or fungi. Preferably the hexosaminidase, e.g. a dispersin, is obtained from Terribacillus, Curtobacterium, Aggregatibacter, Haemophilus or Actinobacillus, preferably Terribacillus. The hexosaminidase may also be a variant of a polypeptide obtained from any of these or other organisms.

[0360] Suitable hexosaminidases include those disclosed in WO 2017 / 186936, WO 2017 / 186937, WO

[0361] 2017 / 186943, WO 2017 / 207770, WO 2018 / 184873, WO 2019 / 086520, WO 2019 / 086528, WO

[0362] 2019 / 086530, WO 2019 / 086532, WO 2019 / 086521 , WO 2019 / 086526, WO 2020 / 002604, WO

[0363] 2020 / 002608, WO 2020 / 007863, WO 2020 / 007875, WO 2020 / 008024, WO 2020 / 070063, WO

[0364] 2020 / 070249, WO 2020 / 088957, WO 2020 / 088958, WO 2020 / 207944 and WO 2023 / 194204.

[0365] Mannanases

[0366] Endo-1 , 4-mannanases (EC 3.2.1.78) are involved in the random hydrolysis of (1^4)-beta-D- mannosidic linkages in mannans, galactomannans, glucomannans and galactoglucomanns and may belong to the glycosyl hydrolase family 5 or glycosyl hydrolase family 26. The mannanase may be of bacterial or fungal origin. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, Paenibacillus illinoisensis, Paeni- bacillus woosongensis, or H. insolens. Suitable GH5 mannanases are described in, e.g., WO 2018 / 206300, WO 2018 / 206302, WO 2018 / 185367, WO 2018 / 184767, WO 2018 / 220273, WO 2018 / 220274, WO 2020 / 207882, WO 2021 / 058452, WO 2017 / 079751 ; suitable GH26 mannanases are described in WO 2019 / 068713, WO 2019 / 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

[0375] Kao.

[0376] Lipoxygenase

[0377] Lipoxygenase (EC 1.13.11.12, linoleate: oxidoreductase, LOX) is an enzyme that catalyzes the oxygenation of polyunsaturated fatty acids such as linoleic acid, linolenic acid and arachidonic acid, which contain a cis,cis-1 ,4-pentadiene unit and produces hydroperoxides of these fatty acids. Lipoxygenase may be able to oxidize substrates containing a cis-cis-pentadienyl moiety and thus act on polyunsaturated fatty acids such as linoleic acid (18 carbon atoms, 2 double bonds), linolenic acid (18:3), arachidonic acid (20:4), eicosapentaenoic acid (EPA, 20:5) and / or docosahexaenoic acid (DHA, 22:6).

[0378] Lipoxygenase may be a 9-lipoxygenase with the ability to oxidize the double bond between carbon atoms 9 and 10 in linoleic acid and linolenic acid, or it may be a 13-lipoxygenase with the ability to oxidize the double bond between carbon atoms 12 and 13 in linoleic acid and linolenic acid, or it may be a 9 / 13-lipoxygenase with the ability to oxidize the double bond both between carbon atoms 9 and 10 and between carbon atoms 12 and 13. Suitable lipoxygenases are described in e.g. WO 2022 / 090320

[0379] Lipases and Cutinases

[0380] 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).

[0381] 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,

[0382] W096 / 00292, W097 / 04079, W097 / 07202, WO00 / 34450, WO00 / 60063, W001 / 92502,

[0383] W007 / 87508 and WO09 / 109500.

[0384] 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).

[0385] 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).

[0386] Amylases

[0387] 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.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] Other examples are amylase variants such as those described in WO2011 / 098531 , WO2013 / 001078 and WO2013 / 001087.

[0392] 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).

[0393] Peroxidases / Oxidases

[0394] 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).

[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 (WO 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.

[0400] Pectate Lyases

[0401] Pectate lyases are hemicellulotic enzymes that catalyze the cleavage of alpha-1 , 4-D- galacturonan (i.e. , homogalacturonan or polygalacturonic 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 and may be isolated from, e.g., Bacillus subtilis. Pectate lyases and their use is disclosed in WO 02 / 092741 , WO 03 / 095638, and WO 2018 / 007435.

[0402] Xanthanases

[0403] Xanthan gum is a polysaccharide consisting of different sugars which are connected by several different bonds, such as beta-D-mannosyl-beta-D-1 ,4-glucuronosyl bonds and beta-D-glucosyl- beta-D-1 ,4-glucosyl bonds. The degradation of xanthan gum requires the presence of both xanthan lyase and xanthan endoglucanase, collectively referred to as xanthanase.

[0404] Xanthanase is disclosed in WO 2013 / 167581 , whereas WO 2018 / 037061 and WO 2019 / 038057 disclose xanthan lyase variants, and WO 2018 / 037062 and WO 2019 / 038058 disclose xanthan endoglucanase variants.

[0405] Uses

[0406] The mannanases variants of the invention may be used for preventing, reducing or removing complex food stains or cosmetic stains from an item.

[0407] Washing method

[0408] The detergent compositions of the present invention are ideally suited for use in laundry applications. The mannanase (as formulated product) may be present in the detergent in a concentration from 0.01-10 wt%, such as in the range of 0.01-5 wt%, such as in the range of 0.0.01- 3 wt%, such as in the range of 0.02-2 wt%, or in the range of 0.02-1 wt%, or even in the range of 0.05-0.2 wt%.

[0409] Accordingly, the present invention includes a method for laundering a fabric. The method comprises the steps of contacting a fabric to be laundered with a cleaning laundry solution comprising the detergent composition according to the invention. The fabric may comprise any fabric capable of being laundered in normal consumer use conditions. The solution preferably has a pH of from about 5.5 to about 9. The compositions may be employed at concentrations of from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5°C to about 90°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C and about 90°C. The water to fabric ratio is typically from about 1 :1 to about 30:1.

[0410] In particular embodiments, the washing method is conducted at a pH of from about 5.0 to about 11.5, or in alternative embodiments, even from about 6 to about 10.5, such as about 5 to about 11 , about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11 , about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5. to about 7, about 6 to about 11 , about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about

[0411] 7, about 6.5 to about 11 , about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11 , about 7 to about 10, about 7 to about 9, or about 7 to about

[0412] 8, preferably about 5.5 to about 9, and more preferably about 6 to about 8.

[0413] In particular embodiments, the washing method is conducted at a degree of hardness of from about 0°dH to about 30°dH, such as about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21 °dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, about 30°dH. Under typical European wash conditions, the degree of hardness is about 15°dH, under typical US wash conditions about 6°dH, and under typical Asian wash conditions, about 3°dH.

[0414] The present invention relates to a method of cleaning a fabric, a dishware or hard surface with a detergent composition comprising a mannanase variants of the invention.

[0415] A preferred embodiment concerns a method of cleaning, said method comprising the steps of: contacting an object with a cleaning composition comprising a mannanase variants of the invention under conditions suitable for cleaning said object. In a preferred embodiment the cleaning composition is a detergent composition and the process is a laundry or a dish wash process.

[0416] Still another embodiment relates to a method for removing stains from fabric which comprises contacting said a fabric with a composition comprising a mannanase variants of the invention under conditions suitable for cleaning said object.

[0417] Low temperature uses

[0418] One embodiment of the invention concerns a method of doing laundry, dish wash or industrial cleaning comprising contacting a surface to be cleaned with a mannanase variants of the invention, and wherein said laundry, dish wash, industrial or institutional cleaning is performed at a temperature of about 40°C or below. One embodiment of the invention relates to the use of a mannanase in laundry, dish wash or a cleaning process wherein the temperature in laundry, dish wash, industrial cleaning is about 40°C or below

[0419] In another embodiment, the invention concerns the use of a mannanase according to the invention in a protein removing process, wherein the temperature in the protein removing process is about 40°C or below.

[0420] In each of the above-identified methods and uses, the wash temperature is about 40°C or below, such as about 39°C or below, such as about 38°C or below, such as about 37°C or below, such as about 36°C or below, such as about 35°C or below, such as about 34°C or below, such as about 33°C or below, such as about 32°C or below, such as about 31°C or below, such as about 30°C or below, such as about 29°C or below, such as about 28°C or below, such as about 27°C or below, such as about 26°C or below, such as about 25°C or below, such as about 24°C or below, such as about 23°C or below, such as about 22°C or below, such as about 21°C or below, such as about 20°C or below, such as about 19°C or below, such as about 18°C or below, such as about 17°C or below, such as about 16°C or below, such as about 15°C or below, such as about 14°C or below, such as about 13°C or below, such as about 12°C or below, such as about 11°C or below, such as about 10°C or below, such as about 9°C or below, such as about 8°C or below, such as about 7°C or below, such as about 6°C or below, such as about 5°C or below, such as about 4°C or below, such as about 3°C or below, such as about 2°C or below, such as about 1°C or below.

[0421] In another preferred embodiment, the wash temperature is in the range of about 5-40°C, such as about 5-30°C, about 5-20°C, about 5-10°C, about 10-40°C, about 10-30°C, about 10-20°C, about 15-40°C, about 15-30°C, about 15-20°C, about 20-40°C, about 20-30°C, about 25-40°C, about 25-30°C, or about 30-40°C. In particular preferred embodiments the wash temperature is about 20°C, about 30°C, or about 40°C.

[0422] Use of mannanases of the invention in preventing, reducing or removing a biofilm

[0423] Biofilm can develop on textile when microorganisms are present on an item and stick together on the item. Some microorganisms tend to adhere to the surface of items such as textiles. Some microorganisms adhere to such surfaces and form a biofilm on the surface. The biofilm may be sticky and the adhered microorganisms and / or the biofilm may be difficult to remove. Furthermore, the biofilm adhere soil due to the sticky nature of the biofilm. The commercial laundry detergent compositions available on the marked do not remove such adhered microorganisms or biofilm.

[0424] Embodiments of the Invention

[0425] The invention is further defined in the following embodiments:

[0426] 1 . A GH26 mannanase, characterized in that the mannanase polypeptide 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 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, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22., and that the Mannanase polypeptide has mannanase activity.

[0427] 2. The mannanase polypeptide according to embodiment 1 , wherein the mannanase polypeptide has a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1 .0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22, wherein the three dimensional structure is calculated by Alphafold.

[0428] 3. The mannanase polypeptide of any of embodiments 1 or 2, wherein the mannanase polypeptide comprises one or more of the following substitutions V17I, S49T, K65R, L71 I, D143E, S168T, V272L, K278R, P308V, S356T, P362A, M389P, or E390D when compared to SEQ ID NO: 1.

[0429] 4. The mannanase polypeptide of any of embodiments 1 , 2 or 3, wherein the mannanase polypeptide comprises one or more of the following substitutions K65R, K278R,or S356T, such as K65R+K278R and K278R+S356T, when compared to SEQ ID NO: 1

[0430] 5. A detergent composition comprising at least one detergent adjunct ingredient, one or more of the mannanase polypeptides of any of embodiments 1 to 4 and optionally one or more additional enzymes.

[0431] 6. The detergent composition according to embodiment 5, wherein the one or more additional enzymes is selected from the group consisting of such as amylase, beta-glucanase, cellulase, xyloglucanase, DNase, hexosaminidase, mannanase, protesase, lipase, cutinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations thereof. 7. The detergent composition according to any of embodiments 5 or 6 comprising a GH5 mannanase, wherein the GH5 mannanase has at least 75%, 80%, 85%, 90%, 91%, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to a GH5 mannanase selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:

[0432] 10, SEQ ID NO: 11 , and SEQ ID NO: 12.

[0433] 8. Use of the polypeptide according to any of embodiments 1 to 4 or the detergent composition of any of embodiment 5 to 7 in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.

[0434] 9. A method of cleaning an item, comprising exposing the item to a wash liquor comprising the mannanase of any of embodiments 1 to 4 or to the detergent composition of any of embodiments 5 to 7, e.g. wherein the item is a textile or a hard surface.

[0435] 10. The use according to embodiment 8 or the method of embodiment 9, wherein the wash liquour comprises from about 0.2 g detergent composition of any of embodiments 5 to7 per liter wash liquour to about about 5 g detergent composition of any of embodiments 5 to7 per liter wash liquour.

[0436] 11 . A polynucleotide encoding the polypeptide of any of embodiments 1 to 4.

[0437] 12. A nucleic acid construct or expression vector comprising the polynucleotide of embodiment

[0438] 11.

[0439] 13. A recombinant host cell transformed with the polynucleotide of embodiment 12.

[0440] 14. A method of producing a polypeptide of any of embodiments 1 to 4, comprising a. Cultivating the host cell of embodiment 13 under conditions suitable for expression of the mannanase; and b. Recovering the mannanase.

[0441] EXAMPLES

[0442] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0443] Materials

[0444] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0445] Commercial raw materials were used, and dosage of raw material was adjusted according to the purity of the individual ingredidents to achieve the listed active content

[0446] The concentration of the mannanase (as AEP) in the wash liquor is typically in the range of 0.005- 20 ppm (mg / L) enzyme protein, such as in the range of 0.01-15 ppm, in the range of 0.01-10 ppm, in the range of 0.01-5 ppm, in the range of 0.01-2 ppm, in the range of 0.01-1 ppm.

[0447] For the test of wash performance standard textile pieces with guar gum was used:

[0448] Table 1 : Test swatch

[0449] The above commercial test materials are available from Center for Testmaterials BV, Stoomlog- gerweg 11 , 3133 KT Vlaardingen, the Netherlands.

[0450] Example 1 : Reducing End Assay for Determination of Mannanase Activity

[0451] For estimating the mannose yield after substrate hydrolysis, a reducing end assay developed by Lever (1972), Anal. Biochem. 47: 273-279, is used. The assay is based on 4-hydroxybenzoic acid hydrazide, which under alkaline conditions reacts with the reducing ends of saccharides. The product is a strong yellow anion, which absorbs at 410 nm.

[0452] Method

[0453] 4-Hydroxybenzhydrazide (PAHBAH) (Sigma, H9882) is diluted in PAHBAH buffer to a concentration of 15 mg / ml. PAHBAH buffer contained: 50 g / L K-Na-tartrate (Merck, 1.08087) and 20 g / L sodium hydroxide(Sigma, S8045).This PAHBAH mix was made just before usage.

[0454] 70 pl PAHBAH mix and MiliQ water are mixed in a 96 well PCR plate (Thermo Scientific). Samples from hydrolysis experiment were added. Samples and MiliQ always reached the total volume of 150 pl, but the dilution of the sample differed. The plate is sealed with Adhesive PCR Sealing Foil Sheets (Thermo Scientific). Plates are incubated at 95 °C for 10 min, cooled down and kept at 10 °C for 1 min in PTC-200 Thermal Cycler (MJ Research). 100 pl sample is transferred to a 96 well microtiter plate, flat bottomed (NuncTM) and color development measured at 405 nm on a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices). Results are compared to mannose standards, that had undergone the same treatment and dilution as the samples to which they were compared.

[0455] Example 2: Variant generation by site-directed mutagenesis

[0456] Site-directed variants of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , or SEQ ID NO: 5 are generated by SOE (Splicing by Overlap Extension) simultaneously adding mutation(s), expression regulatory elements, and bacillus genome homology regions to the PCR product for site-directed integration. Bacillus subtilis are transformed with the PCR product; correct gene sequences are confirmed by NGS. Example 3: Evauation of washperformance in AMSA for laundry

[0457] Experiments were performed to assess the wash performance of mannanases and mannanase variants in laundry detergent compositions. The mannanases and their variants were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA, the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the test sample to be washed (a textile for testing a laundry detergent, or a melamine tile for testing a dishwashing detergent) against the slot openings. During the washing time, the plate, test solutions, test sample and lid are vigorously shaken to bring the test solution into contact with the soiled test sample and to apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 02 / 42740 especially the paragraph "Special method embodiments" at page 23-24.

[0458] The performance of mannanases and mannanase variants were in these examples measured as brightness of coloured textile samples soiled with a galactomannan which can be degraded by the enzymes. Brightness can be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained, the intensity of the reflected light is lower than that of a clean textile. Therefore, the intensity of the reflected light can be used to measure wash performance. Colour measurements are made with a professional flatbed scanner (Epson Expression 10000XL), which is used to capture an image of the washed textile samples. To extract a value for the light intensity from the scanned images, a specially designed software application is used (Novozymes Color Vector Analyzer). The program retrieves the values from the image and converts them into values for red, green and blue (RGB). The intensity value (I nt) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector.

[0459] The wash performance of the mannanases was evaluated using two distinct model detergents: one representing a liquid detergent and the other a powder detergent. The composition of the detergents is shown in the two table below and are is a model detergents without enzymes added.

[0460] Table 2: Composition of Model detergent 1 (powder)

[0461] Table 3: Composition of Model detergent 2 (liquid) Model detergent 2 was produced by mixing water, propylene glycol, triethanolamine and sodium hydroxide. Then topped palm kernel fatty acid, Na-LAS, SLES and AEO was added in portions and stirred for few hours. Trisodium citrate dihydrate, DTPMP Na7 and 2-phenoxyethanol was added and pH adjusted within target after overnight stirring at room temperature. For both model detergents, commercial raw materials were used, and dosage of raw material was adjusted according to the purity of the individual ingredients to achieve the specified active content

[0462] Example 4: Test of WT mannanases in powder detergent

[0463] Wash performance of the mannanases was tested in Model detergent 1 representing a powder detergent using the conditions listed below. Supernatants of culture fermentations of strains were diluted 800 times in 10 mM succinic Acid. 2 mM CaCI2, 0.01% Brij L23, pH 6.5. 20 pL of the diluted supernatants were added to the reaction mixture resulting in a total volume of 160 pL corresponding to a dose of supernatants of 1.25 g supernant / L.

[0464] Table 4: Conditions for AMSA wash

[0465] The intensity value (Int) was determined on textile air dried in the dark overnight and the results shown below.

[0466] Table 5: Wash performance of WT mannanase The tested newly identified wild type mannanases clearly showed performance in a powder detergent. Example 5: Test of mannanase variants in powder and liquid detergent

[0467] Wash performance of Mannanase variants was tested in Model detergent 1 representing a powder detergent and Model detergent 2 representing a liquid detergent. Supernatants of culture fermentations of strains were diluted 800 times in 10 mM succinic Acid. 2 mM CaCI2, 0.01% Brij L23, pH 6.5. 20 pL of the diluted supernatants were added to the reaction mixture resulting in a total volume of 160 pL corresponding to a dose of supernatants of 1.25 g supernant / L.

[0468] Table 6: Conditions for AMSA wash The intensity value (Int) was determined on textile air dried in the dark overnight and the results for model detergent 1 is shown in table 7 and model detergent 2 in table 8.

[0469] Table 7: Wash performance of Mannanase variants in powder detergent (Model detergent 1)

[0470] Table 8: Wash performance of Mannanase variants in liquid detergent (Model detergent 2)

[0471] The tested mannanase variants clearly showed performance in both a liquid and a powder detergent.

Claims

CLAIMS1 . A GH26 mannanase, characterized in that the mannanase polypeptide 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 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, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22., and that the Mannanase polypeptide has mannanase activity.

2. The mannanase polypeptide according to claim 1 , wherein the mannanase polypeptide has a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 22, wherein the three dimensional structure is calculated by Alphafold.

3. The mannanase polypeptide of any of claims 1 or 2, wherein the mannanase polypeptide comprises one or more of the following substitutions V17I, S49T, I57R, K65R, L711, D143E, S168T, V272L, K278R, P308V, S356T, P362A, M389P, or E390D wherein numbering is according to SEQ ID NO: 1.

4. The mannanase polypeptide of any of claims 1 to 3, wherein the mannanase polypeptide comprises one or more of the following substitutions K65R, K278R, or S356T, such as K65R+K278R and K278R+S356T, wherein numbering is according to SEQ ID NO:

15. A detergent composition comprising at least one detergent adjunct ingredient, one or more of the mannanase polypeptides 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 consisting of such as amylase, beta-glucanase, cellulase, xyloglucanase, DNase, hexosaminidase, mannanase, protesase, lipase, cutinase, peroxidase, lipoxygenase, oxidase, and pectate lyase, and combinations thereof.

7. The detergent composition according to any of claims 5 or 6 comprising a GH5 mannanase, wherein the GH5 mannanase has at least 75%, 80%, 85%, 90%, 91 %, such as 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to a GH5 mannanase selected from63the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

8. Use of the polypeptide according to any of claims 1 to 4 or the detergent composition of any of claim 5 to 7 in a cleaning process, such as laundry or hard surface cleaning such as dish- washing.

9. A method of cleaning an item, comprising exposing the item to a wash liquor comprising the mannanase of any of claims 1 to 4 or to the detergent composition of any of claims 5 to 7, e.g. wherein the item is a textile or a hard surface.

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

11. A polynucleotide encoding the polypeptide of any of claims 1 to 4.

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

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

14. A method of producing a polypeptide of any of claims 1 to 4, comprising a. Cultivating the host cell of claim 13 under conditions suitable for expression of the mannanase; and b. Recovering the mannanase.

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