Detergent compositions and uses thereof

Abstract

The present invention relates to polypeptides having aminohexosidase activity and polynucleotides encoding these polypeptides. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising these polynucleotides, as well as methods of producing and using the polypeptides.

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0004] This invention relates to a polypeptide with aminohexosidase activity, a polynucleotide encoding the polypeptide, and a catalytic domain (GH20) belonging to the glycoside hydrolase family 20. www.cazy.org The present invention further relates to compositions comprising such polypeptides, particularly cleaning compositions, the use of polypeptides having aminohexosidase activity in cleaning processes and / or deep cleaning, and methods for deep cleaning. The present invention further relates to nucleic acid constructs, vectors, and host cells comprising these polynucleotides, along with methods for producing and using these polypeptides and catalytic domains. Background Technology

[0006] Polypeptides with aminohexosidase activity include dispersins, such as dispersin B (DspB), which is described as a β-N-acetylglucosidase belonging to the glycoside hydrolase 20 family. WO 04061117 A2 (Kane Biotech INC) describes the use of compositions containing DspB for reducing and preventing biofilms caused by bacteria that produce poly-N-acetylglucosamine, and Kane et al. describe the use of compositions containing dispersins for reducing biofilms on medical devices and for wound care. Biofilms can also be present on clothing items, such as fabrics, and other hard surfaces, such as dishwashing utensils, dishwashers, and washing machines, where they can cause foul odors that are difficult to remove even after washing. Application WO 9850512 (Procter & Gamble) discloses laundry detergent or cleaning products containing one or more aminohexosidases. This invention provides enzymes suitable for detergents and for deep cleaning of items such as laundry detergents and cleaning processes. Summary of the Invention

[0007] This invention provides a polypeptide belonging to the Terribacillus clade, an aminohexosidase derived from the glycoside hydrolase 20 family. This Terribacillus clade is shown in... Figure 1 and 3The polypeptides of the present invention possess aminohexosidase activity. The present invention further provides detergent compositions comprising polypeptides possessing aminohexosidase activity, and the use of polypeptides possessing aminohexosidase activity in cleaning processes. The polypeptides of the present invention possessing aminohexosidase activity have beneficial properties, such as removing and / or reducing biofilm-related components such as EPS and / or PNAG during cleaning processes. Applications include laundry and dishwashing. The polypeptides of the present invention belong to the *Bacillus terreus* clade and are homologous sequences of a specific clade of the glycoside hydrolase 20 family. Therefore, the present invention relates to polypeptides possessing aminohexosidase activity selected from the group consisting of:

[0008] (a) A polypeptide having at least 80% sequence identity with a mature polypeptide of SEQ ID NO 2, 4, 6, 13 or 15;

[0009] (b) A variant of the mature polypeptide of SEQ ID NO 2, 4, 6, 13 or 15, wherein the variant contains substitution, deletion and / or insertion at one or more (e.g., several) positions; and

[0010] (c) Fragments of the polypeptide of (a) or (b) that have aminohexosidase activity.

[0011] This invention also relates to enzymes belonging to the glycoside hydrolase family 20 (GH20, www.cazy.org The present invention relates to a polypeptide having a catalytic domain, and having at least 60% sequence identity with amino acids 1 to 324 of SEQ ID NO 2, at least 60% sequence identity with amino acids 1 to 324 of SEQ ID NO 4, at least 60% sequence identity with amino acids 1 to 324 of SEQ ID NO 6, at least 60% sequence identity with amino acids 1 to 324 of SEQ ID NO 13, or at least 60% sequence identity with amino acids 1 to 324 of SEQ ID NO 15. The present invention also relates to cleaning methods using the polypeptides of the present invention and their use in cleaning processes. The present invention further relates to a method for cleaning or washing articles, the method comprising the following steps:

[0012] a. Exposing an article to a detergent solution containing a polypeptide having aminohexosidase activity or a detergent composition containing such polypeptides, the polypeptide being selected from the group consisting of polypeptides of SEQ ID NO 7, 8, 9, 10 and 11 or polypeptides having at least 60% sequence identity with them.

[0013] b. Complete at least one washing cycle; and

[0014] c. Optional: Rinse the item.

[0015] The item in question is a textile.

[0016] Additionally, protection is claimed for the use of a polypeptide with aminohexosidase activity in deep cleaning articles, the polypeptide being selected from the group consisting of polypeptides of SEQ ID NO 7, 8, 9, 10, 11 or polypeptides having at least 60% sequence identity.

[0017] The present invention further relates to a composition comprising at least 0.001 ppm of a polypeptide having aminohexosidase activity and at least one adjuvant component, wherein the polypeptide is selected from the group consisting of polypeptides having at least 60% sequence identity with the mature polypeptides shown in SEQ ID NO 7, 8, 9, 10 and 11. The present invention further relates to the use of the compositions of the present invention for deep cleaning of articles, wherein the articles are textiles.

[0018] The present invention further relates to a method for washing articles, the method comprising: a) exposing the articles to a detergent solution comprising a polypeptide selected from the group consisting of polypeptides shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, or a polypeptide having at least 60% sequence identity with it, or exposing the articles to a detergent composition according to the invention; b) completing at least one washing cycle; and optionally rinsing the articles, wherein the articles are textiles.

[0019] This invention further relates to the use of the polypeptides or compositions of this invention.

[0020] (i) Used to prevent, reduce or remove the stickiness of the article;

[0021] (ii) Used for pre-treating stains on articles;

[0022] (iii) Used to prevent, reduce or remove the redeposition of dirt during a washing cycle;

[0023] (iv) Used to prevent, reduce or remove dirt from adhering to the article;

[0024] (v) Used to maintain or improve the whiteness of the item;

[0025] (vi) Used to prevent, reduce or eliminate the odor of the item.

[0026] The item in question is a textile. Attached Figure Description

[0027] Figure 1 A phylogenetic tree of the Bacillus terreus clade is shown.

[0028] Figure 2 This is a comparison of the polypeptides of the present invention.

[0029] Figure 3 This is a phylogenetic tree of the polypeptides of this invention.

[0030] Sequence review of Bacillus terrestrialus clades

[0031] SEQ ID NO 1 is the DNA encoding a full-length polypeptide from *Terribacillus saccharophilus*.

[0032] SEQ ID NO 2 is a polypeptide derived from SEQ ID NO 1.

[0033] SEQ ID NO 3 is the DNA encoding a full-length polypeptide from Terribacillus goriensis.

[0034] SEQ ID NO 4 is a polypeptide derived from SEQ ID NO 3.

[0035] SEQ ID NO 5 is the DNA encoding a full-length polypeptide from Bacillus saccharophilus.

[0036] SEQ ID NO 6 is a polypeptide derived from SEQ ID NO 5.

[0037] SEQ ID NO 7 is the mature polypeptide of SEQ ID NO 2.

[0038] SEQ ID NO 8 is the mature polypeptide of SEQ ID NO 4.

[0039] SEQ ID NO 9 is the mature polypeptide of SEQ ID NO 6.

[0040] SEQ ID NO 10 is the mature polypeptide of SEQ ID NO 13.

[0041] SEQ ID NO 11 is the mature polypeptide of SEQ ID NO 15.

[0042] SEQ ID NO 12 is the DNA encoding a full-length polypeptide from Bacillus saccharophilus.

[0043] SEQ ID NO 13 is a polypeptide derived from SEQ ID NO 12.

[0044] SEQ ID NO 14 is the DNA encoding a full-length polypeptide from Bacillus saccharophilus.

[0045] SEQ ID NO 15 is a polypeptide derived from SEQ ID NO 14.

[0046] SEQ ID NO 16 is a secretion signal of Bacillus clausii.

[0047] SEQ ID NO 17 is a His-tag sequence

[0048] SEQ ID NO 18 is the polypeptide motif GXDE.

[0049] SEQ ID NO 19 is a polypeptide motif

[0050] [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN]

[0051] SEQ ID NO 20 is the polypeptide motif [VIM][LIV]G[GAV]DE[VI][PSA]

[0052] SEQ ID NO 21 is the polypeptide motif WND[SQR][IVL][TLVM].

[0053] SEQ ID NO 22 is a peptide motif QSTL

[0054] SEQ ID NO 23 is the polypeptide motif NKFFY.

[0055] SEQ ID NO 24 is the polypeptide motif NLD[DR]S

[0056] definition

[0057] Dispersed proteins: The term "dispersed protein" and the abbreviation "Dsp" refer to polypeptides, such as those found in biological membranes, that have aminohexosidase activity, EC 3.2.1.-, which catalyze the hydrolysis of β-1,6-glycosidic bonds in N-acetyl-glucosamine polymers (poly-N-acetylglucosamine).

[0058] Aminohexosidase: The term "aminohexosidase" refers to polypeptides, for example, those found in biological membranes, that possess aminohexosidase activity and include, according to EC 3.2.1, catalyzing the hydrolysis of N-acetyl-D-aminohexose or N-acetyl-glucosamine polymers. This term includes dispersed proteins and polypeptides that include both N-acetylglucosidase and β-N-acetylglucosidase activities. The term "polypeptide with aminohexosidase activity" is used interchangeably with the term aminohexosidase, and similarly, the term "polypeptide with β-N-acetylglucosaminidase activity" is used interchangeably with the term β-N-acetylglucosaminidase. For the purposes of this invention, aminohexosidase activity is determined according to the procedure described in determination 1 or 2. In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% aminohexosidase activity of the mature polypeptide of SEQ ID NO 2. In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% aminohexosidase activity of the mature polypeptide of SEQ ID NO 4. In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% aminohexosidase activity of the mature polypeptide of SEQ ID NO 6. In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% aminohexosidase activity of the mature polypeptide of SEQ ID NO 13. In another aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% aminohexosidase activity of the mature polypeptide of SEQ ID NO 15.

[0059] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0060] Biofilms: Biofilms can be produced by any group of microorganisms in which cells adhere to one another or to a surface (such as textiles, tableware, or hard surfaces) or another surface. These adherent cells are often embedded within a matrix of extracellular polymers (EPS). Biofilms (EPS) are polymeric aggregates generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms can form on living or non-living surfaces. Microbial cells growing in biofilms are physiologically different from planktonic cells of the same organism (in contrast, planktonic cells are single cells that can float or float in liquid culture media). Bacteria living in biofilms often have significantly different characteristics from planktonic bacteria of the same species because the dense and protected environment of the membrane allows them to cooperate and interact in different ways. One benefit of this environment for microorganisms is increased resistance to detergents and antibiotics because the dense extracellular matrix and the outer layer of the cells protect the interior of the community. On clothing and hard surfaces, biofilm-forming bacteria can be found in the following species: Acinetobacter, Aerobicum, Shortwave Monoclonal, Microbacterium, Micrococcus luteus, Pseudomonas, Streptococcus, and Streptococcus lactis subsp. lactis, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Oligotrophozoites, Enterobacteriaceae, Xanthomonas, Yersinia, Klebsiella, Burkholderia, Oligotrophozoites, Gluttony, Escherichia, and others. Species of the genera *Stonebacterium*, *Achromobacter*, *Luteibacter*, *Citrobacter*, *Xanthomonas*, *Haemophilus*, *Bordetella*, *Lysobacterium*, *Serratia*, *Escherichia*, *Aggregatibacter*, *Listeria monocytogenes*, *Clostridium difficile*, *Mycobacterium*, *Neisseria gonorrhoeae*, *Haemophilus influenzae*, *Haemophilus dulcis*, *Helicobacter pylori*, *Campylobacter jejuni*, and *Enterococcus faecalis*, along with the fungi *Candida albicans*, *Aspergillus flavus*, *Fusarium solani*, and *Cryptococcus neoformans*. In one aspect, the biofilm component, such as poly-N-acetylglucosamine, includes strains of the genus *Bacillus*. In another aspect, the biofilm component, such as poly-N-acetylglucosamine, includes strains of *Pseudomonas basophila* or *Pseudomonas fluorescens*. In yet another aspect, the biofilm component, such as poly-N-acetylglucosamine, includes strains of *Staphylococcus aureus*.

[0061] Catalytic domain: The term "catalytic domain" refers to the region of an enzyme containing the enzyme's catalytic mechanism.

[0062] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. The initial RNA transcript is a precursor to mRNA, which undergoes a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0063] A clade is a group of polypeptides grouped together based on homology traits traced back to a common ancestor. A polypeptide clade can be viewed as a phylogenetic tree, and a clade consists of a group of polypeptides that share a common ancestor and all their direct descendants. Figure 1 The polypeptides of the present invention (e.g., all polypeptides belonging to the Bacillus clade), in Figure 1 and 3 This is represented as a phylogenetic tree. A clade of *Bacillus terrestris*, or a subclade of *Bacillus terrestris*, is a group of enzymes that are all related to a common ancestor and share common characteristics. A group of polypeptides formed within a clade (subclade) of the phylogenetic tree can also share common characteristics and be more closely related to other polypeptides in the clade.

[0064] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by a read frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0065] Control Sequences: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the polypeptide.

[0066] Deep cleaning: The term “deep cleaning” refers to reducing or removing components of biofilms, such as EPS or portions thereof, polysaccharides, PNAG (poly-N-acetylglucosamine), proteins, DNA, dirt, or other components present in the biofilm.

[0067] Detergent adjuvant components: These detergent adjuvant components differ from the aminohexosidase of the present invention. The precise properties of these additional adjuvant components, and their incorporation levels, will depend on the physical form of the composition and the nature of the operation in which the composition will be used. Suitable adjuvant materials include, but are not limited to, the components described below, such as surfactants, builder agents, flocculants, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymerizers, clay detergents / anti-reprecipitants, brighteners, antifoaming agents, dyes, fragrances, structural elastic agents, fabric softeners, carriers, water-soluble additives, builder agents and co-builder agents, fabric colorants, antifoaming agents, dispersants, processing aids, and / or pigments.

[0068] Detergent Composition: The term "detergent composition" refers to a composition used to remove unwanted compounds from items to be cleaned, such as textiles. This detergent composition can be used, for example, to clean textiles, for both household and industrial cleaning. These terms encompass any material / compound chosen for the desired form of the cleaning composition and product (e.g., liquid, gel, powder, granules, paste, or spray composition), and include, but are not limited to, detergent compositions (e.g., liquid and / or solid laundry detergents and delicate fabric detergents; fabric fresheners; fabric softeners; and textile and garment pre-treatments / pre-treatments). In addition to the polypeptides of the present invention, the composition may also contain one or more other enzymes (e.g., proteases, amylases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases, or any mixture thereof), and / or detergent adjuvants, such as surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, optical brighteners, bactericides, fungicides, dirt suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0069] Enzyme Washing Benefits: This article defines the term "enzyme washing benefits" as the advantageous effect of adding an enzyme to a detergent compared to the same detergent without that enzyme. Key washing benefits that enzymes can provide include stain removal with minimal or no visible dirt after washing and / or cleaning; prevention or reduction of redeposition of dirt released during washing (an anti-redeposition effect); and complete or partial restoration of the whiteness of textiles that were originally white but have acquired a grayish or yellowish appearance after repeated use and washing (an anti-whitening effect). Textile care benefits not directly related to the catalytic removal of stains or prevention of redeposition are also important for the benefits of enzyme washing. Examples of such textile care benefits include preventing or reducing dye transfer from one fabric to another or another part of the same fabric (also known as dye transfer inhibition or anti-rebound effect), removing protruding or broken fibers from the fabric surface to reduce pilling tendency or removing existing fuzz or fluff (also known as anti-pilling effect), improving fabric softness, clarifying fabric colors, and removing particulate dirt trapped in the fibers of fabrics or garments. Enzymatic bleaching is another benefit of enzymatic washing, where catalytic activity is typically used to catalyze the formation of bleaching components (such as hydrogen peroxide or other peroxides).

[0070] Expression: 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.

[0071] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operatively linked to a control sequence that provides for its expression.

[0072] Fragment: The term "fragment" means a polypeptide or catalytic domain having one or more (e.g., several) amino acids deleted from the amino and / or carboxyl termini of a mature polypeptide or domain; wherein the fragment has aminohexosidase activity. In one aspect, the fragment contains at least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ ID NO 2), at least 305 amino acid residues (e.g., amino acids 1 to 305 of SEQ ID NO 2), at least 310 amino acid residues (e.g., amino acids 1 to 310 of SEQ ID NO 2), at least 315 amino acid residues (e.g., amino acids 1 to 315 of SEQ ID NO 2), or at least 320 amino acid residues (e.g., amino acids 1 to 320 of SEQ ID NO 2). In one aspect, the fragment contains at least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ ID NO 4), at least 305 amino acid residues (e.g., amino acids 1 to 305 of SEQ ID NO 4), at least 310 amino acid residues (e.g., amino acids 1 to 310 of SEQ ID NO 4), at least 315 amino acid residues (e.g., amino acids 1 to 315 of SEQ ID NO 4), or at least 320 amino acid residues (e.g., amino acids 1 to 320 of SEQ ID NO 4). In another aspect, the fragment contains at least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ ID NO 6), at least 305 amino acid residues (e.g., amino acids 1 to 305 of SEQ ID NO 6), at least 310 amino acid residues (e.g., amino acids 1 to 310 of SEQ ID NO 6), at least 315 amino acid residues (e.g., amino acids 1 to 315 of SEQ ID NO 6), or at least 320 amino acid residues (e.g., amino acids 1 to 320 of SEQ ID NO 6). In one aspect, the fragment contains at least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ ID NO 13), at least 305 amino acid residues (e.g., amino acids 1 to 305 of SEQ ID NO 13), at least 310 amino acid residues (e.g., amino acids 1 to 310 of SEQ ID NO 13), at least 315 amino acid residues (e.g., amino acids 1 to 315 of SEQ ID NO 13), or at least 320 amino acid residues (e.g., amino acids 1 to 320 of SEQ ID NO 13).In one aspect, the fragment contains at least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ ID NO 15), at least 305 amino acid residues (e.g., amino acids 1 to 305 of SEQ ID NO 15), at least 310 amino acid residues (e.g., amino acids 1 to 310 of SEQ ID NO 15), at least 315 amino acid residues (e.g., amino acids 1 to 315 of SEQ ID NO 15), or at least 320 amino acid residues (e.g., amino acids 1 to 320 of SEQ ID NO 15).

[0073] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0074] Isolated: The term "isolated" means a substance in a form or environment not naturally occurring. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, said substance being at least partially removed from one or all of the naturally occurring components associated with its properties; (3) any substance artificially modified relative to a substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples; for example, host cells may be genetically modified to express the polypeptides of the present invention. Fermentation broth from such host cells will contain the isolated polypeptides.

[0075] Improved washing performance: The term “improved washing performance” is defined herein as the washing performance of an enzyme in a detergent composition that demonstrates increased washing performance relative to the same detergent composition without the enzyme, for example, by increasing stain removal or reducing redeposition. The term “improved washing performance” includes washing performance in clothing.

[0076] Washing: The term "washing" encompasses both household and industrial washing and refers to the process of treating textiles with a solution containing the cleaning or detergent composition of the present invention. The washing process can be performed, for example, using a household or industrial washing machine or can be performed manually.

[0077] Odor: The term "odor" refers to an undesirable smell on cleaned items. Cleaned items should smell fresh and clean, without any unpleasant odor adhering to them. One example of odor is a compound with an unpleasant smell, which can be produced by microorganisms. Another example is the smell of sweat or body odor that can adhere to items that have come into contact with humans or animals. Yet another example of odor can be the smell of spices that adhere to items, such as strongly scented curry or other exotic spices.

[0078] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In some aspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 2, and amino acids -41 to -1 of SEQ ID NO 2 are signal peptides. In some aspects, the mature polypeptide has the amino acid sequence of SEQ ID NO 7. In some aspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 4, and amino acids -25 to -1 of SEQ ID NO 4 are signal peptides. In some aspects, the mature polypeptide has the amino acid sequence of SEQ ID NO 8. In some aspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 6, and amino acids -25 to -1 of SEQ ID NO 6 are signal peptides. In some aspects, the mature polypeptide has the amino acid sequence of SEQ ID NO 9. In some aspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 13, and amino acids -24 to -1 of SEQ ID NO 13 are a signal peptide. In some aspects, the mature polypeptide has the amino acid sequence of SEQ ID NO 10. In some aspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 15, and amino acids -24 to -1 of SEQ ID NO 15 are a signal peptide. In some aspects, the mature polypeptide has the amino acid sequence of SEQ ID NO 11.

[0079] It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and therefore a host cell expressing a polynucleotide may produce different mature polypeptides (e.g., with different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide.

[0080] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide having aminohexosidase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 124 to 1095 of SEQ ID NO 1, and nucleotides 1 to 123 of SEQ ID NO 1 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 76 to 1047 of SEQ ID NO 3, and nucleotides 1 to 75 of SEQ ID NO 3 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 76 to 1047 of SEQ ID NO 5, and nucleotides 1 to 75 of SEQ ID NO 5 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 73 to 1044 of SEQ ID NO 12, and nucleotides 1 to 72 of SEQ ID NO 12 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 73 to 1044 of SEQ ID NO 14, and nucleotides 1 to 72 of SEQ ID NO 14 encode a signal peptide.

[0081] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized, and the nucleic acid molecule contains one or more control sequences.

[0082] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence guides the expression of the coding sequence.

[0083] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0084] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS software 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) was used to determine sequence identity between two amino acid sequences. The parameters used were a nick opening penalty of 10, a nick expansion penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The Needle-labeled "Longest Identity" output (obtained using the -nobrief option) was used as the identity percentage and calculated as follows:

[0085] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0086] Strict conditions: the term " Very low stringency conditions This means that for probes at least 100 nucleotides in length, a standard DNA blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0087] the term" Low stringency conditions This means that for probes with a length of at least 100 nucleotides, the standard DNA blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0088] the term" Strict conditions "This refers to the standard DNA blotting procedure for probes at least 100 nucleotides in length, following a pre-hybridization and hybridization process at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time."

[0089] the term" Medium to high strict conditions This means that for probes with a length of at least 100 nucleotides, the standard DNA blotting procedure is followed, with pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0090] The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes at least 100 nucleotides in length, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / mL of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0091] the term" Very high stringent conditions This means that for probes with a length of at least 100 nucleotides, the standard DNA blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0092] Variants: The term "variant" refers to a polypeptide with aminohexosidase activity that contains alterations (i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions). Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0093] name

[0094] For the purposes of this invention, the designation [IV] or [I / V] indicates that the amino acid at that position can be isoleucine (Ile, I) or valine (Val, V). Similarly, for other combinations as described herein, the designations [LVI] and [L / V / I] indicate that the amino acid at that position can be leucine (Leu, L), valine (Val, V), or isoleucine (Ile, I), and so on. Unless further limited otherwise, amino acid X is defined such that it can be any of the 20 naturally occurring amino acids. Detailed Implementation

[0095] Various enzymes have been used in laundry, many of which are involved in deodorization. WO2014 / 087011 describes the use of deoxyribonuclease (DNase) for reducing odors from clothing and / or textiles; WO 9909143 describes the use of one or more oxidoreductases in combination with a medium for reducing odors; and WO 2012 / 112718 describes a method for inhibiting the generation of bacterial odors from clothing using various Bacillus strains. This invention relates to polypeptides and cleaning agents, such as detergent compositions comprising polypeptides from the GH20 family of Bacillus terrestrialus clades with aminohexosidase activity. Washing methods and the use of polypeptides with aminohexosidase activity are also claimed. Polypeptides from the GH20 family of Bacillus terrestrialus clades with aminohexosidase activity can be used to reduce and prevent staining of washed items. The inventors have surprisingly discovered that polypeptides from the GH20 family of Bacillus terrestrialus clades with aminohexosidase activity can be used to reduce clothing-related organic matter, such as EPS and / or PNAG. WO200406117 describes compositions comprising dispersive proteins, such as DspB. These compositions may include detergents, which may be anionic, cationic, or nonionic detergents. WO 9850512 describes enzymes having aminohexosidase activity. The polypeptides of the present invention have aminohexosidase activity, but these polypeptides do not belong to the DspB clade or relate to any enzymes described in WO9850512. The polypeptides of the present invention belong to the Bacillus terreus clade and are therefore distinct from DspB and those disclosed in WO 9850512. Surprisingly, the polypeptides of the present invention can be used to prevent, reduce, and / or remove organic matter, such as biofilm components on hard surfaces in conjunction with laundry, such as polysaccharides, such as PNAG (poly-N-acetylglucosamine). The use of aminohexosidases in cleaning methods such as laundry or in detergent compositions containing, for example, builder agents and / or bleach is not indicated in the art. For enzymes to be useful in the cleaning process, they need to function within the detergent under conditions such as laundry washing, including stability in the presence of detergent components such as surfactants, builders, and bleaching agents. The components of the detergent can significantly affect the performance of enzymes such as aminohexosidases. This application surprisingly demonstrates that polypeptide surfactants belonging to the *Bacillus terreus* clade are particularly stable in the presence of linear alkylbenzene sulfonates (LAS), which are very common surfactants in detergents. Therefore, polypeptides belonging to *Bacillus terreus* are particularly suitable for deep cleaning, such as for textiles or washing machines.

[0096] Figure 1A review of the phylogenetic clades of *Bacillus terrestris* is provided. These clades contain homologous sequences. The polypeptides of this invention having mature amino acid sequences SEQ ID 7, 8, and 9, exhibiting aminohexosidase activity, can be aligned using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453). The percentage of identity resulting from such alignments is shown in Table 1 below.

[0097] Table 1

[0098]

[0099] Table 1 shows the close sequence correlation of the peptides of the present invention. Peptides containing the amino acid sequences of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11 belong to a subclade of the dispersed protein clade. Compared to, for example, more distant DspB peptides (not shown), these peptides share over 90% pairwise sequence identity and are more closely related to each other.

[0100] One aspect of the present invention relates to a polypeptide comprising the amino acid sequence shown in SEQ ID NO 7, or a polypeptide having at least 99.8% or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0101] One aspect of the present invention relates to a polypeptide comprising the amino acid sequence shown in SEQ ID NO 8, or a polypeptide having at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0102] One aspect of the invention relates to a polypeptide comprising the amino acid sequence shown in SEQ ID NO 9, or a polypeptide having at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0103] One aspect of the present invention relates to a polypeptide comprising the amino acid sequence shown in SEQ ID NO 10, or a polypeptide having at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0104] One aspect of the invention relates to a polypeptide comprising the amino acid sequence shown in SEQ ID NO 11, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0105] The polypeptides of this invention are all located within the same clade, the *Bacillus terrestris* clade, and all possess common functional characteristics, including deep cleaning properties in the presence of detergents. The inventors have surprisingly discovered that polypeptides of the *Bacillus terrestris* clade, comprising mature polypeptides having SEQ ID NOs 7, 8, 9, 10, 11 or polypeptides having at least 80% sequence identity with them, possess specific properties. More precisely, the polypeptides contained in this clade all exhibit excellent deep cleaning effects, have a wide range of surfactants such as LAS, and are therefore particularly suitable for detergents containing surfactants, such as those containing anionic, nonionic, cationic, and / or amphoteric surfactants.

[0106] As already described, the polypeptides of the present invention having aminohexosidase activity may contain a Glycohydro20 domain, such as GH20. Polypeptides containing the GH20 domain may contain several motifs, one example being GXDE (SEQ ID NO 18) located at positions 158 to 161 in *Bacillus saccharophilus* (SEQ ID NO 9). Residues D and E are key catalytic residues of GH20 (positions 160 and 161 in SEQ ID NO 9). GH20 polypeptides may be divided into several distinct subclusters or clades, examples of specific domains of which are listed below. Additional domains preferably shared by the polypeptides of the present invention have been identified. This domain, previously undescribed, is referred to as IAS, and polypeptides containing this domain, in addition to having aminohexosidase activity, such as PNAG activity, are characterized by containing certain motifs, such as one or more of [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), corresponding to ESYAIAS at positions 44 to 50 of SEQ ID NO 9. Another domain, preferably shared by the polypeptides of the present invention, was identified. This domain, previously undescribed, is referred to as WND, and polypeptides containing this domain contain a GH20 domain, are of bacterial origin, and, in addition to having PNAG activity, are characterized by containing certain motifs. The polypeptide containing the domain may include the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), corresponding to positions 156 to 163 of SEQ ID NO 9, wherein G (corresponding to position 158 in SEQ ID NO 9) is completely conserved in the *Bacillus terreus* clade, and E is a key catalytic residue of GH20 (positions 160 and 161 in SEQ ID NO 9). Another motif that may be included by the polypeptide of the present invention is WND[SQR][IVL][TLVM] (SEQ ID NO:21), at positions 193 to 198 of SEQ ID NO 9, wherein W (position 193 in SEQ ID NO 9) is part of the active site pocket and is presumed to involve the binding of an N-acetyl group to a PNAG substrate. The polypeptide of the *Bacillus terreus* clade can be further subdivided into a clade called QSTL, which contains bacterial-derived WND domain polypeptides with PNAG activity. The polypeptide of this clade contains a motif instance QSTL (SEQ ID NO:22) corresponding to positions 216 to 219 of SEQ ID NO 9, wherein all four amino groups are completely conserved in the QSTL clade and are presumed to be involved in substrate binding.Another motif that may be contained in a polypeptide of the QSTL clade is NKFFY (SEQ ID NO:23), 273 to 277 of SEQ ID NO 9. Another motif that may be contained in a polypeptide of the QSTL clade is NLD[DR]S (SEQ ID NO:24), 204 to 208 of SEQ ID NO 9. In one aspect, the polypeptide of the present invention contains the GXDE (SEQ ID NO 18) motif. In one aspect, the polypeptide of the present invention contains the [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19) motif. In one aspect, the polypeptide of the present invention contains the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20). In one aspect, the polypeptide of the present invention contains the motif WND[SQR][IVL][TLVM] (SEQ ID NO:21). In one aspect, the polypeptide of the present invention contains the motif QSTL (SEQ ID NO 22). In one aspect, the polypeptide of the present invention comprises the motif NKFFY (SEQ ID NO:23). In another aspect, the polypeptide of the present invention comprises the motif NLD[DR]S (SEQ ID NO:24). Alignments of the polypeptides of the present invention contained in the QSTL clade are shown. Figure 2 middle.

[0107] Some aspects of the present invention relate to polypeptides comprising one or both of the motifs GXDE (SEQ ID NO 18) or [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), wherein the polypeptide comprises an amino acid sequence selected from the polypeptides shown in SEQ ID NO 7, 8, 9, 10 and 11 or has at least 80% sequence identity with the polypeptide.

[0108] Some aspects of the present invention relate to polypeptides comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises an amino acid sequence selected from the polypeptides shown in SEQ ID NO 7, 8, 9, 10 and 11 or has at least 80% sequence identity with the polypeptides shown in SEQ ID NO 11.

[0109] Some aspects of the present invention relate to polypeptides comprising one or more of the motifs QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), or NLD[DR]S (SEQ ID NO:24), wherein the polypeptide comprises an amino acid sequence selected from the polypeptides shown in SEQ ID NO 7, 8, 9, 10, and 11 or has at least 80% sequence identity therewith.

[0110] One aspect of the invention relates to a polypeptide comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO 7, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0111] One aspect of the invention relates to a polypeptide comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO 8, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0112] One aspect of the invention relates to a polypeptide comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO 9, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0113] One aspect of the invention relates to a polypeptide comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO 10, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0114] One aspect of the invention relates to a polypeptide comprising one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO 11, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0115] One aspect of the invention relates to a polypeptide comprising one or more of the motifs QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), or NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide having at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0116] One aspect of the invention relates to a polypeptide comprising one or more of the motifs QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), or NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide having at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0117] One aspect of the invention relates to a polypeptide comprising one or more of the motifs QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), or NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 9, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0118] One aspect of the invention relates to a polypeptide comprising one or more of the motifs QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), or NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 10, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0119] One aspect of the invention relates to a polypeptide comprising one or more of the motifs QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), or NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 11, or a polypeptide having at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity with it, wherein the polypeptide has aminohexosidase activity.

[0120] In one aspect, the use of a polypeptide comprising the amino acid sequences shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11 during a cleaning process, or a polypeptide having at least 84%, for example, at least 85%, for example, at least 86%, for example, at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99%, or 100% sequence identity with the polypeptide, wherein the polypeptide has aminohexosidase activity. Some aspects of the present invention relate to cleaning, for example, detergent compositions comprising a) one or more polypeptides selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity, and b) at least one surfactant, preferably at least one surfactant selected from the group consisting of anionic, nonionic and / or cationic surfactants.

[0121] One aspect of the present invention relates to a composition comprising at least 0.001 ppm of a polypeptide having aminohexosidase activity and at least one adjuvant component, wherein the polypeptide is selected from the group consisting of polypeptides having at least 60% sequence identity with the mature polypeptides shown in SEQ ID NO 7, 8, 9, 10 and 11.

[0122] The amount of polypeptide can be in the range of 0.00004-100 ppm, for example, in the range of 0.00008-50 ppm, in the range of 0.00001-20 ppm, in the range of 0.0002-20 ppm, in the range of 0.0001-50 ppm, in the range of 0.0002-50 ppm, in the range of 0.0004-50 ppm, in the range of 0.0008-50 ppm, in the range of 0.001-50 ppm, 0.01-50 ppm, preferably 0.0001-50 ppm, more preferably 0.0002-20 ppm, more preferably 0.0002-10 ppm, more preferably 0.001-10 ppm, and most preferably 0.002-10 ppm. The amount of aminohexosidase in this invention can correspond to at least 0.00001 ppm, for example at least 0.00002 ppm, at least 0.0001 ppm, at least 0.0002 ppm, at least 0.0005 ppm, at least 0.001 ppm, at least 0.002 mg ppm, at least 0.005 ppm, at least 0.01 ppm, or at least 0.02 ppm. The composition may contain at least 0.00008%, preferably at least 0.00001%, 0.00002%, 0.0001%, 0.0002%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.008%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% aminohexosidase.

[0123] Preferably, the polypeptide has N-acetylglucosidase activity and / or β-N-acetylglucosidase activity, for example, activity against PNAG. In one aspect, the polypeptide comprises one or more of the motifs GXDE (SEQ ID NO: 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ ID NO: 21), QSTL (SEQ ID NO: 22), NKFFY (SEQ ID NO: 23), and NLD[DR]S (SEQ ID NO: 24). In one aspect, the polypeptide has N-acetylglucosidase activity and / or β-N-acetylglucosidase activity, for example, activity against PNAG, and comprises one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), and NLD[DR]S (SEQ ID NO:24). In one aspect, the polypeptide has at least 60%, 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% sequence identity with the polypeptides shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11.

[0124] In one aspect, the polypeptide has N-acetylglucosidase activity and / or β-N-acetylglucosidase activity, for example, activity against PNAG, and comprises one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), and NLD[DR]S (SEQ ID NO:24), wherein the polypeptide is associated with SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO The polypeptide shown in 11 has at least 60%, 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% sequence identity.

[0125] In one aspect, the polypeptide comprises or is composed of a mature polypeptide of SEQ ID NO 7 or SEQ ID NO 2, a mature polypeptide of SEQ ID NO 8 or SEQ ID NO 4, a mature polypeptide of SEQ ID NO 9 or SEQ ID NO 6, a mature polypeptide of SEQ ID NO 10 or SEQ ID NO 13, or a mature polypeptide of SEQ ID NO 11 or SEQ ID NO 15.

[0126] The composition is preferably a cleaning composition, more preferably a laundry or dishwashing composition. Some aspects of the invention relate to compositions comprising the polypeptide of the invention and an adjuvant ingredient, wherein the adjuvant ingredient is selected from...

[0127] a) At least one detergent builder

[0128] b) at least one surfactant, and

[0129] At least one bleaching component.

[0130] Some aspects of the present invention relate to cleaning compositions, such as detergent compositions, comprising a) one or more polypeptides selected from the group consisting of SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10 and SEQ ID NO11, wherein the polypeptide has aminohexosidase activity, and b) at least one surfactant, preferably at least one surfactant selected from the group consisting of anionic, nonionic and / or cationic surfactants.

[0131] Some aspects of the present invention relate to detergent compositions comprising:

[0132] a) At least 0.01 ppm of an active enzyme polypeptide, wherein the enzyme polypeptide is selected from or has at least 80% sequence identity with the group consisting of: SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, wherein the polypeptide has aminohexosidase activity, and

[0133] b) Surfactant from about 2 wt% to about 60 wt%, or surfactant from about 5 wt% to about 60 wt%.

[0134] Some aspects of the present invention relate to detergent compositions comprising:

[0135] a) At least 0.0001 ppm of an active enzyme polypeptide, wherein the enzyme polypeptide is selected from or has at least 80% sequence identity with the group consisting of: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity, and

[0136] b) Surfactant from about 2 wt% to about 60 wt%, or surfactant from about 5 wt% to about 60 wt%, surfactant from about 5 wt% to about 60 wt%.

[0137] The composition preferably comprises from about 2 wt% to about 60 wt%, from about 5 wt% to about 50 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 20 wt%, and from about 5 wt% to about 10 wt% of anionic and / or nonionic surfactants. The surfactant may be selected from the nonionic, anionic, and / or amphoteric surfactants described above; preferably, anionic or nonionic surfactants may also be used, but amphoteric surfactants may also be used. Typically, bleach-stabilized surfactants are preferred. Preferred anionic surfactants are sulfate surfactants, and specifically alkyl ether sulfates, particularly C9-C15 alcohol ether sulfates, C12-15 primary alcohol ethoxylates, C8-C16 ester sulfates, and C10-C14 ester sulfates, such as monododecyl ester sulfates. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), and alcohol ethers. Sulfates (AES, AEOS, or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids, or salts (soaps) of fatty acids, and combinations thereof. Anionic surfactants are preferably added to detergents in the form of salts. Suitable cations in these salts are alkali metal ions, such as sodium, potassium, and lithium, and ammonium salts, such as (2-hydroxyethyl)ammonium, di(2-hydroxyethyl)ammonium, and tri(2-hydroxyethyl)ammonium salts.Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (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 (glucosamide (GA) or fatty acid glucosamide (FAGA)), together with products available under the trade names SPAN and TWEEN, and combinations thereof. Commercially available nonionic surfactants include Plurafac from BASF. TM Lutensol TM and Pluronic TM Class, Dehypon from Cognis TM Series and Genapor from Clariant TM series.

[0138] In some aspects of the present invention, the detergent composition of the present invention comprises:

[0139] a) At least 0.01 ppm of an active enzyme polypeptide, wherein the enzyme polypeptide belongs to the *Bacillus terreus* clade and is selected from or has at least 60% sequence identity with the following group of polypeptides: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity.

[0140] b) at least one surfactant from about 2 wt% to about 60 wt%.

[0141] In a preferred aspect of the invention, the ratio of anionic to nonionic surfactant is greater than 1, meaning the content of anionic surfactant is higher than the amount of nonionic surfactant. Therefore, one aspect of the invention relates to a detergent composition comprising:

[0142] a) At least 0.01 ppm of an active enzyme polypeptide, wherein the enzyme polypeptide belongs to the *Bacillus terreus* clade and is selected from or has at least 60% sequence identity with the following group of polypeptides: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity.

[0143] b) Anionic surfactants from about 5 wt% to about 50 wt%, and

[0144] c) From about 1 wt% to about 8 wt% of nonionic surfactants.

[0145] The polypeptides of the present invention can also be formulated into compositions, for example, liquid laundry compositions optionally comprising a detergent builder, the composition comprising:

[0146] a) At least 0.0001 ppm, for example 0.01 ppm, of an active enzyme polypeptide, wherein the enzyme polypeptide belongs to the *Bacillus terreus* clade and is selected from or has at least 60% sequence identity with the group consisting of: SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10 and SEQ ID NO11, wherein the polypeptide has aminohexosidase activity.

[0147] b) at least one surfactant from about 2 wt% to about 60 wt%, and optionally

[0148] c) At least one detergent builder, such as carbonate, zeolite, phosphate, calcium chelating agent, or complexing agent, in a range of about 5 wt% to about 50 wt%.

[0149] The composition comprises at least one detergent builder, wherein the detergent builder is added in an amount from about 0 to about 65% wt%, from about 40 wt% to about 65 wt%, from about 20 wt% to about 65 wt%, from about 10 wt% to about 50 wt%, or from about 5 wt% to about 50 wt% by weight, wherein the detergent builder is selected from phosphates, sodium citrate builders, sodium carbonate, sodium silicate, sodium, and zeolite.

[0150] One aspect of the present invention relates to a cleaning composition comprising:

[0151] a) At least 0.0001 ppm of a polypeptide belonging to the *Bacillus terreus* clade, containing one or both of the motifs [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide is selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity, and optionally...

[0152] b) at least one surfactant from about 2 wt% to about 60 wt%, and optionally

[0153] c) at least one detergent builder from about 5 wt% to about 50 wt%, and optionally

[0154] d) At least one bleaching component, from about 1 wt% to about 15 wt%.

[0155] The surfactant can be any of those described above.

[0156] Preferably, the detergent builder is selected from phosphates, sodium citrate builders, sodium carbonate, sodium silicate, and sodium aluminosilicate (zeolite). Suitable builders are alkali metals or ammonium phosphates, polyphosphates, phosphates, polyphosphates, carbonates, bicarbonates, borates, citrates, and polycarboxylates. Citrate builders, such as citric acid and its soluble salts (especially sodium salts), are polycarboxylate builders. Citrates can be used in combination with zeolites, silicates such as BRITESIL type, and / or layered silicate builders. The detergent builder is preferably added in an amount of about 0-65% by weight (e.g., about 5% to about 50% by weight). In the composition, the level of the detergent builder is typically about 40%-65% by weight, particularly about 50%-65% by weight, and particularly from 20%-50% by weight. The detergent builder and / or co-builder can be, in particular, chelating agents that form water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art and used in cleaning detergents can be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), and (carboxymethyl) inulin (CMI), and combinations thereof. Further non-limiting examples of builders include citrates; chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphates; and alkyl succinic acids or alkenyl succinic acids.Other specific examples include 2,2',2”-nitrotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine-N,N-diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid, N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), and N-(sulfomethyl)aspartic acid (SM). As, N-(2-sulfoethyl)-aspartic acid (SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and N'-(2-hydroxyethyl)-ethylenediamine-N,N,N'-triacetic acid (HEDTA), diethanolglycine (DEG), and combinations thereof and their salts.

[0157] Suitable phosphates for use in this article include 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta(methylenephosphonic acid) (DTMPA or DTPMPA or DTPMP), nitrotri(methylenephosphonic acid) (ATMP or NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), and hexamethylenediaminetetra(methylenephosphonic acid) (HDTMP).

[0158] The composition may also contain 0-50% by weight, such as about 5% to about 30% of detergent co-cleansing agent.

[0159] The composition may include only a co-adjuvant or in combination with an adjuvant, such as a zeolite adjuvant. Non-limiting examples of co-adjuvants include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers (acrylic acid / maleic acid) (PAA / PMA) or polyaspartic acid.

[0160] Other exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and US5977053.

[0161] In a preferred embodiment, the detergent builder is based on a non-phosphorus builder, such as citric acid and / or methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and / or their salts.

[0162] Some aspects of the present invention relate to compositions comprising at least one enzymatic polypeptide, wherein the enzyme is selected from polypeptides of the following group or polypeptides having at least 60% sequence identity with the group comprising: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide has aminohexosidase activity and a nonphosphate builder selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof.

[0163] In one aspect, the composition is a detergent composition, such as a laundry detergent composition or an automatic dishwashing (ADW) composition, the composition comprising:

[0164] a) At least 0.0001 ppm, for example 0.01 ppm, of an active enzyme polypeptide, wherein the enzyme polypeptide is selected from or has at least 60% sequence identity with the group consisting of: SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10 and SEQ ID NO11, wherein the polypeptide has aminohexosidase activity, and

[0165] b) 10 wt% to 50 wt% of a detergent builder selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof, and optionally...

[0166] c) At least one bleaching component.

[0167] The composition may contain 0-30% by weight, for example, about 1% to about 20%, for example, about 1% to about 10%, for example, about 1% to about 5%, for example, about 10% to about 30%, for example, about 5% to about 10%, or by weight of the bleaching system, for example, about 10% to about 20%. Any bleaching system containing components known in the art for use in cleaning detergents can be utilized. Suitable bleaching system components include hydrogen peroxide sources; peracid sources; and bleaching catalysts or synergists.

[0168] hydrogen peroxide source

[0169] Suitable sources of hydrogen peroxide are inorganic peroxide salts, including alkali metal salts such as sodium percarbonate and sodium perborate (usually monohydrate or tetrahydrate), as well as hydrogen peroxide-urea (1 / 1).

[0170] peracid source

[0171] The peracid can be (a) directly incorporated as a pre-formed peracid, or (b) formed in situ in the washing solution from hydrogen peroxide and bleaching activator (perhydrolysis), or (c) formed in situ in the washing solution from hydrogen peroxide and catalase and a suitable substrate (e.g., ester) of the latter.

[0172] a) Suitable preformed peracids include, but are not limited to: peroxycarboxylic acids such as peroxybenzoic acid and its cyclically substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ε-phthaliminoperoxylanthanic acid [phthaliminoperoxyhexanoic acid (PAP)], and o-carboxybenzoamide peroxylanthanic acid; aliphatic and aromatic disperoxydicarboxylic acids such as disperoxydodecanoic acid, disperoxyazeloic acid, disperoxysedicarboxylic acid, disperoxytridecanoic acid, 2-decyldisperoxysuccinic acid, and disperoxyphthalic acid, disperoxyisophthalic acid and disperoxyterephthalic acid, perimino, peroxymonosulfate, peroxydisulfate, peroxyphosphoric acid, peroxysilicic acid, and mixtures of said compounds. It should be understood that in some cases, the mentioned peracids may preferably be added as a suitable salt, such as an alkali metal salt (e.g., ) or alkaline earth metal salts.

[0173] (b) Suitable bleaching activators include those belonging to the classes of esters, amides, imides, nitriles, or acid anhydrides, and, where applicable, their salts. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), sodium 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), sodium 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), sodium 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A family of bleaching activators of interest is disclosed in EP624154, and in that family, triethyl acetyl citrate (ATC) is particularly preferred. ATC, or short-chain triglycerides (like triacetin), have the advantage of being environmentally friendly. Furthermore, triethyl acetylglucosamine and triacetin exhibit good hydrolytic stability in the product during storage and are effective bleaching activators. Finally, ATC is versatile because the citrate released during the over-hydrolysis reaction can act as a detergent builder.

[0174] Bleaching catalysts and synergists

[0175] The bleaching system may also include a bleaching catalyst or synergist.

[0176] Some non-limiting examples of bleaching catalysts that can be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), specifically Me3-TACN, such as the binuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2”-nitrogenotris(ethane-1,2-diylazalkylyl-κN-methylyl)triphenol-κ3O]manganese(III). These bleaching catalysts can also be other metal compounds, such as iron or cobalt complexes.

[0177] In some embodiments, including those containing sources of excessive acid, an organic bleaching catalyst or bleaching synergist having one of the following formulas may be used:

[0178]

[0179] (iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing 9 to 18 carbons or a straight-chain alkyl group containing 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isothidecyl, isothidecyl, and isopentachidyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO2007 / 087244, WO 2007 / 087259, EP 1867708 (Vitamin K) and WO 2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc phthalocyanine or aluminum phthalocyanine.

[0180] This invention relates to polypeptides of Bacillus terreus evolutionary clades having aminohexosidase activity, compositions such as detergent compositions containing the polypeptides, and the use of detergent compositions containing the polypeptides of this invention for deep cleaning articles such as textiles.

[0181] Therefore, some aspects of the present invention relate to cleaning compositions, such as detergent compositions, comprising:

[0182] a) At least 0.0001 ppm, for example, 0.001 ppm polypeptide of a Bacillus terreus clade, optionally comprising one or more motifs selected from the group consisting of: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the enzyme polypeptide is selected from the group consisting of and polypeptides having at least 60% (e.g., at least 70%, at least 80%, or at least 90%) sequence identity with the group consisting of: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide possesses aminohexosidase activity, and optionally...

[0183] b) From about 10 wt% to about 50 wt% of a building block, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof, and optionally...

[0184] c) From about 5 wt% to about 50 wt% of a surfactant, preferably selected from anionic surfactants such as LAS, AOS, AEOS and / or nonionic surfactants such as AE or AEO, and optionally

[0185] d) At least one bleaching component, preferably selected from percarbonate, persulfate and peracid.

[0186] Therefore, some aspects of the present invention relate to detergent compositions comprising:

[0187] a) At least 0.0001 ppm, for example, 0.001 ppm polypeptide of a Bacillus terreus clade, optionally comprising one or more motifs selected from the group consisting of: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the enzyme polypeptide is selected from the group consisting of and polypeptides having at least 60% (e.g., at least 70%, at least 80%, or at least 90%) sequence identity with the group consisting of: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide possesses aminohexosidase activity, and optionally...

[0188] b) From about 10 wt% to about 50 wt% of a building block, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof, and optionally...

[0189] c) From about 5 wt% to about 50 wt% of a surfactant, preferably selected from anionic surfactants such as LAS, AOS, AEOS and / or nonionic surfactants such as AE or AEO, and optionally

[0190] d) At least one bleaching component, preferably selected from percarbonate, persulfate and peracid.

[0191] Therefore, some aspects of the present invention relate to detergent compositions comprising:

[0192] a) A polypeptide of at least 0.0001 ppm, for example, 0.001 ppm, of a Bacillus terreus clade, optionally comprising one or more motifs selected from the group consisting of: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the enzyme polypeptide is selected from the group consisting of and polypeptides having at least 60% (e.g., at least 70%, at least 80%, or at least 90%) sequence identity with the group consisting of: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, wherein the polypeptide possesses aminohexosidase activity, and optionally...

[0193] b) From about 10 wt% to about 50 wt% of a building block, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof, and optionally...

[0194] c) From about 5 wt% to about 50 wt% of a surfactant, preferably selected from anionic surfactants such as LAS, AOS, AEOS and / or nonionic surfactants such as AE or AEO, and optionally

[0195] d) At least one bleaching component, wherein the bleaching agent is a peroxide and the bleaching catalyst is a manganese compound, wherein the oxygen bleaching agent is preferably a percarbonate and the manganese catalyst is preferably 1,4,7-trimethyl-1,4,7-triazacyclononane or manganese(III) acetate tetrahydrate (MnTACN).

[0196] One aspect relates to the use of the compositions described above for deep cleaning of items, such as textiles.

[0197] The polypeptides of the present invention, possessing aminohexosidase activity, can be used for deep cleaning of articles, such as hard surfaces, textiles, and / or fabrics. In some aspects of the invention, the polypeptides of the present invention, for example, have at least 60%, such as at least 70%, such as at least 80%, or such as at least 90% sequence identity with mature polypeptides of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13, and SEQ ID NO 15, or with mature polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11, and possess β-N-acetylglucosidase activity, and in some aspects, this aminohexosidase activity is β-N-acetylglucosidase activity and the polypeptides of the present invention are β-N-acetylglucosidases.

[0198] One aspect of the invention relates to the use of a polypeptide of the Bacillus tumefaciens clade in cleaning processes (e.g., laundry and / or dishwashing), wherein the polypeptide has aminohexosidase activity, preferably N-acetylglucosidase activity and / or β-N-acetylglucosidase activity.

[0199] One aspect of the present invention relates to the use of a polypeptide comprising one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), and NLD[DR]S (SEQ ID NO:24).

[0200] One aspect of the present invention relates to the use of a polypeptide of the Bacillus tumefaciens clade in cleaning processes (e.g., laundry and / or dishwashing), wherein the polypeptide has aminohexosidase activity, preferably N-acetylglucosidase activity and / or β-N-acetylglucosidase activity, wherein the polypeptide comprises one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), and NLD[DR]S (SEQ ID NO:24).

[0201] One aspect of the invention relates to the use of a polypeptide of the Bacillus subtilis clade for deep cleaning articles, the polypeptide comprising, for example, one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the polypeptide has aminohexosidase activity and wherein the polypeptide is a polypeptide having at least 60%, for example 80%, 85%, 90%, or 95% sequence identity with the mature polypeptide shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, or SEQ ID NO 11, wherein the article is a textile. In one aspect of the invention, polypeptides from the *Bacillus terrestrialus* clade possessing aminohexosidase activity are used to prevent, reduce, or remove stickiness from articles. In another aspect of the invention, polypeptides from the *Bacillus terrestrialus* clade possessing aminohexosidase activity can be further used for pretreatment of textiles, such as stains on textiles.

[0202] One aspect of the present invention relates to the use of polypeptides of Bacillus terreus evolutionary clades having aminohexosidase activity for preventing, reducing or removing redeposition of dirt during a washing cycle.

[0203] Furthermore, one aspect of the invention relates to the use of polypeptides from the *Bacillus terrestrialus* clade with aminohexosidase activity for preventing, reducing, or removing dirt adhesion to articles. In one embodiment, the article is a textile. When the dirt is not adhered to the article, the article appears cleaner. Therefore, the invention further relates to the use of polypeptides from the *Bacillus terrestrialus* clade with aminohexosidase activity for maintaining or improving the whiteness of the article.

[0204] When items (like T-shirts or sportswear) are used, they are exposed to bacteria from the user's body and from the rest of the environment in which they are used. This can cause an unpleasant odor even after washing the item. Therefore, this invention also relates to the removal or reduction of unpleasant odors on textiles. This odor may be caused by bacteria that produce compounds with unpleasant smells. An example of such compounds with unpleasant odors is E-2-nonenal. This odor can be present on freshly washed, still-wet textiles. Or the odor can be present on freshly washed, substantially-dried textiles. The odor can also be present on textiles that have been stored for a period of time after washing. This invention relates to reducing or removing odors, such as E-2-nonenal, from wet or dry textiles.

[0205] One aspect of the invention relates to the use of polypeptides of the Bacillus terrestrialus clade (e.g., comprising one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24)) in the following manner, wherein the polypeptide has aminohexosidase activity, preferably N-acetylglucosidase activity and / or β-N-acetylglucosidase activity, and wherein the polypeptide is associated with SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 18). The mature polypeptide shown in 11 has at least 60%, 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% sequence identity.

[0206] (i) Used to prevent, reduce or remove the stickiness of the article;

[0207] (ii) Used for pre-treating stains on articles;

[0208] (iii) Used to prevent, reduce or remove the redeposition of dirt during a washing cycle;

[0209] (iv) Used to prevent, reduce or remove dirt from adhering to the article;

[0210] (v) Used to maintain or improve the whiteness of the item;

[0211] (vi) Used to prevent, reduce or eliminate the odor of the item.

[0212] The item in question is a textile.

[0213] Cleaning agents, such as detergent compositions according to the invention, may contain at least one adjuvant component, which may be a surfactant and a builder and / or a chelating agent, such as those described above. The adjuvant component may also be any of the following flocculants: dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymerized dispersants, clay detergents / anti-reprecipitants, brighteners, antifoaming agents, dyes, fragrances, structural elastic agents, fabric softeners, carriers, water-soluble additives, builder and co-builder agents, fabric colorants, antifoaming agents, dispersants, processing aids, and / or pigments.

[0214] In one embodiment, the detergent adjuvant is a detergent builder or a clay detergent / anti-redeposition agent.

[0215] In one embodiment, the detergent adjuvant component is an enzyme. The one or more enzymes may be selected from the group consisting of: proteases, lipases, keratins, amylases, glycoses, cellulases, pectins, mannanases, arabinosylases, galactansylases, xylanases, and oxidases.

[0216] In addition to polypeptides having aminohexosidase activity, the present invention comprises polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11, or polypeptides having aminohexosidase activity and having at least 60% sequence identity thereof. The cleaning compositions of the present invention may further comprise cellulases. Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Suitable cellulases include cellulases from the genera *Bacillus*, *Pseudomonas*, *Pythium*, *Fusarium*, *Fusarium*, and *Cladosporium*, such as the fungal cellulases produced by *Pythium*, *Phytophthora*, and *Fusarium* disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259. Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0 495257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase polypeptides, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299. Examples of cellulases exhibiting endo-β-1,4-glucanase activity (EC 3.2.1.4) are those already described in WO 02 / 099091.Other examples of cellulases include family 45 cellulases described in WO 96 / 29397, and particularly those corresponding to SEQ ID NO. 02 / 099091. The polypeptide in 8 has substitutions, insertions, and / or deletions at one or more of the following positions: 2, 4, 7, 8, 10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119, 121, 133, 136, 13 7, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and / or 20, preferably selected from P19A, G20K, Q44K, N48E, Q119H, or Q146R. Commercially available cellulases include Celluzyme. TM Cellulean and Carezyme TM (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM (Kao Corporation).

[0217] In addition to polypeptides having aminohexosidase activity, the clean composition of the present invention also contains SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 or polypeptides having aminohexosidase activity and having at least 60% sequence identity therewith. The clean composition of the present invention may further contain cellulase. Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as plant or microbial origin. Microbial origin is preferred. This includes chemically modified mutants or protein-engineered mutants. It can be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases can be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from the M4 family or other metalloproteinases, such as those from the M5, M7, or M8 families. The term "subtilase" refers to the serine protease subgroup according to Siezen et al., Protein Engng. [Protein Engineering] 4 (1991) 719-737 and Siezen et al., Protein Science [Protein Science] 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by the presence of serine residues at their active sites that form covalent adducts with their substrates. Subtilases can be divided into six subfamilies: the subtilase family, the thermophilic protease family, the proteinase K family, the lanathionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family. Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus tarda*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and subtilisin protease lentus, subtilisin Novo, subtilisin Carlsberg, *Bacillus licheniformis*, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168 as described in WO 89 / 06279, and protease PD138 as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547.Examples of trypsin-like proteases are trypsin (e.g., from porcine or bovine sources) and Fusarium proteases (described in WO89 / 06270, WO94 / 25583 and WO 05 / 040372), as well as chymotrypsin derived from Cellumonas (described in WO05 / 052161 and WO05 / 052146). Further preferred proteases are alkaline proteases from Bacillus tarda DSM 5483 (as described, for example, in WO 95 / 23221), and their variants (described in WO 92 / 21760, WO 95 / 23221, EP1921147 and EP 1921148). Examples of metalloproteinases are neutral metalloproteinases as described in WO07 / 044993 (Genencor Int.), such as those derived from Bacillus amylolyticus.

[0218] Examples of useful proteases are variants described in the following: WO 92 / 19729, WO 96 / 034946, WO98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO 11 / 036264, especially those with SEQ ID NO of WO 2016 / 001449 at one or more of the following locations. 1. Compared to variants with substitutions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268, and 269, where these positions correspond to SEQ ID WO2016 / 001449. NO1 indicates the position of the delayed Bacillus protease. More preferred Bacillus subtilis enzyme variants may contain any of the following mutations: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N 120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, L256D, T268A or R269H. These protease variants are preferably the Bacillus retarder protease represented by SEQ ID NO 1 of WO 2016 / 001449. The variants, or variants of Bacillus amyloliquefaciens protease (BPN') shown in SEQ ID NO: 2 of WO 2016 / 001449. These protease variants preferably have at least 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 of WO 2016 / 001449.

[0219] The substituted protease variant is contained at one or more of the following positions, corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO 1 of WO 2004 / 067737, wherein the protease variant has at least 75% but less than 100% sequence identity with SEQ ID NO 1 of WO 2004 / 067737.

[0220] Suitable commercially available proteases include those sold under the following brand names: Duralase Tm Durazym Tm , Ultra Ultra Ultra Ultra Blzae 100T, Blaze 125T, Blaze 150T and (Novozymes A / S), those sold under the following trademarks: Purafect Purafect Excellenz P1000 TM Excellenz P1250 TM , Preferenz P100 TM Purafect Preferenz P110 TM Effectenz P1000 TM , Effectenz P1050 TM Purafect Effectenz P2000 TM , and (Danisco / DuPont), Axapem) TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US5352604) and its variants (Henkel AG) and KAP (Bacillus alkalophilus protease) from Kao Corporation.

[0221] In addition to polypeptides with aminohexosidase activity, the present invention also contains polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11, or polypeptides with aminohexosidase activity and having at least 60% sequence identity. The cleaning compositions of the present invention may further contain lipases and keratinases, including those of bacterial or fungal origin. Chemically modified or protein-engineered mutant enzymes are also included. Examples include, for instance, lipases from the genus *Thermomyces* (e.g., *T. lanuginosus*, formerly named *Humicola lanuginosa*) described in EP258068 and EP 305216; keratinases from the genus *Humicola* (e.g. *H. insolens* (WO 96 / 13580)); strains from the genus *Pseudomonas* (some of which are now renamed *Burkholderia*) (e.g. *P. alcaligenes* or *P. pseudoalcaligenes* (EP 218272)); *P. cepacia* (EP 331376); and *Pseudomonas* species strain SD705 (WO 96 / 13580). Lipases from *Pseudomonas wisinensis* (WO 96 / 12012), *Streptomyces* genus GDSL (WO 10 / 065455), *Magnaporthe grisea* (WO 10 / 107560), *Pseudomonas mendocina* (US 5,389,536), *Thermobifidafusca* (WO 11 / 084412), *Geobacillus stearothermophilus* (WO 11 / 084417), and *Bacillus subtilis* (WO 95 / 06720 & WO 96 / 27002), *Pseudomonas wisinensis* (WO 96 / 12012), *Streptomyces* genus GDSL (WO 10 / 065455), *Streptomyces* genus GDSL (WO 10 / 065455), *Streptomyces* genus GDSL (WO 10 / 107560), *Streptomyces* genus GDSL (WO 10 / 065455), *Streptomyces* genus GDSL (WO 10 / 107560), *Streptomyces* genus GDSL (WO 10 / 107560), *Streptomyces* genus GDSL (WO 10 / 107560), *Streptomyces* genus GDSL (WO 10 / 06545 ... Lipases from Streptomyces griseus (WO 11 / 084599) and Streptomyces pristinaespiralis (WO 12 / 137147).Other examples are lipase peptides, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508, and WO 09 / 109500. Preferred commercially available lipase products include Lipolase. TM Lipex TM Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades). Other examples are lipases sometimes called acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarctica lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and polypeptides of M. smegmatis perhydrolases (particularly the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd) (WO 10 / 100028).

[0222] In addition to polypeptides having aminohexosidase activity, the clean composition of the present invention also comprises SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, or polypeptides having aminohexosidase activity and having at least 60% sequence identity therewith. The clean composition of the present invention may further comprise an amylase, which can be used in conjunction with the polypeptides of the present invention. The amylase may be α-amylase or glucosylamylase and may be of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. The amylase includes, for example, α-amylase obtained from specific strains of Bacillus, such as Bacillus licheniformis (described in more detail in GB 1,296,839). Suitable amylases include the amylase having SEQ ID NO 3 in WO95 / 10603 or a polypeptide having 90% sequence identity with SEQ ID NO 3. Preferred polypeptides are described in SEQ ID NO 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424, and WO 99 / 019467, for example, polypeptides having 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. Various suitable amylases include amylases having SEQ ID NO 6 of WO 02 / 010355 or polypeptides having 90% sequence identity with SEQ ID NO 6. Preferred polypeptides of SEQ ID NO 6 are those with deletions at positions 181 and 182 and substitutions at position 193. Other suitable amylases are hybrid α-amylases comprising residues 1 to 33 of the α-amylase derived from *Bacillus amylolyticus* as shown in SEQ ID NO 6 of WO 2006 / 066594 and residues 36 to 483 of the α-amylase from *Bacillus licheniformis* as shown in SEQ ID NO 4 of WO 2006 / 066594, or polypeptides thereof having 90% sequence identity. Preferred polypeptides of such hybrid α-amylases are those with substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred polypeptide of the hybrid α-amylase comprising residues 1 to 33 of the α-amylase derived from Bacillus amylolyticus as shown in SEQ ID NO 6 of WO 2006 / 066594 and residues 36 to 483 of SEQ ID NO 4 is those having the following substitutions:

[0223] M197T;

[0224] H156Y+A181T+N190F+A209V+Q264S; or

[0225] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0226] Another suitable amylase is the amylase having SEQ ID NO 6 in WO 99 / 019467, or a polypeptide having 90% sequence identity with SEQ ID NO 6. Preferred polypeptides of SEQ ID NO 6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182 or positions H183 and G184. Other amylases that can be used are those having SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 2, or SEQ ID NO 7 in WO 96 / 023873, or polypeptides having 90% sequence identity with SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 7. Preferred polypeptides of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 7 are those having substitution, deletion, or insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred polypeptides are those having deletions in positions 181 and 182 or positions 183 and 184. The most preferred amylase polypeptide of SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 7 is those having deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304, and 476. Other usable amylases are SEQ ID NO 2 of WO 08 / 153815, SEQ ID NO 10 of WO 01 / 66712, or polypeptides thereof having 90% sequence identity with SEQ ID NO 2 of WO 08 / 153815 or 90% sequence identity with SEQ ID NO 10 of WO 01 / 66712. Preferred polypeptides of SEQ ID NO 10 of WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0227] Further suitable amylases are the amylase of SEQ ID NO 2 having WO 09 / 061380 or a polypeptide thereof having 90% sequence identity with SEQ ID NO 2. Preferred polypeptides of SEQ ID NO 2 are those having C-terminal truncation and / or substitution, deletion or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred polypeptides of SEQ ID NO:2 are those having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those having deletions at positions R180 and / or S181 or T182 and / or G183. Most preferred amylase polypeptides of SEQ ID NO:2 are those having the following substitutions:

[0228] N128C+K178L+T182G+Y305R+G475K;

[0229] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0230] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0231] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these peptides are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0232] Other suitable amylases are α-amylases having SEQ ID NO 12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO 12. Preferred amylase polypeptides are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include polypeptides having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, and additionally having substituted variants at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with most preferred variants having additional substitutions at all of these positions.

[0233] Other examples are amylase peptides, such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0234] Commercially available amylase is Duramyl TM Terminyl TM Fungayl TM Stainzyme TM StainzymePlus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase and Preferenz S100 (from Genencor International Inc. / DuPont).

[0235] In addition to polypeptides having aminohexosidase activity, the present invention comprises polypeptides of SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, or polypeptides having aminohexosidase activity and having at least 60% sequence identity thereof. The cleaning compositions of the present invention may further comprise peroxidases / oxidases, including those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *C. cinereus*, and polypeptides thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guardzyme. TM (Novozymes A / S).

[0236] The cleaning composition or the one or more of these ingredients can be included, for example, in a detergent composition by adding a single additive containing one or more enzymes, or by adding a combination of additives containing all of these enzymes. The detergent additives of the present invention, i.e., single or combined additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, particularly non-dust granules; liquids, particularly stabilized liquids; or slurries.

[0237] Non-dust particles can be generated, for example, as disclosed in US 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols having 12 to 20 carbon atoms and containing 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (e.g., propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP 238,216.

[0238] The detergent compositions of the present invention may also contain 0-10% by weight, for example 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 used. The polymer may function as a co-adjuvant as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or antifoaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), (carboxymethyl)inulin (CMI), and polycarboxylates (e.g., PAA, PAA / PMA), polyaspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxide terephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Further exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium salts of ethoxysulfonic acid. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered.

[0239] The cleaning compositions of the present invention may also include fabric colorants, such as dyes or pigments, which, when formulated in a detergent composition, can deposit on the fabric upon contact with a detergent solution containing the detergent composition, and thus alter the color of the fabric through the absorption / reflection of visible light. Fluorescent brighteners emit at least some visible light when exposed to ultraviolet light. In contrast, fabric colorants alter the color of the surface when they absorb at least a portion of the visible spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling under the Colour Index (CI) classification: 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 WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276 and EP 1876226 (incorporated herein by reference). The detergent composition preferably contains 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% of a fabric toner. The composition may contain from 0.0001 wt% to 0.2 wt% of a fabric toner, which may be particularly preferred when the composition is in the form of a unit-dosage pouch. Suitable toners are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0240] The product may further 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 water-soluble additive. Any water-soluble additive known in the art for use in detergents can be used. Non-limiting examples of water-soluble additives include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthoate, sodium ethylhexyl sulfonate, and combinations thereof.

[0241] The cleaning compositions of the present invention may also contain dispersants. Specifically, powdered detergents may contain dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc.

[0242] The cleaning composition may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole, or mixtures thereof. When present in the subject composition, the dye transfer inhibitor may be present at a level 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.

[0243] Preferably, the cleaning composition may also contain additional components that can color the item being cleaned, such as optical brighteners or fluorescent whitening agents. When present, the brightening agent is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in the clothing composition. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyrene derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents include sodium salts of the following: 4,4'-bis[(4-anilino-6-diethanolamino-s-triazin-2-yl)amino]stilbene-2,2'-disulfonate, 4,4'-bis[(4,6-diphenylamino-s-triazin-2-yl)amino]stilbene-2,2'-disulfonate, 4,4'-bis{4-anilino-6-[methyl(2-hydroxyethyl)amino]-s-triazin-2-ylamino}stilbene-2,2'-disulfonate, 4,4'-bis(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from BASF. Tinopal DMS is the disodium salt of 4,4'-bis[(4-anilino-6-morpholino-s-triazine-2-yl)amino]stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-[diphenyl-4,4'-bis(2,1-vinyldiyl)]dibenzyl ether-1-sulfonate. Also preferred is Parawhite KX, commercially available and supplied by Paramount Minerals and Chemicals in Mumbai, India. Other suitable fluorescent agents include 1,3-diarylpyrazoline and 7-alkylaminocoumarin.

[0244] Suitable levels of optical brighteners range from a low level of about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from a low level of about 0.2 wt% to a high level of 0.5 wt% or even 0.75 wt%.

[0245] The cleaning composition may also include one or more dirt-releasing polymers that help remove dirt from fabrics, such as cotton and polyester-based fabrics, particularly hydrophobic dirt from polyester-based fabrics. Dirt-releasing polymers can be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, or polyester polyamides; see, for example, Powdered Detergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Dekker, Inc. Another type of dirt-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and multiple alkoxylated groups attached to that core structure. The core structure may comprise a polyalkylimide structure or a polyalkanolamine structure, as described in detail, for example, in WO 2009 / 087523 (incorporated herein by reference). Furthermore, randomly bonded copolymers are suitable dirt-releasing polymers. Suitable conjugated copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (incorporated herein by reference). Other fouling-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, for example those described in EP 1867808 or WO 2003 / 040279 (both of which are hereby incorporated by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulose polymers include anionicly modified cellulose, nonionicly modified cellulose, cationicly modified cellulose, zwitterionicly modified cellulose and mixtures thereof.

[0246] The cleaning composition may also include one or more anti-redeposition agents, such as (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. Cellulose-based polymers described above under dirt-releasing polymers may also function as anti-redeposition agents.

[0247] The cleaning composition may also contain one or more excipients. Suitable excipients include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam conditioners, water-soluble solvents, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastic agents for liquid detergents.

[0248] The cleaning composition can be in any conventional form, such as strips, uniform tablets, tablets with two or more layers, bags with one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids.

[0249] The bag can be configured as a single compartment or multiple compartments. It can have any form, shape, and material suitable for containing the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane containing an internal volume. This internal volume can be divided into compartments of the bag. Preferred membranes are polymeric materials, preferably polymers forming films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference No. M8630, as sold by MonoSol LLC, Indiana, USA), plus a plasticizer, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The bag can contain a solid laundry detergent composition or portions thereof, and / or a liquid detergent composition or portions thereof separated by a water-soluble membrane. The chamber for the liquid component may be composed differently from the chamber containing the solid component: US 2009 / 0011970A1.

[0250] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the cleaning solution can cause delayed dissolution of selected components.

[0251] Non-unit-volume liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0 to 30% organic solvents.

[0252] The present invention also relates to methods of using the polypeptides or compositions thereof according to the invention in the washing of textiles and fabrics, such as household laundry and industrial laundry.

[0253] One aspect of the invention further relates to a method for washing articles, the method comprising:

[0254] a) Exposing the article to a detergent solution containing a polypeptide selected from the group consisting of the polypeptides shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, or exposing the article to a detergent composition according to any one of claims 1 to 14;

[0255] b) Complete at least one washing cycle; and

[0256] The item may be rinsed, wherein the item is a textile.

[0257] The present invention also relates to a method for using the polypeptides or combinations thereof according to the invention in cleaning hard surfaces such as floors, tables, walls, roofs, etc., as well as surfaces of hard objects such as automobiles (car washing) and tableware (dishwashing).

[0258] The polypeptides of the present invention can be added to detergent compositions and thus make them components of cleaning compositions. One aspect of the present invention relates to the use of polypeptides having aminohexosidase activity in cleaning processes (e.g., laundry washing and / or hard surface cleaning), the polypeptide optionally comprising one or more motifs selected from the group consisting of: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the polypeptide having aminohexosidase activity is 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 a polypeptide having at least 60% sequence identity with it.

[0259] Therefore, one aspect of the present invention relates to the use of polypeptides having aminohexosidase activity in cleaning processes (e.g., laundry washing and / or hard surface cleaning), the polypeptide optionally comprising one or more motifs selected from the group consisting of: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), NLD[DR]S (SEQ ID NO:24), wherein the polypeptide is 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 a polypeptide having at least 60% sequence identity with it, wherein the polypeptide has improved deep cleaning properties relative to the reference enzyme.

[0260] This cleaning or textile care process can be, for example, a laundry process, a dishwashing process, or the cleaning of hard surfaces (e.g., bathroom tiles, floors, countertops, drains, sinks, and basins). The laundry process can be, for example, household laundry, but it can also be industrial laundry. Furthermore, the present invention relates to a method for washing fabrics and / or clothing, wherein the method comprises treating the fabric with a washing solution containing a cleaning composition and at least one polypeptide of the present invention. For example, the cleaning or textile care process can be performed during machine cleaning or during manual washing. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0261] Therefore, the polypeptides contained in the Bacillus clade are particularly suitable for compositions containing surfactants such as detergent compositions, and the polypeptides of the present invention are preferably used in cleaning processes, such as laundry and dishwashing.

[0262] In some aspects, the present invention relates to polypeptides of clades of Bacillus terrestris that have at least 60%, for example 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% sequence identity with the mature polypeptide of SEQ ID NO 2, and these polypeptides possess aminohexosidase activity. In one aspect, these polypeptides differ from the mature polypeptide shown in SEQ ID NO 7 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0263] In some aspects, the present invention relates to polypeptides of clades of Bacillus terrestris that have at least 60%, for example 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% sequence identity with the mature polypeptide of SEQ ID NO 4, and these polypeptides possess aminohexosidase activity. In one aspect, these polypeptides differ from the mature polypeptide shown in SEQ ID NO 8 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0264] In some aspects, the present invention relates to polypeptides of clades of Bacillus terrestris that have at least 60%, for example, 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% sequence identity with the mature polypeptide of SEQ ID NO 6, and these polypeptides possess aminohexosidase activity. In one aspect, these polypeptides differ from the mature polypeptide shown in SEQ ID NO 9 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0265] In some aspects, the present invention relates to polypeptides of clades of Bacillus terrestris that have at least 60%, for example 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% sequence identity with the mature polypeptide of SEQ ID NO 13, and these polypeptides possess aminohexosidase activity. In one aspect, these polypeptides differ from the mature polypeptide shown in SEQ ID NO 10 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0266] In some aspects, the present invention relates to polypeptides of clades of Bacillus terrestris that have at least 60%, for example 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% sequence identity with the mature polypeptide of SEQ ID NO 14, and these polypeptides possess aminohexosidase activity. In one aspect, these polypeptides differ from the mature polypeptide shown in SEQ ID NO 11 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0267] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO 7 or its allelic variant; or is a fragment thereof having aminohexosidase activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO 2. In yet another embodiment, the polypeptide comprises or consists of amino acids 1 to 324 of SEQ ID NO 2.

[0268] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO 8 or its allelic variant; or is a fragment thereof having aminohexosidase activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO 4. In yet another embodiment, the polypeptide comprises or consists of amino acids 1 to 324 of SEQ ID NO 4.

[0269] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO 9 or its allelic variant; or is a fragment thereof having aminohexosidase activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO 6. In yet another embodiment, the polypeptide comprises or consists of amino acids 1 to 324 of SEQ ID NO 6.

[0270] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO 10 or its allelic variant; or is a fragment thereof having aminohexosidase activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO 13. In yet another embodiment, the polypeptide comprises or consists of amino acids 1 to 324 of SEQ ID NO 13.

[0271] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO 11 or its allelic variant; or is a fragment thereof having aminohexosidase activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO 15. In yet another embodiment, the polypeptide comprises or consists of amino acids 1 to 324 of SEQ ID NO 15.

[0272] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity encoded by a polynucleotide that hybridizes under very low stringent, low stringent, medium stringent, medium-high stringent, high stringent, or very high stringent conditions to: (i) the mature polypeptide coding sequence of SEQ ID NO 1, 3, 5, 12, or 14, or (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0273] Nucleic acid probes can be designed using polypeptides or subsequences thereof from SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 12, or SEQ ID NO 14, together with polypeptides or fragments thereof from SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13, or SEQ ID NO 15, to identify and clone DNA encoding polypeptides with aminohexosidase activity from strains of different genera or species, according to methods well known in the art. Such probes can be used to hybridize with genomic DNA or cDNA of cells of interest, following standard DNA blotting procedures, to identify and isolate the corresponding genes therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, such as at least 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probe is labeled (e.g., with...). 32 P, 3 H, 35 Probes (such as biotin or avidin) are used to detect corresponding genes. Such probes are covered in this invention.

[0274] DNA from genomic DNA or cDNA libraries prepared from other strains of this type that hybridizes to the probes described above and encodes polypeptides with aminohexosidase activity can be screened. Genomic DNA or other DNA from other strains of this type can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier materials. The carrier material is used for DNA blotting to identify clones or DNA that hybridize to SEQ ID NO 1, 3, 5, 12 or 14 or their subsequences.

[0275] For the purposes of this invention, hybridization indicates that the polynucleotide hybridizes with a labeled nucleic acid probe corresponding to any of the following under very low to very high stringency conditions: (i) the coding sequence of the mature polypeptide of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 12, or SEQ ID NO 14; (ii) the coding sequence of the mature polypeptide of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 12, or SEQ ID NO 14; (iii) its full-length complement; or (iv) its subsequence; the hybridization is performed under very low to very high stringency conditions. Molecules hybridized under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0276] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity, the polypeptide being encoded by a polynucleotide having at least 60%, for example 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 1. In another embodiment, the polypeptide has been isolated.

[0277] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity, the polypeptide being encoded by a polynucleotide having at least 60%, for example 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 3. In another embodiment, the polypeptide has been isolated.

[0278] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity, the polypeptide being encoded by a polynucleotide having at least 60%, for example 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 5. In another embodiment, the polypeptide has been isolated.

[0279] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity, the polypeptide being encoded by a polynucleotide having at least 60%, such as 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 12. In another embodiment, the polypeptide has been isolated.

[0280] In another embodiment, the present invention relates to a polypeptide having aminohexosidase activity, the polypeptide being encoded by a polynucleotide having at least 60%, such as 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 14. In another embodiment, the polypeptide has been isolated.

[0281] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13, or SEQ ID NO 15, which include substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one embodiment, up to 10 amino acid substitutions, deletions, and / or insertions are introduced into the mature polypeptide of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, or SEQ ID NO 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1 to 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as methionine residues at the amino terminus; small linker peptides of up to 20 to 25 residues; or small extensions that facilitate purification by altering net charge or another function (e.g., polyhistidine fragments, antigenic epitopes, or binding domains). Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0282] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the thermal stability of the peptide, change its substrate specificity, change its optimal pH, etc. Essential amino acids in a peptide 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, a single alanine mutation is introduced at each residue in the molecule, and the aminohexosidase activity of the resulting molecule is tested to identify the amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations in the amino acids at the putative contact sites. 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 comparison with related polypeptides. The polypeptide of the present invention is an aminohexosidase, for example, a dispersible protein belonging to the Bacillus tumefaciens clade. The Bacillus tumefaciens dispersible protein is a β-aminohexosidase that specifically hydrolyzes the β-1,6-glycosidic bonds of N-acetylglucosamine polymers, which can be found, for example, in biological membranes. Dispersed protein B belongs to a different evolutionary branch than the polypeptide (aminohexosidase) of this invention and contains three highly conserved acidic residues: aspartic acid at residue 183 (D183), glutamic acid at residue 184 (E184), and glutamic acid at residue 332 (E332). Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, 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 Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127). Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of mutagenic peptides expressed by clones from host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the peptide. The peptide can be a hybrid peptide, in which a region of one peptide is fused to the N-terminus or C-terminus of a region of another peptide.

[0283] The polypeptide can be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with the polynucleotide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intronomer technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, EMBO J. [Journal of the European Society for Molecular Biology] 12:2575-2583; Dawson et al., 1994, Science 266:776-779). The fusion polypeptide may further include a cleavage site between the two polypeptides. This site is cleaved upon secretion of the fusion protein, releasing both polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3:568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins; Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0284] Sources of peptides with aminohexosidase activity

[0285] The polypeptides of the present invention having aminohexosidase activity can be obtained from microorganisms of any genus. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0286] In another aspect, the polypeptide is a Bacillus terrestris polypeptide, for example, a polypeptide obtained from Bacillus saccharophilus. In a preferred aspect, the polypeptide is a polypeptide having at least 80% sequence identity with SEQ ID NO 7 and is obtained from Bacillus terrestris, preferably Bacillus saccharophilus.

[0287] In another aspect, the polypeptide is a Terribacillus polypeptide, for example, a polypeptide obtained from Terribacillus goriensis. In a preferred aspect, the polypeptide is a polypeptide having at least 80% sequence identity with SEQ ID NO 8 and is obtained from Terribacillus, preferably Terribacillus goriensis.

[0288] In another aspect, the polypeptide is a Bacillus terrestris polypeptide, for example, a polypeptide obtained from Bacillus saccharophilus. In a preferred aspect, the polypeptide is a polypeptide having at least 80% sequence identity with SEQ ID NO 9 and is obtained from Bacillus terrestris, preferably Bacillus saccharophilus.

[0289] In another aspect, the polypeptide is a Bacillus terrestris polypeptide, for example, a polypeptide obtained from Bacillus saccharophilus. In a preferred aspect, the polypeptide is a polypeptide having at least 80% sequence identity with SEQ ID NO 10 and is obtained from Bacillus terrestris, preferably Bacillus saccharophilus.

[0290] In another aspect, the polypeptide is a Bacillus terrestris polypeptide, for example, a polypeptide obtained from Bacillus saccharophilus. In a preferred aspect, the polypeptide is a polypeptide having at least 80% sequence identity with SEQ ID NO 11 and is obtained from Bacillus terrestris, preferably Bacillus saccharophilus.

[0291] It should be understood that, for the aforementioned species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0292] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial and Cell Culture Collection (DSMZ), the Centralbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0293] The polypeptide can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from natural habitats are known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide has been detected with one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0294] Nucleic acid constructs

[0295] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences, which, under conditions compatible with the control sequences, guide the expression of a coding sequence in a suitable host cell.

[0296] The polynucleotide can be manipulated in many ways to provide polypeptide expression. Depending on the expression vector, manipulating the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art.

[0297] The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expressing a polynucleotide encoding the polypeptide of the present invention. The promoter contains a transcriptional control sequence that mediates the expression of the polypeptide. The promoter may be any polynucleotide exhibiting transcriptional activity in the host cell, including variants, truncated and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0298] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, and Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular). Microbiology [Molecular Microbiology] 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene [Gene] 69:301-315), Streptomyces agar hydrolase gene (dagA) and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, see above. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0299] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters derived from the genes of the following enzymes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amylase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiase I, Trichoderma reesei cellobiase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor, and NA2-tpi Promoters (modified promoters of Aspergillus neutral α-amylase genes, wherein the untranslated leader sequence has been replaced with an untranslated leader sequence of Aspergillus triose phosphate isomerase genes; non-limiting examples include modified promoters of Aspergillus niger neutral α-amylase genes, wherein the untranslated leader sequence has been replaced with an untranslated leader sequence of Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase genes); and variants, truncated and heterozygous promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0300] In yeast hosts, useful promoters are derived from the genes of the following enzymes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, Yeast 8:423-488, describe other useful promoters in yeast host cells.

[0301] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operatively linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in this invention.

[0302] The preferred terminator for bacterial host cells is obtained from the following genes: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0303] Preferred terminators for filamentous fungal host cells are derived from the genes of the following enzymes: Aspergillus nidulans acetamase, Aspergillus nidulans o-aminobenzoic acid synthase, Aspergillus niger glucosylamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor. Preferred terminators for yeast host cells are derived from the genes of the following enzymes: *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells were described by Romanos et al., 1992 (see above). The control sequence can also be a stabilizer region of mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene. Examples of suitable mRNA stabilizer regions are derived from the *Bacillus thuringiensis* cryIIIA gene (WO 94 / 25612) and the *Bacillus subtilis* SP82 gene (Hue et al., 1995, *Journal of Bacteriology* 177:3465-3471). The control sequence can also be a leader, i.e., a non-translated region of mRNA important for translation in the host cell. This leader is operatively linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used. Preferred leader sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Control sequences can also be polyadenylated sequences, signal sequences operatively linked to the 3'-end of a polynucleotide and recognized by the host cell during transcription as adding polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that functions in the host cell can be used. Preferred polyadenylated sequences for filamentous fungal host cells are obtained from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosylamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease. The polyadenylation sequence useful for yeast host cells is described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15:5983-5990.

[0304] The control sequence can also be a signal peptide coding region encoding a signal peptide that is linked to the N-terminus of the polypeptide and guides the polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence naturally linked to a segment of the coding sequence of the polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a foreign signal peptide coding sequence may be required. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. However, any signal peptide coding sequence that guides the secretory pathway of the expressed polypeptide into the host cell can be used.

[0305] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of *Bacillus NCIB 11837* maltose amylase, *Bacillus licheniformis* subtilis protease, *Bacillus licheniformis* β-lactamase, *Bacillus thermophilus* α-amylase, *Bacillus thermophilus* neutral protease (nprT, nprS, nprM), and *Bacillus subtilis* prsA. Additional signal peptides are described by Simonen and Palva, 1993, *Microbiological Reviews* 57:109-137. Effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of the following enzymes: *Aspergillus niger* neutral amylase, *Aspergillus niger* glucosylamylase, *Aspergillus oryzae* TAKA amylase, *Porcine spp.* cellulase, *Porcine spp.* endoglucanase V, *Porcine spp.* lipase, and *Rhizomucor miehei* aspartic protease. Useful signal peptides for yeast host cells are derived from the genes for *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Sequences encoding other useful signal peptides are described above by Romanos et al., 1992.

[0306] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. Propeptide-coding sequences can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae α-factor.

[0307] When both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence.

[0308] Alternatively, regulatory sequences may be added that regulate the expression of host cell growth-related polypeptides. Examples of regulatory sequences are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter and *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide is operatively linked to the regulatory sequence.

[0309] expression carrier

[0310] The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Multiple nucleotides and control sequences may be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0311] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0312] The vector can be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0313] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or protrophic traits such as auxotrophy.

[0314] Examples of selective markers for bacteria include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for filamentous fungal host cells include, but are not limited to: adeA (phosphoribonucleoylaminoimidazolium-succinate formamide synthase), adeB (phosphoribonucleoylaminoimidazolium synthase), amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate acyltransferase), and trpC (o-aminobenzoic acid synthase), and their equivalents. The amdS and pyrG genes of *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene of *Streptomyces hygroscopicus* are preferably used in *Aspergillus* cells. In *Trichoderma* cells, the adeA, adeB, amdS, hph, and pyrG genes are preferred.

[0315] The selective marker can be a biselective marker system as described in WO 2010 / 039889. In one aspect, the biselective marker is the hph-tk biselective marker system.

[0316] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0317] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, the integrating element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have a high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. The integrating element can be any sequence homologous to the target sequence within the host cell genome. Furthermore, the integrating element can be a non-coding polynucleotide or a coding polynucleotide. Alternatively, the vector can be integrated into the host cell genome via non-homologous recombination.

[0318] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0319] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0320] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0321] Examples of origins of replication used in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res 15:9163-9175; WO 00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0322] More than one copy of the polynucleotide of the present invention can be inserted into host cells to increase polypeptide production. An increased 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 selective marker gene along with the polynucleotide, wherein cells containing amplified copies of the selective marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selective reagent.

[0323] The methods for connecting the elements described above to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0324] host cells

[0325] This invention also relates to recombinant host cells containing polynucleotides of the invention operably linked to one or more control sequences, which direct the production of the polypeptides of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend to a large extent on the gene encoding the polypeptide and its origin.

[0326] The host cell can be any cell useful in the recombinant production of the polypeptides of the present invention, such as prokaryotic or eukaryotic cells.

[0327] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Staphylococcus, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0328] The bacterial host cell can be any Bacillus genus cell, including but not limited to: Bacillus alkalophilus, Bacillus albopictus, Bacillus amyloliquefaciens, subspecies of Bacillus amyloliquefaciens plantarum, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sclerotiorum, Bacillus splenicum, Bacillus stenosis, Bacillus licheniformis, Bacillus megaterium, Bacillus methyltrophicus, Bacillus brevis, Bacillus saforticus, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0329] The bacterial host cell can also be any streptococcal cell, including but not limited to: Streptococcus equi, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equi subsp. veterinaryis.

[0330] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces and light blue Streptomyces cells.

[0331] DNA can be introduced into Bacillus cells through the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. [Molecular Genetics and Genomics] 168:111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. [Journal of Bacteriology] 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. [Journal of Molecular Biology] 56:209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques [Biotechnology] 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. [Journal of Bacteriology] 169:5271-5278). DNA can be introduced into E. coli cells by protoplast transformation (see, for example, Hanahan, 1983, J. Mol. Biol. [Journal of Molecular Biology] 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. [Nucleic Acid Research] 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).DNA can be introduced into Streptococcus cells by means of natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0332] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0333] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al., in: Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, International CAB, University Press, Cambridge, UK).

[0334] The fungal host cell may be a yeast cell. As used herein, “yeast” includes *Entomophycetes* (Endomophytes), *Basidiophycetes*, and yeasts belonging to the class Deuteromycetes (Bacillus). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *The Biology and Activity of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0335] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Kluyveromyces*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Saccharomyces douglas*, *Kluyveromyces*, *Nordiya*, *Ovoyces*, or *Yarrowia lipolytica*.

[0336] The host cell of fungi can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms within the subphyla of Eumycota and Oomycota (as described by Hawksworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeast (e.g., Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentative.

[0337] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Pleurotus*, *Pleurotus*, *Schizophyllum*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Plebia*, *Pleurotus*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophila*, *Fusarium*, *Pleurotus*, *Trametes*, or *Trichoderma*.

[0338] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium lucknowense*, and *Chrysosporium foetida*. merdarium), rent spores, Chrysosporium queenslandicum, tropical golden spores, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Fusarium moniliforme, Fusarium cereals, Fusarium kuwai, Fusarium scabra, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium oxy ... radiata), Pleurotus eryngii, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0339] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in the following literature: EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81:1470-1474 and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene [Gene] 78:147-156 and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0340] Generation method

[0341] The present invention also relates to methods for producing the polypeptides of the invention, the methods comprising (a) culturing a cell under conditions favorable to the production of the polypeptide, the cell producing the polypeptide in its wild-type form; and optionally (b) recovering the polypeptide. In one aspect, the cell is a *Bacillus terrestris* cell. In another aspect, the cell is a *Bacillus saccharophilus* cell. In one aspect, the cell is a *Bacillus terrestris* cell. In another aspect, the cell is a *Terribacillus goriensis* cell.

[0342] The present invention also relates to a method for producing the polypeptide of the invention, comprising (a) culturing the recombinant host cell of the invention under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0343] These host cells are cultured in a nutrient medium suitable for producing these peptides using methods known in the art. For example, cells can be cultured by shake-flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, said culture being carried out in a suitable medium and under conditions that allow for peptide expression and / or isolation. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable culture media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the peptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0344] The polypeptide can be detected using methods known in the art that are specific to polypeptides exhibiting aminohexosidase activity. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the polypeptide.

[0345] Peptides can be recovered using methods known in the art. For example, peptides can be recovered from nutrient media by conventional methods, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, fermentation broth containing peptides is recovered.

[0346] Peptides can be purified to obtain substantially pure peptides by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatographic focusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0347] In an alternative aspect, instead of recovering the polypeptide, the host cell of the present invention expressing the polypeptide is used as the source of the polypeptide.

[0348] Detergent product formulations

[0349] The cleaning composition can be in any conventional form, such as strips, uniform tablets, tablets with two or more layers, bags with one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids. The bag can be configured as a single-compartment or multi-compartment bag. It can have any form, shape, and material suitable for containing the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane containing an internal volume. This internal volume can be divided into compartments of the bag. Preferred membranes are polymeric materials, preferably polymers forming films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference No. M8630, such as sold by MonoSol LLC, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may contain solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may differ in composition from those containing solids: US 2009 / 0011970A1.

[0350] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the cleaning solution can cause delayed dissolution of selected components.

[0351] Non-unit-volume liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0 to 30% organic solvents.

[0352] Liquid or gel detergents can be non-aqueous.

[0353] Laundry soap bars

[0354] The polypeptides of this invention can be added to laundry soap bars and used for hand washing, fabric washing, and / or textile washing. The term "laundry soap bar" includes laundry bars, soap bars, combo bars, synthetic detergent bars, and detergent bars. The types of bars are generally distinguished by the type of surfactant they contain, and the term "laundry soap bar" includes those containing soaps derived from fatty acids and / or synthetic soaps. Laundry soap bars have a physical form that is solid at room temperature, rather than liquid, gel, or powder. The term "solid" is defined as a physical form that does not change significantly over time, i.e., if a solid object (e.g., a laundry soap bar) is placed in a container, the solid object will not change in order to fill the container in which it is placed. The bar is typically in the form of a bar when it is solid, but it can also be other solid shapes such as round or oval. The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or sulfide adducts or hemiacetal adducts), boric acid, borates, borax and / or phenylboronic acid derivatives such as 4-carboxylic acid, one or more soaps or synthetic surfactants, polyols such as glycerol, pH-controlling compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or salts of monovalent cations and organic anions, wherein the monovalent cation may be, for example, Na+. + K + or NH4 + Furthermore, the organic anion can be, for example, a formate, acetate, citrate, or lactate, such that the salt of the monovalent cation and the organic anion can be, for example, sodium formate. Laundry soap bars may also contain complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants such as anionic synthetic surfactants, builders, polymerized stain release agents, detergent chelating agents, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachates, bleaching activators, clay detergents, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances, and / or other compounds known in the art. Laundry soap bars can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, pressing machines such as two-stage vacuum pressing machines, extruders, cutters, logo-stamper machines, cooling tunnels, and packaging machines. This invention is not limited to preparing laundry soap bars by any single method. Premixes can be added to the soap at different stages of the process. For example, a premix comprising soap, aminohexosidase, optionally one or more other enzymes, protease inhibitors, and salts of monovalent cations and organic anions can be prepared, and then the mixture can be pressed into strips. Aminohexosidase, as a protease inhibitor in liquid form, and optionally other enzymes can be added simultaneously. In addition to the mixing and pressing steps, the process may further include grinding, extrusion, cutting, molding, cooling, and / or packaging steps.

[0355] Enzyme formulations in co-particles

[0356] The polypeptides of the present invention can be formulated as particles, for example, as co-particles incorporating one or more enzymes. Each enzyme will then be present in multiple particles, ensuring a more uniform distribution of the enzyme in the detergent. This also reduces the physical isolation of different enzymes due to varying particle sizes. A method for producing multi-enzyme co-particles for the detergent industry is disclosed in IP.com Publication IPCOM000200739D. Another example of an enzyme formulation using co-particles is disclosed in WO2013 / 188331, which relates to a detergent composition comprising: (a) multi-enzyme co-particles; (b) less than 10 wt% zeolite (anhydrous base); and (c) less than 10 wt% phosphate (anhydrous base), wherein the enzyme co-particles contain a water pool component from 10 wt% to 98 wt%, and the composition further contains a detergent water pool component from 20 wt% to 80 wt%.

[0357] The multi-enzyme co-particle may contain an aminohexosidase and one or more enzymes selected from the group consisting of: lipase, cellulase, xyloglucanase, hydrolase, peroxidase, lipoxygenase, laccase, hemicellulase, protease, cellobiose dehydrogenase, xylanase, phospholipase, esterase, keratinase, pectinase, mannanase, pectic acid lyase, keratinase, reductase, oxidase, phenol oxidase, ligninase, amylopectinase, tannic acidase, pentosanase, lichen polysaccharidease, glucanase, arabinosidase, hyaluronidase, chondroitinase, amylase, and mixtures thereof.

[0358] The invention is further summarized in the following paragraphs:

[0359] 1. The use of a polypeptide with aminohexosidase activity for deep cleaning articles, the polypeptide preferably comprising one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), or NLD[DR]S (SEQ ID NO:24), wherein the article is a textile.

[0360] 2. As described in paragraph 1, for preventing, reducing or removing the stickiness of an article.

[0361] 3. For the purpose of pretreating stains on the article according to any one of paragraphs 1 or 2.

[0362] 4. For use according to any one of paragraphs 1-3, for preventing, reducing or removing the redeposition of dirt during a washing cycle.

[0363] 5. For the purpose of any one of paragraphs 1-4, for preventing, reducing or removing dirt from adhering to articles.

[0364] 6. For the purpose of maintaining or improving the whiteness of the article as described in any of the preceding paragraphs.

[0365] 7. The use according to any one of the preceding paragraphs, wherein odors are reduced or eliminated from the article.

[0366] 8. The use according to any one of the above composition paragraphs, wherein the surface is a textile surface.

[0367] 9. The use according to any one of the above composition paragraphs, wherein the textile is made of cotton, cotton / polyester, polyester, polyamide, polypropylene and / or silk.

[0368] 10. The use according to any one of the preceding paragraphs, wherein the polypeptide is the polypeptide as described in paragraphs 47-61.

[0369] 11. A composition comprising a polypeptide having aminohexosidase activity, preferably comprising one or more of the following motifs: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), or NLD[DR]S (SEQ ID NO:24), and an adjuvant component.

[0370] 12. The composition according to paragraph 11, wherein the polypeptide is the polypeptide as described in paragraphs 47-61.

[0371] 13. The composition according to any one of the above composition paragraphs, wherein the detergent adjuvant component is selected from the group consisting of: surfactants, detergent builders, flocculants, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymerized dispersants, clay detergents / anti-reprecipitants, brighteners, defoamers, dyes, fragrances, structural elastic agents, fabric softeners, carriers, water-soluble additives, detergent builders and co-detergent builders, fabric colorants, antifoaming agents, dispersants, processing aids, and / or pigments.

[0372] 14. The composition according to any one of the preceding composition paragraphs, wherein the composition comprises from about 5 wt% to about 50 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 20 wt%, from about 5 wt% to about 10 wt% of an anionic surfactant, preferably selected from linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates. Salts, alkyl sulfates (AS) (e.g., sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES, AEOS, or FES), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or fatty acid salts (soaps), and combinations thereof.

[0373] 15. The composition according to any one of the preceding composition paragraphs, wherein the composition comprises from about 10 wt% to about 50 wt% of at least one builder, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and mixtures thereof.

[0374] 16. The composition according to any one of the preceding composition paragraphs, wherein the polypeptide having aminohexosidase activity is selected from the group consisting of: polypeptides having the amino acid sequences of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and polypeptides having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with them.

[0375] 17. The composition according to any one of the preceding composition paragraphs, wherein the polypeptide has aminohexosidase activity, is the amino acid sequence of SEQ ID NO 7 or a polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it.

[0376] 18. The composition according to any one of paragraphs 11-16, wherein the polypeptide has aminohexosidase activity and is the amino acid sequence of SEQ ID NO 8 or a polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it.

[0377] 19. The composition according to any one of paragraphs 11-16, wherein the polypeptide has aminohexosidase activity and is the amino acid sequence of SEQ ID NO 9 or a polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it.

[0378] 20. The composition according to any one of paragraphs 11-16, wherein the polypeptide has aminohexosidase activity and is the amino acid sequence of SEQ ID NO 10 or a polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it.

[0379] 21. The composition according to any one of paragraphs 11-16, wherein the polypeptide has aminohexosidase activity and is the amino acid sequence of SEQ ID NO 11 or a polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98% or 99% sequence identity with it.

[0380] 22. The composition according to any one of the preceding paragraphs comprises from about 5 wt% to about 40 wt% of a nonionic surfactant and from about 0 wt% to about 5 wt% of an anionic surfactant.

[0381] 23. The composition according to paragraph 22, wherein the nonionic surfactant is selected from: alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (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 (glucosamide (GA), or fatty acid glucosamide (FAGA)) and combinations thereof.

[0382] 24. The composition according to any one of the preceding composition paragraphs, wherein the composition further comprises one or more enzymes selected from the group consisting of: proteases, lipases, keratins, amylases, glycoses, cellulases, pectins, mannanases, arabinases, galactans, xylanases, and oxidases.

[0383] 25. The composition according to any one of the above composition paragraphs, wherein the enzyme is a protease of animal, plant or microbial origin.

[0384] 26. The composition according to any one of the above composition paragraphs, wherein the protease is a chemically modified protease or a protein-engineered protease.

[0385] 27. The composition according to any one of the above composition paragraphs, wherein the protease is a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.

[0386] 28. The composition according to any one of the above composition paragraphs, wherein the protease is selected from the group consisting of: Bacillus species, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, subtilisin 168, bovine trypsin, porcine trypsin and Fusarium protease.

[0387] 29. The composition according to any one of the preceding composition paragraphs, wherein the composition is capable of reducing EPS adhesion of bacteria selected from the group consisting of: Acinetobacter species, Aerobicmyli species, Shortwave Monoclonal species, Microbacterium species, Micrococcus flavonoids, Pseudomonas species, Staphylococcus epidermidis, Staphylococcus aureus, and Oligotrophomonas species, or capable of releasing bacteria from the surface to which they adhere.

[0388] 30. The composition according to any one of the preceding composition paragraphs, wherein the composition is a strip, a uniform tablet, a tablet having two or more layers, a bag having one or more chambers, a regular or compressed powder, granules, paste, gel, or a regular, compressed or concentrated liquid.

[0389] 31. The composition according to any one of the preceding composition paragraphs, wherein the composition is a cleaning composition selected from liquid detergent, powder detergent and granular detergent compositions.

[0390] 32. A method for washing articles, the method comprising the following steps:

[0391] a. Exposing an article to a washing solution containing a polypeptide as described in paragraphs 46-56 or a composition according to any one of paragraphs 11-31;

[0392] b. Complete at least one washing cycle; and

[0393] c. Optional: Rinse the item.

[0394] The item in question is a textile.

[0395] 33. A method for deep cleaning an article, preferably a textile, the method comprising: exposing the article to a detergent containing a polypeptide, the polypeptide preferably comprising one or more of the motifs GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), or NLD[DR]S (SEQ ID NO:24), wherein the polypeptide is selected from polypeptides having at least 60%, for example 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with polypeptides shown in the following group, which comprises: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 18, SEQ ID NO 19 ... 10 and SEQ ID NO 11.

[0396] 34. The method according to paragraph 32 or 33, wherein the pH of the washing solution is in the range of 1 to 11.

[0397] 35. The method according to any one of the preceding method paragraphs, wherein the pH of the washing solution is in the range of 5.5 to 11, such as in the range of 7 to 9, in the range of 7 to 8, or in the range of 7 to 8.5.

[0398] 36. The method according to any one of the preceding method paragraphs, wherein the temperature of the washing liquid is in the range of 5°C to 95°C, or in the range of 10°C to 80°C, in the range of 10°C to 70°C, in the range of 10°C to 60°C, in the range of 10°C to 50°C, in the range of 15°C to 40°C, in the range of 20°C to 40°C, in the range of 15°C to 30°C, or in the range of 20°C to 30°C.

[0399] 37. The method according to any one of the preceding method paragraphs, wherein the temperature of the washing liquid is from about 20°C to about 40°C.

[0400] 38. The method according to any one of the preceding paragraphs, wherein the temperature of the washing solution is about 15°C to about 30°C.

[0401] 39. The method according to any one of the preceding paragraphs, wherein the stains present on the article are pretreated with a polypeptide as described in paragraphs 46-60 or a detergent composition according to any one of paragraphs 11-31.

[0402] 40. The method according to any one of the preceding paragraphs, wherein the adhesiveness of the article is reduced.

[0403] 41. The method according to any one of the preceding method paragraphs, wherein redeposition of dirt is reduced.

[0404] 42. The method according to any one of the preceding paragraphs, wherein the adhesion of dirt to the article is reduced or removed.

[0405] 43. The method according to any one of the preceding method paragraphs, wherein the whiteness of the article is maintained or improved.

[0406] 44. The method according to any one of the preceding paragraphs, wherein odor is reduced or removed from the article.

[0407] 45. The method according to any one of the preceding method paragraphs, wherein the concentration of the polypeptide having aminohexosidase activity in the washing solution is at least 0.001 mg of polypeptide per liter of washing solution, for example at least 0.05 mg of protein, or at least 1.0 mg of protein, or at least 1.5 mg of protein, optionally the concentration of the polypeptide in the washing solution is in the range of 0.0002 mg / L to 2 mg / L, for example 0.002 mg / L to 2 mg / L, for example 0.2 mg / L to 2 mg / L, or in the range of 0.00001 mg / L to 10 mg / L. The concentration of the polypeptide of the present invention may be 0.00001% to 2 wt% in the total detergent concentration, for example, 0.0001 to 0.1 wt%, 0.0005 to 0.1 wt%, 0.001 to 0.1 wt%, 0.001 to 0.5 wt%, 0.002 to 0.5 wt%, or 0.0002 to 0.09 wt%.

[0408] 46. ​​A polypeptide having aminohexosidase activity, the polypeptide being selected from the group consisting of:

[0409] a. A polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the mature polypeptide shown in SEQ ID NO 7, 8, 9, 10, or 11;

[0410] b. A polypeptide encoded by the following polynucleotide, which hybridizes with the following under low stringency conditions:

[0411] i. The mature polypeptide coding sequence of SEQ ID NO 1, 3, 5, 12 or 14,

[0412] ii. its cDNA sequence, or

[0413] iii. The full-length complement of (i) or (ii);

[0414] c. A polypeptide having at least 60%, for example 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the coding sequence of a mature polypeptide of SEQ ID NO 1, 3, 5, 12, or 14 or its cDNA sequence;

[0415] d. Variants of the mature polypeptide of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11, wherein the variant comprises substitution, deletion, and / or insertion at one or more sites; and

[0416] e. A fragment of the polypeptide of (a), (b), (c), or (d) that has aminohexosidase activity; and

[0417] f. A polypeptide comprising one or more of the following motifs: GXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO:20), WND[SQR][IVL][TLVM] (SEQ ID NO:21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO:23), and NLD[DR]S (SEQ ID NO:24).

[0418] 47. The polypeptide described in paragraph 46, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13 or SEQ ID NO 15 or with the polypeptide shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11.

[0419] 48. The polypeptide as described in paragraph 46 or 47, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 2 or the mature polypeptide shown in SEQ ID NO 7.

[0420] 49. The polypeptide as described in paragraph 46 or 47, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 4 or the mature polypeptide shown in SEQ ID NO 8.

[0421] 50. The polypeptide as described in paragraph 46 or 47, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 6 or the mature polypeptide shown in SEQ ID NO 9.

[0422] 51. The polypeptide as described in paragraph 46 or 47, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 13 or the mature polypeptide shown in SEQ ID NO 10.

[0423] 52. The polypeptide as described in paragraph 46 or 47, having at least 60%, 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% sequence identity with the mature polypeptide of SEQ ID NO 15 or the mature polypeptide shown in SEQ ID NO 11.

[0424] 53. The polypeptide described in paragraphs 46 to 52, wherein the polypeptide is encoded by a polynucleotide that hybridizes with the following under low-strict, low-to-medium-strict, medium-strict, medium-to-high-strict, high-strict, or very high-strict conditions:

[0425] i. The mature polypeptide coding sequence of SEQ ID NO 1, 3, 5, 12 or 14,

[0426] ii. its cDNA sequence, or

[0427] iii. The full-length complement of (i) or (ii).

[0428] 54. The polypeptide according to any one of paragraphs 46-53, wherein the polypeptide is encoded by a polynucleotide having at least 60%, 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% sequence identity with the mature polypeptide coding sequence of SEQ ID NO 1, 3, 5, 12, 14 or its cDNA sequence.

[0429] 55. The polypeptide according to any one of paragraphs 46 to 54, comprising or consisting of the amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11 or the mature polypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13 or SEQ ID NO 15.

[0430] 56. The polypeptide according to any one of paragraphs 46 to 55, wherein the polypeptide is a variant of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11, the variant comprising substitution, deletion and / or insertion at one or more sites.

[0431] 57. A polynucleotide encoding a polypeptide according to any one of paragraphs 46-56.

[0432] 58. A nucleic acid construct or expression vector comprising a polynucleotide as described in paragraph 57, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide within an expression host.

[0433] 59. A recombinant host cell comprising a polynucleotide as described in paragraph 57, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide.

[0434] 60. A method for producing a polypeptide as described in any one of paragraphs 46-56, the method comprising: culturing cells under conditions conducive to the production of the polypeptide, the cells producing the polypeptide in their wild-type form.

[0435] 61. The method as described in paragraph 60, further comprising recovering the polypeptide.

[0436] 62. A method for producing a polypeptide according to any one of paragraphs 46-56, the method comprising: culturing a host cell as described in paragraph 59 under conditions favorable for the production of the polypeptide.

[0437] 63. The method as described in paragraph 62, further comprising recovering the polypeptide.

[0438] 64. A nucleic acid construct or expression vector comprising a gene operatively linked to a polynucleotide as described in paragraph 57, encoding a protein, wherein the gene is exogenous to the polynucleotide encoding the signal peptide.

[0439] 65. A recombinant host cell comprising a gene encoding a protein operatively linked to a polynucleotide as described in paragraph 57, wherein the gene is exogenous to the polynucleotide encoding the signal peptide.

[0440] 66. A method for producing a protein, the method comprising: culturing a recombinant host cell under conditions conducive to the production of the protein, the recombinant host cell comprising a gene operatively linked to a polynucleotide as described in paragraph 62, wherein the gene is exogenous to the polynucleotide encoding the signal peptide.

[0441] 67. The method as described in paragraph 66, the method further comprising recovering the protein.

[0442] 68. The recombinant host cell as described in paragraph 65 further comprises a polynucleotide encoding a second polypeptide of interest; preferably an enzyme of interest; more preferably a secreted enzyme of interest, even more preferably a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; and most preferably the secreted enzyme is α-galactosidase, α-glucosidase, aminopeptidase, amylase, asparaginase, β-galactosidase, β-glucosidase, β-xylosidase, or glucosidase. Enzymes, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucan transferase, glucosylamylase, invertase, laccase, lipase, mannosidase, polysaccharidase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0443] 69. As in paragraph 65, a recombinant host cell in which the second polypeptide of interest is heterologous or homologous to the host cell.

[0444] 70. The recombinant host cell as described in paragraph 65 or 68, wherein the recombinant host cell is a fungal host cell; preferably a filamentous fungal host cell; more preferably cells of the genera *Aperitifolium*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Phyllostachys*, *Phyllostachys*, *Phyllostachys*, *Pleurotus*, *Schizophyllum*, *Basilella*, *Thermophila*, *Cladosporium*, *Cladosporium*, *Phyllostachys ...The most preferred species are *Aspergillus buergerianus*, *Aspergillus scabra*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis aneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium keratophilum*, and *Chrysosporium loukenowens*. *Chrysosporium merdarium*, *Chrysosporium queenslandicum*, *Chrysosporium zonatum*, *Coprinus cinereus*, *Coriolus hirsutus*, *Fusarium moniliforme*, *Fusarium cerealis*, *Fusarium kuwaiense*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium moniliforme ... *Chrysosporium*, *Phlebia radiata*, *Pleurotus eryngii*, *Clostridium terrestris*, *Trametes villosa*, *Trametes versicolor*, *Trichoderma harzianum*, *Trichoderma cornigeri*, *Trichoderma longifolia*, *Trichoderma reesei*, or *Trichoderma viride* cells.

[0445] 71. The recombinant host cell as described in paragraph 65 or 68, wherein the recombinant host cell is a bacterial host cell; preferably a prokaryotic host cell; more preferably a Gram-positive host cell; even more preferably a host cell of Bacillus spp., Clostridium spp., Enterococcus spp., Bacillus spp., Lactobacillus spp., Lactococcus spp., Marine Bacillus spp., Staphylococcus spp., Streptococcus spp., or Streptomyces spp.; and most preferably a host cell of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausti, Bacillus coagulans, Bacillus sturdierus, Bacillus splendidus, Bacillus tarda, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0446] 72. Articles washed according to any one of paragraphs 32-45.

[0447] Some preferred embodiments are outlined in the following paragraphs:

[0448] 1. A composition comprising at least 0.01 mg of an active enzyme and at least one adjuvant component per gram of the composition, wherein the polypeptide having aminohexosidase activity is selected from polypeptides having at least 60% sequence identity with mature polypeptides of SEQ ID NO:2, 4 and 6.

[0449] 2. The composition as described in paragraph 1, wherein the polypeptide has at least 60%, 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% sequence identity with the mature polypeptides of SEQ ID NO:2, 4, and 6.

[0450] 3. The composition as described in any one of paragraphs 1 or 2, comprising or consisting of the mature polypeptide of SEQ ID NO:7, or SEQ ID NO:2, or SEQ ID NO:8, or the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:9, or the mature polypeptide of SEQ ID NO:6.

[0451] 4. The composition according to any one of paragraphs 1 to 3, wherein the composition is a cleaning composition, such as a laundry or dishwashing composition.

[0452] 5. The composition according to paragraph 4, wherein the adjuvant component is selected from,

[0453] a. At least one detergent builder,

[0454] b. at least one surfactant, and

[0455] c. At least one bleaching component.

[0456] 6. The composition according to paragraph 5, wherein the composition comprises at least one detergent builder, wherein the amount of the detergent builder added is about 0-65% by weight, preferably about 40%-65% by weight, particularly about 20%-65% by weight, particularly from 10% to 50% by weight, and wherein the detergent builder is selected from phosphates, sodium citrate detergent builder, sodium carbonate, sodium silicate, sodium and zeolite.

[0457] 7. The composition according to paragraph 6, wherein the detergent builder is selected from citric acid, methyl glycine-N,N-diacetate (MGDA) and / or glutamic acid-N,N-diacetate (GLDA) and mixtures thereof.

[0458] 8. The composition according to any of the preceding paragraphs comprises 1 wt% to 40 wt%, preferably from 0.5 wt% to 30 wt%, of at least one bleaching component, wherein the bleaching component comprises a percarbonate and a bleaching catalyst, preferably a manganese compound.

[0459] 9. The composition according to paragraph 8, wherein at least one bleaching component is a peroxide, preferably a percarbonate, and a catalyst, preferably a metal-containing bleaching catalyst such as 1,4,7-trimethyl-1,4,7-triazacyclononane or manganese(II) acetate tetrahydrate (MnTACN).

[0460] 10. The composition according to any one of the preceding paragraphs, wherein the composition comprises at least one surfactant, wherein the surfactant is an anionic surfactant and / or a nonionic surfactant.

[0461] 11. The composition according to paragraph 10, wherein the composition comprises from about 5 wt% to about 50 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 20 wt%, and from about 5 wt% to about 10 wt% of anionic surfactant.

[0462] 12. The composition according to any one of paragraphs 10 or 11, wherein the composition comprises from about 5 wt% to about 50 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 30 wt%, from about 5 wt% to about 20 wt%, from about 5 wt% to about 10 wt% of a nonionic surfactant.

[0463] 13. The composition according to any one of paragraphs 10 to 12, wherein the anionic surfactant is selected from linear alkylbenzene sulfonic acid (LAS) isomers of LAS, alcohol ether sulfates (AEO, AEOS), sodium lauryl ether sulfate, and sodium laureth sulfate (SLES).

[0464] 14. The composition according to any one of paragraphs 10 to 13, wherein the nonionic surfactant is selected from: alcohol ethoxylates (AE or AEO), alcohol propoxylates, alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (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, N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)) and combinations thereof.

[0465] 15. Use of the composition according to any one of paragraphs 1 to 14 for deep cleaning articles, wherein the articles are textiles.

[0466] 16. A washing method for washing articles, the method comprising the following steps:

[0467] The article is exposed to a detergent containing a polypeptide selected from the group consisting of: a polypeptide having at least 60% sequence identity with the mature polypeptides of SEQ ID NO: 2, 4 and 6 or a detergent composition according to any one of paragraphs 1 to 14.

[0468] Complete at least one washing cycle; and

[0469] Rinse the item as desired.

[0470] The item in question is a textile.

[0471] 17. Use of peptides from the Bacillus tumefaciens clade in cleaning processes, such as laundry and / or dishwashing, wherein the peptides possess aminohexosidase activity.

[0472] 18. A polypeptide of the Bacillus subtilis clade for use in deep cleaning items, wherein the polypeptide has aminohexosidase activity, and wherein the item is a textile.

[0473] 19. As described in paragraph 17, for preventing, reducing or removing the stickiness of an article.

[0474] 20. The use according to any one of paragraphs 17 or 18, for preventing, reducing or removing the redeposition of dirt during a washing cycle.

[0475] 21. The use according to any one of the preceding paragraphs, wherein the polypeptide is selected from the group consisting of a polypeptide having at least 60% sequence identity with the mature polypeptides of SEQ ID NO:2, 4 and 6 and at least one adjuvant component.

[0476] 22. The use as described in paragraph 21, wherein the polypeptide has at least 60%, 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% sequence identity with the mature polypeptides of SEQ ID NO:2, 4, and 6.

[0477] It should be understood that every maximum numerical limit given throughout this specification includes every lower numerical limit, as such lower numerical limits are explicitly stated herein. Every minimum numerical limit given throughout this specification will include every higher numerical limit, as such higher numerical limits are explicitly stated herein. Every numerical range given in this specification will include every narrow range falling within such a broader range, as if all such narrow numerical ranges were expressed in writing herein.

[0478] Measurement and detergent composition

[0479] Detergent composition

[0480] The detergent compositions mentioned below can be used in conjunction with the enzymes of the present invention.

[0481] Biotex Black (Liquid)

[0482] 5%-15% anionic surfactants, <5% nonionic surfactants, fragrances, enzymes, DMDM ​​and hydantoin.

[0483] Tide Sensitive White and Colored Laundry Detergent Composition, Liquid Detergent Composition

[0484] Water, alcohol ethoxysulfate, alcohol ethoxylate, amino oxide, citric acid, C12-18 palm kernel fatty acid, protease, glycosidase, amylase, ethanol, 1,2-propylene glycol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, transsulfate EHDQ (these components are listed in descending order).

[0485] WFK IEC-A standard detergent composition (powder)

[0486] Ingredients: Sodium linear alkylbenzene sulfonate 8.8%, ethoxylated fatty alcohol C12-18(7EO) 4.7%, sodium soap 3.2%, defoamer DC2-4248S 3.9%, sodium aluminum silicate zeolite 4A 28.3%, sodium carbonate 11.6%, sodium salt of copolymer of acrylic acid and maleic acid (Sokalan CP5) 2.4%, sodium silicate 3.0%, carboxymethyl cellulose 1.2%, Dequest 2066 2.8%, optical brightener 0.2%, sodium sulfate 6.5%, protease 0.4%.

[0487] Standard Detergent A Composition (Liquid)

[0488] Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 5% AEOS (SLES), 6% MPG (propylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerin, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTMPA and 0.2% PCA (all percentages are w / w).

[0489] Standard detergent NA composition (liquid)

[0490] Ingredients: NaOH 0.87%, MPG (propylene glycol) 6%, glycerin 2%, soap-soy 2.75%, soap-cocoa 2.75%, PCA (Sokalon CP-5) 0.2%, AEO Biosoft N25-7 (NI) 16%, sodium formate 1%, sodium formate 2%, DTMPA 0.2%, ethanol (96%) 3%, adjust pH with NaOH or citric acid and add water to 100% (all percentages are w / w (weight / volume)).

[0491] The composition of Baoying universal gel

[0492] Ingredients: 15%-30% anionic surfactant, 5%-15% nonionic surfactant, <5% phosphate, soap, pentyl cinnamaldehyde, butyl phenyl methyl propionaldehyde, limonene, geraniol, optical brightener, enzyme.

[0493] Tide Actilift composition (liquid)

[0494] Ingredients: 5%-15% anionic surfactants; <5% nonionic surfactants, phosphates, soaps; enzymes, optical brighteners, benzisothiazolinone, methylisothiazolinone, fragrances, α-isomethylionone, citronellol, geraniol, linalool.

[0495] Tide Actilift Color & Style Combination (Liquid)

[0496] Ingredients: 5%-15% anionic surfactants; <5% nonionic surfactants, phosphates, soaps; enzymes, fragrances, benzisothiazolinone, methylisothiazolinone, α-isomethylionone, butylphenyl methylpropionaldehyde, citronellol, geraniol, linalool.

[0497] Bao Ying Small & Powerful Detergent Combination (Liquid)

[0498] Ingredients: 15%-30% anionic surfactants, nonionic surfactants, 5%-15% soap, <5% polycarboxylates, fragrances, phosphates, optical brighteners

[0499] Bao Ying 2-in-1 with Comfort Passion Fruit Powder

[0500] Sodium sulfate, sodium carbonate, sodium dodecylbenzenesulfonate, bentonite, sodium carbonate peroxide, sodium silicate, zeolite, water, citric acid, TAED, C12-15Pareth-7, stearic acid, fragrance, sodium acrylate / MA copolymer, cellulose gum, modified corn starch, sodium chloride, tetrasodium hydroxyethyl phosphate, EDTMP calcium sodium, disodium aniline triazine-aminobenzenesulfonate, sodium bicarbonate, phenylpropyl ethyl polymethylsiloxane, butylphenyl methylpropionaldehyde, glyceryl stearate, calcium carbonate, sodium polyacrylate, α-isomethyl ionone, disodium stilbene diphenyl disulfonate, cellulose, protease, limonene, PEG-75, titanium dioxide, dextrin, sucrose, sodium polyarylsulfonate, CI 12490, CI 45100, CI 42090, sodium thiosulfate, CI 61585.

[0501] Baoying Biopowder

[0502] Sucrose, sorbitol, aluminum silicate, polyoxymethylene melamine, sodium polyarylsulfonate, CI 61585, CI 45100, lipase, amylase, xanthocyanin, hydroxypropyl methylcellulose, CI 12490, disodium stilbene diphenyl disulfonate, sodium thiosulfate, CI 42090, mannanase, CI 11680, hydroxyethyl phosphate, tetrasodium EDTA.

[0503] Baoying Biological Tablets

[0504] Sodium carbonate, sodium carbonate peroxide, sodium bicarbonate, zeolite, water, sodium silicate, sodium lauryl sulfate, cellulose, TAED, sodium dodecylbenzene sulfonate, hemicellulose, lignin, lauryl glucoside, sodium acrylate / MA copolymer, bentonite, sodium chloride, fragrance, tetrasodium hydroxyethyl phosphate, sodium sulfate, sodium polyacrylate, dimethicone, disodium aniline morpholinotriazine aminostilbene sulfonate, dodecylbenzene sulfonic acid, trimethylsiloxysilicate, calcium carbonate, cellulose, PEG-75, titanium dioxide, dextrin, protease, modified corn starch, sucrose, CI 12490, sodium polyaryl sulfonate, sodium thiosulfate, amylase, kaolin.

[0505] Bao Ying Color Care Bio Powder

[0506] Subtilisin, imidazolinone, hexylcinnamaldehyde, sucrose, sorbitol, aluminum silicate, polyoxymethylene melamine, CI 61585, CI 45100, lipase, amylase, xanthocyanin, hydroxypropyl methylcellulose, CI 12490, disodium stilbene diphenyl disulfonate, sodium thiosulfate, CI 42090, mannanase, CI 11680, hydroxyethyl phosphate, tetrasodium EDTA.

[0507] Bao Ying Color Care Biological Tablets

[0508] Sodium bicarbonate, sodium carbonate, zeolite, water, sodium silicate, sodium dodecyl sulfate, cellulose gum, sodium dodecylbenzenesulfonate, lauryl glucoside, sodium chloride, sodium acrylate / MA copolymer, fragrance, sodium thioacetate, PVP, sodium sulfate, tetrasodium hydroxyethyl phosphate, sodium polyacrylate, dimethicone, bentonite, dodecylbenzenesulfonic acid, trimethylsiloxysilicate, calcium carbonate, cellulose, PEG-75, titanium dioxide, dextrin, protease, modified corn starch, sucrose, sodium thiosulfate, amylase, CI74160, kaolin.

[0509] Baoying Dual-Effect Capsules Biological Products

[0510] MEA-dodecylbenzenesulfonic acid, MEA-hydrogenated coconut oil acid, C12-15Pareth-7, dipropylene glycol, water, tetrasodium hydroxyethyl phosphate, polyvinyl alcohol, glycerol, aziridine, ethoxylated homopolymer, propylene glycol, fragrance, sodium diethylenetriaminepentamethylene phosphate, sorbitol, MEA-sulfuric acid, ethanolamine, subtilisin, ethylene glycol, butylphenyl methylpropionaldehyde, boric acid, (4-formylphenyl), hexylcinnamaldehyde, limonene, linalool, disodium stilbene diphenyl disulfonate, α-isomethyl ionone, geraniol, amylase, polymerized blue colorant, polymerized yellow colorant, talc, sodium chloride, benzisothiazolinone, mannanase, benzylammonium benzoate.

[0511] Bao Ying 2-in-1 with Comfort Sunny Powder

[0512] Sodium sulfate, sodium carbonate, sodium dodecylbenzenesulfonate, bentonite, sodium carbonate peroxide, sodium silicate, zeolite, water, citric acid, TAED, C12-15Pareth-7, fragrance, stearic acid, sodium acrylate / MA copolymer, cellulose gum, modified corn starch, sodium chloride, tetrasodium hydroxyethyl phosphate, EDTMP calcium sodium, disodium aniline triazine-aminobenzenesulfonate, sodium bicarbonate, phenylpropyl ethyl polymethylsiloxane, butylphenyl methylpropionaldehyde, glyceryl stearate, calcium carbonate, sodium polyacrylate, geraniol, disodium stilbene diphenyl disulfonate, cellulose, protease, PEG-75, titanium dioxide, dextrin, sucrose, sodium polyarylsulfonate, CI 12490, CI 45100, CI 42090, sodium thiosulfate, CI 61585.

[0513] Bao Ying Small & Powerful 2-in-1 Plus Comfortable Sunny Day

[0514] Water, C12-15Pareth-7, Sodium dodecylbenzenesulfonate, Propylene glycol, Sodium hydrogenated cocoate, Triethanolamine, Glycerin, TEA-hydrogenated cocoate, Fragrance, Sodium chloride, Polyquaternium-10, PVP, Polymerized pink colorant, Sodium sulfate, Disodium stilbene diphenyl disulfonate, Butylphenyl methylpropionaldehyde, Styrene / acrylate copolymer, Hexylcinnamaldehyde, Citronellol, Eugenol, Polyvinyl alcohol, Sodium acetate, Isopropanol, Polymerized yellow colorant, Sodium dodecyl sulfate.

[0515] Baoying Small & Powerful Biological Products

[0516] Water, MEA-dodecylbenzenesulfonic acid, propylene glycol, sodium lauryl ether sulfate, C12-15Pareth-7, TEA-hydrogenated cocoate, MEA-citric acid, aziridine, ethoxylated homopolymer, MEA-hydroxyethyl phosphate, triethanolamine, fragrance, acrylate copolymer, sorbitol, MEA-sulfuric acid, sodium sulfite, disodium stilbene diphenyl disulfonate, butylphenyl methylpropionaldehyde, styrene / acrylate copolymer, citronellol, sodium sulfate, peptides, salts, sugars from fermentation (process), subtilisin, glycerol, boric acid, (4-formylphenyl), geraniol, pectin lyase, amylase, sodium dodecyl sulfate, mannanase, CI42051.

[0517] Baoying Small & Powerful Capsule Biological Products

[0518] MEA-dodecylbenzenesulfonic acid, MEA-hydrogenated coconut oil acid, C12-15Pareth-7, dipropylene glycol, water, glycerin, polyvinyl alcohol, fragrance, aziridine, ethoxylated homopolymer, sodium diethylenetriaminepentamethylene phosphate, propylene glycol, sorbitol, MEA-sulfuric acid, ethanolamine, subtilisin, ethylene glycol, butylphenyl methylpropionaldehyde, hexylcinnamaldehyde, starch, boric acid, (4-formylphenyl), limonene, linalool, disodium stilbene diphenyl disulfonate, α-isomethylionone, geraniol, amylase, talc, polymerized blue colorant, sodium chloride, benzisothiazolinone, benzylammonium benzoate, polymerized yellow colorant, mannanase.

[0519] Bao Ying Small & Powerful Capsule Color Care

[0520] MEA-dodecylbenzenesulfonic acid, MEA-hydrogenated coconut oil acid, C12-15Pareth-7, dipropylene glycol, water, glycerin, polyvinyl alcohol, fragrance, aziridine, ethoxylated homopolymer, diethylenetriamine pentamethyl phosphate sodium, propylene glycol, MEA-sulfuric acid, ethanolamine, PVP, sorbitol, butylphenyl methylpropionaldehyde, subtilisin, hexylcinnamaldehyde, starch, limonene, linalool, boric acid, (4-formylphenyl), α-isomethylionone, geraniol, talc, polymerized blue colorant, benzyl benzoate ammonium amide, polymerized yellow colorant.

[0521] Bao Ying Small & Powerful Color Care

[0522] Water, MEA-dodecylbenzenesulfonic acid, propylene glycol, sodium lauryl ether sulfate, C12-15Pareth-7, TEA-hydrogenated cocoate, MEA-citric acid, aziridine, ethoxylated homopolymer, MEA-hydroxyethyl phosphate, triethanolamine, fragrance, acrylate copolymer, sorbitol, MEA-sulfuric acid, sodium sulfite, glycerol, butylphenyl methylpropionaldehyde, citronellol, sodium sulfate, peptide, salt, sugar from fermentation (process), styrene / acrylate copolymer, subtilisin, boric acid, (4-formylphenyl), geraniol, pectin lyase, amylase, sodium dodecyl sulfate, mannanase, CI 61585, CI 45100.

[0523] Fairy Non-Biological Product Composition (Liquid)

[0524] Ingredients: 15%-30% anionic surfactant, 5%-15% nonionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, fragrance

[0525] Standard Detergent Composition T (Powder)

[0526] Ingredients: 11% LAS, 2% AS / AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelating agent, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP (all percentages are w / w).

[0527] Standard Detergent X Composition (Powder)

[0528] Ingredients: 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% sokalan, 35.5% sodium sulfate (all percentages are w / w).

[0529] Tide Actilift Color & Style Combination (Powder)

[0530] Ingredients: 15%-30% anionic surfactant, <5% nonionic surfactant, phosphate, polycarboxylate, zeolite; enzyme, fragrance, hexylcinnamaldehyde.

[0531] Tide Actilif composition (powder)

[0532] Ingredients: 5%-15% anionic surfactants, oxygen-based bleaching agents, <5% nonionic surfactants, phosphates, polycarboxylates, zeolites, optical brighteners, enzymes, fragrances, butylphenyl methylpropionaldehyde, coumarins, hexyl cinnamaldehyde

[0533] Megaperls composition (powder)

[0534] Ingredients: 15%-30% of the following: anionic surfactants, oxygen-based bleaching agents and zeolites; less than 5% of the following: nonionic surfactants, phosphates, polycarboxylates, soaps; additional ingredients: fragrances, hexyl cinnamaldehyde, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol.

[0535] Jiasheng Liquid, Original Version:

[0536] Ingredients: Water, Alcohol Ethoxysulfate, Diethylene Glycol, Alcohol Ethoxide, Ethanolamine, Linear Alkylbenzene Sulfonate, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Citric Acid, Borax, Sodium Cucurbitacin Sulfonate, Propylene Glycol, DTPA, Sodium Diaminostilbene Disulfonate, Dipropylethyltetramine, Sodium Hydroxide, Sodium Formate, Calcium Formate, Dimethicone, Amylase, Protease, Liquidint™, Hydrogenated Castor Oil, Fragrance.

[0537] Tide liquid, original version:

[0538] Ingredients: Linear alkylbenzene sulfonates, propylene glycol, citric acid, sodium hydroxide, borax, ethanolamine, ethanol, alcohol sulfates, polyethyleneimine ethoxylate, sodium fatty acid, diquaternary ammonium ethoxysulfate, protease, diethylene glycol, lauryl ether-9, alkyl dimethylamine oxide, fragrance, amylase, sodium diaminostyrene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquidint TM Blue, dimethicone.

[0539] Liquid Tide, Free & Mild:

[0540] Water, sodium ethoxysulfate, propylene glycol, borax, ethanol, sodium linear alkylbenzene sulfonate, salt, polyethyleneimine ethoxylate, diethylene glycol, trans-sulfated and ethoxylated hexamethylenediamine, ethoxide, linear alkylbenzene sulfonates, MEA salts, sodium formate, sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diaminostilbene, disulfonate, amylase, protease, dimethicone, benzisothiazolinone

[0541] Tide Cold Water Liquid, Fresh Scent:

[0542] Water, alcohol ethoxylates, linear alkylbenzene sulfonates, diethylene glycol, propylene glycol, ethanolamine, citric acid, borax, alcohol sulfates, sodium hydroxide, polyethyleneimine, ethoxylates, sodium fatty acids, ethanol, protease, lauryl ether-9, diquaternary ammonium ethoxylate, laurylamine oxide, sodium isopropylbenzene, sulfonates, fragrance, DTPA, amylase, disodium, diaminostilbene, disulfonates, sodium formate, disodium stilbene diphenyl disulfonate, calcium formate, polyethylene glycol 4000, mannanase, pectinase, Liquidint TM Blue, dimethicone

[0543] Tide Total Care TM Liquid, cold cotton:

[0544] Water, alcohol ethoxylate, propylene glycol, sodium fatty acid, lauryl trimethylammonium chloride, ethanol, sodium hydroxide, sodium isopropylbenzenesulfonate, citric acid, ethanolamine, diethylene glycol, polyether siloxane, borax, fragrance, polyethyleneimine, ethoxylate, protease, lauryl ether-9, DTPA, polyacrylamide quaternary ammonium chloride, sodium diaminostilbene disulfonate, sodium formate, Liquidint TM Orange, dipropyltetramine, dimethicone, cellulase,

[0545] Liquid Tide with bleach (Alternative) TM Vibrant white and vivid, original and clean breeze:

[0546] Water, Sodium Alkyl Ethoxysulfate, Sodium Alkyl Sulfate, MEA Citric Acid, Linear Alkylbenzene Sulfate, MEA Salt, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Sodium Fatty Acid, Ethanolamine, Laurylamine Oxide, Borax, Lauryl Ether-9, DTPA, Sodium Cucurbita Sulfate, Sodium Formate, Calcium Formate, Linear Alkylbenzene Sulfate, Sodium Salt, Alkyl Sulfate, Sodium Hydroxide, Diquaternary Ammonium Ethoxysulfate, Fragrance, Amylase, Protease, Mannanase, Pectinase, Sodium Diaminostilbene Disulfonate, Benzo[a]isothiazolinone, Liquidint TM Blue, dimethicone, dipropyltetramine.

[0547] Liquid Tide HE, Original Flavor:

[0548] Water, Sodium Alkyl Ethoxysulfate, MEA Citric Acid, Sodium Alkyl Sulfate, Alkyl Ethoxide, Linear Alkylbenzene Sulfate, MEA Salt, Sodium Fatty Acid, Polyethylene Imine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternary Ammonium Ethoxysulfate, Borax, Polyethylene Imine, Ethoxylate Propylene, Ethanol, Sodium Cucurbita Sulfonate, Fragrance, DTPA, Sodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Amylase, Sodium Formate, Calcium Formate, Laurylamine Oxide, Liquidint™ Blue, Dimethicone / Dimethyl Silicone.

[0549] Tide Totalcare HE Liquid, Renewing Rain:

[0550] Water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, alcohol ethoxide, citric acid, ethanolamine, sodium fatty acid, diethylene glycol, propylene glycol, sodium hydroxide, borax, polyethyleneimine ethoxylate, polyether siloxane, ethanol, protease, sodium cumene sulfonate, diquaternary ammonium ethoxysulfate, lauryl ether-9, fragrance, amylase, DTPA, sodium diaminostyrene disulfonate, disodium styrene-biphenyl disulfonate, sodium formate, calcium formate, mannanase, Liquidint™ orange, dimethicone, polyacrylamide quaternary ammonium chloride, cellulase, dipropylethylenetetramine.

[0551] Tide Liquid HE Freedom:

[0552] Water, alcohol ethoxysulfate, diethylene glycol, monoethanolamine citric acid, sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, amylase, benzisothiazoline, borax, calcium formate, citric acid, sodium ethylenediaminepentaacetate, dimethicone, diquaternary ammonium ethoxysulfate, sodium diaminostyrene disulfonate, lauryl ether-9, mannanase, protease, sodium cumene sulfonate, sodium fatty acid.

[0553] Tide Cold Water HE Liquid, Light Scent:

[0554] Water, alcohol ethoxylates, MEA citric acid, alcohol sulfates, alcohol ethoxides, linear alkylbenzene sulfonates (MEA), sodium fatty acids, polyethyleneimine ethoxylates, diethylene glycol, propylene glycol, diquaternary ammonium ethoxylates, borax, polyethyleneimine ethoxylate propoxylates, ethanol, sodium cumene sulfonate, fragrances, DTPA, sodium diaminostilbene disulfonate, protease, mannanase, cellulase, amylase, sodium formate, calcium formate, laurylamine oxide, liquidint TM Blue, dimethicone.

[0555] Tide Cold Water HE Free Liquid:

[0556] Water, sodium ethoxysulfate, MEA citric acid, linear alkylbenzene sulfonates: sodium salt, ethoxylates, linear alkylbenzene sulfonates: MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, borax, protease, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, amylase, citric acid, DTPA, sodium diaminostilbene disulfonate, sodium formate, calcium formate, dimethicone.

[0557] Tide: Simple, clean, and fresh.

[0558] Water, alcohol ethoxide sulfate, sodium linear alkylbenzene sulfonate / Mea salt, propylene glycol, diethylene glycol, sodium formate, ethanol, borax, sodium fatty acid, fragrance, laurylamine oxide, DTPA, polyethyleneamine ethoxylate, calcium formate, sodium diaminostyrene disulfonate, dimethicone, tetraamine, Liquidint TM blue.

[0559] Tide capsule, sea fog, mysterious forest, spring pasture:

[0560] Linear alkylbenzene sulfonates, C12-16 Pareth-9, propylene glycol, alcohol ethoxylates, water, polyethyleneimine ethoxylate, glycerol, fatty acid salts, PEG-136 polyvinyl acetate, ethylenediamine succinate, monoethanolamine citric acid, sodium bisulfite, sodium ethylenediaminepentaacetate, disodium stilbene diphenyl disulfonate, calcium formate, mannanase, xyloglucanase, sodium formate, hydrogenated castor oil, natalase, dyes, termamyl, subtilisin, benzisothiazoline, fragrances.

[0561] Tide to Go Stain Remover Pen:

[0562] Deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyl dimethylamine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, fragrance.

[0563] Tide Stain Release Liquid:

[0564] Water, alkyl ethoxylates, linear alkylbenzene sulfonates, hydrogen peroxide, diquaternary ammonium ethoxysulfate, ethanolamine, disodium stilbene diphenyl disulfonate, tetrabutyl ethylidene bisphenol, F&DC Yellow 3, fragrance.

[0565] Tide Stain Releasing Powder:

[0566] Sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzenesulfonate, sodium polyacrylate, water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodium palmitate, amylase, protease, modified starch, FD&C Blue 1, fragrance.

[0567] Tide Stain Release, Pre-treatment Spray:

[0568] Water, alkyl ethoxylates, MEA borate, linear alkylbenzene sulfonates, propylene glycol, diquaternary ammonium ethoxysulfate, calcium chloride enzyme, protease, ethanolamine, benzisothiazolinone, amylase, sodium citrate, sodium hydroxide, fragrance.

[0569] Tide Stain Remover Eraser:

[0570] Water, alkylamine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, sodium ethylenediamine disuccinate, sodium alkyl sulfate, fragrance.

[0571] Tide oxidation enhancement:

[0572] Sodium bicarbonate, sodium carbonate, sodium percarbonate, ethylene oxide, sodium chloride, maleic acid / acrylic acid copolymer, nonanoyloxybenzene sulfonate, sodium sulfate, colorant, sodium ethylenediaminepentaacetate, hydrated aluminosilicate (zeolite), polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate, starch, water, fragrance.

[0573] Tide Stain Release Enhanced Double Pac:

[0574] Polyvinyl alcohol (PVA) bags containing both liquid and powder portions:

[0575] Liquid ingredients: dipropylene glycol, diquaternary ammonium ethoxysulfate, water, glycerin, Liquidint™ orange; Powder ingredients: sodium percarbonate, nonanoyloxybenzenesulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzenesulfonate, maleic acid / acrylic acid copolymer, water, amylase, polyethylene glycol, sodium palmitate, modified starch, protease, glycerin, DTPA, fragrance.

[0576] Tide Super Stain Release:

[0577] Water, Sodium Alkyl Ethoxysulfate, Linear Alkylbenzene Sulfate, Sodium / MEA Salt, MEA Citric Acid, Propylene Glycol, Polyethylene Imine Ethoxylate, Ethanol, Diethylene Glycol, Polyethylene Imine Propylene Ethoxylate, Sodium Fatty Acid, Protease, Borax, Sodium Cucurbitacin Sulfate, DTPA, Fragrance, Amylase, Sodium Diaminostilbene Disulfonate, Calcium Formate, Sodium Formate, Dextranase, Dimethicone, Liquidin TM Blue, mannanase.

[0578] With a few Super Tide powder detergent, April Freshness / Cleansing Breeze / April Essence:

[0579] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicates, alkyl sulfates, nonanoyloxyphenol sulfonic acid, DTPA, polyethylene glycol 4000, silica gel, ethoxylates, fragrance, polyethylene oxide, palmitic acid, sodium diaminostilbene disulfonate, protease, Liquidint TM Red, FD&C Blue 1, cellulase.

[0580] Super Tide with a touch of Downy Clean Breeze:

[0581] Water, Sodium Alkyl Ethoxysulfate, MEA Citric Acid, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Polyethylene Imine Ethoxylate, Ethanol, Diethylene Glycol, Polyethylene Imine, Propylene Ethoxylate, Diquaternary Ammonium Ethoxysulfate, Alkyl Sulfate, Dimethicone, Fragrance, Borax, Sodium Fatty Acid, DTPA, Protease, Sodium Bisulfite, Sodium Diaminostilbene Disulfonate, Amylase, Dextranase, Castor Oil, Calcium Formate, MEA, Styrene-propylene Ester Copolymer, Sodium Formate, Liquidint TM blue.

[0582] Super Tide with Downy Sunflower:

[0583] Water, Sodium Alkyl Ethoxysulfate, MEA Citric Acid, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Ethanol, Diethylene Glycol, Polyethyleneimine Propylene Ethoxylate, Polyethyleneimine Ethoxylate, Alkyl Sulfate, Dimethicone, Fragrance, Borax, Sodium Fatty Acid, DTPA, Protease, Sodium Bisulfite, Sodium Diaminostilbene Disulfonate, Amylase, Castor Oil, Calcium Formate, MEA, Styrene-propylene Copolymer, Propionamide, Dextranase, Sodium Formate, Liquidint TM blue.

[0584] Super Tide, with a Downy April freshness / sweet dream:

[0585] Water, Sodium Alkyl Ethoxysulfate, MEA Citric Acid, Linear Alkylbenzene Sulfonates: Sodium / MEA Salts, Propylene Glycol, Polyethylene Imine Ethoxylate, Ethanol, Diethylene Glycol, Polyethylene Imine Propylene Ethoxylate, Diquaternary Ammonium Ethoxysulfate, Alkyl Sulfate, Dimethicone, Fragrance, Borax, Sodium Fatty Acid, DTPA, Protease, Sodium Bisulfite, Sodium Diaminostilbene Disulfonate, Amylase, Dextranase, Castor Oil, Calcium Formate, MEA, Styrene-propylene Ester Copolymer, Propionamide, Sodium Formate, Liquidint TM blue.

[0586] Super Tide Free Powder Detergent:

[0587] Sodium carbonate, sodium aluminosilicate, alkyl sulfate, sodium sulfate, linear alkylbenzene sulfonate, water, sodium polyacrylate, silicate, ethoxylate, sodium percarbonate, polyethylene glycol 4000, protease, sodium diaminostilbene disulfonate, silica gel, cellulase.

[0588] Super Tide Powder Detergent, cleans Breeze / Spring Lavender / Forest Spring:

[0589] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicate, nonanoyloxyphenol sulfonic acid, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, sodium diaminostilbene disulfonate, palmitic acid, protease, silica gel, cellulase.

[0590] Super Tide HE (High Efficiency) Powder Detergent, Cleans with a Gentle Breeze:

[0591] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, water, nonanoyloxyphenol sulfonic acid, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, sodium diaminostilbene disulfonate, protease, silica gel, cellulase.

[0592] Super Tide Cold Water Powder Detergent, Fresh Scent:

[0593] Sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfates, linear alkylbenzene sulfonates, water, nonanoyloxyphenol sulfonic acid, sodium polyacrylate, silicates, ethoxylates, polyethylene glycol 4000, DTPA, fragrance, Natalase, palmitic acid, protease, disodium, diaminostilbene disulfonate, FD&C Blue 1, silica gel, cellulase, alkyl ether sulfates.

[0594] Super Tide, a detergent with bleach powder, cleans like a gentle breeze:

[0595] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxyphenol ester sulfonic acid, alkyl sulfate, water, silicate, sodium polyacrylate ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, protease, sodium diaminostilbene disulfonate, silica gel, FD&C Blue 1, cellulase, alkyl ether sulfate.

[0596] With Febreze Freshness TM Super Tide, a powdered detergent, brings a fresh start to spring:

[0597] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxyphenol sulfonic acid, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, protease, sodium diaminostilbene disulfonate, silica gel, FD&C Blue 1.

[0598] Liquid Tide Plus with Febreze Freshness - Sporty & Active Fresh:

[0599] Water, sodium ethoxysulfate, MEA citric acid, linear alkylbenzene sulfonate, sodium salt, linear alkylbenzene sulfonate: MEA salt, ethoxylate, sodium fatty acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate propoxylate, diquaternary ammonium ethoxysulfate,

[0600] Ethanol, sodium cumene sulfonate, borax, fragrance, DTPA, sodium bisulfate, sodium diaminostilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, laurylamine oxide, Liquidint TM Blue, dimethicone / polydimethylsilicone.

[0601] Tide plus Febreeze Freshness:

[0602] Water, Sodium Alkyl Ethoxysulfate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, MEA Citric Acid, Propylene Glycol, Polyethylene Imine Ethoxylate, Fragrance, Ethanol, Diethylene Glycol, Polyethylene Imine Propoxyethoxylate, Protease, Alkyl Sulfate, Borax, Sodium Fatty Acid, DTPA, Sodium Diaminostilbene Disulfonate, MEA, Mannanase, Dextranase, Sodium Formate, Dimethicone, Liquidint TM Blue, tetraamine.

[0603] Liquid Tide Plus with Febreze Freshness, for sporty and victorious freshness:

[0604] Water, Sodium Alkyl Ethoxysulfate, MEA Citric Acid, Linear Alkylbenzene Sulfonate, Sodium Salt, Linear Alkylbenzene Sulfonate: MEA Salt, Alkyl Ethoxide, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Prooxylate, Diquaternary Ammonium Ethoxysulfate, Ethanol, Sodium Cucurbita Sulfate, Borax, Fragrance, DTPA, Sodium Bisulfite, Sodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Amylase, Sodium Formate, Calcium Formate, Laurylamine Oxide, Liquidint TM Blue, dimethicone / polydimethylsilicone.

[0605] Tide Vivid White + Brightening, Original Version:

[0606] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxyphenol ester sulfonic acid, alkyl sulfate, water, silicates, sodium polyacrylate ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, protease, sodium diaminostilbene disulfonate, silica gel, FD&C Blue 1, cellulase, alkyl ether sulfate.

[0607] Washing test

[0608] Mini Launder-O-Meter (Mini LOM) Mode Washing System

[0609] MiniLOM is a miniature washing system in which washing is performed in 50ml test tubes placed within a Stuart rotator. Each test tube simulates a small washing machine, and in one experiment, each test tube will contain a specific detergent / enzyme system to be tested, along with soiled and unsoiled fabrics on which the test is being conducted. Mechanical stress is obtained through rotation (typically 20 rpm), and the temperature is controlled by placing the rotator in a heated cabinet / chamber.

[0610] Terg-O-toMeter (TOM) Washing Determination

[0611] The Terg-O-toMeter (TOM) is a medium-sized standard washing system that can be used to simultaneously test 12 different washing conditions. TOM is essentially a large, temperature-controlled water bath containing up to 12 open metal beakers submerged within it. Each beaker constitutes a small top-loaded washing machine, and during the experiment, each contains a solution of a specific detergent / enzyme system, testing the performance of both soiled and unsoiled fabrics. Mechanical stress is achieved by rotating agitator arms that agitate the liquid within each beaker. Because the TOM beakers are lidless, it is possible to retrieve samples during TOM experiments and analyze information online during washing.

[0612] The TOM standard washing system is primarily used for medium-scale testing of detergents and enzymes under conditions such as US or LA / AP washing. In a TOM experiment, factors such as the ballast-to-dirt ratio and the fabric-to-wash ratio can vary. Therefore, TOM provides a link between small-scale experiments (such as AMSA and microwashing) and more time-consuming full-scale experiments in top-loading washing machines. Equipment: A water bath with 12 steel beakers, each with a rotating arm, each beaker containing 500 mL or 1200 mL of detergent solution. Temperature range: 5°C to 80°C. The water bath must be filled with deionized water. Rotation speed can be set up to 70 to 120 rpm / min. Set the temperature in the Terg-O-toMeter and begin rotation in the water bath. Allow the temperature to adjust (tolerance is + / - 0.5°C). All beakers should be cleaned and free of trace amounts of previously tested substances. Prepare a washing solution in a bucket with the desired amount of detergent, temperature, and water hardness. Dissolve the detergent during magnetic stirring for 10 min. The washing solution should be used within 30 to 60 min after preparation. Add 800 ml of washing solution to a TOM beaker. Stir the washing solution at 120 rpm and optionally add one or more enzymes to the beaker. Sprinkle the cloth sample into the beaker, followed by the ballast loading. Start time measurement when the cloth sample and ballast loading are added to the beaker. Wash the cloth sample for 20 min, after which stop stirring. Then, transfer the washing loading from the TOM beaker to a sieve and rinse with cold tap water. Separate the solid cloth sample from the ballast loading. Transfer the soiled cloth sample to a 5 L beaker containing cold tap water under running water for 5 min. Store the ballast loading separately for the upcoming inactivation. Gently squeeze out the water from the cloth sample by hand and place it on a tray lined with paper. Place another sheet of paper on top of the cloth sample. Before undergoing analysis, allow small pieces of fabric to dry overnight, for example, by measuring color intensity using Color Eye as described herein.

[0613] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0614] Measurement

[0615] Determination 1: Aminohexosidase activity

[0616] The aminohexosidase activity of mature peptides having SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 was determined using 4-nitrophenyl N-acetyl-β-D-aminoglucosinolate (Sigma-Aldrich) as a substrate. Enzymatic reactions were performed in triplicate in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) under the following conditions: 50 mM 2-(N-morpholino)ethanesulfonic acid pH 6 buffer, 1.5 mg / ml 4-nitrophenyl N-acetyl-β-D-aminoglucosinolate, and 20 μg / ml purified enzyme sample in a total reaction volume of 100 μl. Blank samples without enzyme were run in parallel. Reactions were performed at 37 °C in a Thermomixer comforter (Eppendorf). After 10 min of incubation, 5 μl of 1M NaOH was added to each reaction mixture to stop the enzymatic reaction. Absorbance was read at 405 nm using a POLARstarOmega plate reader (BMG LABTECH) to estimate the formation of 4-nitrophenol ions released due to the enzymatic hydrolysis of the 4-nitrophenyl N-acetyl-β-D-aminoglucosinolate substrate. The results are summarized in the table below. This table shows the average absorbance measured at 405 nm for each reaction performed in triplicate. It can be seen that the reactions performed with SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 exhibited higher absorbance compared to the blank without enzyme, demonstrating that SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 exhibited aminohexosidase activity.

[0617] Table 2. Aminohexosidase activities of SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9.

[0618]

[0619] Determination 2: Aminohexosidase activity

[0620] The aminohexosidase activity of mature peptides having SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10 was determined using 4-methylumbelliferone N-acetyl-β-D-aminoglucosinolate (Sigma-Aldrich) as a substrate. Enzymatic reactions were performed in triplicate in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) under the following conditions: 20 mM 3-morpholinylpropane-1-sulfonic acid pH 7 buffer, 5 mM 4-methylumbelliferone N-acetyl-β-D-aminoglucosinolate, and 20 nM purified enzyme sample in a total reaction volume of 200 μL. A blank sample without enzyme was run in parallel. Reactions were carried out at ambient temperatures of 20°C–25°C. Reaction kinetics were monitored immediately after mixing the enzyme and substrate using a SpectraMax M2e plate reader. The excitation wavelength was set to 368 nm, and fluorescence emission readings were taken at 448 nm. The reaction was tracked at 60-second intervals for 30 min. The increase in fluorescence signal was used to estimate the formation of 4-methylumbelliferone ions released due to the enzymatic hydrolysis of the 4-methylumbelliferone N-acetyl-β-D-aminoglucosinolate substrate. The results are summarized in the table below. This shows the average initial reaction rate in relative fluorescence units per minute (RFU / min) measured using excitation at 368 nm and the fluorescence emission at 448 nm for each reaction performed in triplicate. It can be seen that the initial reaction rates of the reactions performed with SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10 were higher than those of the blank without enzyme, demonstrating that SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10 exhibited aminohexosidase activity.

[0621] Table 3. Aminohexosidase activities of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10. ΔInitial reaction rate (RFU / min) = (Initial reaction rate 样品 -Initial reaction rate 空白 )

[0622]

[0623] Example

[0624] Example 1: Expression and Cloning of Aminohexosidase

[0625] The DNA encoding aminohexosidase, containing the polypeptide in SEQ ID NO 2, was isolated from a *Bacillus saccharophilus* strain (ATCC12327) obtained from ATCC in 1969. The DNA encoding aminohexosidase, containing the polypeptides in SEQ ID NO 4 and 6, was isolated from environmental soil samples collected in the United States, specifically from *Terribacillus goriensis* and *Bacillus saccharophilus* strains, respectively. The DNA encoding aminohexosidase, containing the polypeptides in SEQ ID NO 13 and 15, was subsequently isolated from two *Bacillus saccharophilus* strains isolated from environmental samples collected in the United States. Chromosomal DNA from four *Bacillus saccharophilus* strains and *Terribacillus goriensis* strain was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), and whole-genome sequencing was performed using Illumina technology. Genomic sequencing was performed targeting proteins with glycosyl hydrolase domains (GH20, www.cazy.org). Three GH20 genes and their corresponding sequences SEQ ID NO: 1, 3, 5, 12, and 14 were identified from *Bacillus saccharophilus* and *Terribacillus goriensis* strains. Codon-optimized synthetic DNA encoding the mature peptide sequence of the aminohexosidase was ordered from Geneart. The mature polypeptide is shown in SEQ ID NO: 7, 8, 9, 10, and 11.

[0626] Table 4

[0627] SEQ ID NO 7 Bacillus saccharophilus SEQ ID NO 8 Terribacillus goriensis SEQ ID NO 9 Bacillus saccharophilus SEQ ID NO 10 Bacillus saccharophilus SEQ ID NO 11 Bacillus saccharophilus

[0628] Example 2: Cloning and expression of aminohexosidase

[0629] Codon-optimized synthetic genes encoding mature peptide sequences of aminohexosidase or polypeptides with aminohexosidase activity are shown in SEQ ID NOs 7, 8, 9, 10, and 11. The sequences were inserted into a Bacillus expression vector as described in WO 12 / 025577. In short, the DNA frame encoding the mature peptide of the aminohexosidase gene was cloned into the Bacillus clauri secretory signal (BcSP; having the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO 16)). BcSP replaces the native secretory signal in this gene. Downstream of the BcSP sequence, an affinity tag sequence is introduced to facilitate the purification process (His-tag; having the following amino acid sequence: HHHHHHPR (SEQ ID NO 17)). Therefore, the expressed gene contains the BcSP sequence, followed by the His-tag sequence, and then the mature wild-type GH20 sequence, i.e., the polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11. The final expression plasmid (BcSP-His-tag-GH20) is transformed into a Bacillus subtilis expression host. During transformation, the GH20BcSP fusion gene is integrated into the Bacillus subtilis host cell genome via homologous recombination.

[0630] The gene construct was expressed under the control of a triple promoter system (as described in WO 99 / 43835). The gene encoding chloramphenicol acetyltransferase was used as a marker (as described in (Diderichsen et al., 1993, Plasmid [plasmid] 30:312-315)). Transformants were selected on LB agar supplemented with 6 mg chloramphenicol per ml. A recombinant Bacillus subtilis clone containing the GH20 expression construct was selected and cultured on a rotary shaker in 500 mL baffled Erlenmeyer flasks, each containing 100 mL of yeast extract-based medium. After 3–5 days of culture at 30–37 °C, the supernatant containing the enzymes was harvested by centrifugation and the enzymes were purified by His-tag purification.

[0631] Example 3: His-tagged purification method

[0632] On a 5 mL HisTrap Excel column (GE Healthcare LifeSciences), using Ni 2+All His-tagged GH20 aminohexosidases were purified as metal ions using immobilized metal chromatography (IMAC). Purification was performed at pH 7, with imidazole used to elute the binding proteins. The purity of the purified enzymes was assessed by SDS-PAGE, and enzyme concentration was determined by absorbance at 280 nm after buffer exchange in 50 mM HEPES, 100 mM NaCl, and pH 7.0.

[0633] Example 4: Biofilm assay

[0634] Staphylococcus aureus Lasa (Valle et al., Mol Microbiol. [Molecular Microbiology] May 2003; 48(4):1075-87) kindly provided. The strain was grown overnight at 37°C on trypsin-digested casein-soybean agar (TSA). The next day, single colonies were transferred to 15 ml of trypsin-digested casein-soybean broth (TSB) and incubated at 37°C with shaking for 5 hours. The culture was diluted 1:100 in TSB + 1% glucose, and 100 μL of the bacterial suspension was transferred to each well of a 96-well microtiter plate (Thermo Scientific, Nunclon Delta Surface, catalog 167008) and incubated at 37°C without shaking for 24 hours. Aspirate the supernatant, wash the wells with 100 μL of 0.9% sodium chloride, and fill with 100 μL of hard water or 3.3 g / L standard detergent A containing 0 (control) or 20, 10, 5, 2.5, 1.25, 0.62, 0.31, 0.16, 0.08, 0.04, 0.02, and 0.01 μg / mL of enzyme (the mature polypeptide has SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11). After incubation at 37°C for 1 hour, wash the wells with water and stain with 100 μL of 0.095% crystal violet solution (SIGMA V5265) for 15 min. Then rinse the wells twice with 100 μL of water, dry, and scan the plate.

[0635] In the presence and absence of detergent, the minimum concentration of each enzyme that could reduce visible formation of Staphylococcus aureus biofilm after 1 hour of incubation was determined (see Table 5). All enzymes were measured in each replicate, yielding similar results.

[0636] Table 5. The lowest concentration of enzymes that can be reduced to visible formation in Staphylococcus aureus after 1 hour of incubation in hard water or standard detergent A.

[0637]

[0638] Example 5: Deep Cleaning with Aminohexosidase in Liquid Standard Detergent

[0639] Staphylococcus aureus 15981 (by Lasa (Valle et al., Mol Microbiol. [Molecular Microbiology] May 2003; 48(4):1075-87) was kindly provided) and used as the standard microorganism in this example. Staphylococcus aureus was streaked again on tryptone soybean agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd., Basingstock, UK) and incubated at 37°C for 1 day. Single colonies were inoculated into 10 mL of TSB and the culture was incubated at 37°C with shaking (200 rpm) for 16 hours. After propagation, Staphylococcus aureus cultures were diluted (1:100) in fresh TSB + 1% glucose (24563; Roquette Freres), and 2 mL aliquots were added to the wells of 12-well polystyrene flat-bottom microplates (3512; Corning Incorporated, Corning, NY, USA) containing small circular pieces of sterile polyester (WFK30A) fabric (2 cm in diameter). Sterile TSB + 1% glucose was added to the control wells. After 48 h of static incubation at 37°C, the fabric pieces were rinsed twice with 15°dH water. Five small, rinsed cloth samples (sterile or containing Staphylococcus aureus) were placed in 50 mL test tubes, and 10 mL of washing buffer (15°dH water and 0.2 g / L iron(III) oxide nanoparticles (544884; Sigma-Aldrich) containing 3.33 g / L liquid standard A detergent) and 0.2 ppm of enzyme (mature polypeptides with SEQ ID NO 7, 8, and 9) were added to each tube. Washing without the enzyme was included as a control. The tubes were placed in a Stuart vortex mixer and incubated at 37°C at 20 rpm for 1 hour. The washing buffer was then removed, and the cloth samples were rinsed twice with 15°dH water and dried overnight on filter paper.

[0640] The color difference (L) values ​​were measured using a Handheld Minolta CR-300 and are shown in Table 6. The Δ value (L) is also indicated. (用酶洗涤的小块布样) -L (没有用酶洗涤的小块布样) ).

[0641] Table 6. Deep cleaning effect of aminohexosidase in standard detergent A

[0642] No enzymes 0 102.9 SEQ ID NO 7 0.2 115.1 12.3 SEQ ID NO 8 0.2 115.5 12.7 SEQ ID NO 9 0.2 115.2 12.3

[0643] The results showed that aminohexosidase exhibited deep cleaning properties in standard detergent A.

[0644] Example 6: Deep cleaning effect of aminohexosidase in liquid standard detergent

[0645] As described in Example 5, Staphylococcus aureus biofilms were grown on textile fabric samples (WFK30A). Fabric samples incubated with sterile medium were included as controls. To test the deep cleaning properties of the aminohexosidase, five rinsed fabric samples (sterile or containing Staphylococcus aureus) were placed in 50 mL conical centrifuge tubes and 10 mL of washing buffer (15°dH water and 0.2 g / L iron(III) oxide nanoparticles (544884; Sigma-Aldrich) containing 3.33 g / L liquid standard A detergent) were added to each tube, along with 0.2 ppm (or 2 ppm) of the enzyme. No enzyme was added to the control. The tubes were placed on a Stuart rotator and incubated at 37°C at 20 rpm for 1 hour. The washing buffer was then removed, and the fabric samples were rinsed twice with 15°dH water and dried overnight on filter paper.

[0646] The color difference (L) value was measured using a Handheld Minolta CR-300, and the relative washing efficiency ((L) was calculated) 有酶 -L 无酶 ) / (L 无菌小块布样 -L 无酶 )*100) and indicated in Table 7.

[0647] Table 7. Percentage of washing efficiency of liquid standard detergent in small cloth samples of Staphylococcus aureus biofilm.

[0648] Staphylococcus aureus biofilm 0 0.0 Staphylococcus aureus biofilm SEQ ID NO 9 0.2 83.7 Staphylococcus aureus biofilm SEQ ID NO 10 0.2 82.1 Staphylococcus aureus biofilm SEQ ID NO 11 0.2 62.5 Staphylococcus aureus biofilm SEQ ID NO 11 2 89.1

[0649] Combined with previous examples, the results show that all peptides of the present invention exhibit deep cleaning properties compared to samples without enzymes.

[0650] Example 7: Deep cleaning effect of aminohexosidase on small cloth samples of *Pseudomonas fluorescens* biofilm.

[0651] *Pseudomonas fluorescens* isolated from Iceland was used as the standard microorganism in this example. *Pseudomonas fluorescens* was streaked again on tryptone soybean agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd., Basingstoke, UK) and incubated at ambient temperature for 3 days. Single colonies were inoculated into 10 mL of TSB, and the culture was incubated at ambient temperature with shaking (Tetramax 1000, 460 rpm) for 16 hours. After propagation, the culture was diluted 1:100 in fresh TSB, and 1.65 mL aliquots were added to the wells of a 12-well polystyrene flat-bottom microplate (3512; Corning Incorporated, Corning, NY, USA) containing small circular pieces of sterile polyester (WFK20A) fabric (2 cm in diameter). Sterile TSB was added to the control wells. After incubation at ambient temperature (static) for 48 hours, the small cloth samples were rinsed twice with 0.9% (w / v) NaCl. Five rinsed cloth samples (sterile or with fluorescein biofilm) were placed in 50 mL tubes and 10 mL of washing buffer (15°dH water and 0.2 g / L iron(III) oxide nanoparticles (544884; Sigma-Aldrich) containing 3.33 g / L liquid standard A detergent) and 0.2 ppm or 2 ppm of enzyme were added to each tube. Washes without enzyme were included as controls. The tubes were placed in a Stuart vortex mixer and incubated at 30°C and 20 rpm for 1 hour. The washing buffer was then removed, and the cloth samples were rinsed twice with 15°dH water and dried overnight on filter paper.

[0652] The intensity (Y) values ​​of the three-color stimuli were measured using the DigiEYE color measurement and imaging system (VeriVide) equipped with a Nikon D90 digital camera and are shown in Table 8. The Δ values ​​(Y) are also indicated. (用酶洗涤的小块布样) -Y (没有用酶洗涤的小块布样) ).

[0653] Table 8. Deep cleaning effect of aminohexosidase on Pseudomonas fluorescens biofilm on textiles

[0654]

[0655] Example 8: Deep cleaning effect of aminohexosidase on EPS from different microorganisms in liquid standard detergents

[0656] Crude extracts of extracellular polymeric biofilm (EPS) were obtained from Staphylococcus aureus 15981 (by... Lasa (Valle, J., A. Toledo-Arana, C. Berasain, JMGhigo, B. Amorena, JRPenades, and I. Lasa. 2003, Mol. Microbiol. [Molecular Microbiology] 48:1075-1087) kindly provided), Staphylococcus aureus (textile isolate, Denmark, O437F4), Pseudomonas fluorescens (isolated from Iceland), and Acinetobacter rofibrinosus (isolated from textiles in Denmark). For Staphylococcus aureus, the extract was prepared as follows: the strain was streaked again on tryptone soybean agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd., Basingstoke, UK) and incubated at 37°C for 3 days. Cells were then inoculated with 500 mL of TSB + 1% glucose (24563; Roquette Freres), aliquoted into 50 mL conical centrifuge tubes (339652; Thermo Scientific Nunc) (33 mL each time), and incubated at 37°C with shaking (200 rpm) for 24 hours. Cells were then pelleted by centrifugation (10 min, 6000 g, 25°C), collected, and resuspended in a total of 5 mL of 3M NaCl. The suspension was vigorously vortexed and incubated at ambient temperature for 15 min to extract surface-associated EPS. Cells were then reprecipitated (10 min, 5000 g, 25°C), and the supernatant containing EPS was recovered. The supernatant was aseptically filtered twice (0.45 μm, then 0.2 μm), sterility was tested, and the supernatant was stored at -20°C until further use.

[0657] Staphylococcus coli isolates were streaked again on TSA, incubated at 37°C for 3 days, and used to inoculate overnight cultures (10 ml TSB + 1% glucose, incubated at 37°C, 200 rpm). This culture was then diluted (1:100) to a container with VentCap (product number 3293). Add 250 ml of fresh culture medium (TSB + 1% glucose) to a 225 cm² angled neck cell culture flask and incubate at 37°C for 2 days. Precipitate the biofilm culture by centrifugation (10 min, 8000 g, 25°C), resuspend in 2.5 ml of 3M NaCl, and incubate at room temperature for 30 min. Then reprecipitate the cells (10 min, 5000 g, 25°C), recover the supernatant containing EPS, aseptically filter (0, 20 μm, 16534-K, Minisart, Sartorius Stedim) and store at -20°C until use.

[0658] Streaking of *Pseudomonas fluorescens* onto TSA and incubating at 20°C for 1 day. Inoculating the strain into 10 mL of TSB and incubating the culture statically at 20°C for 16 hours. After propagation, the culture was diluted 1:100 in 400 mL of M63 supplemented medium (15 mM (NH4)2SO4, 100 mM KH2PO4, 1.8 μM FeSO4, 1 mM MgSO4·7H2O, 0.4% (w / v) glycerol, 0.2% (w / v) caseinate, and 0.0001% (w / v) thiamine) and added to a container with Vent Cap. Cell culture flasks with 225 cm² angled necks were statically incubated at 20°C for 3 days. The biofilm culture was then precipitated by centrifugation (10 min, 8000 g, 25°C), and the cells were resuspended in 4 ml of 3M NaCl and incubated at 30°C for 30 min to extract surface-associated EPS. The supernatant containing EPS obtained after centrifugation (10 min, 5000 g, 25°C) was then aseptically filtered and stored at -20°C until further processing.

[0659] The crude EPS extract from *Acinetobacter lofeta* was prepared as follows: The strain was streaked again on TSA plates and incubated at 30°C for 3 days. Then, a single colony was inoculated into 10 ml of LB medium (L3152, Fluka) and incubated at 30°C, 200 rpm for 48 h. The culture was diluted (1:100) in 300 ml of fresh LB medium and added to a container with Vent Cap. 225cm 2 The cells were cultured in angled neck flasks and statically incubated at 30°C for 4 days. The biofilm culture was then precipitated by centrifugation (10 min, 8000 g, 25°C), and the precipitate was resuspended in 3 ml of 3M NaCl and incubated at room temperature for 30 min. The supernatant containing EPS obtained after centrifugation (10 min, 5000 g, 25°C) was aseptically filtered (0, 20 μm, 16534-K, Minisart, Sartorius Stedim) and stored at -20°C until use.

[0660] For the washability test, 50 μl aliquots of different crude EPS extracts were spotted onto sterile textile samples (WFK20A) and incubated at ambient temperature for 15 min. The samples (sterile or with EPS) were placed in 50 mL tubes and 10 mL of washing buffer (15°dH water and 0.2 g / L iron(III) nanoparticles (544884; Sigma-Aldrich) containing 3.33 g / L liquid standard A detergent) and 0.2 μg / mL or 2 μg / mL enzyme was added to each tube. Washing without enzyme was included as a control. The tubes were placed in a Stuart vortex mixer and incubated at 37°C at 20 rpm for 1 hour. The washing buffer was then removed, and the samples were rinsed twice with 15°dH water and dried overnight on filter paper. The intensity (Y) values ​​of the three-color stimulus light were measured using the DigiEYE color measurement and imaging system (VeriVide) equipped with a Nikon D90 digital camera and are shown in Table 9.

[0661] It also indicates the Δ value (Y) (用酶洗涤的小块布样) -Y (没有用酶洗涤的小块布样) ) and relative washing efficiency value ((L) 有酶 -L 无酶 ) / (L 无菌小块布样 -L 无酶 )*100)).

[0662] Table 9. Demonstrates the deep cleaning efficacy of the aminohexosidase in SEQ ID NO 9 against EPS from different microorganisms in liquid standard detergents.

[0663]

[0664] The data clearly demonstrate that aminohexosidase exhibits deep cleaning properties against biofilm EPS from a variety of microorganisms, including Gram-positive and Gram-negative bacteria.

[0665] Example 9: Reduction of odor by aminohexosidase in liquid standard detergents

[0666] EPS was purified as described in Example 8, for instance. After purification, 50 μL aliquots of EPS were added to the wells of a 12-well polystyrene flat-bottom microplate (3512; Costa, Corning Incorporated, Corning, NY, USA), in which a small circular piece of sterile polyester (WFK30A) fabric sample (2 cm in diameter) was placed. The fabric sample was incubated for 15 min (static incubation) before proceeding. Six rinsed fabric samples were placed in 50 mL tubes and 10 mL of washing buffer (15°dH water containing 3.33 g / L liquid standard A detergent) and 0.2 ppm, 2.0 ppm, or 20.0 ppm of enzyme (SEQ ID NO 9) were added to each tube. Washes without enzyme were included as controls. The tubes were placed in a Stuart spinner and incubated at 37°C and 20 rpm for 1 hour. Then remove the washing solution, rinse the small cloth sample twice with 20 mL of 15°dH water, and dry it on filter paper overnight.

[0667] An odor chamber using an electronic nose is used to set up and analyze volatile substances on textiles.

[0668] To test the binding of gaseous volatiles, all dry small pieces of cloth were randomly distributed in a square plastic box (30.7cm x 21.9cm x 6.0cm - internal measurement). 5mL of a mixture of various aldehydes (pentanal 160mM, hexanal 40mM, heptanal 80mM, (E)-2-heptanal 20mM, octanal 30mM, nonanal 10mM, decanal 8mM, (E)-2-decenal 10mM, and (E,E)-2-decadienal 12mM - all chemicals purchased from Sigma-Aldrich) was added to a 10mL glass beaker in the center. The odor chamber was closed with the lid and... Wrap. Incubate small pieces of fabric at room temperature for 16 h, then place them individually at the bottom of a 20 mL GC-MS vial (Mikrolab Aarhus, Aarhus, Denmark) and seal with a silicone screw cap (Mikrolab Aarhus, Aarhus, Denmark). Analyze 5 mL of the headspace from each sample.

[0669] • After incubation at 40°C for 20 minutes, samples were run in a Heracles II electronic nose system from Alpha MOS (a dual-column gas chromatograph with two FIDs: column 1: Restek MXT-5 10m x 0.18mm x 0.2μm and column 2: Restek MXT-1701 10m x 0.18mm x 0.2μm). Samples were run in random order.

[0670] Table 10. Deodorization of Type A standard detergent by aminohexosidase.

[0671]

[0672] The results showed that aminohexosidase exhibited odor-reducing properties in standard detergent A.

[0673] Example 10: Deep cleaning of aminohexosidase in different detergents

[0674] As described in Example 8, crude EPS (extracellular polymeric substances) extract was prepared from *Pseudomonas fluorescens* (isolated from Iceland). For washing performance testing, 50 μl aliquots of the extract were spotted onto sterile textile samples (WFK20A) and immersed at ambient temperature for 15 min. The samples (sterile or with EPS) were placed in 50 mL conical centrifuge tubes and 10 mL of washing solution (15°dH water and 0.2 g / L iron(III) oxide nanoparticles (544884; Sigma-Aldrich)) was added, along with detergent containing 3.33 g / L liquid standard A, 2.0 g / L standard N, or 4.6 g / L Baoying General Purpose detergent. Add enzyme to a final concentration of 0.2 μg / ml. Test tubes without enzyme were included as controls. Place the test tubes in a Stuart vortex mixer and incubate at 37°C and 20 rpm for 1 hour. Then remove the washing solution, rinse the small cloth sample twice with 15°dH water, and dry on filter paper overnight. The intensity (Y) values ​​of the three-color stimuli were measured using a DigiEYE color measurement and imaging system (VeriVide) equipped with a Nikon D90 digital camera and are shown in Table 11.

[0675] It also indicates the Δ value (Y) (用酶洗涤的小块布样) -Y (没有用酶洗涤的小块布样) ).

[0676] Table 11. Depth of cleaning by aminohexosidase in different detergents on small EPS fabric samples of *Pseudomonas fluorescens*.

[0677]

[0678] Example 11: Washing performance in TOM

[0679] As described in Example 8, crude EPS extract was prepared from *Pseudomonas fluorescens* (isolated from Iceland). The extract was aseptically filtered before use, and 100 μl aliquots were spotted onto small pieces of textile fabric (wfk20A (polyester / cotton blend (65% / 35%)), 50 mm x 50 mm) and incubated at room temperature for 10 min. Terg-o-tometer washing was performed as follows: 0.4 g / L iron(III) oxide nanoparticles (544884; Sigma-Aldrich) and 3.33 g / L liquid standard detergent A were weighed and soaked for 1 h before washing. The soiled detergent was then added to 1000 ml of preheated hot water (15°dH) in a TOM beaker and allowed to dissolve for 10 min (with maximum stirring). The stirring level was then reduced to 90 rpm, and small textile samples were added (two EPS samples along with ballast material (WFK10A, WFK30A) to a total of 10 g textile / beaker) and washed at 30°C for 35 min (0.2 ppm) (with or without enzyme). After washing, all samples were rinsed twice with tap water and dried overnight on filter paper. Reflectance (REM) was measured using a Macbeth Color-Eye 7000 (CE7000). 460nm The values ​​are shown in Table 12.

[0680] It also indicates the Δ value (REM) 460nm (用酶洗涤的小块布样) -REM 460nm (没有用酶洗涤的小块布样) ).

[0681] Table 12: Deep cleaning properties of aminohexosidase on small EPS fabric samples after washing in TOM.

[0682]

[0683] Example 12: Washing performance of full-scale washing

[0684] As described in Example 8, crude EPS extract was prepared from *Pseudomonas fluorescens* (isolated from Iceland). The extract (200 μl aliquots) was spotted onto pre-washed, half-cut T-shirts (Anvil Sustainable (50% polyester / 50% cotton)) and the spots were dried at room temperature for 30 min prior to washing. Both halves of the T-shirts were washed independently in a Miele washing machine (Miele Softtronic, W2245) using tap water and 0.16 g / L iron oxide (III) nanoparticles (544884; Sigma-Aldrich) in 3.33 g / L Liquid Standard A detergent. A color program at 40°C (1 h, 26 min) was used. After washing, both halves of the T-shirts were hung to dry overnight at room temperature. Reflectance (REM) was measured using a Macbeth Color-Eye 7000 (CE7000). 460nm The values ​​are shown in Table 13. The Δ value (REM) is also indicated. 460nm (用酶洗涤的小块布样) -REM 460nm (没有用酶洗涤的小块布样) ).

[0685] Table 13. Deep Cleaning Effects of Aminohexosidase in Full-Scale Washing Machines

[0686]

[0687] Example 13: Measurement of EPS hydrolysis using WGA-Alexa fluor488 fluorescence assay

[0688] A strain of *Pseudomonas fluorescens* isolated from Iceland was used as the standard microorganism in this example. The *P. fluorescens* strain was streaked again on tryptone soybean agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd., Basingstoke, UK) and incubated at 23°C for 1 day. The strain was inoculated into 10 mL of TSB, and the culture was incubated with shaking at 23°C for 16 hours. The overnight culture was diluted 1:100 in 200 mL of M63 supplemental medium, and (15 mM (NH4)2SO4, 100 mM KH2PO4, 1.8 μM MFeSO4, 1 mM MgSO4·7H2O, 0.4% (w / v) glycerol, 0.2% (w / v) caseinate and 0.0001% (w / v) thiamine) was added to a medium containing Vent Cap (product number 3293). Cell culture was carried out in 225 cm² angled neck flasks and statically incubated at 23°C for 5 days. Each biofilm culture was then transferred to four 50 ml Falcon tubes (Corning #430820) and centrifuged (10 min, 8000 g, 25°C), discarding the supernatant completely. The remaining particles from each of the four Falcon tubes were resuspended in 0.450 ml of 3M NaCl to extract surface-associated EPS (extracellular polymeric substances) and collected in a 5 ml tube (Eppendorf No. 0030119401). The suspension was centrifuged at 5000 g for 10 min at 25°C, and 1.8 ml of the supernatant was transferred as the EPS fraction to a new tube and stored at -20°C until further use (referred to as crude EPS). Dispense 150 μL of crude EPS into three Eppendorf tubes and mix with 150 μL of enzyme solution containing 80 ppm of SEQ ID NO 9 or 50 mM Hepes 100 mM NaCl buffer alone, and incubate at room temperature for 1 hour. 50 μL of aliquots were recovered from each tube, and then a nuc Maxisorp black microtiter plate (Thermo Scientific, No. 437111) containing a circular piece of sterile, pre-washed fabric (WFK 20A, 65% / 35% polyester / cotton) was added to each well. The plate was incubated at room temperature for 15 minutes. The supernatant was then removed from each well, and the fabric piece was washed with 100 μL of water. Next, 50 μL of WGA-Alexafluor 488 dye (10 μg / ml; maximum excitation / reflection approximately 495 / 519 nm; Thermo Fischer Scientific, No. W11261) was added to each well. 488WGA binds to sialic acid and N-acetylglucosamine residues. The plate was incubated at room temperature for 15 min. The sample was then washed with 100 μl of water and measured using a SpectraMax M3 instrument. Each of the three samples corresponding to the crude EPS mixed with the enzyme solution and buffer control was tested on eight small cloth samples placed in eight wells. The obtained measurements, including standard deviation, are listed in Table 14 below.

[0689] Table 14. Fluorescence measurements of crude EPS from *Pseudomonas fluorescens* strains treated with buffer or SEQ ID NO 9 using WGA-Alexa Fluor488 dye.

[0690] Buffer control 416 SEQ ID NO 9 121

[0691] Fluorescence measurements showed that EPS extracted from the *Pseudomonas fluorescens* strain was labeled with WGA-Alexa Fluor488, indicating that EPS contained N-acetylglucosamine residues and was sensitive to hydrolysis by aminohexosidase (SEQ ID NO 9).

[0692] Example 14: Wheat germ lectin (WGA) - Stains on a small piece of washed EPS fabric.

[0693] Crude EPS extract was prepared from Staphylococcus aureus 15981 (by... Lasa (Valle, J., A. Toledo-Arana, C. Berasain, J. M. Ghigo, B. Amorena, J. R. P. Nades, and I. Lasa. 2003, Mol. Microbiol. [Molecular Microbiology] 48:1075-1087) (a gift from Lasa). As described in Example 8 (with minor modifications): Before EPS extraction, the culture was incubated at 37°C in TSB + 1% glucose for 24 h. For washing, 100 μl aliquots of EPS were spotted onto sterile textile samples (pre-washed WFK20A fabric, 65% polyester / 35% cotton) and incubated at ambient temperature for 20 min. The samples (sterile or with EPS) were placed in 50 mL tubes and 10 mL of washing buffer (15°dH water containing 3.33 g / L liquid standard A detergent) and 20 μg / mL enzyme were added to each tube. Enzyme-free washing was included as a control. Test tubes were placed in a Stuart spinner and incubated at 37°C and 20 rpm for 1 hour. The washing solution was then removed, and small fabric samples were rinsed twice with 15°dH water and placed in 12-well polystyrene flat-bottom microplates (3512; Costa, Corning Incorporated, Corning, NY, USA). The fabric samples were then processed using Alexa... 488 wheat germ lectin (WGA) (10 μg / ml in 1X PBS) was stained at room temperature for 30 min, washed twice with 1X PBS, and measured using a BMG CLARIOstar microplate reader (excitation at 488 nm, emission at 525 nm). Fluorescence intensity values ​​are shown in Table 15.

[0694] It also shows a comparison with the control (FIU) 有酶 -FIU 没有酶 Compared to the relative substrate removal.

[0695] Table 15. Wheat germ lectin (WGA) - EPS fabric samples with or without aminohexosidase washing (SEQ ID NO9).

[0696]

[0697] When WGA lectins showed affinity for N-acetylglucosamine residues, results indicated that aminohexosidase treatment improved substrate removal from textiles during washing.

[0698] Example 15: Construction of clades and phylogenetic trees

[0699] The Glyco_hydro_20 domain includes the polypeptide of the present invention having aminohexosidase activity such as PNAG and contains the WND domain along with clusters such as clades.

[0700] As defined in PFAM (PF00728, PFAM version 31.0, Finn (2016). Nucleic Acids Research, Database Issue 44: D279-D285), construct a phylogenetic tree of peptide sequences containing a Glyco-hydro-20 domain. The phylogenetic tree is constructed from multiple alignments of mature peptide sequences containing at least one Glyco-hydro-20 domain. The sequences were aligned using MUSCLE algorithm version 3.8.31 (Edgar, 2004. Nucleic Acids Research 32(5):1792-1797), and the tree was constructed using FastTree version 2.1.8 (Price et al., 2010. PLOS ONE 5(3)) and visualized using iTOL (Letunic & Bork, 2007. Bioinformatics 23(1):127-128). The polypeptide sequences containing the Glyco-hydro-20 domain contain several motifs; one example is GXDE (SEQ ID NO 18), located at positions 158 to 161 in Bacillus saccharophilus (SEQ ID NO 9). Residues D and E are key catalytic residues of the Glyco-hydro-20 enzyme (positions 160 to 161 in SEQ ID NO 9).

[0701] The polypeptide in Glyco_hydro_20 can be divided into several different subclusters or clades, as listed below. The different motifs of each clade are described in detail below.

[0702] Generation of IAS domains

[0703] A domain preferably shared by the peptides of the present invention was identified. This domain has not been previously described. This domain is referred to as IES, and the peptide of this domain comprises a Glyco_hydro_20 domain peptide, which is of bacterial origin and, in addition to having PNAG activity, is characterized by containing certain motifs. The peptide of this domain comprises motif examples [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), corresponding to ESYAIAS at positions 44 to 50 of SEQ ID NO 9.

[0704] Generation of WND structural domain

[0705] A domain preferably shared by the peptides of the present invention was identified. This domain has not been previously described. This domain is referred to as WND, and the peptide of this domain comprises a Glyco_hydro_20 domain peptide, which is bacterial in origin and, in addition to having PNAG activity, is characterized by containing certain motifs. The peptide of the domain comprises the motif example [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20), corresponding to positions 156 to 163 of SEQ ID NO 9, wherein G and DE (corresponding to positions 158 and 160-161 of SEQ ID NO 9) are completely conserved in the DSP3 clade and some active sites. Residues D and E are key catalytic residues of the Glyco_hydro_20 enzyme (positions 160 to 161 in SEQ ID NO 9). Another motif that may be contained in a polypeptide by a WND domain is WND[SQR][IVL][TLVM] (SEQ ID NO 21), 193 to 198 in SEQ ID NO 9, wherein W (position 193 in SEQ ID NO 9) is part of the active site pocket and is presumed to involve the binding of the N-acetyl group of the PNAG substrate.

[0706] QSTL clade generation

[0707] The QSTL clade contains a bacterial-derived WND domain polypeptide with aminohexosidase activity, such as PNAG. The polypeptide of this clade contains the motif example QSTL (SEQ ID NO 22), corresponding to positions 216-219 of SEQ ID NO 9, wherein all four amino groups are completely conserved in the QSTL clade. This motif is part of the putative “lid” of the enzyme’s TIM barrel structure and is presumed to involve substrate binding. Another motif that may be contained in the polypeptide of the QSTL clade is NKFFY (SEQ ID NO 23), 273-277 of SEQ ID NO 9. Another motif that may be contained in the polypeptide of the QSTL clade is NLD[DR]S (SEQ ID NO 24), corresponding to amino acids 204-208 of SEQ ID NO 9.

[0708] The comparison of the peptides of the present invention is shown in Figure 2 The phylogenetic tree of the polypeptides of this invention is shown in [the diagram]. Figure 3 middle. sequence list <110> Novozymes A / S <120> Detergent compositions and their uses <130> 14181-WO-PCT <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 1095 <212> DNA <213> Bacillus saccharophilus <220> <221> signal peptide <222> (1)..(123) <220> <221> CDS <222> (1)..(1095) <220> <221> Mature peptides <222> (124)..(1095) <400> 1 atg gta tct cgc acg atg ctt ttc atc aag cgc gag aag ggc aag acg 48 Met Val Ser Arg Thr Met Leu Phe Ile Lys Arg Glu Lys Gly Lys Thr -40 -35 -30 gtt ctt atc aag ttc ctt tct atc acg aca gta tct atc ctt ctt ttc 96 There Will Be Lys Phe You Will Be There Thr Thr There Will Be You Will Be -25 -20 -15 -10 ctt acg atg gcg aac act gcg CA gcg CA Gac CA GAG AAG ggc atc 144 Leu Thr Met Ala Asn Thr Gln Ala Gln Ala Gln Asp Gln Glu Lys Gly Ile -5 -1 1 5 aca atc ​​gac atc tct cgc aag tac tac agc atc aag acg ctt aag gcg 192 Thr Asp Ile Ser Arg Lys Tyr Tyr Ser Ile Lys Thr Leu Lys Ala 10 15 20 atc gtt gac gag atc tca gcg at ggt ggc gac tac ctt caa ctt cac 240 Ile Val Asp Glu Ile Ser Ala Asn Gly Gly Asp Tyr Leu Gln Leu His 25 30 35 ttc tct gac aac gag agc tac gcg atc gcg tct gag ttc ctt ggc caa 288 Phe Ser Asp Asn Glu Ser Tyr Ala Ile Ala Ser Glu Phe Leu Gly Gln 40 45 50 55 aac tct gag aac cca aac tct gcg tac ctt aca aag aag gag ctt ctt 336 Asn Ser Glu Asn Pro Asn Ser Ala Tyr Leu Thr Lys Lys Glu Leu 60 65 70 tca ctt atc gcg tac tct aac gac cgc aac atc atg gtt atc ccg gac 384 Ser Leu Ile Ala Tyr Ser Asn Asp Arg Asn Ile Met Val Ile Pro Asp 75 80 85 atc gac ctt cct gcg cac tca aag ggc tgg ctt aac atc atg aag gag 432 The Asp Leu Pro Ala has Lys Gly Trp and Leu Asn has Lys Glu 90 95 100 aag gac tct ggc ctt tac aca gac atc gta act gac tac tca gaa gac 480 Lys Asp Ser Gly Leu Tyr Thr Asp Ile Val Thr Asp Tyr Ser Glu Asp 105 110 115 act ctt gac tac cac aac aac gcg gtt gcg ctt tac acg gcg aac caa 528 Thr Leu Asp Tyr His Asn Asn Ala Val Ala Leu Tyr Thr Ala Asn Gln 120 125 130 135 ctt ctt gac gag gtt ctt gac ctt ttc tac caa cct aag ttt gct ggc 576 Leu Leu Asp Glu Val Leu Asp Leu Phe Tyr Gln Pro Lys Phe Ala Gly 140 145 150 aag caa cgc atc gtt ctt ggt ggc gac gag gtt cct ggc tct ggt gcg 624 Lys Gln Arg Ile Val Leu Gly Gly Asp Glu Val Pro Gly Ser Gly Ala 155 160 165 cac caa act gac ttc atc cgc ttc atg aac cag atc gcg aag aca gcg 672 His Gln Thr Asp Phe Ile Arg Phe Met Gln Ile Ala Lys Thr Ala 170 175 180 aag gcg tct aac tac gag caa atg tg aac gac agc atc act cct 720 Lys Only Serves Asn Tyr Glu Pro Gln Met Trp Asn Asp Served Thr Pro 185 190 195 gag ggc atc caa aac ctt gac cgc tct ttc tct atc ctt tac tgg aag 768 Glu Gly with Gln Asn and Asp Arg Ser Phe Ser with Tyr Tyr Lys 200 205 210 215 caa tca acg ctt tca aat ggt gcg caa tct ctt gac gta caa gac ttc 816 Gln Ser Thr Leu Ser Asn Gly Ala Gln Ser Leu Asp Val Gln Asp Phe 220 225 230 gag gag aac ggc ctt tca gtt tac aac tac aac gct tac agc ctt tac 864 Glu Glu Asn Gly Leu Ser Val Tyr Asn Tyr Asn Ala Tyr Ser Leu Tyr 235 240 245 ttc ctt cct agc act cgc ttc acg caa gag gac atc acg gag caa atc ​​912 Phe Leu Pro Ser Thr Arg Phe Thr Gln Glu Asp Ile Thr Glu Gln Ile 250 255 260 gac tac atg aag tgg gcg tat gcg tac aac aag ttc ttc tac atc tct 960 Asp Tyr Met Lys Trp Ala Tyr Ala Tyr Asn Lys Phe Phe Tyr Ile Ser 265 270 275 gac tac tac aag CA gta gac aca cca aac gta aag ggc tca tct ctt 1008 Asp Tyr Tyr Lys Gln Val Asp Thr Pro Asn Val Lys Gly Ser Ser Leu 280 285 290 295 gta ttc tgg ggt gag cac gcg aac gac ctt tct caa gag ggc ctt ctt 1056 Val Phe Trp Gly Glu His Ala Asn Asp Leu Ser Gln Glu Gly Leu Leu 300 305 310 aag caa gag aag cca ctt atc CAAAC ttc ctt ggc ctt 1095 Lys Gln Glu Lys Pro Leu Ile Gln Asn Phe Leu Gly Leu 315 320 <210> 2 <211> 365 <212> PRT <213> Bacillus glycinifermentans <400> 2 Met Val Ser Arg Thr Met Leu Phe Ile Lys Arg Glu Lys Gly Lys Thr -40 -35 -30 Val Leu Ile Lys Phe Leu Ser Ile Thr Thr Val Ser Ile Leu Leu Phe -25 -20 -15 -10 Leu Thr Met Ala Asn Thr Ala Gln Ala Gln Asp Gln Glu Lys Gly Ile -5 -1 1 5 Thr Ile Asp Ile Ser Arg Lys Tyr Tyr Ser Ile Lys Thr Leu Lys Ala 10 15 20 Ile Val Asp Glu Ile Ser Ala Asn Gly Gly Asp Tyr Leu Gln Leu His 25 30 35 Phe Ser Asp Asn Glu Ser Tyr Ala Ile Ala Ser Glu Phe Leu Gly Gln 40 45 50 55 Asn Ser Glu Asn Pro Asn Ser Ala Tyr Leu Thr Lys Lys Glu Leu Leu 60 65 70 Ser Leu Ile Ala Tyr Ser Asn Asp Arg Asn Ile Met Val Ile Pro Asp 75 80 85 Ile Asp Leu Pro Ala His Ser Lys Gly Trp Leu Asn Ile Met Lys Glu 90 95 100 Lys Asp Ser Gly Leu Tyr Thr Asp Ile Val Thr Asp Tyr Ser Glu Asp 105 110 115 Thr Leu Asp Tyr His Asn Asn Ala Val Ala Leu Tyr Thr Ala Asn Gln 120 125 130 135 Leu Leu Asp Glu Val Leu Asp Leu Phe Tyr Gln Pro Lys Phe Ala Gly 140 145 150 Lys Gln Arg Ile Val Leu Gly Gly Asp Glu Val Pro Gly Ser Gly Ala 155 160 165 His Gln Thr Asp Phe Ile Arg Phe Met Asn Gln Ile Ala Lys Thr Ala 170 175 180 Lys Ala Ser Asn Tyr Glu Pro Gln Met Trp Asn Asp Ser Ile Thr Pro 185 190 195 Glu Gly Ile Gln Asn Leu Asp Arg Ser Phe Ser Ile Leu Tyr Trp Lys 200 205 210 215 Gln Ser Thr Leu Ser Asn Gly Ala Gln Ser Leu Asp Val Gln Asp Phe 220 225 230 Glu Glu Asn Gly Leu Ser Val Tyr Asn Tyr Asn Ala Tyr Ser Leu Tyr 235 240 245 Phe Leu Pro Ser Thr Arg Phe Thr Gln Glu Asp Ile Thr Glu Gln Ile 250 255 260 Asp Tyr Met Lys Trp Ala Tyr Ala Tyr Asn Lys Phe Phe Tyr Ile Ser 265 270 275 Asp Tyr Tyr Lys Gln Val Asp Thr Pro Asn Val Lys Gly Ser Ser Leu 280 285 290 295 Val Phe Trp Gly Glu His Ala Asn Asp Leu Ser Gln Glu Gly Leu Leu 300 305 310 Lys Gln Glu Lys Pro Leu Ile Gln Asn Phe Leu Gly Leu 315 320 <210> 3 <211> 1047 <212> DNA <213> Terribacillus goriensis <220> <221> Signal peptide <222> (1)..(75) <220> <221> CDS <222> (1)..(1047) <220> <221> Mature peptide <222> (76)..(1047) <400> 3 atg ctt atc aag ttc ctt tct atc acg aca gtt tca atc ctt ctt ttc 48 Met Leu Ile Lys Phe Leu Ser Ile Thr Thr Val Ser Ile Leu Leu Phe -25 -20 -15 -10 ctt acg atg tct aac act gcg CA gcg CA Gac CA GAG AAG ggc atc 96 Leu Three Met Ser Asn Three Ala Gln Ala Gln Asp Gln Glu Lys Gly Ile -5 -1 1 5 aca atc ​​gac atc tct cgc aag tac tac tct atc gag aca ctt aag tct 144 Thr Asp Server with Arg Lys Tyr Tyr Ser with Glu Thr Leu Lys Ser 10 15 20 atc atc gac gag atc tca gcg aat ggt ggc gac tac ctt caa ctt cat 192 Ile Ile Asp Glu Ile Ser Ala Asn Gly Gly Asp Tyr Leu Gln Leu His 25 30 35 ttc tct gac aac gag cgc tac gcg atc gcg tca gag ttc ctt ggc caa 240 Phe Ser Asp Asn Glu Arg Tyr Ala Ile Ala Ser Glu Phe Leu Gly Gln 40 45 50 55 aac ggc gag aac cca aac tct act tac ctt aca aag aag gag ctt ctt 288 Asn Gly Glu Asn Pro Asn Ser Thr Leu Thr Lys Lys Leu Glu 60 65 70 tct ctt atc gcg tac tct aac gac cgc gac atc atg gta atc ​​cca gac 336 Ser Leu Ile Ala Tyr Ser Asn Asp Arg Asp Ile Met Val Ile Pro Asp 75 80 85 atc gac ctt cca gcg cat tct cgt ggc tgg ctt aac atc atg aag gag 384 Ile Asp Leu Pro Ala His Ser Arg Gly Trp Leu Asn Ile Met Lys Glu 90 95 100 aag gac tca ggc ctt tac act gac atc gta acg gac tac tct gag gac 432 Lys Asp Ser Gly Leu Tyr Thr Asp Ile Val Thr Asp Tyr Ser Glu Asp 105 110 115 aca ctt gac tac cat aac aac gct gta gcg ctt tac aca gca aac caa 480 Thr Leu Asp Tyr His Asn Asn Ala Val Ala Leu Tyr Thr Ala Asn Gln 120 125 130 135 ctt ctt gac gag gta ctt gac ctt ttc tac caa ccg aag ttt gct ggc 528 Leu Leu Asp Glu Val Leu Asp Leu Phe Tyr Gln Pro Lys Phe Ala Gly 140 145 150 aag cag cgc atc gta ctt ggt ggc gac gag gtt cct ggc tct ggc gta 576 Lys Gln Arg Ile Val Leu Gly Gly Asp Glu Val Pro Gly Ser Gly Val 155 160 165 cac cag act gac ttc atc cgc ttc atg aac caa atc ​​gcg gag act gcg 624 Historical History of Gln Thr Asp With Arg Phe Met Gln Thr Asp With Glu Thr Ala 170 175 180 aag gct tct aac tac aag cca caa atg tg tg tg aac gac tct atc aca cct Lys Ala Serves Asn Tyr Lys Pro Gln Met Trp Asn Asp Serves Ile Thr Pro 185 190 195 gag ggc atc caa aac ctt gac cgc tca ttc atc ctt tac tgg aag 720 Glu Gly with Gln Asn and Asp Arg Ser Phe Ser with Tyr Tyr Lys 200 205 210 215 caa tct aca ctt agc aat ggt gcg caa ggc ctt gac gta caa gac ttc 768 Gln Ser Thr Leu Ser Asn Gly Ala Gln Gly Leu Asp Val Gln Asp Phe 220 225 230 gag gag aac ggc ctt agc gtt tac aac tac aat gcg tac tca ctt tac 816 Glu Glu Asn Gly Leu Ser Val Tyr Asn Tyr Asn Ala Tyr Ser Leu Tyr 235 240 245 ttc ctt cct gcg aca cgc ttc act caa gag gac atc act gag caa atc ​​864 Phe Leu Pro Ala Thr Arg Phe Thr Gln Glu Asp Ile Thr Glu Gln Ile 250 255 260 gac tac atg aag tgg gcg tat gcg tac aac aag ttc ttc tac atc tct 912 Asp Tyr Met Lys Trp Ala Tyr Ala Tyr Asn Lys Phe Phe Tyr Ile Ser 265 270 275 gac tac tac aag caa gta gac aca agc aac gtt aag ggc tct tct ctt 960 Asp Tyr Tyr Lys Gln Val Asp Thr Ser Asn Val Lys Gly Ser Ser Leu 280 285 290 295 gta ttc tgg ggt gag cat gcg aac gac ctt agc caa gag ggc ctt ctt 1008 Val Phe Trp Gly Glu His Ala Asn Asp Leu Ser Gln Glu Gly Leu Leu 300 305 310 aag caa gag aag cca ctt atc CA acac ttc ctt ggc ctt 1047 Lys Gln Glu Lys Pro Leu And Gln Asn Phe Leu Gly Leu 315 320 <210> 4 <211> 349 <212> PRT <213> Terribacillus goriensis <400> 4 Met And Lys Phe Will Be Thr Thr Val Will Be Thr -25 -20 -15 -10 Leu Thr Met Ser Asn Thr Ala Gln Ala Gln Asp Gln Glu Lys Gly Ile -5 -1 1 5 Thr Ile Asp Ile Ser Arg Lys Tyr Tyr Ser Ile Glu Thr Leu Lys Ser 10 15 20 Ile Ile Asp Glu Ile Ser Ala Asn Gly Gly Asp Tyr Leu Gln Leu His 25 30 35 Phe Ser Asp Asn Glu Arg Tyr Ala Ile Ala Ser Glu Phe Leu Gly Gln 40 45 50 55 Asn Gly Glu Asn Pro Asn Ser Thr Tyr Leu Thr Lys Lys Glu Leu Leu 60 65 70 Ser Leu Ile Ala Tyr Ser Asn Asp Arg Asp Ile Met Val Ile Pro Asp 75 80 85 Ile Asp Leu Pro Ala His Ser Arg Gly Trp Leu Asn Ile Met Lys Glu 90 95 100 Lys Asp Ser Gly Leu Tyr Thr Asp Ile Val Thr Asp Tyr Ser Glu Asp 105 110 115 Thr Leu Asp Tyr His Asn Asn Ala Val Ala Leu Tyr Thr Ala Asn Gln 120 125 130 135 Leu Leu Asp Glu Val Leu Asp Leu Phe Tyr Gln Pro Lys Phe Ala Gly 140 145 150 Lys Gln Arg Ile Val Leu Gly Gly Asp Glu Val Pro Gly Ser Gly Val 155 160 165 His Gln Thr Asp Phe Ile Arg Phe Met Asn Gln Ile Ala Glu Thr Ala 170 175 180 Lys Ala Ser Asn Tyr Lys Pro Gln Met Trp Asn Asp Ser Ile Thr Pro 185 190 195 Glu Gly Ile Gln Asn Leu Asp Arg Ser Phe Ser Ile Leu Tyr Trp Lys 200 205 210 215 Gln Ser Thr Leu Ser Asn Gly Ala Gln Gly Leu Asp Val Gln Asp Phe 220 225 230 Glu Glu Asn Gly Leu Ser Val Tyr Asn Tyr Asn Ala Tyr Ser Leu Tyr 235 240 245 Phe Leu Pro Ala Thr Arg Phe Thr Gln Glu Asp Ile Thr Glu Gln Ile 250 255 260 Asp Tyr Met Lys Trp Ala Tyr Ala Tyr Asn Lys Phe Phe Tyr Ile Ser 265 270 275 As...

Claims

1. A cleaning composition comprising at least 0.001 ppm of *Bacillus terrestrialis* with aminohexosidase activity. Terribacillus The polypeptide of the evolutionary branch, and at least one auxiliary component, wherein the polypeptide has at least 99% sequence identity with the polypeptide shown in SEQ ID NO: 10, and wherein the polypeptide comprises the motifs GXDE (SEQ ID NO: 18), WND[SQR][IVL][TLVM] (SEQ ID NO: 21) and QSTL (SEQ ID NO: 22), wherein the polypeptide is derived from Bacillus saccharophilus (Saccharophilus terrestris). Terribacillus saccharophilus ).

2. The composition according to claim 1, wherein the polypeptide has N-acetylglucosidase activity and / or β-N-acetylglucosidase activity.

3. The composition according to claim 1 or 2, wherein the polypeptide further comprises one or more of the motifs [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), NKFFY (SEQ ID NO: 23), and NLD[DR]S (SEQ ID NO: 24).

4. The composition according to claim 1 or 2, wherein the polypeptide has 100% sequence identity with the polypeptide shown in SEQ ID NO:

10.

5. The composition according to claim 1 or 2, wherein the polypeptide is composed of SEQ ID NO:

10.

6. The composition according to claim 1 or 2, wherein the cleaning composition is a laundry or dishwashing composition.

7. The composition according to claim 1 or 2, wherein the auxiliary agent is selected from, a) At least one detergent builder b) at least one surfactant, and c) At least one bleaching component.

8. The composition according to claim 7, wherein the composition comprises at least one detergent builder, wherein the detergent builder is added in an amount from 5% to 65% wt%, wherein the detergent builder is selected from phosphates, sodium citrate detergent builder, sodium carbonate, sodium silicate and zeolite.

9. The composition of claim 7, wherein the composition comprises at least one bleaching component from 1 wt% to 40 wt%, or from 0.5 wt% to 30 wt%, wherein the bleaching component comprises a percarbonate and a bleaching catalyst.

10. The composition of claim 7, wherein the composition comprises anionic surfactant and / or nonionic surfactant in amounts of 2 wt% to 60 wt%, 5 wt% to 50 wt%, 5 wt% to 40 wt%, 5 wt% to 30 wt%, 5 wt% to 20 wt%, or 5 wt% to 10 wt%.

11. Use of the composition according to any one of claims 1 to 10 for deep cleaning articles, wherein the articles are textiles.

12. A method for washing articles, the method comprising: a) Exposing the article to a washing solution containing a polypeptide of a Bacillus terreus clade having at least 99% sequence identity with the polypeptide shown in SEQ ID NO: 10, wherein the polypeptide has aminohexosidase activity and contains the motifs GXDE (SEQ ID NO: 18), WND[SQR][IVL][TLVM] (SEQ ID NO: 21) and QSTL (SEQ ID NO: 22), wherein the polypeptide is derived from Bacillus terreus (Saccharophilus). Terribacillus saccharophilus Or expose the article to the composition according to any one of claims 1 to 10; b) Complete at least one washing cycle; and c) Optionally rinse the article, wherein the article is a textile.

13. Use of a polypeptide from the *Bacillus terreus* clade in a cleaning process, wherein the polypeptide has at least 99% sequence identity with the polypeptide shown in SEQ ID NO: 10, and wherein the polypeptide is derived from *Bacillus saccharophilus*. Terribacillus saccharophilus The polypeptide has aminohexosidase activity and contains the motifs GXDE (SEQ ID NO: 18), WND[SQR][IVL][TLVM] (SEQ ID NO: 21) and QSTL (SEQ ID NO: 22).

14. The use according to claim 13, wherein the polypeptide has N-acetylglucosidase activity and / or β-N-acetylglucosidase activity.

15. The use according to claim 13 or 14, wherein the polypeptide further comprises one or more of the motifs [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), NKFFY (SEQ ID NO: 23), and NLD[DR]S (SEQ ID NO: 24).

16. The use according to claim 13 or 14, wherein the use is for deep cleaning articles, wherein the articles are textiles.

17. The use according to claim 13 or 14, wherein the use (i) Used to prevent, reduce or remove the stickiness of the article; (ii) Used for pre-treating stains on the item; (iii) Used to prevent, reduce or remove the redeposition of dirt during a washing cycle; (iv) Used to prevent, reduce or remove dirt from adhering to the article; (v) Used to maintain or improve the whiteness of the article; or (vi) Used to prevent, reduce or eliminate the odor of the item. The item in question is a textile.

18. The use according to claim 13 or 14, wherein the polypeptide has 100% sequence identity with the polypeptide shown in SEQ ID NO: 10.

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