Composition containing a polypeptide having alkaline phosphatase activity

Abstract

The present invention relates to a cleaning composition comprising a polypeptide having alkaline phosphatase activity, a polypeptide having alkaline phosphatase activity, and optionally a polypeptide having DNase activity, and to the use of the said composition in a cleaning process such as washing.

Description

[Technical Field]

[0001] Reference to sequence list This application includes a computer-readable list of sequences, which is incorporated herein by reference.

[0002] The present invention relates to a polypeptide having alkaline phosphatase activity, a composition such as a washing composition comprising a polypeptide having alkaline phosphatase activity and optionally a polypeptide having DNase activity, and a washing method using the polypeptide and the composition. [Background technology]

[0003] For example, in laundry, the cleaning solution contains a mixture of various dirt particles, including both organic and inorganic molecules of biological origin. Such cleaning solutions invariably contain phosphate ions. Because phosphate ions play diverse roles, they are particularly important in the physiology of various organisms. For instance, phosphorylation is a unique mechanism for signaling the activation or inactivation of enzymes and other peptides.

[0004] Organophosphate esters (OPs) are a class of compounds whose general structure is O=P(OR)3. They are considered esters of phosphoric acid. Biomolecules such as ATP, RNA, and DNA, as well as phospholipids, are included in the class of organophosphate esters, along with industrially produced chemicals such as insecticides, herbicides, and nerve agents.

[0005] Phospholipids are essential components of cell membranes, and phosphate groups are included in the structural framework of the DNA backbone. Both types of molecules are present in human cells, microbial cells, and cell fragments.

[0006] These compounds are involved in adsorption and precipitation by mineral surfaces. In washing situations, most are dissolved by surfactants and retained in the solution, but in some cases, organophosphate esters are exposed to the surrounding water hardness ions such as calcium and magnesium. These ions, as well as similar divalent and trivalent cations, tend to sequester with organophosphate esters. This occurs particularly through ionic crosslinking with other phosphate groups or with acids of other types of functional groups in proteins and fatty acids.

[0007] Bacteria and other microorganisms that grow on various types of surfaces, including textiles and hard surfaces, tend to be embedded in the self-generating matrix of extracellular polymer material (EPS). Biofilm EPS is generally a polymer aggregate composed of extracellular DNA, proteins, and polysaccharides. In many biofilms, extracellular DNA (eDNA) containing large amounts of 5'-phosphate forms an essential part of the EPS matrix.

[0008] DNase enzymes are known to be used, for example, in laundry detergents. One situation in which organophosphate esters are exposed during washing is related to the degradation of DNA by DNase enzymes. Hydrolysis by DNase produces 5' ends of DNA fragments, which tend to form ionic crosslinks with other functional groups. When fatty acids are released from dirt or suspended in the washing solution as part of the detergent formulation, they can precipitate along with the DNA degraded by DNase. This is mainly because large molecules are formed that are difficult to retain in the solution. Such precipitation can be problematic and may lead to reduced cleaning performance and / or re-deposition of dirt onto laundry items.

[0009] International Publication No. 99 / 10466 is said to disclose a washing composition containing phosphatases, but this document does not contain any examples and does not provide any information about the properties of the phosphatases other than stating that it includes alkaline phosphatase EC3.1.3.1 and acid phosphatase 3.1.3.2. [Overview of the project]

[0010] The inventors discovered that adding alkaline phosphatase (ALP) to a washing solution, such as a solution containing organophosphate groups, yields remarkable advantages, including improved washing performance and reduced redeposition. In particular, synergistic effects have been observed when ALP is used in combination with other enzymes such as DNases.

[0011] In one embodiment, the present invention provides a cleaning composition comprising a polypeptide having DNase activity, a polypeptide having alkaline phosphatase activity, and at least one detergent auxiliary component.

[0012] In another aspect, the present invention provides a cleaning composition comprising a polypeptide having alkaline phosphatase activity and at least one detergent auxiliary component, wherein the polypeptide having alkaline phosphatase activity is selected from the group consisting of: (a) SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 polypeptides having at least 70% sequence identity with SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0013] Further aspects of the present invention relate, for example, to washing and laundry methods using the polypeptides and compositions of the present invention, and to the polypeptide having alkaline phosphatase activity.

[0014] Array Overview Sequence ID No. 1 is a polypeptide obtained from Neobacillus bataviensis, which possesses alkaline phosphatase activity.

[0015] Sequence ID No. 2 is a polypeptide obtained from Parageobacillus caldoxylosilyticus, which possesses alkaline phosphatase activity.

[0016] Sequence ID No. 3 is a polypeptide obtained from Geobacillus thermoleovorans, which possesses alkaline phosphatase activity.

[0017] Sequence ID No. 4 is a polypeptide obtained from a Sporolactobacillus species that possesses alkaline phosphatase activity.

[0018] Sequence ID No. 5 is a polypeptide obtained from Anoxybacillus caldiproteolyticus, which possesses alkaline phosphatase activity.

[0019] Sequence ID No. 6 is a polypeptide obtained from Geobacillus thermoleovorans, which possesses alkaline phosphatase activity.

[0020] Sequence ID 7 is a polypeptide obtained from Paenibacillus xylanexedens, which possesses alkaline phosphatase activity.

[0021] Sequence ID No. 8 is a polypeptide obtained from a Sporolactobacillus species that possesses alkaline phosphatase activity.

[0022] SEQ ID NO: 9 is a polypeptide obtained from Paenibacillus panacisoli having alkaline phosphatase activity.

[0023] SEQ ID NO: 10 is a polypeptide obtained from Collimonas pratensis having alkaline phosphatase activity.

[0024] SEQ ID NO: 11 is a polypeptide obtained from Paenibacillus illinoisensis having alkaline phosphatase activity.

[0025] SEQ ID NO: 12 is a polypeptide obtained from Aeromonas salmonicida subsp. salmonicida having alkaline phosphatase activity.

[0026] SEQ ID NO: 13 is a polypeptide obtained from Paenibacillus amylolyticus having alkaline phosphatase activity.

[0027] SEQ ID NO: 14 is a polypeptide obtained from Serratia plymuthica having alkaline phosphatase activity.

[0028] SEQ ID NO: 15 is a polypeptide obtained from Cytobacillus firmus having alkaline phosphatase activity.

[0029] SEQ ID NO: 16 is a polypeptide obtained from Priestia megaterium having alkaline phosphatase activity.

[0030] Sequence ID No. 17 is a polypeptide obtained from Trichoderma citrinoviride, which possesses alkaline phosphatase activity.

[0031] Sequence ID No. 18 is a polypeptide obtained from Truncatella angustata, which possesses alkaline phosphatase activity.

[0032] Sequence ID No. 19 is a polypeptide obtained from Morchella semilibera, which possesses alkaline phosphatase activity.

[0033] Sequence ID No. 20 is a polypeptide obtained from Serratia ficaria, which possesses alkaline phosphatase activity.

[0034] Sequence ID No. 21 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0035] Sequence ID No. 22 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0036] Sequence ID No. 23 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0037] Sequence ID No. 24 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0038] Sequence ID No. 25 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0039] Sequence ID No. 26 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0040] Sequence ID No. 27 is a polypeptide obtained from Sporormia fimetaria, which possesses alkaline phosphatase activity.

[0041] Sequence ID No. 28 is a polypeptide obtained from Thermoascus crustaceus, which possesses alkaline phosphatase activity.

[0042] Sequence ID No. 29 is a polypeptide obtained from Thielavia australiensis, which possesses alkaline phosphatase activity.

[0043] Sequence ID No. 30 is a polypeptide obtained from Chaetomium thermophilum var. thermophilum, a variety of Chaetomium thermophilum that possesses alkaline phosphatase activity.

[0044] Sequence ID No. 31 is a polypeptide obtained from an Aspergillus species that possesses alkaline phosphatase activity.

[0045] Sequence ID No. 32 is a polypeptide obtained from a bacterial species of the genus Colletotrichum that possesses alkaline phosphatase activity.

[0046] Sequence ID No. 33 is a polypeptide obtained from chicken cecal pouches that possess alkaline phosphatase activity.

[0047] Sequence ID No. 34 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0048] Sequence ID No. 35 is a polypeptide obtained from a species of Caulobacter that possesses alkaline phosphatase activity.

[0049] Sequence ID No. 36 is a polypeptide obtained from Caulobacter vibrioides, which possesses alkaline phosphatase activity.

[0050] Sequence ID No. 37 is a polypeptide obtained from Serratia nematodiphila, which possesses alkaline phosphatase activity.

[0051] Sequence ID No. 38 is a polypeptide obtained from Loktanella salsilacus, which possesses alkaline phosphatase activity.

[0052] Sequence ID No. 39 is a polypeptide obtained from Sphingopyxis chilensis, which possesses alkaline phosphatase activity.

[0053] Sequence ID No. 40 is a polypeptide obtained from a bacterial species of the genus Tolypocladium that possesses alkaline phosphatase activity.

[0054] Sequence ID No. 41 is a polypeptide obtained from Penicillium vasconiae, which possesses alkaline phosphatase activity.

[0055] Sequence ID No. 42 is a polypeptide obtained from a Cladobotryum species that possesses alkaline phosphatase activity.

[0056] Sequence ID No. 43 is a polypeptide obtained from a Taifanglania species that possesses alkaline phosphatase activity.

[0057] Sequence ID No. 44 is a polypeptide obtained from a bacterial species of the genus Achaetomium that possesses alkaline phosphatase activity.

[0058] Sequence ID No. 45 is a polypeptide obtained from a Chaetomium species that possesses alkaline phosphatase activity.

[0059] Sequence ID No. 46 is a polypeptide obtained from Fontibacillus aquaticus, which possesses alkaline phosphatase activity.

[0060] Sequence ID No. 47 is a polypeptide obtained from a Paenibacillus species that possesses alkaline phosphatase activity.

[0061] Sequence ID No. 48 is a polypeptide obtained from a Paenibacillus species that possesses alkaline phosphatase activity.

[0062] Sequence ID No. 49 is a polypeptide obtained from Paenibacillus woosongensis, which possesses alkaline phosphatase activity.

[0063] Sequence ID No. 50 is a polypeptide obtained from Lederbergia lenta that possesses alkaline phosphatase activity, and is the polypeptide described in U.S. Patent Application Publication No. 2018 / 0326020.

[0064] Sequence ID No. 51 is a polypeptide obtained from a metagenomic that possesses alkaline phosphatase activity.

[0065] Sequence ID No. 52 is a polypeptide obtained from a Paenibacillus species that possesses alkaline phosphatase activity.

[0066] Sequence ID No. 53 is a polypeptide obtained from Paenibacillus castaneae, which possesses alkaline phosphatase activity.

[0067] Sequence ID No. 54 is a polypeptide obtained from Paenibacillus taohuashanense, which possesses alkaline phosphatase activity.

[0068] Sequence ID No. 55 is a polypeptide obtained from Hyphomonas hirschiana, which possesses alkaline phosphatase activity.

[0069] Sequence ID No. 56 is a polypeptide obtained from Hyphomonas oceanitis, which possesses alkaline phosphatase activity.

[0070] Sequence ID No. 57 is a polypeptide obtained from Deinococcus radiodurans, which possesses alkaline phosphatase activity.

[0071] Sequence ID No. 58 is a polypeptide obtained from Deinococcus gobiensis, which possesses alkaline phosphatase activity.

[0072] Sequence ID No. 59 is a polypeptide obtained from Deinococcus pimensis, which possesses alkaline phosphatase activity.

[0073] Sequence ID No. 60 is a polypeptide obtained from a Deinococcus species that possesses alkaline phosphatase activity.

[0074] Sequence ID 61 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0075] Sequence ID 62 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0076] Sequence ID 63 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0077] Sequence ID 64 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0078] Sequence ID 65 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0079] Sequence ID 66 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0080] Sequence ID 67 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0081] Sequence ID 68 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0082] Sequence ID 69 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0083] Sequence ID 70 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0084] Sequence ID 71 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0085] Sequence ID 72 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0086] Sequence ID 73 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0087] Sequence ID 74 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0088] Sequence ID 75 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0089] Sequence ID 76 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0090] Sequence ID 77 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0091] Sequence ID 78 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0092] Sequence ID 79 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0093] Sequence ID 80 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0094] Sequence ID 81 is a variant of Sequence ID 1 that possesses alkaline phosphatase activity.

[0095] Sequence ID No. 82 is a polypeptide with DNase activity obtained from Metabacillus indicus.

[0096] Sequence ID 83 is a variant of Sequence ID 82 that possesses DNase activity.

[0097] Sequence ID 84 is a secretion signal from Bacillus clausii.

[0098] Sequence ID 85 is the His tag.

[0099] Sequence ID No. 86 is a polypeptide with DNase activity obtained from Aspergillus oryzae.

[0100] definition In accordance with the detailed explanation herein, the following definitions apply. Note that unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” also include the plural forms.

[0101] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0102] Alkaline phosphatase: The term “alkaline phosphatase” refers to a polypeptide having alkaline phosphatases activity that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Alkaline phosphatases belong to the esterase subgroup (EC number 3.1), which is a subgroup of hydrolytic enzymes, and specifically to phosphate monoester hydrolases (EC number 3.1.3). Alkaline phosphatases are classified under EC 3.1.3.1 and remove phosphate groups from various types of molecules. For the purposes of this invention, alkaline phosphatase activity can be measured according to the alkaline phosphatase activity assays in the examples. The terms “alkaline phosphatase” (abbreviated as “ALP”) and “polypeptide having alkaline phosphatases activity” can be used interchangeably throughout this application.

[0103] Alkaline phosphatase variant: An "alkaline phosphatase variant" is a variant of any of the alkaline phosphatase sequences disclosed herein, which has alkaline phosphatase activity and has one or more distinct mutations (substitutions, deletions, insertions, or extensions), typically substitutions or combinations of substitutions, compared to the parent sequence.

[0104] DNase: The term "DNase" refers to a polypeptide having DNase (deoxyribonuclease) activity, which catalyzes the hydrolysis of phosphodiester bonds in DNA and degrades DNA. DNases belong to the esterase (EC number 3.1), a subgroup of hydrolytic enzymes. DNases are classified under EC 3.1.21. For the purposes of this invention, DNase activity can be measured according to the procedure described in the DNase activity assay of the Examples. The terms "DNase" and "polypeptide having DNase activity" can be used interchangeably throughout this application.

[0105] DNase variant: A "DNase variant" is a variant of any parent DNase that has one or more distinct mutations (substitutions, deletions, insertions, or extensions), typically substitutions or combinations of substitutions, compared to the parent sequence.

[0106] Expression: The term "expression" includes, but is not limited to, any of the processes involved in the production of an enzyme, including, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0107] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence encoding an enzyme, the encoding sequence being operably ligated to appropriate regulatory sequences capable of producing DNA expression in a suitable host. Such regulatory sequences may include promoters that perform transcription, desired operator sequences for controlling transcription, sequences encoding appropriate ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0108] Elongation: The term "elongation" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of an alkaline phosphatase or DNase, and the "elongated" enzyme has alkaline phosphatase activity or DNase activity.

[0109] Fragment: The term "fragment" refers to an enzyme having one or more amino acids missing from the amino-terminus and / or carboxyl-terminus, and this fragment possesses enzymatic activity, such as alkaline phosphatase activity.

[0110] Host cell: A “host cell” is an organism into which an expression vector, phage, virus, or other DNA construct containing an enzyme-encoding polynucleotide has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, fungi, and yeast) capable of expressing the polypeptide of interest.

[0111] Improved Properties: The term "improved properties" refers to properties associated with a variant that have been improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermal stability. In the present invention, improved properties are particularly improvements in stability, such as improved storage stability in detergent compositions, improved stability in use in detergent compositions, and / or improved thermal stability. Furthermore, the variant may also have other improved properties, such as improved alkaline phosphatase or DNase activity, such as improved specific activity and / or improved cleaning performance.

[0112] Transfection: In the context of inserting a nucleic acid sequence into a cell, the term "transfection" means "transfection," "transformation," or "transduction," as is well known in the art.

[0113] Isolated: The term “isolated” means that a polypeptide, nucleic acid, cell, or other specific material or component has been separated from at least one other material or component, including but not limited to other proteins, nucleic acids, or cells. Therefore, isolated polypeptides, nucleic acids, cells, or other materials are forms that do not occur naturally. Isolated polypeptides include, but are not limited to, culture broth containing secreted polypeptides expressed in host cells. Typically, the isolated polypeptides of the present invention are separated from other components in the culture broth using known protein purification methods.

[0114] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal processing and / or C-terminal processing (e.g., removal of signal peptides).

[0115] It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known that different host cells can process polypeptides in different ways, and therefore, one host cell expressing a polynucleotide can produce different mature polypeptides (e.g., having different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide.

[0116] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having the relevant enzymatic activity, namely alkaline phosphatase activity or DNase activity as used herein.

[0117] Mutant: The term "mutant" refers to a polynucleotide that codes for a variant.

[0118] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0119] Nucleic acids: The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids can be single-stranded or double-stranded and may be chemically modified. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, multiple codons may be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction.

[0120] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from naturally occurring genes, modified to include nucleic acid segments in a manner not found in nature, or synthesized, and contains one or more regulatory sequences operably ligated to the nucleic acid sequence.

[0121] Operablely linked: The term "operably linked" means that certain components are in a relationship (including, but not limited to, juxtaposition) that allows them to function as intended. For example, a control sequence is operably linked to a code sequence such that the expression of the code sequence is under the control of the control sequence.

[0122] Parent: The terms “parent,” “parent enzyme,” or “parent polypeptide” refer to an enzyme, particularly an alkaline phosphatase or DNase, that has been modified to produce a variant with the same enzymatic function. For example, an alkaline phosphatase variant will have alkaline phosphatase activity. Modifications to the parent resulting in a variant typically include one or more substitutions, and may also include one or more insertions and / or deletions, and / or elongation or shortening of the N-terminus or C-terminus.

[0123] Polypeptides: Enzymes disclosed herein, including alkaline phosphatases and DNases, may be interchangeably referred to as "polypeptides" or "enzymes."

[0124] Recombination: The term "recombination," used in its ordinary sense, refers to the formation of sequences different from those found in nature, through operations such as cleaving and rejoining nucleic acid sequences. The term "recombination" refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their native state. Therefore, for example, a recombinant cell may express genes that are not present in the cell's native (non-recombinant) form, or it may express native genes at different levels or under different conditions than those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0125] Sequence Identity: The relationship between two amino acid sequences or two nucleotide sequences is represented by a parameter called "sequence identity."

[0126] For the purposes of this invention, sequence identity between two amino acid sequences is determined as the “longest identity” output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Needle program to report the longest identity, the “-nobrief option” must be specified on the command line. The Needle output labeled “longest identity” is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment).

[0127] Signal peptides: Signal peptides are sequences of amino acids attached to the N-terminus of a protein that promote the secretion of proteins outside the cell. Mature extracellular proteins lack signal peptides and are cleaved during the secretion process.

[0128] Variant: The term "variant" refers to a polypeptide having alkaline phosphatase activity or DNase activity that includes substitutions, insertions (including extensions), and / or deletions (e.g., truncation) at one or more positions. Substitution means replacing an amino acid at a given position with a different amino acid, deletion means removing an amino acid at a given position, and insertion means adding 1 to 5 amino acids (e.g., 1 to 3 amino acids, especially 1 amino acid) adjacent to or immediately following an amino acid at a given position.

[0129] Wild-type: With respect to an amino acid sequence or nucleic acid sequence, the term “wild-type” means that the amino acid sequence or nucleic acid sequence is natural or naturally occurring. As used herein, “naturally occurring” refers to anything found in nature (e.g., proteins, amino acids, nucleic acid sequences). Conversely, the term “not naturally occurring” refers to something that does not exist in nature (e.g., recombinant nucleic acids and protein sequences produced in a laboratory or by modification of a wild-type sequence).

[0130] Biofilms: Biofilms are any group of microorganisms whose cells adhere to each other on the surface of textiles, tableware, or other hard surfaces. These adherent cells are often embedded in a self-generated extracellular polymer (EPS) matrix. Biofilm EPS is generally a polymer aggregate composed of extracellular DNA, proteins, and polysaccharides. Biofilms can form on biological or non-biological surfaces. Microbial cells that grow within biofilms are physiologically different from plankton cells of the same organism, which, in contrast, are single cells that float or swim in a liquid medium. Bacteria that survive within biofilms usually have significantly different characteristics from planktonic bacteria of the same species. This is because the dense, protected environment of the biofilm allows bacteria to cooperate and interact in various ways. One advantage of this environment is that the high-density extracellular matrix and outer layer of cells protect the inside of the community, resulting in increased resistance to detergents and antibiotics. The following species of bacteria are found to form biofilms on laundry: Acinetobacter, Aeromicrobium, Brevundimonas, Microbacterium, Micrococcus luteus, Pseudomonas, Staphylococcus epidermidis, and Stenotrophomonas.

[0131] Stability: The term "stability" includes storage stability and stability during use (e.g., during the washing process), and reflects the stability of an enzyme (e.g., alkaline phosphatase or its variants, or DNase or its variants) as a function of time. For example, it refers to the degree to which the activity of an enzyme is retained when it is kept in a solution (especially a detergent solution). Stability is affected by many factors, including pH, temperature, and the composition of the detergent (e.g., the amount of builders or surfactants). Stability can be measured and expressed by, for example, the melting point (Tm) or the half-life improvement factor (HIF) compared to the parent enzyme or reference sequence. The terms "improved stability" and "increased stability" also include the stability of the detergent.

[0132] Improved cleaning performance: The term "improved cleaning performance" can be defined as an improvement in cleaning power, such as an improvement in the thorough cleaning of textile products.

[0133] Cleaning performance can be expressed as the remission value of the stain swatch. After cleaning and rinsing, spread the swatch flat and allow it to air dry at room temperature overnight. All cleaned swatches are evaluated the day after cleaning. The light reflectance of the swatches is evaluated using appropriate equipment such as a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. Measurements are performed with incident light that does not contain UV, and the reflectance value at 460 nm is typically extracted.

[0134] Laundry: The term "laundry" refers to both household and industrial laundry, and means the process of treating textile products with a solution containing, for example, the cleaning composition or detergent composition of the present invention. The laundry process can be carried out, for example, using a household or commercial washing machine, or by hand.

[0135] Thorough Cleaning: The term "thorough cleaning" refers to the reduction or removal of organic matter, including biofilm components, such as EPS or parts thereof, polysaccharides, PNGA (poly-N-acetylglucosamine), proteins, DNA, dirt, or other components present within the biofilm. Thorough cleaning encompasses not only the removal of visible dirt on textile products, but also the removal of organic matter or odors within textile products that cannot be removed by conventional detergent compositions and enzymes. Such organic matter includes, for example, dead cell material, necrotic skin tissue fragments, sebum, sweat, grease, and other dirt originating from, for example, humans (body dirt) or microorganisms.

[0136] Detergent Compositions: The term “detergent composition” (or “cleaning composition”) includes all forms of detergents or cleaning compositions unless otherwise specified. These include: multipurpose or strong detergents, especially cleaning detergents, in granular or powder form; multipurpose detergents, especially cleaning detergents, in liquid, gel or paste form; multipurpose cleaning agents, especially so-called strong liquid (HDL) type, in liquid, gel or paste form; single-dose (SUD) compositions, such as pods, capsules, or tabs, with one or more chambers; liquid high-grade laundry detergents; hand-washing or low-load dishwashing detergents, especially high-foaming types; dishwasher detergents, including various tablet, granule, liquid, and rinse-type detergents for household and commercial use; liquid detergents and disinfectants (such as antibacterial hand washes, cleaning bars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners, etc.); hair shampoos and hair rinses; shower gels, bubble baths; metal cleaners; and cleaning aids such as bleach additives and “stain remover sticks” or pre-treatment types. The terms “detergent composition” and “detergent formulation” are used in reference to mixtures intended for use as a cleaning medium for cleaning soiled objects. In some embodiments, the terms are used in reference to washing fabrics and / or clothing (e.g., “laundry detergent”). In other embodiments, the terms refer to other detergents, such as detergents used for washing dishes, cutlery, etc. (e.g., “dishwashing detergent”). The present invention is not limited to any particular detergent formulation or composition. The term “detergent composition” is not intended to be limited to compositions containing surfactants. In addition to the polypeptides of the present invention, the term is intended to encompass detergents that may include, for example, surfactants, builders, chelating agents or chelating agents, bleaching agents or bleaching components, polymers, softeners, foaming accelerators, foam inhibitors, dyes, fragrances, anti-yellowing agents, optical glossing agents, disinfectants, antifungal agents, dirt suspenders, rust inhibitors, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolytic enzymes, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0137] Textiles: The term "textiles" encompasses all fibrous materials. Therefore, this term is intended to include not only clothing, but also woven fabrics, yarns, fibers, nonwovens, natural materials, synthetic materials, and other fibrous materials.

[0138] Textile products: The term “textile products” refers to woven fabrics, as well as short fibers and filaments suitable for conversion or use into yarns, woven fabrics, knitted fabrics, and nonwoven fabrics. This term includes yarns made from synthetic fibers (e.g., artificial fibers) as well as natural fibers. The term “textile materials” is a general term for fibers, yarn intermediates, yarns, woven fabrics, and products made from woven fabrics (clothing and other products).

[0139] Non-fabric detergent compositions: The term "non-fabric detergent compositions" includes, but is not limited to, detergent compositions for non-fabric surfaces, including, hard surface cleaning compositions such as dishwashing detergent compositions including manual dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and personal cleansing compositions.

[0140] Cleaning hard surfaces: The term “cleaning hard surfaces” includes, but is not limited to, hard surfaces in household or industrial settings, such as floors, walls, tabletops, kitchen surfaces including kitchen appliance surfaces, and bathroom surfaces, in addition to dishwashing.

[0141] Effective amount of enzyme: The term "effective amount of enzyme" refers to the amount of enzyme required to achieve the enzymatic activity needed for a particular application, such as a defined detergent composition. Such an effective amount can be easily determined by those skilled in the art and is based on many factors, including the specific enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry composition (e.g., granular, rod) is required.

[0142] Relevant cleaning conditions: In this specification, the term “relevant cleaning conditions” is used to refer to the conditions actually used in households within the detergent market segment, in particular the cleaning temperature, time, cleaning mechanism, detergent concentration, detergent type, and water hardness.

[0143] Cleaning solution: The term “cleaning solution” (or “cleaning water”) refers to an aqueous solution containing alkaline phosphatase according to the present invention. The cleaning solution is a solution used in a washing machine or dishwasher and comprises water and a detergent composition containing alkaline phosphatase. The detergent composition may be in any suitable form, such as a liquid or powder, as described elsewhere in this specification, before being mixed with water to form the cleaning solution.

[0144] Water hardness: As used herein, the terms “water hardness,” “degree of hardness,” “dH,” or “°dH” refer to the German degree of hardness. One degree is defined as 10 milligrams of calcium oxide per liter of water.

[0145] Auxiliary Materials: The term “auxiliary materials” or “auxiliary components” refers to liquid, solid, or gaseous materials selected to suit the specific type of desired detergent composition and product form (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, or foam composition), which are preferably compatible with the alkaline phosphatase enzyme used in the composition. Further information regarding auxiliary materials is provided below.

[0146] Low detergent concentration: The term "low detergent concentration" includes detergents in which the detergent component in the wash water is less than approximately 800 ppm. Asian detergents, such as Japanese detergents, are typically considered to be in the low detergent concentration category.

[0147] Medium detergent concentration: The term "medium detergent concentration" includes detergents that contain approximately 800 ppm to 2000 ppm of detergent components in the wash water. North American detergents are generally considered to be in the medium detergent concentration category.

[0148] High detergent concentration: The term "high detergent concentration" includes detergents that contain detergent components at a concentration of approximately 2000 ppm or more in the wash water. European detergents are generally considered to be of the high detergent concentration type.

[0149] Rules for designating variants For ease of reference when describing enzyme variants in relation to the present invention, the following nomenclature is used. Certified IUPAC single-letter amino acid abbreviations are employed.

[0150] Substitutions. The following nomenclature is used for amino acid substitutions: original amino acid, position, substituted amino acid. Therefore, a substitution of threonine at position 226 with alanine is designated as "T226A". Multiple mutations are separated by an additional sign ("+"). For example, "G205R+S411F" indicates that glycine (G) at positions 205 and 411 is substituted with arginine (R), and serine (S) is substituted with phenylalanine (F), respectively. Multiple mutations can also be indicated by spaces, commas, or plus signs (e.g., "G205R S411F", "G205R,S411F", or "G205R+S411F").

[0151] Deletion. In the case of amino acid deletions, the following nomenclature is used: original amino acid, position, *. Therefore, a deletion of glycine at position 195 is designated as "G195*". Multiple deletions are separated by an additional sign ("+") (for example, "G195*+S411*").

[0152] Insertion. In the case of amino acid insertion, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is designated as "G195GK". Insertions of multiple amino acids are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, if lysine and alanine are inserted after glycine at position 195, it is indicated as "G195GKA".

[0153] Multiple Modifications. Variants containing multiple modifications are separated by an additional sign ("+") as described above. For example, "R170Y+G195E" indicates that arginine and glycine at positions 170 and 195 are replaced with tyrosine and glutamic acid, respectively. Alternatively, multiple modifications can be separated by spaces or commas as described above.

[0154] Various modifications. When different modifications can be introduced at a certain position, the different modifications are separated by commas. For example, "R170Y,E" indicates that the arginine at position 170 is replaced with tyrosine or glutamic acid. Therefore, "Y167G,A+R170G,A" represents the following variants: "Y167G+R170G", "Y167G+R170A", "Y167A+R170G", and "Y167A+R170A". [Modes for carrying out the invention]

[0155] This invention demonstrates the remarkable effect of adding a polypeptide with alkaline phosphatase activity to a cleaning solution containing a relatively large amount of organophosphate (OP) groups. For example, OP groups of 5'-DNA precipitate with soap or surfactants in aqueous solutions containing water of moderate to high hardness, approximately 5–30°dH. This can occur even at normal to low concentrations in the detergent solution if the detergent does not contain a strong chelating agent capable of binding ions such as calcium ions. This precipitation can typically be suppressed or mitigated by adding alkaline phosphatase to the cleaning solution as part of the detergent.

[0156] While not intended to be bound by any particular theory, Ca 2+ In the presence of divalent metal ions such as , dirt and body grime are thought to be trapped on textiles via exposed phosphate groups bound to phosphorylated polymers such as DNA. These metal ions can originate from stains on laundry, particularly from hard water. When laundry detergents contain DNase, the DNase degrades DNA, thereby exposing more 5' phosphate groups. In particular, under certain difficult washing conditions (e.g., dilution conditions, low detergent levels), the effectiveness of DNase may be reduced. The inventors have discovered that phosphatases can improve washing performance, especially in the presence of DNase. This is thought to be because alkaline phosphatases cleave native and free phosphate groups from DNase-degraded DNA, thereby promoting dirt removal and preventing it from being trapped on textiles.

[0157] Therefore, the present invention provides a washing composition and method for using a polypeptide having alkaline phosphatase activity, preferably together with a polypeptide having DNase activity.

[0158] Alkaline phosphatase polypeptide In one embodiment, the present invention provides a polypeptide having alkaline phosphatase activity, for example, in an isolated and / or purified form. The polypeptide having alkaline phosphatase activity is suitable for use in the washing compositions and methods disclosed herein. Therefore, in another embodiment, the present invention provides a washing composition comprising an alkaline phosphatase polypeptide, and a washing or laundry method using an alkaline phosphatase polypeptide. The polypeptide having alkaline phosphatase activity is preferably selected from the group consisting of: (a) SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 polypeptides having at least 70% sequence identity with SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0159] The polypeptide having alkaline phosphatase activity is more specifically: SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The following polypeptides may be selected from the group having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with respect to sequence number 48, sequence number 49, sequence number 50, sequence number 51, sequence number 52, sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65, sequence number 66, sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 74, sequence number 75, sequence number 76, sequence number 77, sequence number 78, sequence number 79, sequence number 80, or sequence number 81.

[0160] In a preferred embodiment, the polypeptide having alkaline phosphatase activity has 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%, or at least 98% sequence identity with respect to SEQ ID NO: 1.

[0161] In another preferred embodiment, the alkaline phosphatase-active polypeptide is a variant of SEQ ID NO: 1 having one or more substitutions, deletions, or insertions, preferably substitutions. The alkaline phosphatase-active polypeptide is, for example, a variant of SEQ ID NO: 1 and may have substitutions at least one position selected from the group consisting of positions 77, 90, 94, 117, 119, 148, 156, 208, 212, 216, 220, 224, 225, 228, 245, 246, 248, 270, 271, 291, 292, 303, and 381, for example, two or more of these positions.

[0162] Preferred polypeptide variants having alkaline phosphatase activity include at least one substitution selected from the group consisting of A77T, N90I, A94P, I117L, H119A, N148F, M156F, K208A, R212M, M216V, K220P, K220E, E224D, T225E, D228R, Q245L, I246F, W248K, K270G, A271R, S291G, T292A, Y303L, and G381S, for example, a variant of Sequence ID No. 1 containing two or more of the above substitutions.

[0163] In one embodiment, the polypeptide having alkaline phosphatase activity is a variant of SEQ ID NO: 1 having 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 SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.

[0164] In a preferred embodiment, the polypeptide having alkaline phosphatase activity is a variant of SEQ ID NO: 1 having 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 SEQ ID NO: 79.

[0165] In one embodiment, the alkaline phosphatase polypeptide has sequence identity with respect to SEQ ID NO: 2 of at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%. The polypeptide includes, for example, the amino acid sequence of SEQ ID NO: 2 or its mature polypeptide, or essentially consists of or consists of the same.

[0166] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 3. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 3 or its mature polypeptide.

[0167] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 4. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 4 or its mature polypeptide.

[0168] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 5. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 5 or its mature polypeptide.

[0169] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 6. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 6 or its mature polypeptide.

[0170] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 7. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 7 or its mature polypeptide.

[0171] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 8. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 8 or its mature polypeptide.

[0172] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 9. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 9 or its mature polypeptide.

[0173] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 10. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 10 or its mature polypeptide.

[0174] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 11. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 11 or its mature polypeptide.

[0175] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 12. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 12 or its mature polypeptide.

[0176] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 13. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 13 or its mature polypeptide.

[0177] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 14. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 14 or its mature polypeptide.

[0178] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 15. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 15 or its mature polypeptide.

[0179] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 16. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 16 or its mature polypeptide.

[0180] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 17. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 17 or its mature polypeptide.

[0181] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 18. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 18 or its mature polypeptide.

[0182] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 19. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 19 or its mature polypeptide.

[0183] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 20. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 20 or its mature polypeptide.

[0184] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 21. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 21 or its mature polypeptide.

[0185] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 22. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 22 or its mature polypeptide.

[0186] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 23. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 23 or its mature polypeptide.

[0187] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 24. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 24 or its mature polypeptide.

[0188] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 25. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 25 or its mature polypeptide.

[0189] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 26. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 26 or its mature polypeptide.

[0190] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 27. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 27 or its mature polypeptide.

[0191] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 28. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 28 or its mature polypeptide.

[0192] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 29. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 29 or its mature polypeptide.

[0193] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 30. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 30 or its mature polypeptide.

[0194] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 31. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 31 or its mature polypeptide.

[0195] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 32. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 32 or its mature polypeptide.

[0196] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 33. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 33 or its mature polypeptide.

[0197] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 34. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 34 or its mature polypeptide.

[0198] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 35. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 35 or its mature polypeptide.

[0199] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 36. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 36 or its mature polypeptide.

[0200] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 37. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 37 or its mature polypeptide.

[0201] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 38. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 38 or its mature polypeptide.

[0202] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 39. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 39 or its mature polypeptide.

[0203] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 40. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 40 or its mature polypeptide.

[0204] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 41. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 41 or its mature polypeptide.

[0205] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 42. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 42 or its mature polypeptide.

[0206] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 43. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 43 or its mature polypeptide.

[0207] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 44. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 44 or its mature polypeptide.

[0208] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 45. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 45 or its mature polypeptide.

[0209] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 46. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 46 or its mature polypeptide.

[0210] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 47. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 47 or its mature polypeptide.

[0211] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 48. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 48 or its mature polypeptide.

[0212] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 49. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 49 or its mature polypeptide.

[0213] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 50. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 50 or its mature polypeptide.

[0214] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 51. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 51 or its mature polypeptide.

[0215] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 52. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 52 or its mature polypeptide.

[0216] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 53. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 53 or its mature polypeptide.

[0217] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 54. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 54 or its mature polypeptide.

[0218] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 55. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 55 or its mature polypeptide.

[0219] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 56. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 56 or its mature polypeptide.

[0220] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 57. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 57 or its mature polypeptide.

[0221] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 58. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 58 or its mature polypeptide.

[0222] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 59. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 59 or its mature polypeptide.

[0223] In one embodiment, the alkaline phosphatase polypeptide has at least 70%, for example, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 respect to SEQ ID NO: 60. The polypeptide includes, essentially consists of, or comprises, the amino acid sequence of SEQ ID NO: 60 or its mature polypeptide.

[0224] DNase polypeptide The washing composition of the present invention, which contains alkaline phosphatase, preferably also contains a polypeptide having DNase activity. While various polypeptides having DNase activity can be used together with the polypeptide having alkaline phosphatase activity, specific DNase polypeptides are preferred.

[0225] In one embodiment, the polypeptide having the DNase activity may be selected from the group consisting of the following: a) Polypeptides having 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 respect to sequence number 83; b) Polypeptides having 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 respect to sequence number 82; and c) A variant of sequence number 82 having 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%, or at least 95% sequence identity with sequence number 82 or sequence number 83, and compared to sequence number 82, T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V, S1 3N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M, T22P, T22A, T22V, T22D, T2 2R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22I, G24Y, S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S3 0D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42C, S42L, S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S, D5 6I, D56L, D56T, S57W, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T6 5L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F, T7 7N, T77Y, T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R, N80K, S82L, S82E,S82K、S82R、S82H、D83C、D83F、D83L、L92T、A93G、E94N、G99S、S101D、S101A、S102M、S102L、S102V、S102A、S102K、S102T、S102R、T104P、T104A、T105V、T105I、K107L、K107C、K107R、K107H、K107S、K107M、K107E、K107A、K107D、Q109R、Q109S、A112S、S116D、S116R、S116Q、S116H、S116V、S116A、S116E、S116K、A125K、S126I、S126E、S126A、S126C、T127C、T127V、S130E、G132R、D135R、T138Q、W139R、R143E、R143K、S144Q、S144H、S144L、S144P、S144E、S144K、G145V、G145E、G145D、G145A、A147H、A147Q、A147W、A147S、G149S、K152H、K152R、S156C、S156G、S156K、S156R、S156T、S156A、T157S、Y159F、K160V、W161L、W161Y、G162Q、G162D、G162M、G162R、G162A、G162S、G162E、G162L、G162K、G162V、G162H、S164R、S164T、Q166D、S167M、S167L、S167F、S167W、S167E、S167A、S167Y、S167H、S167C、S167I、S167Q、S167V、S167T、S168V、S168E、S168D、S168L、K170S、K170L、K170F、K170R、T171D、T171E、T171A、T171C、A172G、A172S、L173T、L173A、L173V、Q174L、G175D、G175E、G175N、G175R、M176H、L177I、N178D、N178E、N178T、N178S、N178A、S179E、S181R、S181E、S181D、S181F、S181H、S181W、S181L、S181M、S181Y、S181Q、S181G、S181A、Y182M、Y182C、Y182K、Y182G、Y182A、Y182S、Y182V、Y182D、Y182Q、Y182F、Y182L、Y182N、Y182I、Y182E、A variant comprising one or more substitutions selected from the group consisting of Y182T and Y182W, preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more substitutions.

[0226] In one embodiment, the polypeptide having DNase activity may be a variant of SEQ ID NO: 82, the variant having 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%, or at least 95% sequence identity with SEQ ID NO: 82 or SEQ ID NO: 83, and compared to SEQ ID NO: 82, T1I The substitutions include one or more substitutions selected from the group consisting of S13Y, T22P, S25P, S27L, S39P, D56I, S57W, S59V, T65V, V76L, T77Y, Q109R, S116D, T127V, S144P, A147H, G149N, S167L, G175D, and S181L, preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more substitutions.

[0227] In another embodiment, the polypeptide having DNase activity may be a variant of SEQ ID NO: 82, the variant having 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%, or at least 95% sequence identity with SEQ ID NO: 82 or SEQ ID NO: 83, and including two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S, for example, three, four, five, or more substitutions. In this embodiment, the polypeptide having DNase activity may further optionally include at least one substitution selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W.

[0228] Examples of preferred combinations of substitutions for Sequence ID No. 82 in this embodiment include: a) G149N with at least one substitution from N61D, T65I, T65V, S82R, K107Q, T127S, T127V, S164D, and L181S; b) T65I or T65V with at least two substitutions from N61D, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S; c) N61D with at least two substitutions from T65I / V, S82R, K107Q, T127S / V, G149N, S164D, and L181S, preferably at least two substitutions from T65I / V, S82R, K107Q, T127S, and S164D; d) S82R with at least two substitutions from N61D, T65I, T65V, K107Q, T127S, T127V, G149N, S164D, and L181S; e) K107Q with at least two substitutions from N61D, T65I, T65V, S82R, T127S, T127V, G149N, S164D, and L181S; f) T127S with substitution of at least two of N61D, T65I, T65V, S82R, K107Q, G149N, S164D, and L181S; and g) S164D with at least one substitution from N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, and L181S.

[0229] Further information regarding the above DNase polypeptide and its variants based on Sequence ID No. 82 (obtained from Metabacillus indicus, formerly known as Bacillus cibi) is provided, for example, in International Publication No. 2017 / 060475, International Publication No. 2018 / 011277, International Publication No. 2019 / 081724, and International Publication No. 2022 / 194668, which are incorporated herein by reference.

[0230] Alternatively, the polypeptide having DNase activity in the washing composition of the present invention may be a polypeptide having 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 polypeptide of Sequence ID No. 86, which contains, for example, 206 or 204 amino acid residues instead of 221 amino acid residues, by shortening the N-terminus by up to about 20 amino acid residues, for example, by shortening the N-terminus by 15 to 17 amino acid residues.

[0231] In one embodiment, the polypeptide having DNase activity may be a variant of SEQ ID NO: 86, and the variant has 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%, or at least 95% sequence identity with SEQ ID NO: 86, and is S69V, Q102E, S26*, D32Q, K36C,H, G37R, F43W, D46G, A55I, N68D, A76I, K82S,T, P84D,T, K86G,L,N,Q,T,V,Y, A91R, L92E, K95I, P97E,N,A 101E, K105N,G,Q,T,D, F112Y,W, L129K, N133Q, V138C, N140H, G141Q,R, S144E, N146A , K147N,E, V148I, A149D,E,F, Q150D, P153D,V, S154E, K155E,F,L,S,T, Q157D,E, Q158 The modification includes at least two changes selected from the group consisting of D, T159Q, K160D, T170Q, A172D,E,H,R, K185*, V187N,Y, N191*, K192A, D197K,S, G199Q, Q208V, E211Y,T,P, N213S, N214D, N217A, and Y218D,E.

[0232] In another embodiment, the polypeptide having DNase activity may be a variant of SEQ ID NO: 86, the variant having 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%, or at least 95% sequence identity with SEQ ID NO: 86, and comprising at least two substitutions, for example, at least three substitutions, selected from the group consisting of A111P, D32E, V35I, S69V, Q102E, K105N, and G181N. In this embodiment, the variant may further include two or more substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0233] Further information regarding these DNase polypeptides and their variants based on Sequence ID No. 86 (obtained from Aspergillus oryzae) is provided, for example, in International Publication Brochures 2015 / 155350, 2017 / 064269, 2022 / 189521, and International Application PCT / EP2023 / 054907, which are incorporated herein by reference.

[0234] Alternatively, in the cleaning composition of the present invention, polypeptides having DNase activity other than those described above may be used together with alkaline phosphatase polypeptides. Non-limiting examples of other suitable polypeptides having DNase activity are described in International Publication Nos. 2017 / 060493, 2017 / 060505, 2018 / 177203, 2018 / 177936, 2018 / 177938, and 2020 / 069670, which are incorporated herein by reference.

[0235] Polynucleotides The present invention also relates to polynucleotides.

[0236] The polynucleotides encoding alkaline phosphatases disclosed herein may be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0237] In one embodiment, the polynucleotide is isolated and preferably purified.

[0238] Nucleic acid construct The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding an alkaline phosphatase disclosed herein, operably linked to one or more control sequences that induce the expression of a coding sequence in a suitable host cell under conditions compatible with the control sequences. Examples of control sequences that can be used include promoters, terminators, mRNA stabilizers, leader sequences, polyadenylated sequences, signal peptides, propeptides, regulatory sequences, and transcription factors, all of which are well known in the art.

[0239] The aforementioned polynucleotides can be manipulated in various ways to provide polypeptide expression. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotides before insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0240] Expression vector The present invention also relates to a recombinant expression vector comprising a polynucleotide encoding an alkaline phosphatase, a promoter, and transcription and translation stop signals disclosed herein. Various nucleotide sequences and control sequences can be linked to generate a recombinant expression vector comprising one or more convenient restriction sites that allow insertion or substitution of a polynucleotide encoding a polypeptide. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. When the expression vector is prepared, the coding sequence is located within the vector and is operably linked to a suitable control sequence for expression.

[0241] The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be easily used in recombinant DNA procedures and can result in the expression of the polynucleotide. The selection of the vector is typically based on the compatibility between the vector and the host cell into which the vector is introduced. The vector may be a linear plasmid or a closed circular plasmid. Expression vectors suitable for recombinant expression, as well as methods for introducing them into host cells, are well known in the art.

[0242] host cell The present invention also relates to recombinant host cells comprising polynucleotides encoding alkaline phosphatases disclosed herein, which are operably linked to one or more control sequences that induce the production of polypeptides of the present invention.

[0243] A construct or vector containing a polynucleotide is introduced into a host cell, and the construct or vector is maintained as a chromosomal integration or as a self-replicating extrachromosomal vector, as described above. The selection of the host cell largely depends on the gene encoding the polypeptide and its source. The recombinant host cell may contain a single copy of the polynucleotide of the present invention, or at least two copies, for example, three, four, five, or more copies.

[0244] The host cell may be any microbial cell useful for recombinant production of the polypeptide of the present invention, such as a prokaryotic cell or a fungal cell.

[0245] The host cells of prokaryotes may be either Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0246] The bacterial host cells include Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and Bacillus stearothermophilus. This can be any Bacillus genus cell, including but not limited to Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0247] The fungal host cell may be a yeast cell or a filamentous fungal cell.

[0248] Examples of filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, and Myceliophthor. a) Examples include cells of the genera Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In preferred embodiments, the filamentous fungal host cells are cells of the genera Aspergillus, Trichoderma, or Fusarium. In even more preferred embodiments, the filamentous fungal host cells are cells of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum.

[0249] In one embodiment, the host cells are isolated and preferably purified.

[0250] Production method The present invention also relates to a method for producing an alkaline phosphatase disclosed herein, the method comprising (a) culturing recombinant host cells of the present invention under conditions suitable for polypeptide production, and optionally (b) recovering the polypeptide.

[0251] Host cells are cultured in a nutrient medium suitable for polypeptide production using methods known in the art. For example, the cells may be cultured in a suitable medium under conditions in which polypeptides can be expressed and / or isolated, by shaking flask culture in a laboratory or industrial fermenter, or by small or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation). Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., the American Type Culture Collection catalog). If polypeptides are secreted into the nutrient medium, they can be recovered directly from the medium. If polypeptides are not secreted, they can be recovered from cell lysates.

[0252] Polypeptides can be detected using polypeptide-specific methods known in the art, including but not limited to the use of specific antibodies, the formation of enzyme products, the disappearance of enzyme substrates, or enzyme assays that measure the relative or specific activity of polypeptides.

[0253] The polypeptide can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one embodiment, the entire fermentation culture broth is recovered. In another embodiment, a cell-free fermentation broth containing the polypeptide is recovered.

[0254] The polypeptide can be purified by various procedures known in the art to obtain substantially pure polypeptides and / or fragments (see, for example, Wingfield, 2015, Current Protocols in Protein Science; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0255] In another embodiment, the polypeptide is not recovered.

[0256] composition The present invention provides a washing composition comprising at least one alkaline phosphatase, optionally at least one DNase, and at least one washing aid component.

[0257] The cleaning composition comprises one or more cleaning aids selected from the group consisting of surfactants, builders, flocculants, chelating agents, dye migration inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalysts, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, preform peracids, polymer dispersants, clay stain removers / redeposition inhibitors, glossing agents, foam inhibitors, dyes, fragrances, structural elastics, softeners, carriers, hydrotropes, builders and co-builders, fabric colorants, defoamers, dispersants, processing aids, and / or pigments.

[0258] The cleaning composition typically includes at least a surfactant and other cleaning aids, usually a builder or clay / stain remover / anti-redeposition agent, additional enzymes, etc.

[0259] Therefore, the cleaning aid component may be one or more enzymes other than DNase. The one or more enzymes may be selected from the group consisting of, for example, protease, amylase, lipase, cutinase, cellulase, endoglucanase, xyloglucanase, pectinase, pectin lyase, xanthanase, peroxidase, haloperoxygenase, catalase, and mannanase. Specific enzymes suitable for the detergent composition of the present invention are described below.

[0260] The cleaning composition can be formulated in any suitable form, such as a bar, homogeneous tablet, tablet with two or more layers, pouch with one or more compartments, ordinary powder or compact powder, granules, paste, gel, or ordinary liquid, compact liquid, or concentrated liquid. Therefore, the cleaning composition may be, for example, a liquid detergent or a powder or granular detergent, and may be in a "concentrated" or "compact" form as desired. It may also be in the form of a single unit dose composition.

[0261] The amount of alkaline phosphatase in the washing composition may vary depending on factors such as the concentration or density of the composition and the desired enzyme concentration in the washing solution. Alkaline phosphatase is typically included in the washing composition at a maximum of about 10,000 ppm, typically at a maximum of about 5,000 ppm, or at a maximum of about 2,000 ppm. Alkaline phosphatase can be included in the washing composition at levels such as 1 ppm to 10,000 ppm, for example, 10 ppm to 5,000 ppm, 20 ppm to 2,000 ppm, 50 ppm to 1,000 ppm, 80 ppm to 600 ppm, or 100 ppm to 500 ppm. In this context, the unit "ppm" refers to mg / L for enzymes added to liquid compositions (e.g., liquids, gels, etc.) or mg / kg for enzymes added to solid compositions (e.g., powders, granules, tablets, etc.).

[0262] If the washing composition contains DNase, the amount of alkaline phosphatase disclosed above also applies to DNase. Therefore, DNase may be present in the washing composition in amounts up to about 10,000 ppm, typically up to about 5,000 ppm, or up to about 2,000 ppm, for example, at levels of 1 ppm to 10,000 ppm, for example, 10 ppm to 5,000 ppm, 20 ppm to 2,000 ppm, 50 ppm to 1,000 ppm, 80 ppm to 600 ppm, or 100 ppm to 500 ppm.

[0263] In some embodiments, the detergent composition is a liquid or powder laundry detergent suitable for washing at high temperatures and / or high pH, ​​such as 40°C or above and / or pH 8 or above. In some embodiments, the detergent composition is a liquid or powder laundry detergent suitable for washing at low temperatures and / or low pH, such as 20°C or below and / or pH 6. The detergent may also be formulated as a unit-dose detergent and / or compact detergent with a minimum amount of water or no water, if desired. The detergent may also be a dishwashing detergent. The laundry detergent and dishwashing detergent may be phosphorus-free.

[0264] surfactants As mentioned above, the surfactant can be selected from nonionic, anionic, and / or amphoteric surfactants, and preferably anionic or nonionic surfactants, but amphoteric surfactants can also be used. Generally, surfactants that are stable with respect to bleach are preferred. Preferred anionic surfactants are sulfate surfactants, particularly alkyl ether sulfates, especially C9-C15 alcohol ether sulfates, C12-C15 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, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diyrbis(sulfate), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS), such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates ( Examples include AES, AEOS, FES (also known as alcohol ethoxysulfates or aliphatic alcohol ether sulfates), der-alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, α-sulfo fatty acid methyl esters (α-SFMe or SES) including methyl ester sulfonates (MES), alkyl or alkenyl succinates, dodecenyl / tetradecenyl succinates (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinates or salts of fatty acids (soaps), and combinations thereof.

[0265] The anionic surfactant is preferably added to the detergent in the form of a salt. Suitable cations for these salts include alkali metal ions such as sodium, potassium, and lithium, as well as ammonium salts, such as (2-hydroxyethyl)ammonium, bis(2-hydroxyethyl)ammonium, and tris(2-hydroxyethyl)ammonium salts. Non-exclusive 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 polyglycosides (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 (glucamide, GA, or fatty acid glucamide, FAGA), as well as products marketed under the trademark names SPAN and TWEEN, and combinations thereof. Commercially available nonionic surfactants include BASF's Plurafac®, Lutensol®, and Pluronic®, Cognis' Dehypon® series, and Clariant's Genapol® series.

[0266] builder The builder is preferably selected from among phosphates, sodium citrate, sodium carbonate, sodium silicate, and sodium aluminosilicate (zeolite). Suitable builders include alkali metal or ammonium phosphates, polyphosphates, phosphonates, 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 zeolite builders, silicate builders such as BRITESIL type, and / or layered silicate builders. The builder is preferably added in an amount of about 0 to 65% by weight, for example, about 5% to about 50% by weight. In laundry detergents, the builder content is typically about 40 to 65% by weight, particularly about 50 to 65% by weight, and especially 20 to 50% by weight. The builder and / or co-builder may be chelating agents that form water-soluble complexes with calcium (Ca) and magnesium (Mg), in particular. Any builder and / or co-builder known in the art for use in cleaning detergents may 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., Hoechst's SKS-6), and (carboxymethyl)inulin (CMI), and combinations thereof. Further non-limiting examples of builders include chelating agents such as citrates, aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl or alkenyl succinates.Additional specific examples include 2,2',2”-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), 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 acid-N-monoacetic acid (ASMA), aspartic acid-N,N- Diacetic acid (ASDA), aspartic acid-AND-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(sulfomethyl)aspartic acid (SMAS), 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- Examples of suitable phosphonates for the present invention include N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and diethanolglycine (DEG), as well as combinations thereof and salts. Suitable phosphonates for the present invention include 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA), and diethylenetriaminepentakis(methylene Examples include methylenephosphonic acid (DTMPA or DTPMPA or DTPMP), nitrilotris(methylenephosphonic acid) (ATMP or NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), and hexamethylenediaminetetrakis(methylenephosphonic acid) (HDTMP). The composition may also contain a detergent cobuilder in an amount of 0 to 50% by weight, for example, about 5% to about 30%. The detergent composition may contain the cobuilder alone or in combination with a builder such as a zeolite builder.Non-limiting examples of cobuilders include homopolymers or copolymers thereof of polyacrylates, such as polyacrylic acid (PAA), copolyacrylic acid / maleic acid (PAA / PMA), or polyaspartic acid. Further exemplary builders and / or cobuilders are described, for example, in International Publication No. 09 / 102854 and U.S. Patent No. 5977053. In some embodiments, the builder is 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 salts thereof. The liquid composition may be phosphorus-free, in which case preferred builders include citrates and / or methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and / or salts thereof.

[0267] Bleaching agent The cleaning composition may contain 0 to 30% by weight of a bleaching system, for example, about 1% to about 20%. Any bleaching system containing components known in the art for use in cleaning detergents can be used. Suitable bleaching system components include a hydrogen peroxide source, a peracid source, and a bleaching catalyst or accelerator.

[0268] Sources of hydrogen peroxide: Suitable sources of hydrogen peroxide include inorganic perates containing alkali metal salts such as sodium percarbonate and sodium perborate (usually monohydrate or tetrahydrate), as well as hydrogen peroxide-urea (1 / 1).

[0269] Sources of peracids: Peracids can be (a) directly incorporated as preformed peracids, (b) formed in situ in the washing solution from hydrogen peroxide and a bleaching agent (hyperhydrolysis), or (c) formed in situ in the washing solution from hydrogen peroxide, hyperhydrolase, and a suitable substrate of the latter (e.g., an ester).

[0270] a) Suitable preform peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP)], and o-carboxybenzamideperoxycaproic acid; diperoxidedecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassic acid, 2-decyldiperoxybutanedioic acid, and aliphatic and aromatic diperoxydicarboxylic acids such as diperoxyphthalic acid, diperoxyisophthalic acid, and diperoxyterephthalic acid; perimidoic acid; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphate; peroxysilicic acid; and mixtures thereof. It is understood that, in some cases, the peracids mentioned are best added as appropriate salts, such as alkali metal salts (e.g., Oxone®) or alkaline earth metal salts.

[0271] b) Suitable bleaching agents include those belonging to the classes of esters, amides, imides, nitriles, or anhydrides, and, where applicable, their salts. Suitable examples include tetraacetylethylenediamine (TAED), 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate sodium (ISONOBS), 4-(dodecanoyloxy)benzene-1-sulfonate sodium (LOBS), 4-(decanoyloxy)benzene-1-sulfonate sodium, 4-(decanoyloxy)benzoic acid (DOBA), 4-(nonanoyloxy)benzene-1-sulfonate sodium (NOBS), and / or those disclosed in International Publication No. 98 / 17767. A particular family of bleaching agents is disclosed in European Patent No. 624154, of which acetyltriethyl citrate (ATC) is particularly preferred. Short-chain triglycerides such as ATC or triacetin have the advantage of being less environmentally harmful. Furthermore, acetyltriethyl citrate and triacetin exhibit good hydrolysis stability in the product during storage and are efficient bleaching activators. ATC is also multifunctional, and the citric acid released by hyperhydrolysis can function as a builder.

[0272] Bleaching catalyst and accelerator: The bleaching system may include a bleaching catalyst or an accelerator. 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 to be 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), especially with Me3-TACN, for example, the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2 and [2,2',2''-nitrilotris(ethane-1,2-diylazanylylidene-κN-metanylylidene)triphenolate-κ3O]manganese(III). The bleaching catalyst may be an iron or other metal compound such as a cobalt complex. Other suitable bleaching catalysts include acylhydrazone catalysts, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0323381.

[0273] In some embodiments, when a source of peracid is included, an organic bleaching catalyst or bleaching accelerator having one of the following formulas can be used: [ka] (iii) and mixtures thereof; each R 1 Each R is independently a branched alkyl group containing 9 to 24 carbon atoms or a linear alkyl group containing 11 to 24 carbon atoms, preferably each R 1 Each R is independently a branched alkyl group containing 9 to 18 carbon atoms or a linear alkyl group containing 11 to 18 carbon atoms, more preferably each R 1 This is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl, and isopentadecyl.

[0274] Other exemplary bleaching systems are described, for example, in International Publication No. 2007 / 087258, International Publication No. 2007 / 087244, International Publication No. 2007 / 087259, European Patent No. 1867708 (Vitamin K), and International Publication No. 2007 / 087242. Suitable photobleaching agents include, for example, zinc sulfonate or aluminum phthalocyanine.

[0275] When caring for textile products, the selection of detergent ingredients should take into account the type of textile product to be cleaned, the type and / or degree of soiling, the washing temperature, and the formulation of the detergent product. The ingredients listed below are classified by a general header according to their functionality, but this should not be interpreted as limiting; the ingredients may include additional functionality as understood by those skilled in the art, including the exemplary non-limiting ingredients listed below.

[0276] Hydrotrope The detergent composition may contain 0 to 10% by weight, for example, 0 to 5% by weight, for example, about 0.5 to about 5%, or about 3 to about 5% of hydrotropes. Any hydrotrope known in the art for use in detergents can be used. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0277] polymer The detergent composition may contain 0 to 10% by weight of a polymer, for example, 0.5 to 5%, 2 to 5%, 0.5 to 2%, or 0.2 to 1%. Any polymer known in the art for use in detergents can be used. The polymer may function as a cobuilder as described above, or it may exhibit anti-redeposition, fiber protection, stain removal, dye migration inhibition, grease cleaning, and / or defoaming properties. Some polymers may have one or more of the above properties and / or one or more of the following motifs. Examples of 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), PAA, PAA / PMA, polyaspartic acid, polycarboxylates such as lauryl methacrylate / acrylic acid copolymers, hydrophobic modified CMC (HM-CMC), and silicones, copolymers of terephthalic acid and oligomeric glycol, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxides and polypropylene oxides (PEO-PPO), and diquaternium ethoxysulfates. Other exemplary polymers are disclosed, for example, in International Publication No. 2006 / 130575. Salts of the above polymers are also considered.

[0278] Fabric color adjuster The detergent composition of the present invention may also contain fabric colorants such as dyes and pigments. When incorporated into the detergent composition, they deposit on the fabric when the fabric comes into contact with the washing liquid containing the detergent composition, and can change the color tone of the fabric by absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants change the surface color tone because they absorb at least a portion of the visible light spectrum. Suitable fabric colorants include dyes and dye-clay complexes, and may include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include, for example, those falling within the Color Index (C.I.) classifications of direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet, and basic red described in WO 2005 / 03274 pamphlet, WO 2005 / 03275 pamphlet, WO 2005 / 03276 pamphlet, and European Patent No. 1876226 (incorporated herein by reference), or small molecule dyes selected from the group consisting of mixtures thereof. The detergent composition preferably contains from about 0.00003% to about 0.2% by weight, from about 0.00008% to about 0.05% by weight, or from about 0.0001% to about 0.04% by weight of the fabric colorant. The composition may contain from 0.0001% to 0.2% by weight of the fabric colorant, which may be particularly preferred when the composition is in the form of a unit dose pouch. Suitable colorants are also disclosed, for example, in WO 2007 / 087257 pamphlet and WO 2007 / 087243 pamphlet.

[0279] Enzyme The detergent composition may also contain one or more additional enzymes such as protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, hexosaminidase, licheninase, xyloglucanase, oxidase such as laccase, and / or peroxidase.

[0280] Generally, the properties of the selected enzyme need to be compatible with the selected detergent (i.e., the pH is optimal, compatibility with other enzyme components and non-enzyme components, etc.), and the enzyme needs to be present in an effective amount.

[0281] Cellulase Suitable cellulases include those derived from bacteria or fungi. Chemically modified or protein-engineered variants are also included. Suitable cellulases include cellulases derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum disclosed in U.S. Patent Nos. 4,435,307, 5,648,263, 5,691,178, and 5,776,757 and International Publication No. 89 / 09259 pamphlet.

[0282] Particularly suitable cellulases are alkaline or neutral cellulases with a color care effect. Examples of such cellulases include those described in European Patent No. 0495257, European Patent No. 0531372, International Publication No. 96 / 11262 pamphlet, International Publication No. 96 / 29397 pamphlet, International Publication No. 98 / 08940 pamphlet. Other examples include cellulase variants as described in International Publication No. 94 / 07998 pamphlet, European Patent No. 0531315, U.S. Patent No. 5,457,046, U.S. Patent No. 5,686,593, U.S. Patent No. 5,763,254, International Publication No. 95 / 24471 pamphlet, International Publication No. 98 / 12307 pamphlet, and International Publication No. 99 / 001544 pamphlet.

[0283] Other cellulases are family 44 xyloglucanases, which are endo-beta-1,4-glucanase enzymes having at least 97% identity with the amino acid sequence of positions 1-773 of Sequence ID No. 2 in International Publication No. 2002 / 099091, or xyloglucanase enzymes having at least 60% identity with positions 40-559 of Sequence ID No. 2 in International Publication No. 2001 / 062903.

[0284] Commercially available cellulases include Celluzyme® and Carezyme® (Novozymes A / S), Carezyme Premium® (Novozymes A / S), Celluclean® (Novozymes A / S), Celluclean Classic® (Novozymes A / S), Cellusoft® (Novozymes A / S), Whitezyme® (Novozymes A / S), Clazinase®, Puradax HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

[0285] protease The appropriate protease may be of any origin, but is preferably of bacterial or fungal origin and may be in the form of a protein-engineered or chemically modified variant as desired. The protease may be an alkaline protease such as a serine protease or a metalloprotease. The serine protease may be, for example, from the S1 family such as trypsin, or from the S8 family such as subtilisin. The metalloprotease may be, for example, thermolysin from the M4 family, or a metalloprotease from the M5, M7, or M35 family.

[0286] According to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523, the term "subtilase" refers to a subgroup of serine proteases. Serine proteases are a subgroup of proteases characterized by having serine in their active site and forming covalent adducts with substrates. Subtilases can be classified into six subphyla: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family.

[0287] Proteases suitable for detergent applications can be obtained from a variety of organisms, including fungi such as Aspergillus, but detergent proteases are generally obtained from bacteria, especially Bacillus. Examples of Bacillus species from which subtilases are derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and Bacillus gibsonii. Specific subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147, subtilisin 168, and, for example, protease PD138 (described in International Publication No. 93 / 18140). Other useful proteases are described, for example, in International Publication No. 01 / 16285 and International Publication No. 02 / 16547.

[0288] Examples of trypsin-like proteases include Fusarium proteases described in International Publication No. 94 / 25583 and International Publication No. 2005 / 040372, and chymotrypsin proteases from the genus Cellulomonas described in International Publication No. 2005 / 052161 and International Publication No. 2005 / 052146.

[0289] Examples of metalloproteinases include neutral metalloproteinases (e.g., those derived from Bacillus amyloricephasiensis) described in International Publication No. 2007 / 044993, as well as metalloproteinases described in International Publication Nos. 2015 / 158723 and International Publication Nos. 2016 / 075078.

[0290] Examples of useful proteases include International Publication No. 89 / 06279, International Publication No. 92 / 19729, International Publication No. 96 / 34946, International Publication No. 98 / 20115, International Publication No. 98 / 20116, International Publication No. 99 / 11768, International Publication No. 01 / 44452, International Publication No. 03 / 006602, and International Publication No. Examples of protease variants include those described in Brochure No. 2004 / 003186, International Publication No. 2004 / 041979, International Publication No. 2007 / 006305, International Publication No. 2011 / 036263, International Publication No. 2014 / 207227, International Publication No. 2016 / 087617, and International Publication No. 2016 / 174234. Preferred protease variants include, for example, S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S9 9E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G1 16V, G116R, H118D, H118N, A120S, S126L, P127Q, S128A, S154D, A156E, G157D, G1 57P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Q200L, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, S253D, N255W, N255D, N255E, L256E, L256D, T268A and R269H (The location numbers are for Bacillus lentus as shown in Sequence ID 1 of International Publication No. 2016 / 001449). It may contain one or more mutations selected from the group consisting of (lentus) (corresponding to the location of the protease).Protease variants having one or more of these mutations are preferably variants of Bacillus lentus protease (also known as Sabinase®, subtilisin 309) as shown in Sequence ID No. 1 of International Publication No. 2016 / 001449, or variants of Bacillus amyloricephaciens protease (BPN') as shown in Sequence ID No. 2 of International Publication No. 2016 / 001449. Such protease variants preferably have at least 80% sequence identity with Sequence ID No. 1 or Sequence ID No. 2 of International Publication No. 2016 / 001449.

[0291] Other target proteases include, for example, the alkaline protease derived from Bacillus lentus DSM5483 described in International Publication No. 91 / 02792, and its variants described, for example, in International Publication No. 92 / 21760, International Publication No. 95 / 23221, European Patent No. 1921147, European Patent No. 1921148, and International Publication No. 2016 / 096711.

[0292] Alternatively, the protease may be a variant of the TY145 protease having SEQ ID NO: 1 in International Publication No. 2004 / 067737, for example, a variant including substitutions at one or more positions corresponding to positions 27, 109, 111, 171, 173, 174, 175, 180, 182, 184, 198, 199, and 297 of SEQ ID NO: 1 in International Publication No. 2004 / 067737, wherein the protease variant has at least 75% but less than 100% sequence identity with respect to SEQ ID NO: 1 in International Publication No. 2004 / 067737. The TY145 variants covered are described, for example, in International Publication No. 2015 / 014790, International Publication No. 2015 / 014803, International Publication No. 2015 / 014804, International Publication No. 2016 / 097350, International Publication No. 2016 / 097352, International Publication No. 2016 / 097357, and International Publication No. 2016 / 097354.

[0293] Examples of preferred proteases include: (a) Variants of Sequence ID 1 in International Publication No. 2016 / 001449 that include two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D, and L262E, for example, a variant having the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E, or a variant having the substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D, and L262E, the position number is based on the numbering of Sequence ID 2 in International Publication No. 2016 / 001449: (b) A variant of the polypeptide of Sequence ID No. 1 in International Publication No. 2016 / 001449, with the S99SE mutation, the positional number based on the numbering of Sequence ID No. 2 in International Publication No. 2016 / 001449; (c) A variant of the polypeptide of Sequence ID No. 1 in International Publication No. 2016 / 001449, with the S99AD mutation, the positional number is based on the numbering of Sequence ID No. 2 in International Publication No. 2016 / 001449; (d) A variant of the polypeptide of Sequence ID 1 in International Publication No. 2016 / 001449, with the substitution Y167A+R170S+A194P, the position number based on the numbering of Sequence ID 2 in International Publication No. 2016 / 001449; (e) A variant of the polypeptide of Sequence ID 1 in International Publication No. 2016 / 001449, with the substitution S9R+A15T+V68A+N218D+Q245R, the position number based on the numbering of Sequence ID 2 in International Publication No. 2016 / 001449; (f) Variants of the polypeptide of Sequence ID 1 in International Publication No. 2016 / 001449, with the substitution S9R+A15T+G61E+V68A+A194P+V205I+Q245R+N261D, the position number based on the numbering of Sequence ID 2 in International Publication No. 2016 / 001449; (g) Variants of the polypeptide sequence number 1 in International Publication No. 2016 / 001449, with the substitution S99D+S101R / E+S103A+V104I+G160S, for example, a variant of sequence number 1 in International Publication No. 2016 / 001449, with the substitution S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, the position number based on the numbering of sequence number 2 in International Publication No. 2016 / 001449; (h) Variants of the polypeptide of Sequence ID No. 2 of International Publication No. 2016 / 001449, with the substitution S24G+S53G+S78N+S101N+G128A / S+Y217Q, the position number is based on the numbering of Sequence ID No. 2 of International Publication No. 2016 / 001449; (i) The polypeptide disclosed in GENESEQP under accession number BER84782, corresponding to Sequence ID 302 in International Publication No. 2017 / 210295; (j) A variant of the polypeptide of Sequence ID 1 in International Publication No. 2016 / 001449, with the substitution S99D+S101E+S103A+V104I+S156D+G160S+L262E, the position number based on the numbering of Sequence ID 2 in International Publication No. 2016 / 001449; (k) A variant of the polypeptide of Sequence ID No. 1 in International Publication No. 2016 / 001449, with the substitution S9R+A15T+G61E+V68A+N76D+S99G+N218D+Q245R, the position number based on the numbering of Sequence ID No. 2 in International Publication No. 2016 / 001449; (l) Variants of the polypeptide of Sequence ID No. 1 in International Publication No. 2016 / 001449, with the substitution V68A+S106A, the position number based on the numbering of Sequence ID No. 2 in International Publication No. 2016 / 001449; and (m) A variant of the polypeptide of Sequence ID No. 1 in International Publication No. 2004 / 067737, with the substitution S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199+T297P, the position number is based on the numbering of Sequence ID No. 1 in International Publication No. 2004 / 067737.

[0294] Commercially available protease enzymes include Alcalase(registered trademark), Duralase(trademark), Durazym(trademark), Relase(registered trademark), Relase(registered trademark) Ultra, Savinase(registered trademark), Savinase(registered trademark) Ultra, Primase(trademark), Polarzyme(registered trademark), Kannase(registered trademark), Liquanase(registered trademark), Liquanase(registered trademark) Ultra, Ovozyme(registered trademark), Coronase(registered trademark), Coronase(registered trademark) Ultra, Blaze(registered trademark), Blaze Evity(registered trademark) 100T, Blaze Evity(registered trademark) 125T, Blaze Evity(registered trademark) 150T, and Blaze Products sold under the trademark names Evity(registered trademark)200T, Neutrase(registered trademark), Everlase(registered trademark), Esperase(registered trademark), Progress(registered trademark)Uno, Progress(registered trademark)In, Progress(registered trademark)Key, and Progress(registered trademark)Excel (Novozymes A / S), as well as Maxatase(trademark), Maxacal(trademark), Maxapem(registered trademark), Purafect(registered trademark)Ox, Purafect(registered trademark)OxP, Puramax(registered trademark), FN2(trademark), FN3(trademark), FN4ex(trademark), Excellase(registered trademark), Excellenz(trademark)P1000, Excellenz(trademark)P1250,Examples include products marketed under the trademarks Eraser®, Preferenz® P100, Preferenz® P300, Purafect Prime, Preferenz P110®, Effectenz P1000®, Purafect®, Effectenz P1050®, Purafect® Ox, Effectenz® P2000, Purafast®, Properase®, Opticlean®, and Optimase® (Danisco / DuPont), as well as BLAP (the sequence shown in Figure 29 of U.S. Patent No. 5352604) and its variant (Henkel AG), and Kao's KAP (Bacillus alkalophilus subtilisin).

[0295] Lipase and cutinase Suitable lipases and cutinases include those derived from bacteria or fungi. Chemically modified or protein-engineered mutant enzymes are also included.Examples include lipases derived from Thermomyces (e.g., those derived from T. lanuginosus (formerly known as Humicola lanuginosa) as described in European Patent No. 258068 and European Patent No. 305216), cutinases derived from Humicola (e.g., H. insolens (International Publication No. 96 / 13580 pamphlet)), lipases derived from Pseudomonas strains (some of these have now been renamed to the Burkholderia genus) (e.g., Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (European Patent No. 218272)), and Pseudomonas cepacia (P. Pseudomonas cepacia (European Patent No. 331376), Pseudomonas strain SD705 (International Publication No. 95 / 06720 and International Publication No. 96 / 27002), Pseudomonas wisconsinensis (International Publication No. 96 / 12012), GDSL-type streptomyces lipase (International Publication No. 10 / 065455), cutinase derived from Magnaporthe grisea (International Publication No. 10 / 107560), cutinase derived from Pseudomonas mendocina (US Patent No. 5,389,536), Thermobifida fusca Examples include lipase derived from *Bacillus fusca* (International Publication No. 11 / 084412), lipase from *Diobacillus stearothermophilus* (International Publication No. 11 / 084417), lipase derived from *Bacillus subtilis* (International Publication No. 11 / 084599), lipase derived from *Streptomyces griseus* (International Publication No. 11 / 150157), and lipase from *Streptomyces pristinaespiralis* (International Publication No. 12 / 137147).

[0296] Other examples include lipase variants described in European Patent No. 407225, WO 92 / 05249 pamphlet, WO 94 / 01541 pamphlet, WO 94 / 25578 pamphlet, WO 95 / 14783 pamphlet, WO 95 / 30744 pamphlet, WO 95 / 35381 pamphlet, WO 95 / 22615 pamphlet, WO 96 / 00292 pamphlet, WO 97 / 04079 pamphlet, WO 97 / 07202 pamphlet, WO 00 / 34450 pamphlet, WO 00 / 60063 pamphlet, WO 01 / 92502 pamphlet, WO 07 / 87508 pamphlet, and WO 09 / 109500 pamphlet.

[0297] Commercially available lipase products that are recommended include Lipolase™, Lipex™, Lipolex™, Lipoclean™ (Novozymes A / S), Lumafast (formerly made by Genencor), and Lipomax (formerly made by Gist-Brocades).

[0298] Still other examples include lipases also known as acyltransferases or perhydrolases, such as acyltransferases homologous to Candida antarctica lipase A (WO 10 / 111143 pamphlet), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782 pamphlet), perhydrolases from the CE7 family (WO 09 / 67279 pamphlet), and variants of Mycobacterium smegmatis perhydrolase, particularly the S54V variant used in the commercially available product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028 pamphlet), etc.

[0299] amylase Suitable amylases that can be used with the alkaline phosphatase and desired DNase of the present invention are alpha-amylases or glucoamylases, which may be of bacterial or fungal origin. Chemically modified or protein-engineered variants are also included. Examples of amylases include α-amylases obtained from Bacillus (e.g., specific strains of Bacillus licheniformis, as described in more detail in British Patent No. 1,296,839).

[0300] Suitable amylases include the amylase having sequence number 2 in International Publication No. 95 / 10603, or a variant having 90% sequence identity with sequence number 1. Preferred variants are described in Sequence ID No. 4 of International Publication No. 94 / 02597, International Publication No. 94 / 18314, International Publication No. 97 / 43424, and International Publication No. 99 / 019467, and include, for example, variants in which one or more of the following positions are replaced: 15th, 23rd, 105th, 106th, 124th, 128th, 133rd, 154th, 156th, 178th, 179th, 181st, 188th, 190th, 197th, 201st, 202nd, 207th, 208th, 209th, 211th, 243rd, 264th, 304th, 305th, 391st, 408th, and 444th.

[0301] Suitable amylases include the amylase having sequence number 6 in International Publication No. 02 / 010355, or a variant thereof having 90% sequence identity with sequence number 6. Preferred variants of sequence number 6 have deletions at positions 181 and 182 and a substitution at position 193.

[0302] Other suitable amylases are hybrid α-amylases containing residues 1-33 of the α-amylase obtained from Bacillus amyloliquefaciens (B. amyloliquefaciens), shown in SEQ ID NO. 6 of International Publication No. 2006 / 066594, and residues 36-483 of the α-amylase from Bacillus licheniformis (B. licheniformis), shown in SEQ ID NO. 4 of International Publication No. 2006 / 066594, or variants having 90% sequence identity thereof. Preferred variants of this hybrid α-amylase have substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264.

[0303] Other examples include the amylase variants described in International Publication No. 2011 / 098531, International Publication No. 2013 / 001078, and International Publication No. 2013 / 001087.

[0304] Commercially available amylases include Duramyl™, Teramyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, BANTM (manufactured by Novozymes A / S), Rapidase™, Purastar™ / Effectenz™, Powerase, and Preferenz S100 (manufactured by Genencor International Inc. / DuPont).

[0305] Hexosaminidase The detergent composition of the present invention, comprising alkaline phosphatase and a desired DNase, may also contain one or more hexosaminidases. The term hexosaminidase includes "dispersin" and the abbreviation "Dsp," which refers to a polypeptide having hexosaminidase activity (EC3.2.1.-) and catalyzes the hydrolysis of β-1,6-glycosidic bonds of N-acetylglucosamine polymers present in biofilms, etc. The term hexosaminidase includes polypeptides having N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity.

[0306] Polypeptides possessing hexosaminidase activity can be obtained from microorganisms of any genera, particularly bacteria or fungi. Preferably, hexosaminidases, such as dispersin, are obtained from the genera Terribacillus, Curtobacterium, Aggregatibacter, Haemophilus, or Actinobacillus, and are preferably obtained from the genus Terribacillus. The hexosaminidase may be a variant of polypeptides obtained from these organisms or other organisms.

[0307] Appropriate hexosaminidases include International Publication Nos. 2017 / 186936, 2017 / 186937, 2017 / 186943, 2017 / 207770, 2018 / 184873, 2019 / 086520, 2019 / 086528, 2019 / 086530, 2019 / 086532, 2019 / 086521, and 2019 Examples include those disclosed in Pamphlet No. / 086526, International Publication No. 2020 / 002604, International Publication No. 2020 / 002608, International Publication No. 2020 / 007863, International Publication No. 2020 / 007875, International Publication No. 2020 / 008024, International Publication No. 2020 / 070063, International Publication No. 2020 / 070249, International Publication No. 2020 / 088957, International Publication No. 2020 / 088958, and International Publication No. 2020 / 207944.

[0308] Peroxidase / Oxidase Peroxidases may be included in the enzyme classification EC1.11.1.7 as defined by the International Union of Biochemistry and Molecular Biology (IUBMB) Nomenclature Committee, or in any fragment exhibiting peroxidase activity derived therefrom. Suitable peroxidases include those derived from plants, bacteria, or fungi. Chemically modified or protein-engineered variants are also included. Examples of useful peroxidases include peroxidases from the genus Coprinopsis (e.g., Coprinopsis cinerea) (European Patent No. 179,486) and their variants described in International Publications 93 / 24618, 95 / 10602, and 98 / 15257. Peroxidases may also include haloperoxidase enzymes such as chloroperoxidases, bromoperoxidases, and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (EC 1.11.1.10) catalyze the formation of hypochlorites from chloride ions. Haloperoxidases can be chloroperoxidases. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method, the vanadate-containing haloperoxidase is combined with a chloride ion source. Haloperoxidases have been isolated from many different fungi, particularly from the dematiaceous hyphomycetes such as the genera Cardariomyces (e.g., Cardariomyces fumago), Alternaria, Curvularia (e.g., Curvularia verruculosa and Curvularia inaequalis), Drecslera, Ulocladium, and Botrytis.Haloperoxidases have also been isolated from bacteria of the genera Pseudomonas (e.g., Pseudomonas pyrrocinia) and Streptomyces (e.g., Streptomyces aureofaciens). In a preferred embodiment, the haloperoxidase is derived from a species of the genus Curvularia, particularly Curvularia verruculosa or Curvularia inaequalis (for example, Curvularia inaequalis CBS 102.42 as described in International Publication No. 95 / 27046, or Curvularia verruculosa CBS 147.63 or Curvularia verruculosa CBS 444.70 as described in International Publication No. 97 / 04102), or from Drecslera hartlebii as described in International Publication No. 01 / 79459, or Dendryphiella salina as described in International Publication No. 01 / 79458. It is derived from *Phaeotrichoconis crotalarie*, described in International Publication No. 01 / 79461, or from the genus *Geniculosporium*, described in International Publication No. 01 / 79460.

[0309] Oxidases include any laccase enzymes included in the enzyme classification EC 1.10.3.2, or any fragments obtained therefrom that exhibit laccase activity, or compounds exhibiting similar activity such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0310] A preferred laccase enzyme is an enzyme derived from a microorganism. The enzyme can be obtained from plants, bacteria, or fungi (including filamentous fungi and yeasts).

[0311] Suitable examples from fungi include the genera Bacillus, Neurospora (e.g., Neurospora crassa), Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes villosa and Trametes versicolor, Rhizoctonia (e.g., Rhizoctonia solani), Coprinopsis (e.g., Coprinopsis cinerea, Coprinopsis comatus, Coprinopsis friesii, and Coprinopsis plicatilis). Plicatilis, Psathyrella (e.g., P. condelleana), Panaeolus (e.g., Panaeolus papilionaceus), Miseriophsora (e.g., Miseriophsora thermophila), Schytalidium (e.g., Styllium thermophilum), Polyporus (e.g., Polyporus pinsitus), Phlebia (e.g., Phlebia radiata) (International Publication No. 92 / 01046), or Trametes genus (e.g., Coriolus hirsutus (C. Examples include laccase derived from the *Hirsuta* strain (Japanese Patent No. 2238885).

[0312] A suitable example from bacteria is laccase obtained from Bacillus strains.

[0313] Laccases obtained from the genera Coprinopsis or Miseriophthora are preferred. Laccases obtained from Coprinopsis cinerea, as disclosed in International Publication No. 97 / 08325, or laccases obtained from Miseriophthora thermophylla, as disclosed in International Publication No. 95 / 33836, are preferred.

[0314] Dispersant The detergent composition of the present invention may also contain a dispersant. Powder detergents may contain a dispersant. Suitable water-soluble organic materials include homopolymeric acids or copolymeric acids or salts thereof, and polycarboxylic acids contain at least two carboxyl groups separated from each other by two or fewer carbon atoms. Suitable dispersants are described, for example, in "Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc."

[0315] Dye transfer inhibitor The cleaning composition of the present invention may also include one or more dye migration inhibitors. Suitable polymer dye migration inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, and polyvinylimidazole, or mixtures thereof. When present in the composition, the dye migration inhibitor may be present at a level of about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% by weight of the composition.

[0316] fluorescent whitening agent The detergent composition may preferably also contain additional components that can color the article being washed, such as a fluorescent whitening agent or an optical glossing agent. If the glossing agent is present, it is preferably at a level of about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents belong to the classes of diaminostilbenesulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyldistyrill derivatives. Examples of diaminostilbenesulfonic acid derivative type fluorescent whitening agents include 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydro Examples include sodium salts of oxyethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazole-2-yl)stilbene-2,2'-disulfonic acid, and sodium salts of 5-(2H-naphtho[1,2-d][1,2,3]triazole-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate sodium. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis(phenylstyryl)disulfonic acid. Additionally, Parawhite KX, commercially available from Paramount Minerals and Chemicals (Mumbai, India), is also preferred as a fluorescent whitening agent. Tinopal CBS-X is 4,4'-bis-(sulfostyryl)-biphenyl disodium salt, also known as disodium distylylbiphenyl disulfonic acid.Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazoline and 7-alkylaminocoumarin.

[0317] Appropriate levels of fluorescent gloss include lower limits of approximately 0.01% by weight, 0.05% by weight, 0.1% by weight, or 0.2% by weight, and upper limits of 0.5% by weight or 0.75% by weight.

[0318] Dirt-releasing polymer The detergent composition may also include one or more dirt-releasing polymers that help remove dirt from fabrics such as cotton and polyester-based fabrics, and hydrophobic dirt from polyester-based fabrics. Examples of such dirt-releasing polymers include nonionic or anionic terephthalate polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, and polyester polyamides. See, for example, Volume 71, Chapter 7 of *Powdered Detergents*, Surfactant science series (Marcel Dekker, Inc.). Another type of dirt-releasing polymer is an amphiphilic alkoxylated grease-cleaning polymer comprising a core structure and a plurality of alkoxylate groups bonded to the core structure. The core structure may include a polyalkylene imine structure or a polyalkanolamine structure, as described in detail in International Publication No. 2009 / 087523 (incorporated herein by reference). Furthermore, random graft copolymers are suitable as dirt-releasing polymers. Suitable graft copolymers are described in more detail in International Publication No. 2007 / 138054, International Publication No. 2006 / 108856, and International Publication No. 2006 / 113314 (both incorporated herein by reference). Other fouling-releasing polymers are substituted polysaccharide structures, in particular substituted cellulose structures such as modified cellulose derivatives as described in European Patent No. 1867808 or International Publication No. 2003 / 040279 (both incorporated herein by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, amphoterically modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0319] Anti-redeposition agent The detergent composition of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above as dirt-releasing polymers may also function as anti-redeposition agents.

[0320] Rheological modifier The detergent composition of the present invention may also include, in addition to the viscosity-reducing agent, one or more rheological modifiers, structuring agents, or thickeners. The rheological modifier is selected from the group consisting of nonpolymeric crystalline, hydroxy-functionalized polymeric rheological modifiers that impart shear-thinning properties to the aqueous liquid matrix of the liquid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example, as described in European Patent No. 2169040.

[0321] Other suitable auxiliary materials include, but are not limited to, shrinkage inhibitors, wrinkle inhibitors, disinfectants, binders, carriers, dyes, enzyme stabilizers, softeners, fillers, foam modifiers, hydrotropes, fragrances, pigments, foam inhibitors, solvents, and structuring agents and / or structural elastics for liquid detergents.

[0322] Other materials Any detergent component known in the art for use in the cleaning composition of the present invention may also be used. Other desired detergent components include rust inhibitors, shrinkage inhibitors, antifouling and re-deposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants, dyes, enzyme stabilizers (including polyols such as boric acid, borates, CMC, and / or propylene glycol), softeners (including clay), fillers / processing aids, fluorescent whitening agents / optical gloss agents, foaming accelerators, foaming (foam) modifiers, fragrances, dirt suspenders, softeners, foam inhibitors, discoloration inhibitors, and absorbents, which may be used alone or in combination. Any component known in the art for use in detergents may be used. The selection of such components is within the scope of the art of those skilled in the art.

[0323] detergent product formulations The detergent composition may be in any convenient form, such as a bar, homogeneous tablet, tablet with two or more layers, ordinary powder or compact powder, granules, paste, gel, or ordinary liquid, compact liquid, or concentrated liquid. Other detergent formulations include layered forms and single-unit dosage forms such as pouches.

[0324] The pouch can be configured as one or more compartments. These can be any form, shape, and material suitable for holding the composition without the composition being released from the pouch before, for example, contact with water. The pouch is made from a water-soluble film that surrounds the internal volume and can be divided into compartments. The preferred film is a polymer material, preferably a polymer formed into a film or sheet. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, and polymethacrylate, most preferably polyvinyl alcohol copolymer and hydroxypropyl methylcellulose (HPMC). Preferably, the level of polymer in the film is at least about 60%. The preferred average molecular weight is typically about 20,000 to about 150,000. The film may be a blended composition comprising a hydrolyzable water-soluble polymer blend such as polylactic acid and polyvinyl alcohol, in addition to plasticizers such as glycerol, ethylene glycerol, propylene glycol, and sorbitol, and mixtures thereof. The pouch may contain a solid laundry composition or a portion thereof separated by the water-soluble film and / or a liquid laundry composition or a portion thereof. The compartment for the liquid component may have a different configuration from the compartment containing the solid. See, for example, U.S. Patent Application Publication No. 2009 / 0011970(A1).

[0325] Detergent components can be physically separated from each other by compartments within a water-soluble pouch or by tablets in different layers, thus avoiding storage interactions that could negatively affect the components. Different dissolution profiles in each compartment may result in slower dissolution of selected components in the cleaning solution.

[0326] Liquid detergents or gel detergents that are not in unit doses may be aqueous and typically contain at least 20% to a maximum of 95% water, for example, up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water. Concentrated liquid detergents may have a lower water content, for example, up to about 30% or less, or up to about 20% or less, for example, in the range of about 1% to about 20%, for example, about 2% to about 15%. Other types of liquids, including but not limited to alkanols, amines, diols, ethers, and polyols, may be contained in aqueous liquids or gels. Aqueous liquid detergents or gel detergents may contain 0 to 30% organic solvents. Liquid detergents or gel detergents may also be non-aqueous.

[0327] The liquid detergent composition may be formulated to have a moderate pH of about 6 to about 10, for example, about pH 7, about pH 8, or about pH 9, or it may be formulated to have a higher pH of about 10 to about 12, for example, about pH 10, about pH 11, or about pH 12.

[0328] Unless otherwise stated, the term “liquid” as used herein should be understood to include all types of liquid detergent compositions, such as concentrated liquids, gels, or the liquid or gel portion of a pouch having, for example, one or more compartments.

[0329] Detergent enzymes can be included in a detergent composition by adding a separate additive containing one or more enzymes, or by adding a composite additive containing these enzymes. Detergent additives, i.e., single additives or composite additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, non-dusting granules, liquids, stabilized liquids, and slurries.

[0330] Non-dusty granules can be manufactured, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, and can be coated, if desired, by methods known in the art. Examples of waxy coating materials include poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols (the alcohol contains 12 to 20 carbon atoms and 15 to 80 ethylene oxide units); fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. An example of a film-forming coating material suitable for coating by fluidized bed technology is described in British Patent No. 1,483,591. For example, liquid enzyme preparations can be stabilized by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid, or boric acid, according to established methods. The protected enzyme can be prepared according to the method disclosed in European Patent No. 238216.

[0331] Contains granular detergent Granular enzymes comprising an enzyme-containing core and optionally one or more coatings are commonly used in granular (powdered) detergents. Various methods for preparing the core are well known in the art and include, for example, a) spray drying of an enzyme-containing liquid; b) production of a layered product by coating pre-formed inert core particles with an enzyme in layers, for example using a fluidized bed apparatus; c) absorption of an enzyme onto and / or inside a pre-formed core; d) extrusion of an enzyme-containing paste; e) obtaining a granular product by suspending an enzyme-containing powder in molten wax and atomizing it; f) mixer granulation by adding an enzyme-containing liquid to a dry powder composition of granulation components; g) reduction of the size of the enzyme-containing core by crushing or pulverizing larger particles, pellets, etc.; and h) fluidized bed granulation. The enzyme-containing core can be dried, for example, using a fluidized bed dryer or other known methods for drying granules in the feed or enzyme industry, resulting in a typical moisture content of 0.1-10% (w / w).

[0332] The enzyme-containing core may be optionally coated to improve storage stability and / or reduce dust formation. One type of coating commonly used for enzyme granules in detergents is a salt coating, usually an inorganic salt coating, which can be applied as a salt solution using, for example, a fluidized bed. Other coating materials that can be used include, for example, polyethylene glycol (PEG), methylhydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). The granules may include multiple coatings, such as an additional coating with PEG, MHPC, or PVA after the salt coating.

[0333] For further details regarding enzyme granules and their manufacture, please refer to International Publication No. 2013 / 007594, as well as, for example, International Publication No. 2009 / 092699, European Patent No. 1705241, European Patent No. 1382668, International Publication No. 2007 / 001262, U.S. Patent No. 6,472,364, International Publication No. 2004 / 074419, and International Publication No. 2009 / 102854.

[0334] Enzyme composition in cogranules DNases can be formulated as granules, such as cogranules combining one or more enzymes. Each enzyme is present in a greater number of granules, ensuring more uniform dispersion of the enzymes within the detergent. This also reduces the physical separation of different enzymes due to differences in particle size. A method for producing multi-enzyme cogranules for the detergent industry is disclosed in IP.com disclosure: IPCOM000200739D.

[0335] Another example of an enzyme formulation using cogranules is disclosed in International Publication No. 2013 / 188331, which relates to a detergent composition comprising (a) a polyenzyme cogranule, (b) less than 10% by weight of zeolite (anhydrous basis), and (c) less than 10% by weight of phosphate (anhydrous basis), wherein the enzyme cogranule contains 10 to 98% by weight of moisture sink components, and the composition further contains 20 to 80% by weight of detergent moisture sink components. International Publication No. 2013 / 188331 also relates to a method for treating and / or cleaning a surface, preferably a fabric surface, comprising (i) contacting the surface with a detergent composition of an aqueous cleaning solution claimed and described herein, and (ii) rinsing and / or drying the surface.

[0336] Liquid formulation The present invention also relates to a liquid composition comprising the alkaline phosphatase and a desired DNase of the present invention. The composition may contain an enzyme stabilizer (for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid, or a boric acid derivative, such as an aromatic boric acid ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid).

[0337] In some embodiments, fillers or carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, and similar materials. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water, or low molecular weight primary or secondary alcohols containing polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain about 5% to about 90% of such materials.

[0338] In one embodiment, the liquid formulation contains 20-80% (w / w) of polyol. In another embodiment, the liquid formulation contains 0.001-2% (w / w) of preservative.

[0339] In another embodiment, the present invention relates to a liquid formulation comprising: (A) 0.001 to 25% (w / w), for example 0.001 to 5% (w / w), of the alkaline phosphatase disclosed herein, and optionally 0.001 to 25% (w / w), for example 0.001 to 5% (w / w), of the DNase disclosed herein; (B) 20-80% (w / w) polyols; (C) Preservatives of 0.001-2% (w / w) as desired; and (D) Water.

[0340] In another embodiment, the present invention relates to a liquid formulation comprising: (A) 0.001 to 25% (w / w), for example 0.001 to 5% (w / w), of the alkaline phosphatase disclosed herein, and optionally 0.001 to 25% (w / w), for example 0.001 to 5% (w / w), of the DNase disclosed herein; (B) 0.001-2% (w / w) of preservatives; (C) 20-80% (w / w) polyols as desired; and (D) Water.

[0341] In another embodiment, the liquid formulation comprises one or more formulations selected from the group consisting of polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate, and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0342] In another embodiment, the liquid formulation contains 20-80% polyol (i.e., total amount of polyol), for example, 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation contains 20-80% polyol, for example, 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, the polyol being selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation contains 20-80% polyol (i.e., the total amount of polyol), for example, 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0343] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation contains 0.02 to 1.5% (w / w) of a preservative, for example, 0.05 to 1% (w / w) of a preservative or 0.1 to 0.5% (w / w) of a preservative. In one embodiment, the liquid formulation contains 0.001 to 2% (w / w) of a preservative (i.e., the total amount of preservative), for example, 0.02 to 1.5% (w / w) of a preservative, 0.05 to 1% (w / w) of a preservative, or 0.1 to 0.5% (w / w) of a preservative, and the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, potassium benzoate, or any combination thereof.

[0344] In another embodiment, the liquid formulation further comprises, for example, one or more additional enzymes as described above.

[0345] Use of DNase polypeptides The alkaline phosphatase and desired DNase of the present invention are suitable for use, for example, in washing or cleaning hard surfaces, particularly in washing processes for laundry. Therefore, one aspect of the present invention is a method for washing an article, wherein the article is a textile product. a) Exposing an article to a washing solution containing a polypeptide having alkaline phosphatase activity and a polypeptide having DNase activity. b) Complete at least one cleaning cycle, and if desired c) A method including rinsing the article.

[0346] In related embodiments, the present invention relates to a method for washing an article, wherein the article is a textile product. a) Exposing the article to a washing solution comprising a polypeptide having alkaline phosphatase activity as defined elsewhere in this specification, and optionally further comprising a polypeptide having DNase activity; b) Complete at least one cleaning cycle, and if desired c) A method comprising rinsing the article.

[0347] The polypeptide having alkaline phosphatase activity used in the methods of this specification is preferably an alkaline phosphatase polypeptide as disclosed elsewhere in this specification. Similarly, the polypeptide having DNase activity is preferably a DNase polypeptide as defined elsewhere in this specification.

[0348] The pH of the liquid cleaning solution is typically in the range of about 5.5 to about 10, more typically in the range of about 7 to about 9, for example, in the range of about 7 to about 8.5 or about 7 to about 8.

[0349] The temperature of the cleaning solution may be within the range of 5°C to 95°C, or within the range of 10°C to 80°C, 10°C to 70°C, 10°C to 60°C, 10°C to 50°C, 15°C to 40°C, or 20°C to 30°C.

[0350] The concentration of alkaline phosphatase in the washing solution is typically within the range of 0.0001 mg / L to 10 mg / L, 0.0002 mg / L to 10 mg / L, 0.001 mg / L to 10 mg / L, 0.002 mg / L to 10 mg / L, 0.01 mg / L to 10 mg / L, 0.02 mg / L to 10 mg / L, 0.1 mg / L to 10 mg / L, 0.2 mg / L to 10 mg / L, or 0.2 mg / L to 5 mg / L as enzyme protein. The concentration of DNase contained in the washing solution is typically within the same range as alkaline phosphatase.

[0351] The present invention is further defined by the following numbered paragraphs. 1. A cleaning composition comprising a polypeptide having DNase activity, a polypeptide having alkaline phosphatase activity, and at least one detergent auxiliary component.

[0352] 2. The washing composition according to paragraph 1, wherein the polypeptide having alkaline phosphatase activity is selected from the group consisting of the following: (a) SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 polypeptides having at least 70% sequence identity with SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0353] 3. The polypeptide having alkaline phosphatase activity corresponds to Sequence IDs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, Sequence ID No. A cleaning composition according to paragraph 1 or 2, selected from the group consisting of polypeptides having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with respect to No. 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.

[0354] 4. The washing composition according to paragraph 3, wherein the polypeptide having alkaline phosphatase activity has 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%, or at least 98% sequence identity with respect to SEQ ID NO: 1.

[0355] 5. The washing composition according to paragraph 4, wherein the polypeptide having alkaline phosphatase activity is a variant having a substitution at at least one position selected from the group consisting of positions 77, 90, 94, 117, 119, 148, 156, 208, 212, 216, 220, 224, 225, 228, 245, 246, 248, 270, 271, 291, 292, 303, and 381 of SEQ ID NO: 1.

[0356] 6. The washing composition according to paragraph 5, wherein the polypeptide having alkaline phosphatase activity comprises at least one substitution selected from the group consisting of A77T, N90I, A94P, I117L, H119A, N148F, M156F, K208A, R212M, M216V, K220P, K220E, E224D, T225E, D228R, Q245L, I246F, W248K, K270G, A271R, S291G, T292A, Y303L, and G381S.

[0357] 7. The washing composition according to any one paragraph of paragraphs 4 to 6, wherein the polypeptide having alkaline phosphatase activity is a variant of SEQ ID NO: 1 having 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 SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.

[0358] 8. The washing composition according to paragraph 7, wherein the polypeptide having alkaline phosphatase activity has 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 respect to SEQ ID NO: 79.

[0359] 9. A washing composition according to any one paragraph from paragraphs 1 to 8, comprising a polypeptide having DNase activity selected from the group consisting of the following: a) Polypeptides having 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%, or at least 95% sequence identity with respect to sequence number 83; b) Polypeptides having 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%, or at least 95% sequence identity with respect to SEQ ID NO: 82; and c) A variant of sequence number 82 having 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%, or at least 95% sequence identity with sequence number 82 or sequence number 83, and compared to sequence number 82, T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V, S1 3N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M, T22P, T22A, T22V, T22D, T2 2R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22I, G24Y, S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S3 0D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42C, S42L, S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S, D5 6I, D56L, D56T, S57W, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T6 5L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F, T7 7N, T77Y, T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R, N80K, S82L, S82E,S82K、S82R、S82H、D83C、D83F、D83L、L92T、A93G、E94N、G99S、S101D、S101A、S102M、S102L、S102V、S102A、S102K、S102T、S102R、T104P、T104A、T105V、T105I、K107L、K107C、K107R、K107H、K107S、K107M、K107E、K107A、K107D、Q109R、Q109S、A112S、S116D、S116R、S116Q、S116H、S116V、S116A、S116E、S116K、A125K、S126I、S126E、S126A、S126C、T127C、T127V、S130E、G132R、D135R、T138Q、W139R、R143E、R143K、S144Q、S144H、S144L、S144P、S144E、S144K、G145V、G145E、G145D、G145A、A147H、A147Q、A147W、A147S、G149S、K152H、K152R、S156C、S156G、S156K、S156R、S156T、S156A、T157S、Y159F、K160V、W161L、W161Y、G162Q、G162D、G162M、G162R、G162A、G162S、G162E、G162L、G162K、G162V、G162H、S164R、S164T、Q166D、S167M、S167L、S167F、S167W、S167E、S167A、S167Y、S167H、S167C、S167I、S167Q、S167V、S167T、S168V、S168E、S168D、S168L、K170S、K170L、K170F、K170R、T171D、T171E、T171A、T171C、A172G、A172S、L173T、L173A、L173V、Q174L、G175D、G175E、G175N、G175R、M176H、L177I、N178D、N178E、N178T、N178S、N178A、S179E、S181R、S181E、S181D、S181F、S181H、S181W、S181L、S181M、S181Y、S181Q、S181G、S181A、Y182M、Y182C、Y182K、Y182G、Y182A、Y182S、Y182V、Y182D、Y182Q、Y182F、Y182L、Y182N、Y182I、Y182E、A variant comprising one or more substitutions selected from the group consisting of Y182T and Y182W, preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more substitutions.

[0360] 10. The polypeptide having DNase activity is a variant of SEQ ID NO: 82, and the variant has 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%, or at least 95% sequence identity with respect to SEQ ID NO: 82 or SEQ ID NO: 83, and compared to SEQ ID NO: 82, it has T1I, S13Y, T22P The cleaning composition according to paragraph 9, comprising one or more substitutions selected from the group consisting of S25P, S27L, S39P, D56I, S57W, S59V, T65V, V76L, T77Y, Q109R, S116D, T127V, S144P, A147H, G149N, S167L, G175D, and S181L, preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more substitutions.

[0361] 11. The washing composition according to paragraph 9, wherein the polypeptide having DNase activity is a variant of SEQ ID NO: 82, and the variant has 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%, or at least 95% sequence identity with respect to SEQ ID NO: 82 or SEQ ID NO: 83, and comprises two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S, for example, three, four, five, or more substitutions.

[0362] 12. The washing composition according to paragraph 11, wherein the polypeptide having DNase activity further comprises at least one substitution selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W.

[0363] 13. The washing composition according to paragraph 11 or 12, wherein the polypeptide having DNase activity comprises a set of substitutions selected from the group consisting of: a) G149N with at least one substitution from N61D, T65I, T65V, S82R, K107Q, T127S, T127V, S164D, and L181S; b) T65I or T65V with at least two substitutions from N61D, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S; c) N61D with at least two substitutions from T65I / V, S82R, K107Q, T127S / V, G149N, S164D, and L181S, preferably at least two substitutions from T65I / V, S82R, K107Q, T127S, and S164D; d) S82R with at least two substitutions from N61D, T65I, T65V, K107Q, T127S, T127V, G149N, S164D, and L181S; e) K107Q with at least two substitutions from N61D, T65I, T65V, S82R, T127S, T127V, G149N, S164D, and L181S; f) T127S with substitution of at least two of N61D, T65I, T65V, S82R, K107Q, G149N, S164D, and L181S; and g) S164D with at least one substitution from N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, and L181S.

[0364] 14. The washing composition according to any one paragraph of paragraphs 1 to 8, wherein the composition comprises a polypeptide having DNase activity having 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%, or at least 95% sequence identity with respect to SEQ ID NO: 86, or to a polypeptide of SEQ ID NO: 86 whose N-terminus is shortened by up to about 20 amino acid residues, for example, a polypeptide of SEQ ID NO: 86 whose N-terminus is shortened by 15 to 17 amino acid residues.

[0365] 15. The polypeptide having DNase activity is a variant of SEQ ID NO: 86, and the variants are S69V, Q102E, S26*, D32Q, K36C,H, G37R, F43W, D46G, A55I, N68D, A76I, K82S,T, P84D,T, K86G,L,N,Q,T,V,Y, A91R, L92E, K95I, P97E,N, A101E, K105N,G,Q,T,D, F112Y,W, L129K, N133Q, V138C, N140H, G141Q,R, S144E, N14 The cleaning composition according to paragraph 14, comprising at least two modifications selected from the group consisting of 6A, K147N,E, V148I, A149D,E,F, Q150D, P153D,V, S154E, K155E,F,L,S,T, Q157D,E, Q158D, T159Q, K160D, T170Q, A172D,E,H,R, K185*, V187N,Y, N191*, K192A, D197K,S, G199Q, Q208V, E211Y,T,P, N213S, N214D, N217A, and Y218D,E.

[0366] 16. The polypeptide having DNase activity is a variant of SEQ ID NO: 86, wherein the variant comprises at least two substitutions selected from the group consisting of A111P, D32E, V35I, S69V, Q102E, K105N, and G181N, for example, at least three substitutions, and optionally the variant comprises S26H, D32E, V35I, K65E, The cleaning composition according to paragraph 14, further comprising two or more substitutions selected from the group consisting of K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0367] 17. A cleaning composition comprising a polypeptide having alkaline phosphatase activity and at least one detergent auxiliary component, wherein the polypeptide having alkaline phosphatase activity is selected from the group consisting of the following: (a) SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 polypeptides having at least 70% sequence identity with SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0368] 18. The washing composition according to paragraph 17, wherein the polypeptide having alkaline phosphatase activity is selected from the group consisting of the following: (a) A polypeptide having at least 70% sequence identity with respect to Sequence ID No. 1, for example, 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%, or at least 98% sequence identity; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0369] 19. The washing composition according to paragraph 18, wherein the polypeptide having alkaline phosphatase activity is a variant having a substitution at at least one position selected from the group consisting of positions 77, 90, 94, 117, 119, 148, 156, 208, 212, 216, 220, 224, 225, 228, 245, 246, 248, 270, 271, 291, 292, 303, and 381 of SEQ ID NO: 1.

[0370] 20. The washing composition according to paragraph 19, wherein the polypeptide having alkaline phosphatase activity comprises at least one substitution selected from the group consisting of A77T, N90I, A94P, I117L, H119A, N148F, M156F, K208A, R212M, M216V, K220P, K220E, E224D, T225E, D228R, Q245L, I246F, W248K, K270G, A271R, S291G, T292A, Y303L, and G381S.

[0371] 21. The washing composition according to any one paragraph of paragraphs 18 to 20, wherein the polypeptide having alkaline phosphatase activity is a variant of SEQ ID NO: 1 having 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 SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81.

[0372] 22. A cleaning composition according to any one paragraph of paragraphs 1 to 21, comprising one or more cleaning aids selected from the group consisting of surfactants, builders, flocculants, chelating agents, dye migration inhibitors, other enzymes, enzyme stabilizers, enzyme inhibitors, catalysts, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, preform peracids, polymer dispersants, clay stain removers / redeposition inhibitors, glossing agents, foam inhibitors, dyes, fragrances, structural elastics, softeners, carriers, hydrotropes, builders and cobuilders, fabric colorants, defoamers, dispersants, processing aids, and / or pigments.

[0373] 23. The cleaning composition according to any one paragraph of paragraphs 1 to 22, wherein the composition is in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a pouch having one or more compartments, a typical powder or compact powder, granules, a paste, a gel, or a typical liquid or concentrated liquid.

[0374] 24. The cleaning composition according to paragraph 23, wherein the composition is in the form of a liquid detergent, a powder or granular detergent, or a single-dose composition.

[0375] 25. A method for washing an article, wherein the article is a textile product, a) Exposing an article to a washing solution containing a polypeptide having alkaline phosphatase activity and a polypeptide having DNase activity. b) Complete at least one cleaning cycle, and if desired c) A method comprising rinsing the article.

[0376] 26. The method according to paragraph 25, wherein the washing solution comprises a polypeptide having DNase activity as defined in any one of paragraphs 9 to 16.

[0377] 27. A method for washing an article, wherein the article is a textile product, a) Exposing the article to a washing solution containing a polypeptide having alkaline phosphatase activity as defined in any one of paragraphs 17 to 21; b) Complete at least one cleaning cycle, and if desired c) A method comprising rinsing the article.

[0378] 28. A method for washing or cleaning a hard surface, comprising exposing a textile product or a hard surface to a cleaning composition described in any one paragraph of paragraphs 1 to 24.

[0379] 29. Use of a polypeptide having alkaline phosphatase activity as defined in any one of paragraphs 1 to 8 in a cleaning process such as washing clothes or cleaning hard surfaces such as dishes.

[0380] 30. Use of polypeptides having alkaline phosphatase activity to reduce redeposition in the washing process, particularly in laundry.

[0381] 31. The use described in paragraph 30, wherein the polypeptide having alkaline phosphatase activity is used in combination with the polypeptide having DNase activity.

[0382] 32. Polypeptides having alkaline phosphatase activity, selected from the group consisting of the following: (a) SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 polypeptides having at least 70% sequence identity with SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) The polypeptide of (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity.

[0383] 33. A polypeptide having alkaline phosphatase activity as described in paragraph 32, wherein the polypeptide is as defined in any one of paragraphs 3 to 8.

[0384] 34. A polynucleotide encoding a polypeptide having alkaline phosphatase activity as defined in any one of paragraphs 2 to 8, a nucleic acid construct comprising the polynucleotide, or an expression vector comprising the polynucleotide.

[0385] 35. Recombinant host cells comprising a polynucleotide encoding a polypeptide having alkaline phosphatase activity as defined in any one of paragraphs 2 to 8, the polynucleotide being operably linked to one or more regulatory sequences that induce the production of the polypeptide.

[0386] 36. A method for producing a polypeptide having alkaline phosphatase activity, comprising (a) culturing recombinant host cells as described in paragraph 35 under conditions suitable for polypeptide production, and optionally (b) recovering the polypeptide.

[0387] The present invention will be further illustrated by the following embodiments, but this should not be construed as limiting the scope of the invention. [Examples]

[0388] Materials and methods Phosphatase activity assay Dispense 75 μL of phosphatase-containing enzyme solution (diluted with MQ water containing 0.01% (v / w) Triton X-100) into the wells of a microtiter plate (e.g., NUNC 269620 96-well plate) and add 75 μL of substrate (to prepare the substrate, dissolve 5 mg of p-nitrophenyl phosphate tablet (Sigma-Aldrich, catalog no. 20-106) in 10 mL of 50 mM Hepes, 100 mM sodium chloride (NaCl), 1 mM calcium chloride (CaCl2), 1 mM magnesium chloride (MgCl2), 1 mM zinc chloride (ZnCl2), pH 7.0). Seal the plate and incubate at 37°C for 15 minutes and shake at 750 rpm. After incubation, 75 μL of reaction stop reagent (0.5 M sodium hydroxide (NaOH)) is added, and the absorbance at 405 nm is measured using a microtiter plate spectrophotometer. A blank (75 μL of 0.01% (v / w) Triton X-100 + 75 μL of substrate solution) is also incubated for 15 minutes, 75 μL of stop reagent is added, and the absorbance at 405 nm is measured. This value is subtracted from the phosphatase reading. One phosphatase unit is defined as the enzyme activity that releases 1 μmol of phosphate / min under the given reaction conditions (value after subtracting the blind buffer). The absorbance of 1 μmol of p-nitrophenol is defined as 56 AU (absorbance units) under analytical conditions.

[0389] DNase activity assay DNase activity can be measured using methyl green-containing DNase test agar (BD, Franklin Lakes, New Jersey, USA) prepared according to the supplier's instructions. Briefly, 21 g of agar is dissolved in 500 mL of water and autoclaved at 121°C for 15 minutes. The autoclaved agar is warmed to 48°C in a water bath, 20 mL of the agar is poured into a Petri dish, and incubated overnight at room temperature to solidify. 5 μL of enzyme solution is added to the solidified agar medium, and DNase activity is observed as a colorless region around the spotted enzyme solution.

[0390] Mechanical action assay for laundry (Mini-Terg-O-ToMeter) The Terg-O-ToMeter (TOM) is a device that simulates a "top-loader / vertical drum" washing machine, using a series of metal beakers, typically with capacities of 1-2 liters. Each beaker is fitted with an agitator that rotates back and forth at a controlled speed (e.g., 120 rpm), simulating the agitation mode that occurs in commercially available top-loader washing machines. The Mini-TOM functions as a TOM device, but with a reduced capacity of 0.2 liters.

[0391] This assay is used to test several conditions on several swatches (fabric samples) at once. Cut the swatches to a size of 2 cm in diameter and place them in a beaker containing water and detergent. Control the stirring speed and temperature during the washing time. After washing, rinse the swatches, dry them for 24 hours, and measure the intensity of the swatches (reflectance, determined as described in Example 5 below).

[0392] Example 1 Cloning, expression, and fermentation of fungal alkaline phosphatases Alkaline phosphatase genes derived from fungal strains were isolated from environmental samples using standard microbiological isolation techniques.

[0393] Chromosomal DNA from individual strains was isolated using the DNeasy® Plant Maxi Kit (Qiagen, Hilden, Germany). 5 μg of chromosomal DNA was sent for whole-genome sequencing using Illumina technology. Genome sequencing, subsequent read assembly, and gene discovery (i.e., gene function annotation) are publicly known to those skilled in the art, and this service is commercially available. The genome sequence of putative ALP from the PFAM database family PF00245 was analyzed. This analysis identified the gene encoding putative ALP, which was then cloned and recombinantly expressed in Aspergillus oryzae.

[0394] The ALP gene was amplified by PCR from the isolated genomic DNA described above. The purified PCR product was cloned into pre-digested pCaHj505 or pDAU724 cells by ligation using the IN-FUSION® CF Dry-down Cloning Kit (Clontech Laboratories, Inc., Mountain View, California, USA) according to the manufacturer's instructions. The ligation mixture was used to transform E. coli TOP10 chemically competent cells. Colonies containing the corresponding ALP gene were selected and validated by DNA sequencing (SinoGenoMax Company Limited, Beijing, China). Colonies containing the correct ALP were cultured overnight in 3 mL of LB soil supplemented with 100 μg of ampicillin per mL. Plasmid DNA was purified using the Qiagen Spin Miniprep Kit (catalog no. 27106) (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions.

[0395] Protoplasts of Aspergillus oryzae MT3568 were prepared according to International Publication No. 95 / 002043. Protoplasts of Aspergillus oryzae DAU785 were prepared according to International Publication No. 2018 / 113745. 100 μL of protoplasts were gently mixed with an Aspergillus expression vector containing 2.5–10 μg of ALP gene (plasmid extracted above) and 250 μL of 60% PEG 4000, 10 mM calcium chloride (CaCl2), and 10 mM Tris-HCl (pH 7.5). The mixture was incubated at 37°C for 30 minutes, and the protoplasts were spread on a COVE sucrose plate for selection. After culturing at 37°C for 4-7 days, spores from four transformants were inoculated into 3 mL of Dap4C medium.

[0396] After culturing at 30°C for 3 days, the culture broth was analyzed by SDS-PAGE using Novex® 4-20% Tris-Glycine Gel (Invitrogen Corporation, Carlsbad, California, USA) to identify the transformant that produced the maximum amount of recombinant ALP according to the estimated mature peptide size. Spores from the best-expressing transformant were spread onto a COVE-2 plate and re-isolated, and single colonies were isolated. Next, the single colonies were spread onto a COVE-2 tube until spore formation. Spores from the best-expressing transformant were cultured in 1600-2400 mL of Dap4C medium in a shaking flask at 30°C for 3 days with stirring at 80 rpm. The culture broth was collected by filtration using a 0.22 μm filter. The filtered fermentation broth was used for enzyme characterization.

[0397] Example 2 Cloning and expression of bacterial alkaline phosphatase The DNA encoding sequence of the alkaline phosphatase maturation peptide was ordered as a synthetic gene from Twist Bioscience. The synthetic DNA fragment was orientedly assembled into a Bacillus expression vector as described in International Publication No. 2012 / 025577 using the standard Golden Gate cloning method with BsaI and T4 DNA ligase enzymes. Briefly, the DNA encoding the gene's maturation peptide was cloned in-frame into the Bacillus clausii secretion signal (BcSP, with the amino acid sequence below): MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 84). BcSP replaced the innate secretion signal within the gene. Downstream of the BcSP sequence, an affinity tag sequence was introduced to facilitate the purification process (His tag, amino acid sequence below: HHHHHHPR (SEQ ID NO: 85)). Thus, the expressed gene consisted of the BcSP sequence, followed by the His tag sequence, and then the mature wild-type alkaline phosphatase gene sequence. The final expression plasmid (BcSP-His-tag-alkaline phosphatase) was used to transform Bacillus subtilis-expressing hosts. The alkaline phosphatase BcSP fusion gene was incorporated into the Bacillus subtilis host cell genome by homologous recombination upon transformation. The gene construct was expressed under the control of a triple promoter system (as described in International Publication No. 99 / 43835). A gene encoding chloramphenicol acetyltransferase was used as a marker (as described in Diderichsen et al., 1993, Plasmid 30: 312-315). Transformants were selected on LB agar supplemented with 6 micrograms of chloramphenicol per mL. One recombinant Bacillus subtilis clone containing an alkaline phosphatase-expressing construct was selected and cultured on a rotating shaker in a 500 mL baffled Erlenmeyer flask containing 100 mL of yeast extract-based medium.After culturing at 30°C for 3 days, the supernatant containing the enzyme was collected by centrifugation, and the enzyme was purified by His-tagged purification.

[0398] Example 3 Purification of alkaline phosphatase Typically, the purification process for alkaline phosphatase (ALP) from culture broth was first performed using hydrophobic interaction chromatography with an AKTA chromatography system (Cytiva), followed by ion exchange chromatography as needed. The differences in all molecules were due to the type of buffer, pH, and salt concentration.

[0399] The conductivity of the recombinant ALP culture supernatant was adjusted to approximately 190 mS / cm by adding ammonium sulfate. The culture broth was then loaded onto a phenyl Sepharose high-performance column (Cytiva, 17108203) equilibrated with 20 mM Tris-HCl at pH 7.0 containing 2.0 M ammonium sulfate. The elution condition was set to a gradient decrease in ammonium sulfate concentration from 2.0 M to 0. The eluted and flow-through fractions were analyzed by SDS-PAGE. ALP activity was measured as follows.

[0400] For further purification, an ion exchange chromatography process was applied. The ALP-active fractions were pooled together, dialyzed with 20 mM Tris-HCl at pH 8.0, and then loaded onto MonoQ HR16 / 10 (Cytiva, 17050601) or CaptoQ column (Cytiva, 17547003) equilibrated with 20 mM Tris-HCl at pH 8.0. For the elution process, a gradient of increasing sodium chloride (NaCl) concentration from 0 M to 1 M was set using 20 mM Tris-HCl at pH 8.0. The eluted and flow-through fractions were analyzed for ALP activity by SDS-PAGE.

[0401] The enzyme-active fractions were then pooled and dialyzed with 20 mM PBS at pH 7.0. Protein concentration was measured using the Qubit® Protein Assay Kit (Invitrogen, Q33212).

[0402] Example 4 His tag purification method His-tagged alkaline phosphatase was collected in a 5 mL HisTrap Excel column (GE Healthcare Life Sciences) using Ni 2+ The enzyme was purified as a metal ion by immobilized metal chromatography (IMAC). Purification was performed at pH 7, and the bound protein was eluted with imidazole. The purity of the purified enzyme was confirmed by SDS-PAGE, and the enzyme concentration was measured by absorbance at 280 nm after buffer exchange with 50 mM HEPES, 100 mM sodium chloride (NaCl), and pH 7.0.

[0403] Example 5 Washing performance assay Using the Mini-TOM instrument described above, cleaning performance assays were performed to test the effectiveness of alkaline phosphatases and DNases individually and in combination. The assays were carried out using one or two of two detergents: a conventional detergent (Detergent 1) and a detergent containing plant-derived surfactants (Detergent 2), with various commercially available stain swatches. Information on the swatches used and other details of the assay setup are shown in Table 1 below. Information on the composition of Detergent 1 and Detergent 2 is shown in Tables 2 and 3.

[0404] method The stained sheet was cut into small pieces with a diameter of 2 cm. 200 mL of detergent solution was placed in a Mini-TOM beaker and set up in the TOM apparatus. Then, the mechanical rotary device and temperature control device were started. When the washing temperature was reached, the enzyme and swatch were added. After washing for 30 minutes, the swatch was transferred to a strainer to drain the water from the washed material. After rinsing quickly with running water, the swatch was squeezed and transferred to a 5 L beaker filled with cold tap water. The swatch washed under blank conditions without enzyme was rinsed separately from the sample containing enzyme. The swatch was placed on blotting paper at room temperature in a dark place and dried for 24 hours.

[0405] Dry swatches were evaluated using VeriVide's Digi-Eye color measurement and imaging system. Digi-Eye is a controlled digital imaging system for measuring color and capturing repeatable images.

[0406] [Table 1]

[0407] [Table 2]

[0408] [Table 3]

[0409] result The results of the Mini-TOM assay are shown in Tables 4, 5, 6, 7, 8, and 9 below, as the emission values ​​for each enzyme treatment and the delta emission (Δ emission) values ​​based on the reference treatment in each table. The delta emission value is calculated by subtracting the emission value of the reference treatment in the relevant table from the emission value of the specific enzyme treatment (typically ALP alone, or a combination of ALP and DNase and / or Medley® Brilliant). The reference treatment can be a blank (no enzyme) or another enzyme treatment shown in the table.

[0410] Table 4 shows the cleaning performance of conventional detergents (conventional detergent 1 containing a surfactant) against two types of stains. The effects of different doses of alkaline phosphatase (ALP, SEQ ID NO: 79) are compared with DNase (SEQ ID NO: 82) administered at 0.2 ppm and Medley® Brilliant enzyme mixture administered at 0.025 mg / L (0.025 ppm). The Δ-remission values ​​in Table 4 are calculated based on the blank (no enzyme).

[0411] [Table 4]

[0412] Table 5 shows the cleaning performance of conventional detergents against two different types of stains. The effects of alkaline phosphatase (ALP, SEQ ID NO: 79) alone or in combination with a Medley® Brilliant enzyme mixture administered at 0.025 mg / L (0.025 ppm) were measured. The Δ-remission values ​​in Table 5 are calculated based on the emission of Medley® Brilliant treatment alone.

[0413] [Table 5]

[0414] Table 6 shows the cleaning performance of conventional detergents for a single stain. The effects of combining different doses of alkaline phosphatase (ALP, SEQ ID NO: 79) with DNase (SEQ ID NO: 82) administered at 0.2 ppm and a Medley® Brilliant enzyme mixture administered at 0.025 mg / L (0.025 ppm) were measured. The Δ-remission values ​​in Table 6 were calculated based on the emission of treatment using DNase and Medley® Brilliant without alkaline phosphatase.

[0415] [Table 6]

[0416] Table 7 shows the cleaning performance of plant-derived detergents against two different types of stains. The effects of alkaline phosphatase (ALP, SEQ ID NO: 79) alone or in combination with 0.2 ppm DNase (SEQ ID NO: 82) were measured. The Δ-remission values ​​in Table 7 are calculated based on the emission of DNase treatment alone.

[0417] [Table 7]

[0418] Table 8 shows the cleaning performance of plant-derived detergents against two different types of stains. The effects of different doses of alkaline phosphatase (ALP, SEQ ID NO: 79) and a Medley® Brilliant enzyme mixture administered at 0.025 mg / L (0.025 ppm) were measured. The Δ-remission values ​​in Table 8 are calculated based on the remission of treatment with Medley® Brilliant alone.

[0419] [Table 8]

[0420] Example 6 The effect of preventing the redeposition of alkaline phosphatase in liquid detergents. In this example, a strain of Pseudomonas fluorescens isolated from Iceland was used as a model microorganism. Pseudomonas fluorescens was re-streaked onto Tryptone Sawyer agar (TSA) (pH 7.3) (CM0131, Oxoid Ltd., Basingstoke, UK) and cultured at 30°C for 3 days. A single colony was inoculated into 10 mL of TSB, and the culture was incubated with shaking at 30°C for 19 hours (BenchTop Orbital Shaker MaxQ 4000, 200 rpm). After growth, the culture was diluted to an OD value of 0.03 with fresh TSB, and 1.65 mL aliquots were added to the wells of a 12-well polystyrene flat-bottom culture plate (3512, Costar, Corning Incorporated, Corning, New York, USA) on which a circular swatch (2 cm in diameter) of sterile fiber (WFK10A, 100% cotton) was placed. Sterile TSB was added to the control well. The plate was incubated in an orbital shaker (Titramax1000, Heidolph Instruments GmbH & Co. KG, Germany) at 30°C and 150 rpm. After 72 hours, the swatch was rinsed twice with 2 mL of 0.9% (w / v) sodium chloride (NaCl).

[0421] Sebum swatches (sebum tracers) were prepared by dropping 50 μL of 1% (w / v) sebum solution onto a 2 cm diameter circular swatch (WFK20A) of a textile product and drying it overnight. The 1% (w / v) sebum solution was prepared by adding molten Sebum Bey (CFT, Center For Testmaterials BV, Netherlands) to MiliQ water containing 0.4% (w / v) Tween® 80.

[0422] Three rinsed switches with Pseudomonas fluorescens attached, two sebum switches, and two clean switches (clean tracers) were placed in a 50 mL test tube, and 10 mL of cleaning solution containing 6°dH water with 0.7 g / L of dirt (pigment dirt (Pigmentschmutz), 09V, wfk contaminated cloth, clay felt, Germany) and 0.8 g / L of liquid model J detergent (Table 9) was added.

[0423]

Table 9

[0424] 100 μL of enzyme stock (containing alkaline phosphatase and / or DNase) was added to each tube to make the cleaning concentration 1 ppm DNase, 5 ppm alkaline phosphatase, or 1 ppm DNase + 5 ppm alkaline phosphatase. Alkaline phosphatase was the polypeptide of SEQ ID NO: 79, and DNase was the polypeptide of SEQ ID NO: 82.

[0425] The cleaning solution without enzyme was also included as a control. The test tubes were placed in a Stuart rotator and incubated at 20 rpm and 30 °C for 1 hour. Then, the cleaning solution was removed, and the switches were rinsed twice with 20 mL of 6°dH water and dried overnight on filter paper. The reflectance (REM 460nm ) values were measured using a Datacolor® 800 spectrophotometer. These values are shown in Table 10.

[0426] The Δ value (REM 460nm (酵素で洗浄したスワッチ) -REM 460nm (酵素なしで洗浄したスワッチ) ) is also shown.

[0427]

Table 10

[0428] As shown in Table 10, combining alkaline phosphatase and DNase yields superior anti-redeposition properties in Model J detergent compared to the individual enzymes. The anti-redeposition performance of the alkaline phosphatase and DNase mixture (ΔREM = 7.3) clearly exceeds the sum of the ΔREM values ​​of the individual enzymes on the clean tracer (= 4.0). Similarly, in tracers containing sebum, the anti-redeposition effect of the alkaline phosphatase and DNase mixture (ΔREM = 10.6) is higher than the sum of the ΔREM values ​​of the individual enzymes (= 6.8). This indicates a synergistic cleaning effect when using alkaline phosphatase and DNase in combination.

Claims

1. A cleaning composition comprising a polypeptide having DNase activity, a polypeptide having alkaline phosphatase activity, and at least one detergent auxiliary component.

2. The polypeptide having alkaline phosphatase activity is comprised of the following group: (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 , polypeptide having at least 70% sequence identity with SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) A polypeptide according to (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity, A cleaning composition according to claim 1, selected from the following.

3. The polypeptide having alkali phosphatase activity corresponds to Sequence IDs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 4 9. A cleaning composition according to claim 1 or 2, selected from the group consisting of polypeptides having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with respect to SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO:

81.

4. The washing composition according to claim 3, wherein the polypeptide having alkaline phosphatase activity has sequence identity of 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%, or at least 98% with respect to SEQ ID NO:

1.

5. The cleaning composition according to claim 4, wherein the polypeptide having alkaline phosphatase activity is a variant having a substitution at at least one position selected from the group consisting of positions 77, 90, 94, 117, 119, 148, 156, 208, 212, 216, 220, 224, 225, 228, 245, 246, 248, 270, 271, 291, 292, 303, and 381 of SEQ ID NO:

1.

6. The cleaning composition according to claim 5, wherein the polypeptide having alkaline phosphatase activity comprises at least one substitution selected from the group consisting of A77T, N90I, A94P, I117L, H119A, N148F, M156F, K208A, R212M, M216V, K220P, K220E, E224D, T225E, D228R, Q245L, I246F, W248K, K270G, A271R, S291G, T292A, Y303L, and G381S.

7. The cleaning composition according to any one of claims 4 to 6, wherein the polypeptide having alkaline phosphatase activity is a variant of SEQ ID NO: 1 having 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 SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO:

81.

8. The washing composition according to claim 7, wherein the polypeptide having alkaline phosphatase activity has 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 respect to SEQ ID NO:

79.

9. The following group: a) Polypeptides having 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%, or at least 95% sequence identity with respect to Sequence ID No. 83; b) polypeptides having 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%, or at least 95% sequence identity with respect to SEQ ID NO: 82; and c) A variant of sequence number 82 having 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%, or at least 95% sequence identity with sequence number 82 or sequence number 83, and having T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V, S1 3N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M, T22P, T22A, T22V, T22D, T2 2R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22I, G24Y, S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S3 0D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42C, S42L, S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S, D5 6I, D56L, D56T, S57W, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T6 5L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F, T7 7N, T77Y, T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R, N80K, S82L, S82E,S82K、S82R、S82H、D83C、D83F、D83L、L92T、A93G、E94N、G99S、S101D、S101A、S102M、S102L、S102V、S102A、S102K、S102T、S102R、T104P、T104A、T105V、T105I、K107L、K107C、K107R、K107H、K107S、K107M、K107E、K107A、K107D、Q109R、Q109S、A112S、S116D、S116R、S116Q、S116H、S116V、S116A、S116E、S116K、A125K、S126I、S126E、S126A、S126C、T127C、T127V、S130E、G132R、D135R、T138Q、W139R、R143E、R143K、S144Q、S144H、S144L、S144P、S144E、S144K、G145V、G145E、G145D、G145A、A147H、A147Q、A147W、A147S、G149S、K152H、K152R、S156C、S156G、S156K、S156R、S156T、S156A、T157S、Y159F、K160V、W161L、W161Y、G162Q、G162D、G162M、G162R、G162A、G162S、G162E、G162L、G162K、G162V、G162H、S164R、S164T、Q166D、S167M、S167L、S167F、S167W、S167E、S167A、S167Y、S167H、S167C、S167I、S167Q、S167V、S167T、S168V、S168E、S168D、S168L、K170S、K170L、K170F、K170R、T171D、T171E、T171A、T171C、A172G、A172S、L173T、L173A、L173V、Q174L、G175D、G175E、G175N、G175R、M176H、L177I、N178D、N178E、N178T、N178S、N178A、S179E、S181R、S181E、S181D、S181F、S181H、S181W、S181L、S181M、S181Y、S181Q、S181G、S181A、Y182M、Y182C、Y182K、Y182G、Y182A、Y182S、Y182V、Y182D、Y182Q、Y182F、Y182L、Y182N、Y182I、Y182E、A variant comprising one or more substitutions selected from the group consisting of Y182T and Y182W, preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more substitutions. A cleaning composition according to any one of claims 1 to 8, comprising a polypeptide having DNase activity selected from the above.

10. The washing composition according to any one of claims 1 to 8, wherein the polypeptide having DNase activity is a polypeptide having 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%, or at least 95% sequence identity with respect to SEQ ID NO: 86, or to a polypeptide of SEQ ID NO: 86 with its N-terminus shortened by up to approximately 20 amino acid residues.

11. A method for washing an article, wherein the article is a textile product, a) Exposing the article to a washing solution containing a polypeptide having alkaline phosphatase activity and a polypeptide having DNase activity, b) Complete at least one cleaning cycle, and optionally c) A method comprising rinsing the article.

12. A method for washing or cleaning a hard surface, comprising exposing a textile product or a hard surface to a cleaning composition according to any one of claims 1 to 10.

13. A cleaning composition comprising a polypeptide having alkaline phosphatase activity and at least one detergent auxiliary component, wherein the polypeptide having alkaline phosphatase activity is comprised of the following: (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 , polypeptide having at least 70% sequence identity with SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) A polypeptide according to (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity, A cleaning composition selected from the following.

14. The polypeptide having alkaline phosphatase activity is comprised of the following group: (a) A polypeptide having at least 70% sequence identity with respect to Sequence ID No. 1, for example, 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%, or at least 98% sequence identity; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) A polypeptide according to (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity, A cleaning composition according to claim 13, selected from the above.

15. The polypeptide having alkaline phosphatase activity is substituted at at least one position selected from the group consisting of positions 77, 90, 94, 117, 119, 148, 156, 208, 212, 216, 220, 224, 225, 228, 245, 246, 248, 270, 271, 291, 292, 303, and 381 of Sequence ID No. 1, for example, A77T, N90 The cleaning composition according to claim 14, which is a variant having at least one substitution selected from the group consisting of I, A94P, I117L, H119A, N148F, M156F, K208A, R212M, M216V, K220P, K220E, E224D, T225E, D228R, Q245L, I246F, W248K, K270G, A271R, S291G, T292A, Y303L, and G381S.

16. Polypeptides having alkaline phosphatase activity, comprising the following group: (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 , polypeptide having at least 70% sequence identity with SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81; (b) Polypeptides of (a) in which 1 to 120 amino acids, for example 1 to 100, 1 to 80, 1 to 60, or 1 to 40 amino acids are substituted, deleted, or added; (c) A polypeptide according to (a) or (b) wherein the N-terminus and / or C-terminus are elongated by the addition of 1 to 50 amino acids, for example 1 to 40, 1 to 30, or 1 to 20 amino acids; and (d) A fragment of the polypeptide of (a), (b), or (c) having alkaline phosphatase activity, A polypeptide selected from the following.

17. The polypeptide having alkaline phosphatase activity according to claim 16, wherein the polypeptide is as defined in any one of claims 3 to 8.

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