DNase variants and compositions

Abstract

The present invention relates to novel DNase variants that exhibit alterations compared to parent DNases that have been found to provide improved stability, such as improved detergent stability.The present invention also relates to detergent compositions that include the DNase variants, as well as methods of using and producing the DNase variants, and isolated DNA sequences, expression vectors, and host cells that code for the variants.The variants of the present invention are suitable for use in cleaning processes and detergent compositions.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to novel DNase (deoxyribonuclease) variants that exhibit alterations compared to the parent DNase that have been found to provide improved stability, e.g., improved detergent stability. The invention also relates to detergent compositions comprising the DNase variants, as well as methods of using and producing the DNase variants, and isolated DNA sequences, expression vectors, and host cells encoding the variants. The variants of the invention are suitable for use in cleaning processes and detergent compositions, such as laundry and dishwashing compositions. [Background technology]

[0003] Microorganisms commonly live and adhere to surfaces in many natural, industrial, and medical environments, encapsulated by extracellular substances, including biopolymers and macromolecules. The resulting layer of microbial organisms with a sticky capsule is called a biofilm. Biofilms are the predominant growth method for bacteria in natural environments, and bacteria growing within biofilms exhibit unique physiological properties. Compared to their planktonically growing counterparts, bacteria within biofilms are more resistant to antibiotics, ultraviolet radiation, detergents, and host immune responses.

[0004] Biofilms may contain one or more microorganisms, including gram-positive and gram-negative bacteria, marine algae, protozoa, and / or yeast or filamentous fungi, as well as viruses and / or bacteriophages. Examples of problematic biofilms include dental plaque, infection of medical implants, but also the initial contamination of ship hulls. Biofilms are responsible for many infectious diseases in humans and are a significant problem in industry with regard to biofouling of exposed surfaces, where biofilm colonization can form a base component of the local ecosystem, which can disrupt and interfere with industrial processes and components.

[0005] When laundry items, such as T-shirts and sportswear, as well as other clothing items, are used, they are exposed to bacteria originating from the user's body and not from the environment in which they are used. Some of these bacteria can adhere to the laundry items and form biofilms on the items. The presence of bacteria means that the laundry items become sticky, which causes dirt to adhere to the sticky areas. This dirt has proven difficult to remove with commercially available detergent compositions and causes difficult problems such as malodor, yellowing, and darkening of the items. Malodor resulting from bacteria can be difficult to remove and may persist even after washing because bacteria can remain a source of malodor by adhering to fabric surfaces. Furthermore, when very soiled laundry items are washed together with less soiled laundry items, dirt present in the wash liquor tends to adhere to the biofilm. As a result, the laundry items may be subject to "redeposition" of dirt during washing.

[0006] WO 2014 / 087011 discloses bacterial DNase polypeptides and methods for disrupting and preventing biofilms. WO 2015 / 155350 discloses detergent compositions containing specific DNase polypeptides obtained from fungal sources. WO 2017 / 064269 discloses variants of fungal DNase from Aspergillus oryzae with improved stability. Despite these disclosures, there remains a need for further improving DNase enzymes in detergent compositions, for example, further improving the stability of the enzymes. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides DNase variants with improved properties, particularly improved stability, compared to the parent DNase, obtained by combining specific substitutions discovered by the inventors in a particularly advantageous manner. [Means for solving the problem]

[0008] The present invention relates to isolated DNase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 having substitutions at two or more of positions 32, 35, 69, 102, 105, 111 and 181, where the position numbers are based on SEQ ID NO: 1.

[0009] The invention also relates to compositions, such as detergent compositions, comprising the DNase variants disclosed herein, as well as isolated polynucleotides encoding the variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of producing the variants. The invention further relates to methods of using the DNase variants and compositions for cleaning, such as for laundry. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is an alignment of the polypeptides of SEQ ID NOs: 1, 2, and 3. DETAILED DESCRIPTION OF THE INVENTION

[0011] Sequence Listing Free Text SEQ ID NO: 1 is the mature DNase polypeptide obtained from Aspergillus oryzae.

[0012] SEQ ID NO: 2 is the mature N-terminally truncated DNase polypeptide obtained from Aspergillus oryzae.

[0013] SEQ ID NO: 3 is the mature N-terminally truncated DNase polypeptide obtained from Aspergillus oryzae.

[0014] definition In accordance with this detailed description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0015] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] DNase: The term "DNase" refers to a polypeptide having DNase (deoxyribonuclease) activity, which degrades DNA by catalyzing the hydrolytic cleavage of phosphodiester bonds in DNA. DNases belong to the esterases (EC number 3.1), a subgroup of hydrolases. DNases are classified under EC 3.1.21. For the purposes of the present invention, DNase activity can be determined according to the procedure described in Assay I. The terms "DNase" and "polypeptide having DNase activity" can be used interchangeably throughout this application.

[0017] DNase variant: A "DNase variant" is a variant of the DNase of SEQ ID NO: 1, 2, or 3 having one or more individual mutations, typically substitutions, or a combination of substitutions.

[0018] Expression: The term "expression" includes all steps involved in the production of a variant (e.g., but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0019] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a variant, the coding sequence operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter for effecting transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0020] Extended: The term "extended" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a variant, such that the "extended" variant has DNase activity.

[0021] Fragment: The term "fragment" refers to a variant in which one or more amino acids are absent from the amino and / or carboxyl terminus of the variant, which fragment has DNase activity. Fragments in the context of the present invention include variants of SEQ ID NO: 1 in which one or more amino acids are absent from the amino terminus, for example SEQ ID NO: 2 in which the 15 N-terminal amino acids are absent or SEQ ID NO: 3 in which the 17 N-terminal amino acids are absent.

[0022] Host cell: A "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct (containing a polynucleotide encoding a variant) has been introduced. Exemplary host strains include microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest.

[0023] Improved property: The term "improved property" refers to a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, improved catalytic efficiency, catalytic rate, chemical stability, oxidative 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 context of the present invention, improved properties are, in particular, improved stability, e.g., improved storage stability, including improved storage stability in a detergent composition, improved stability during use in a detergent composition, and / or improved thermal stability. In addition, variants of the present invention may also have other improved properties, e.g., improved DNase activity, e.g., improved specific activity, and / or improved cleaning performance.

[0024] Isolated: The term "isolated" refers to a polypeptide variant, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including, but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell, or other substance is therefore in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted variants expressed in host cells. Typically, isolated variants of the invention will be separated from other components in the culture broth using known protein purification methods.

[0025] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0026] It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known that different host cells can process polypeptides differently, such that one host cell expressing a polynucleotide can produce a different mature polypeptide (e.g., with different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide. In the context of the present invention, because the mature polypeptide is typically N-terminally processed by the host cell, the mature polypeptide can be fewer than the 221 amino acid residues of the mature polypeptide set forth in SEQ ID NO: 1, for example, the 206 amino acids set forth in SEQ ID NO: 2 or the 204 amino acids set forth in SEQ ID NO: 3.

[0027] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having DNase activity.

[0028] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0029] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0030] Nucleic Acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids may 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, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' orientation.

[0031] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that contains one or more regulatory sequences operably linked to a nucleic acid sequence that is isolated from a naturally occurring gene, or that has been modified to contain a segment of nucleic acid in a way that is not normally found in nature, or that is synthetic.

[0032] Operably linked: The term "operably linked" means that the specified components are in a relationship (including, but not limited to, juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0033] Parent or Parent DNase: The terms "parent" or "parent DNase" refer to a DNase polypeptide that is altered to produce a DNase variant of the present invention. A parent is thus a DNase polypeptide that has the same amino acid sequence of a variant disclosed herein but does not have the specified alterations, typically substitutions, disclosed herein.

[0034] In certain embodiments, the DNase parent is a DNase having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 99.5%, or 100% identity to a polypeptide having SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0035] The parent DNase may, for example, be obtained from a fungal species, such as an Aspergillus species, for example Aspergillus oryzae.

[0036] In one embodiment, the parent DNase is a DNase having the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a preferred embodiment, the parent DNase is the polypeptide of SEQ ID NO: 1.

[0037] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and recombining, of nucleic acid sequences to form a constellation different from that found in nature. The term "recombinant" refers to a cell, nucleic acid, variant, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0038] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0039] For purposes of the present 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) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0040] Percent sequence identity may be referred to herein with reference to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. As explained above, it is known that host cells can produce mixtures of two or more different mature polypeptides, and that different host cells process polypeptides differently. In the case of the mature polypeptide of SEQ ID NO:1, it has been found that this can be expressed, with different N-terminal truncations, as, for example, the polypeptide of SEQ ID NO:2 (206 amino acid residues, corresponding to amino acids 16-221 of SEQ ID NO:1) or SEQ ID NO:3 (204 amino acid residues, corresponding to amino acids 18-221 of SEQ ID NO:1). Because SEQ ID NOs:1, 2, and 3 are identical except for the N-terminal truncations, any reference herein to a polypeptide having a given percent sequence identity to SEQ ID NO:1 should also be understood to include a polypeptide having a percent sequence identity to SEQ ID NO:2 or SEQ ID NO:3.

[0041] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0042] Variant: The term "variant" refers to a polypeptide having DNase activity, which contains a substitution, insertion (including extension), and / or deletion (e.g., truncation) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position. Variants of the present invention include various substitutions compared to SEQ ID NO: 1, as described in detail herein.

[0043] Wild-type: The term "wild-type" in reference to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acid and protein sequences that are produced in the lab or by modification of a wild-type sequence).

[0044] Biofilm: A biofilm is any community of microorganisms in which cells adhere to one another on a surface, such as a fabric, dish, or other hard surface. These adherent cells are often embedded in a self-produced matrix of extracellular polymeric substances (EPS). Biofilm EPS is generally a polymeric mass composed of extracellular DNA, proteins, and polysaccharides. Biofilms can form on living or non-living surfaces. Microbial cells growing within a biofilm are physiologically distinct from planktonic cells of the same organism, which, in contrast, are single cells that can float or swim in a liquid medium. Bacteria living within a biofilm typically have properties that are quite different from planktonic bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, due to the dense extracellular matrix and outer layer of cells protecting the interior of the community. In laundry, biofilm-producing bacteria can be found in the following species: Acinetobacter spp., Aeromicrobium spp., Brevundimonas spp., Microbacterium spp., Micrococcus luteus, Pseudomonas spp., Staphylococcus epidermidis, and Stenotrophomonas spp.

[0045] Improved DNase activity: The term "improved DNase activity" is defined herein as altered DNase activity (DNase variants that exhibit altered activity compared to the activity of a parent DNase, e.g., compared to the DNase having SEQ ID NO: 1), for example, due to increased catalysis of the hydrolytic cleavage of phosphodiester bonds in DNA. The improved activity can be, for example, an improved specific activity.

[0046] Stability: The term "stability" includes storage stability and stability during use, e.g., during a washing process, and reflects the stability of a DNase variant according to the present invention as a function of time, e.g., the extent to which activity is retained when the DNase variant is maintained in solution, particularly in a detergent solution. Stability is affected by many factors, such as pH, temperature, and detergent composition, e.g., the amount of builder, surfactant, etc. DNase stability can be measured as described in the Examples and expressed as a melting temperature (Tm) or half-life improvement factor (HIF), e.g., compared to the parent DNase or a reference sequence, e.g., SEQ ID NO: 1. The terms "improved stability" or "increased stability" are defined herein as a variant DNase that exhibits increased stability in solution compared to the stability of the parent DNase without substitutions in the variant and / or compared to SEQ ID NO: 1. The terms "improved stability" and "increased stability" include detergent stability.

[0047] Improved cleaning performance: The term "improved cleaning performance" can be defined as an improved deep cleaning effect ("deep cleaning" refers to the destruction or removal of biofilms or their components) of the DNase variants according to the present invention compared to the DNase parent or the DNase having SEQ ID NO: 1. The DNase variants may also improve malodor removal. The term "malodor" refers to an undesirable odor of a clean item. A cleaned item should smell fresh and clean, free of malodors adhering to the item. One example of a malodor is a compound with an unpleasant odor that can be produced by microorganisms. Another example is the unpleasant odor caused by sweat or body odor adhering to an item that has been in contact with humans or animals. Another example of a malodor can be the odor from spices, such as curry, or other strong-smelling spices adhering to the item. One way to measure the ability of an item to adsorb malodors is by using Assay II disclosed herein.

[0048] Wash performance can be expressed as the recovery value of the stained swatches. After washing and rinsing, the swatches are laid out flat and allowed to air dry overnight at room temperature. All washed swatches are evaluated the day after washing. Evaluation of the light reflectance of the swatches is carried out using a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. Measurements are taken without UV in the incident light, and reflectance at 460 nm is extracted.

[0049] Laundry: The term "laundry" refers to both domestic and institutional laundering, and refers to the process of treating fabrics with a solution containing the cleaning or detergent composition of the present invention. The laundering process can be carried out, for example, using a domestic or institutional washing machine, or can be carried out by hand.

[0050] Detergent Composition: The term "detergent composition" (or "cleaning composition"), unless otherwise specified, includes all forms of detergent or cleaning compositions. These include granular or powdered all-purpose or heavy-duty detergents, especially cleaning detergents; liquid, gel, or paste all-purpose detergents, especially so-called heavy-duty liquid (HDL) types; single unit dose (SUD) compositions, such as pods, capsules, and tubs, having one or more compartments; liquid detergents for delicate fabrics; hand dishwashing detergents or light-duty dishwashing detergents, especially high-foaming types; dishwashing detergents, including various tablet, granular, liquid, and rinse-aid types for household and industrial use; liquid cleaning and disinfecting agents, including antibacterial handwashing types, cleaning bars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos, and bathroom cleaners; hair shampoos and hair rinses; shower gels, foam baths; metal cleaners; and cleaning aids, such as bleach additives and "stain sticks" or pre-treatment types. The terms "detergent composition" and "detergent formulation" are used in reference to mixtures intended for use in a washing medium for cleaning soiled objects. In some embodiments, the term is used in reference to laundering fabrics and / or clothing (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents (e.g., "dishwashing detergents"), such as those used to clean dishes, cutlery, and the like. It is not intended that the present invention be 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 variants in accordance with the present invention, the term is intended to encompass detergents that may contain, for example, surfactants, builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric softeners, suds boosters, suds suppressors, dyes, fragrances, yellowing inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing and fluorescent dyes, antioxidants, and dissolving agents.

[0051] Fabric: The term "fabric" includes any textile material. Thus, the term is intended to include garments, as well as fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material.

[0052] Fabric: The term "fabric" refers to woven fabrics and staple fibers and filaments suitable for conversion into or use as yarns, woven fabrics, knitted fabrics, and nonwoven fabrics. The term encompasses yarns made from natural and synthetic (e.g., manufactured) fibers. The term "textile material" is a general term for fibers, yarn intermediates, yarns, fabrics, and products (e.g., clothing and other articles) made from fabrics.

[0053] Non-fabric detergent compositions: The term "non-fabric detergent compositions" includes detergent compositions for surfaces other than fabrics, including hard surface cleaning compositions such as dishwashing detergent compositions, including, but not limited to, manual dishwashing compositions, mouthwash compositions, denture cleaner compositions, and personal cleansing compositions.

[0054] Effective amount of enzyme: The term "effective amount of enzyme" refers to the amount of enzyme necessary to achieve the enzymatic activity required for a particular application, e.g., a defined detergent composition. Such an effective amount is readily determined by one skilled in the art and is based on many factors, such as the particular enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granular, bar) composition is required. The term "effective amount" of a DNase variant refers to the amount of the DNase variant described above that achieves a desired level of enzymatic activity, e.g., in a defined detergent composition.

[0055] Relevant washing conditions: The term "relevant washing conditions" is used herein to indicate the conditions actually used in households in the detergent market segment, in particular washing temperature, time, washing machine, detergent concentration, detergent type and water hardness.

[0056] Washing liquor: The term "washing liquor" (or "wash water") refers to an aqueous solution comprising a DNase variant of the invention. A washing liquor is a solution (e.g. found in a washing machine or dishwasher) containing water and a detergent composition comprising a DNase variant. The detergent composition may be in any suitable form as described elsewhere herein, e.g., liquid or powder, before being mixed with water to form the washing liquor.

[0057] Water Hardness: As used herein, the term "water hardness" or "hardness" or "dH" or "° dH" refers to the German hardness scale, where 1 degree is defined as 10 milligrams of calcium oxide per liter of water.

[0058] Adjunct Materials: The term "adjunct materials" or "adjunct ingredients" refers to any liquid, solid, or gaseous material selected for the particular type of detergent composition and product form desired (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, or foam composition), which materials are also preferably compatible with the DNase variant enzyme used in the composition. More detailed information regarding adjunct materials is provided further below.

[0059] Low detergent concentration: The term "low detergent concentration" system includes detergents in which less than about 800 ppm of detergent ingredients are present in the wash water. Asian, e.g., Japanese, detergents are typically considered to be low detergent concentration systems.

[0060] Mid Detergent Concentration: The term "mid detergent concentration" system includes detergents in which from about 800 ppm to about 2000 ppm of detergent ingredients are present in the wash water. North American detergents are generally considered to be mid detergent concentration systems.

[0061] High Detergent Concentration: The term "high detergent concentration" systems includes detergents in which more than about 2000 ppm of detergent ingredients are present in the wash water. European detergents are generally considered to be high detergent concentration systems.

[0062] Rules for naming variants For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid position in another DNase. The amino acid sequence of another DNase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0063] In describing the variants of the present invention, the nomenclature described below is preferred for ease of reference: Recognized IUPAC single-letter or three-letter amino acid abbreviations are employed.

[0064] Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine at position 226 with alanine is designated "T226A" (or "Thr226Ala" using the three-letter code). Multiple mutations may be separated by a plus sign ("+"), e.g., "G205R+S411F" represents the substitution of arginine (R) with glycine (G) and phenylalanine (F) with serine (S) at positions 205 and 411, respectively. Alternatively, multiple mutations may be indicated by a space, comma, or plus sign (e.g., "G205R S411F," "G205R,S411F," or "G205R+S411F").

[0065] Deletions. For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 would be named "G195*." Multiple deletions are separated by a plus sign ("+") (e.g., "G195*+S411*").

[0066] Insertions. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after a glycine at position 195 would be designated "G195GK." Multiple amino acid insertions are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of a lysine and an alanine after a glycine at position 195 would be designated as "G195GKA."

[0067] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence is:

[0068] [Table 1]

[0069] Multiple Alterations. Variants containing multiple alterations are separated by a plus sign ("+") as described above, e.g., "R170Y+G195E" represents the substitution of arginine and glycine with tyrosine and glutamic acid, respectively, at positions 170 and 195. Alternatively, multiple alterations may be separated by spaces or commas as described above.

[0070] Differential modifications. When different modifications can be introduced at a certain position, the different modifications are separated by a comma, for example, "R170Y,E" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Y167G,A+R170G,A" represents the following variants: "Y167G+R170G," "Y167G+R170A," "Y167A+R170G," and "Y167A+R170A."

[0071] The present invention provides novel DNase variants, particularly variants of DNase obtained from the genus Aspergillus, including Aspergillus oryzae. The DNase of the present invention has at least 60% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and contains at least two substitutions, for example at least three substitutions, at positions selected from the group consisting of 111, 32, 35, 69, 102, 105, and 181 in SEQ ID NO: 1 described herein.

[0072] In one embodiment, the invention provides a DNase variant comprising three or more substitutions at positions selected from the group consisting of 26, 32, 35, 65, 67, 69, 98, 102, 105, 111, 115, 150, 157, 159, 161, 172, 181, 182, 185, 187, 192, 206, 208, and 212, wherein the position numbers are based on the numbering of SEQ ID NO: 1, and wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0073] More particularly, the variant may comprise three or more substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, where the position numbers are based on the numbering of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and wherein the variant has DNase activity. In one aspect, the variant comprises two or more substitutions, such as three or more substitutions, selected from the group consisting of A111P, D32E, V35I, S69V, Q102E, K105N, and G181N, and optionally one or more of the other substitutions listed above.

[0074] The present invention also provides compositions, particularly detergent compositions, such as laundry compositions, that include the DNase variants, and methods of using the DNase variants. Also provided are isolated DNA sequences, expression vectors, and host cells encoding the variants, and methods of producing the DNase variants.

[0075] variant As mentioned above, in one embodiment the DNase variant of the invention comprises three or more substitutions at positions selected from the group consisting of 26, 32, 35, 65, 67, 69, 98, 102, 105, 111, 115, 150, 157, 159, 161, 172, 181, 182, 185, 187, 192, 206, 208 and 212, in particular S26H, D32E, V35I, K65 E, K67A, S69E, S69V, S98R, Q102E, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, where numbering is based on SEQ ID NO: 1. In some embodiments, variants may include four, five, or more of said substitutions.

[0076] More particularly, the present invention in one aspect relates to a DNase variant comprising at least two substitutions, such as at least three substitutions, in particular at least two substitutions, such as at least three of said substitutions, selected from the group consisting of A111P, D32E, V35I, S69V, Q102E, K105N and G181N, at positions selected from the group consisting of 111, 32, 35, 102, 105 and 181, wherein the position numbers are based on the numbering of SEQ ID NO: 1, and wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0077] In certain preferred embodiments, the variant comprises the substitution A111P. Preferably, the variant of this embodiment comprises the substitution A111P and two or more substitutions selected from the group consisting of S26H, D32E, V351, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0078] Among such DNase variants with the substitution A111P, preferred variants include those containing the substitutions D32E+V351 and / or the substitution G181N. Other preferred embodiments of variants with the substitution A111P include those containing the substitutions S69V and / or Q102E, as well as those containing the substitution K105N.

[0079] In another embodiment, the variant comprises the substitution K105N, preferably in combination with the substitution A111P. Variants of this embodiment may further comprise at least one additional substitution from those listed above, i.e. one or more of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, such as two or more of said additional substitutions.

[0080] In yet another embodiment, the variant comprises the substitutions S69V and / or Q102E, preferably S69V+Q102E. Variants of this embodiment may further comprise at least one additional substitution from those listed above, i.e. one or more of S26H, D32E, V35I, K65E, K67A, S98R, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, such as two or more of said additional substitutions.

[0081] In one preferred embodiment, the variant comprises the substitutions D32E+V35I+A111P. Variants of this embodiment may further comprise at least one additional substitution from those listed above, i.e. one or more of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, such as two or more of said additional substitutions.

[0082] In another preferred embodiment, the variant comprises the substitutions A111P+G181N. Variants of this embodiment may further comprise at least one additional substitution from those listed above, i.e. one or more of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E, such as two or more of said additional substitutions.

[0083] In a further preferred embodiment, the variant comprises the substitutions D32E+V35I+A111P+G181N. Variants of this embodiment may further comprise at least one additional substitution from those listed above, namely one or more of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0084] In another embodiment, the variant comprises the substitutions D32E+V35I+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0085] In another embodiment, the variant comprises the substitutions D32E+V35I+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0086] In another embodiment, the variant comprises the substitutions D32E+V35I+S69V+Q102E+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0087] In another embodiment, the variant comprises the substitutions D32E+V35I+S69V+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0088] In another embodiment, the variant comprises the substitutions S69V+Q102E+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0089] In another embodiment, the variant comprises the substitutions S69V+Q102E+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0090] In another embodiment, the variant comprises the substitutions S69V+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0091] In another embodiment, the variant comprises the substitutions Q102E+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0092] In another embodiment, the variant comprises the substitutions S69V+Q102E+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0093] In another embodiment, the variant comprises the substitutions D32E+V35I+K105N+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0094] Examples of preferred DNase variants of the present invention include those that contain one of the following sets of substitutions, and optionally one or more additional substitutions disclosed herein: D32E+V35I+A111P D32E+V35I+A111P+G181N · D32E+V35I+Q102E+A111P+G181N · D32E+V35I+K105N+A111P+G181N D32E+V35I+S69V+Q102E+A111P · D32E+V35I+S69V+Q102E+A111P+G181N S69V+Q102E+A111P S69V+Q102E+G181N S69V+Q102E+A111P+G181N Q102E+K105N+A111P+G181N · S69V+Q102E+K105N+A111P+G181N D32E+V35I+K105N+A111P

[0095] A variant may, for example, comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 with one of these substitution sets.

[0096] In any of the embodiments disclosed herein above or elsewhere, the DNase variant may further comprise one or more additional modifications, such as one or more additional substitutions other than those disclosed above. Non-limiting examples of such additional substitutions include any one or more of K18S, T19P, D32P, K86E, A101E, A101T, K105D, K105E, K105T, G137R, N146H, K147N, K155E, A172D, K178S, K192A, N214D, and N217A.

[0097] Examples of specific DNase variants of the present invention include variants that comprise a set of substitutions selected from the group consisting of: · S69V+K86E+Q102E+K147N+K155E+A172D+K192A+Q208V; G20C+K30C+D32E+V35I+A111P; · S69V+K86E+Q102E+K147N+K155E+A172D+G181N+K192A+Q208V; · D32E+V35I+N77E+A111P+G181N+S182V; · D32E+E34N+V35I+K86E+A111P+G181N; · S69V+K86E+Q102E+K105T+K147N+K155E+A172D+K192A+Q208V; · S69V+Q102E+Q157E+T159Q+N214D+N217A; · S26H+D32E+V35I+A111P+K155L; · K18S+D32E+V35I+A111P+K185I+K215T; · D32E+V35I+A111P+K185I+K215T; · D32E+V35I+K82G+A111P+G181N+S182V; · D32E+V35I+A111P+K178M+K212T; · K18S+D32E+V35I+A111P+K178S+K212L; · S69V+Q102E+V138A+S144R+Q157E+T159Q+A172E+G181N; · D32E+V35I+A111P+K204L+K215I; · K18F+D32E+V35I+A111P+K185Q; · D32E+V35I+A111P+K147N+K204L; · S69V+K86E+Q102E+K105D+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+S69V+K86E+Q102E+A111P+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P+K147N; · S69V+K86E+Q102E+S115T+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P+N140H; · D32E+V35I+K65Q+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+K178S+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P; · E6D+A101E+Q150E+G181N+Q208V; · S26H+D32E+V35I+A111P; · K18Q+D32E+V35I+K105E+A111P+K155F; · D32E+V35I+A111P+G199E; · S69V+Q102E+Q157E+T159Q+G181N; · D32E+V35I+K67A+A111P+P175N+S182V; · D32E+V35I+A111P+N214D; · S69V+Q102E+Q157E+T159Q+A172E; · E6D+A101E+Q150E+G181N+Q208V; · D32E+V35I+S69E+A111P; · D32E+V35I+Q85E+A111P; · K18Q+D32E+V35I+A111P; · D32E+V35I+A101E+K105T+A111P+G181N+K212E+Y218E; · D32E+V35I+A111P+G181N+S182V+K185E; · D32E+V35I+S98R+A111P+G181N+S182Y; · D32E+V35I+A101E+Q102E+A111P+G181N; · T19P+D32E+V35I+Q102E+K105E+A111P; · D32E+V35I+D72V+A111P; · S69V+K86E+Q102E+F112W+K147N+K155E+A172D+K192A+Q208V; · S69V+Q102E+A111P+Q157E+T159Q+A172E; · K67A+S69V+Q102E+K147N+Q157E+T159Q+A172E+D197S; · S69V+Q102E+S115T+Q157E+T159Q+A172E; · S69V+K86E+Q102E+G136E+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+K67A+A111P+K204G; · A101E+A111P+Q150E+Q208V; · D32E+V35I+A101E+K105E+A111P+S182V; · D32E+V35I+A101E+K105E+A111P+S182V; · D32E+V35I+A101T+K105N+A111P+K147E+G181N+S182Y; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+Q102E+A111P; · S26H+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+K67A+K105T+A111P+K178L+K185T; · K67A+K105T+G181N; · T19P+D32E+V35I+A101E+K105T+A111P+K160A+G181N; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+Q102E+A111P; · K18S+D32E+V35I+Q102E+K105E+A111P; · D32E+V35I+K65E+A101E+K105T+A111P+N217A; · D32E+V35I+K105T+A111P+G181N+K204W+K215L; · D32E+V35I+K105T+A111P+K178L+G181N+K212T; · S26H+S69V+Q102E+A111P+N146H+Q150E+Q157E+T159Q+A172E; · K18Q+D32E+V35I+K67A+K105T+A111P+K215V; · D32E+V35I+Q85E+A111P; · K18S+T19P+D32E+V35I+A101E+K105T+A111P+G181N; · D32E+V35I+A101E+K105N+A111P; · K18M+D32E+V35I+K67A+K105T+A111P+K178S; · D32E+V35I+K105T+A111P+G181N; · D32E+V35I+K67A+K105T+A111P+K185G+K212M; · D32E+V35I+A101T+K105N+A111P+G181N+S182Y+N214D; · T19P+D32E+V35I+A101E+K105T+A111P+K178S+G181N; · D32E+V35I+K105T+A111P+G181N+K185L+K192V+K204W; · D32E+V35I+S98R+K105N+A111P+K147N+G181N+S182Y; · D32E+V35I+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+K105T+A111P; · D32E+V35I+K53F+K105N+A111P+G181N+S182Y+K212E; · K18S+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+S98R+K105N+A111P+Q158D+G181N+S182Y; · D32E+V35I+K67A+K105T+A111P+K190V+K204G; · N4K+D32E+V35I+S98R+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+K67A+K105T+A111P+K147N+K190N; · D32E+V35I+S98R+K105N+A111P+G181N+S182Y+Q208V; · D32E+V35I+A101E+K105T+A111P+G181N+K212E+N214D; · D32E+V35I+K82T+A101E+K105T+A111P+G181N+K212E; · D32E+V35I+A111P+S115T; · S26H+S69V+Q102E+A111P+K147N+A149S+Q150E+Q157E+T159Q+A172E; · K18S+D32E+V35I+K67A+K105T+A111P+K185L+K192F; · V12L+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+S69E+K105N+A111P+K147E+G181N+S182Y; · K18S+D32E+V35I+A101E+K105T+A111P+G181N+K212E; · D32E+V35I+K65E+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+S98R+K105N+A111P+G181N+S182Y+D189N+N214D; · D32E+V35I+S98R+A101T+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+A111P+Q157E+T159Q+G181N+A206G+Q208V; · D32E+V35I+K67A+S69R+A111P+G181N+K212E; · D32E+V35I+K67A+A111P+G181N+A206G+Q208V; · D32E+V35I+A101E+K105T+A111P+G181N; · D32E+V35I+K67A+A101E+K105D+A111P+N217A; · D32E+V35I+K67A+A111P+Q157E+G181N+Q208V; · D32E+V35I+K67A+A111P+Q157E+A172E+G181N+A206G+Q208V; · S26H+D32E+V35I+K67A+S69R+A111P+G181N+K212E; · D32E+V35I+S69E+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+K105D+A111P+S115T; · D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; · D32E+V35I+K65E+K67A+K105N+A111P+G181N+S182Y; · S69V+Q102E+K105N+A111P+S115T+V138M+Q150E+G161R+G181N+V187N+K192I; · D32E+V35I+K67A+S69V+K105T+A111P+Q157E+G181N+A206G+Q208V; · S69V+Q102E+K105N+A111P+S115T+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+Q150E+K160T+G161R+G181N+V187N+K192I; · S69V+D72S+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; · S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+S134H+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · D32P+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+T120H+N146H+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+Q157E+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · S26H+D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · K65Q+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · S69A+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+A172D+G181N+V187N+K192I; · D32P+S69A+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+K192I; D32P+S69V+Q102E+K105D+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; and · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I.

[0098] A variant may, for example, comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 with one of these substitution sets.

[0099] In a preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+A111P.

[0100] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+K105N+A111P+G181N+S182Y.

[0101] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions S26H+S69V+Q102E+A111P+Q157E+T159Q+A172E.

[0102] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I.

[0103] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+K67A+A111P+G181N+A206G+Q208V.

[0104] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+A111P+G181N+S182V+K185E.

[0105] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions S69V+Q102E+K105N+A111P+S115T+G161R+G181N+V187N+K192I.

[0106] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+S69E+S98R+K105N+A111P+G181N+S182Y.

[0107] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+K65E+K105N+A111P+G181N+S182Y+K212E.

[0108] In another preferred embodiment, the DNase variant of the invention comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 with the substitutions D32E+V35I+K65E+K67A+K105N+A111P+G181N+S182Y.

[0109] The DNase variants disclosed herein have at least 60% sequence identity to the parent DNase without substitutions within the variant. The DNase variants may, for example, have at least 65%, at least 70%, at least 75%, or at least 80% sequence identity to the parent DNase. The DNase variants may, for example, have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, e.g., at least 96%, at least 97%, or at least 98%, but less than 100% sequence identity to the parent DNase.

[0110] In particular, any of the DNase variants disclosed herein have at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. A DNase variant may, for example, have at least 65%, at least 70%, at least 75%, or at least 80% sequence identity to any of SEQ ID NO: 1, 2, or 3. A DNase variant may, for example, have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, such as at least 96%, at least 97%, or at least 98%, and less than 100% sequence identity to any of SEQ ID NO: 1, 2, or 3.

[0111] In one aspect, the number of changes in a variant of the invention compared to SEQ ID NO: 1, 2 or 3 is 1 to 20, for example 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes.

[0112] Variants of the present invention may, in some cases, be minor in nature, e.g., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or other amino acid changes that may be small extensions that facilitate purification by altering net charge or another function, e.g., a polyhistidine tail, an antigenic epitope, or a binding domain.

[0113] Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0114] Other amino acid changes may be of such a nature that they alter the physicochemical properties of the polypeptide, for example, they may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0115] Essential amino acids within a polypeptide can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for DNase activity to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structure, as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Additionally, the identity of essential amino acids can be inferred from alignments with related polypeptides and / or sequence homology with related polypeptides or conserved catalytic mechanisms within polypeptide or protein families, typically with polypeptides / proteins derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or the active site of a polypeptide. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0116] A variant of the present invention may consist of, for example, 195 to 230 amino acids, preferably 200 to 225 amino acids, e.g., 204 to 221 amino acids. A variant may optionally include an extension of one or more amino acids at the N-terminus and / or C-terminus, or a truncation of one or more amino acids at the N-terminus and / or C-terminus. For example, a variant may include a fragment in which some N-terminal amino acids are absent compared to SEQ ID NO: 1, e.g., a variant of SEQ ID NO: 2 or SEQ ID NO: 3.

[0117] In one embodiment, the variant has improved stability, in particular improved storage stability, i.e., improved stability under storage in a detergent composition, compared to the parent enzyme. As noted above, improved stability may be expressed, for example, by melting temperature (Tm) or half-life improvement factor (HIF), as described in the examples herein.

[0118] In one embodiment, the DNase variant has improved stability, expressed as thermostability, compared to the parent DNase. Preferably, the variant has a melting temperature (Tm) at least 1°C higher than that of the parent, e.g., at least 2°C, at least 3°C, or at least 4°C higher than the parent Tm, determined, e.g., as described in the examples herein. In preferred embodiments, the variant may have a melting temperature at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, or at least 10°C higher than the parent Tm.

[0119] In one embodiment, the DNase variant has improved stability compared to the parent DNase, expressed as a half-life improvement factor (HIF), i.e., a HIF value greater than 1. Preferably, the variant has a half-life improvement factor relative to the parent of at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2.0, determined, for example, as described in the examples herein. In preferred embodiments, the variant may have a half-life improvement factor relative to the parent of at least 2.5, at least 3.0, at least 3.5, or at least 4.0.

[0120] The DNase variants of the present invention are preferably isolated, and more preferably purified, using standard protein purification methods known in the art.

[0121] Generating variants The present invention also relates to a method for obtaining a variant having DNase activity and having the substitutions disclosed herein, in one embodiment, the method comprises: (a) introducing into a parent DNase at least two substitutions, e.g., at least three substitutions, at positions selected from the group consisting of 111, 32, 35, 69, 102, 105, and 181, where the position numbers are based on the numbering of SEQ ID NO: 1, and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the variant has DNase activity; and (b) recovering the variant.

[0122] In another embodiment, the method comprises: (a) introducing three or more substitutions into the parent DNase at positions selected from the group consisting of 26, 32, 35, 65, 67, 69, 98, 102, 105, 111, 115, 150, 157, 159, 161, 172, 181, 182, 185, 187, 192, 206, 208, and 212, wherein the position numbers are based on the numbering of SEQ ID NO: 1, and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity; and (b) recovering the variant.

[0123] In another embodiment, the method comprises: (a) introducing into the parent DNase three or more substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E. wherein the position numbers are based on the numbering of SEQ ID NO:1, and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the variant has DNase activity; and (b) recovering the variant.

[0124] In another embodiment, the method comprises: (a) introducing into the parent DNase at least two substitutions, such as at least three substitutions selected from the group consisting of A111P, D32E, V351, S69V, Q102E, K105N, and G181N, where the position numbers are based on the numbering of SEQ ID NO: 1, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity; and (b) recovering the variant.

[0125] In other embodiments, the method comprises: (a) introducing into the parent DNase any of the sets of substitutions listed above under the heading "Variant", where the position numbers are based on the numbering of SEQ ID NO:1, and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the variant has DNase activity; and (b) recovering the variant.

[0126] Variants can be prepared using any mutagenesis technique known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, DNA shuffling, and the like.

[0127] Polynucleotides The present invention also relates to polynucleotides encoding the variants of the invention.

[0128] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0129] In one embodiment, the polynucleotide is isolated, preferably purified.

[0130] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention, operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Examples of control sequences that may be used include promoters, terminators, mRNA stabilizers, leader sequences, polyadenylation sequences, signal peptides, propeptides, regulatory sequences, and transcription factors, all of which are well known in the art.

[0131] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. It may be desirable or necessary to manipulate the polynucleotide according to the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0132] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences can be ligated together to generate a recombinant expression vector that includes one or more convenient restriction sites, allowing for insertion or substitution of a polynucleotide encoding a variant at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide, or a nucleic acid construct comprising the polynucleotide, into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0133] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA techniques and that can result in expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid. Expression vectors suitable for recombinant expression are well known in the art, as are methods for introducing the vector into a host cell.

[0134] host cell The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the variants of the present invention.

[0135] Once the construct or vector containing the polynucleotide is introduced into a host cell, the construct or vector may be maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The choice of host cell will largely depend on the gene encoding the variant and its source. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or at least two copies, e.g., three, four, five, or more copies.

[0136] The host cell can be any microbial cell, such as a prokaryotic or fungal cell, useful for the recombinant production of the polypeptides of the invention.

[0137] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, the genera Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0138] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus spp. The cell may be any Bacillus cell, including cells of Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0139] The fungal host cell may be a yeast cell or a filamentous fungal cell.

[0140] Filamentous fungal host cells include, for example, those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, and Myceliophthora. , Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell, and in a more preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0141] In one embodiment, the host cell is isolated, preferably purified.

[0142] Generation method The present invention also relates to a method for producing a variant of the invention, comprising (a) culturing a recombinant host cell of the invention under conditions conducive to the production of the variant; and optionally (b) recovering the variant.

[0143] The host cells are cultured in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells can be cultured in shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors in an appropriate medium and under conditions that allow for expression and / or isolation of the variant. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in catalogs of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0144] Variants can be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzyme assays to determine the relative or specific activity of the variant.

[0145] The variant may 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 whole fermentation broth is recovered. In another embodiment, the cell-free fermentation broth containing the polypeptide is recovered.

[0146] Variants can be purified by various procedures known in the art to obtain substantially pure variants and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0147] In an alternative embodiment, the variants are not recovered.

[0148] composition The present invention further relates to cleaning compositions comprising at least one DNase variant according to the invention and at least one cleaning adjunct ingredient. The cleaning compositions may be used to improve deep cleaning efficacy, including but not limited to, deep cleaning of items, to prevent and / or reduce item stickiness, to pretreat items for stains, to prevent and / or reduce redeposition of soil during the wash cycle, to prevent and / or reduce soil adhesion to items, to maintain or improve the whiteness of items, and to prevent and / or reduce malodors from items. The DNase variants of the present invention are useful in powder and liquid cleaning compositions, and in, for example, single-unit dose compositions.

[0149] The compositions may contain one or more cleaning adjunct ingredients selected from the group consisting of surfactants, builders, flocculating aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, mud stain removal / anti-redeposition agents, brighteners, suds suppressors, dyes, fragrances, structural elasticizers, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, defoamers, dispersants, processing aids, and / or pigments.

[0150] Cleaning compositions will typically contain surfactants, and usually other cleaning adjunct ingredients such as builders or mud / soil removal / anti-redeposition agents.

[0151] The cleaning adjunct ingredient 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 compositions of the present invention are described below.

[0152] The cleaning composition can be formulated into any suitable form, such as a bar, a homogeneous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compressed powder, a granule, a paste, a gel, or a regular, compressed, or concentrated solution. Thus, the cleaning composition can be, for example, a liquid detergent, or a powder or granular detergent, optionally in "concentrated" or "compressed" form. It can also be in the form of a single unit dose composition.

[0153] The amount of DNase in a cleaning composition can vary depending on factors such as the degree of concentration or compactness of the composition and the desired concentration of DNase in the cleaning solution. DNase will usually be included in the cleaning composition in an amount of up to about 10,000 ppm, typically up to about 5000 ppm, or up to about 2000 ppm. DNase may, for example, be included in the cleaning composition at a level of 1 ppm to 10,000 ppm, e.g., 10 ppm to 5000 ppm, 20 ppm to 2000 ppm, 50 ppm to 1000 ppm, 80 ppm to 600 ppm, or 100 ppm to 500 ppm. The unit "ppm" in this context is intended to refer to mg / L for enzyme added to a liquid composition (e.g., liquid, gel, etc.) or mg / kg for enzyme added to a solid composition (e.g., powder, granules, tablet, etc.).

[0154] In some embodiments, the detergent composition is, for example, a liquid or powder laundry detergent suitable for washing at high temperatures and / or pH, for example, 40°C or higher and / or a pH of 8 or higher. In some embodiments, the detergent composition is, for example, a liquid or powder laundry detergent suitable for washing at low temperatures and / or pH, for example, 20°C or lower and / or a pH of 6 or lower. The detergent can also be formulated as a unit dose detergent and / or compact detergent, optionally with minimal or no water. The detergent can also be a dishwashing detergent. The laundry detergent and dishwashing detergent can be phosphate-free.

[0155] surfactants The surfactant may be selected from the nonionic, anionic, and / or amphoteric surfactants described above; preferably, anionic or nonionic surfactants are used, although amphoteric surfactants may also be used. Generally, bleach-stable surfactants are preferred. Preferred anionic surfactants include sulfate surfactants, particularly alkyl ether sulfates, especially C9-C15 alcohol ether sulfates, C12-C15 primary alcohol ethoxylates, C8-C16 sulfate esters, and C10-C14 sulfate esters, such as monododecyl sulfate ester. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfofatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and monoesters of sulfosuccinic acid, or salts of fatty acids (soaps), and combinations thereof.

[0156] Anionic surfactants are preferably added to detergents in the form of salts, in which suitable cations are alkali metal ions such as sodium, potassium, lithium, and ammonium salts, for example, (2-hydroxyethyl)ammonium, bis(2-hydroxyethyl)ammonium, and tris(2-hydroxyethyl)ammonium salts. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters such as 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 (glucamides, GA or fatty acid glucamides, FAGA), and products available under the trade names SPAN and TWEEN®, and combinations thereof. Commercially available nonionic surfactants include Plurafac™, Lutensol™, and Pluronic™ from BASF, the Dehypon™ series from Cognis, and the Genapol™ series from Clariant.

[0157] builder The builder is preferably selected from phosphates, sodium citrate builders, sodium carbonate, sodium silicate, and sodium aluminosilicate (zeolites). 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 the sodium salt), are polycarboxylate builders. Citrates can be used in combination with zeolites, silicates such as Britesil types, 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 level 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 a chelating agent, particularly one that forms water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in cleaning detergents may be utilized. 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 SKS-6), and (carboxymethyl)inulin (CMI), and combinations thereof. Further non-limiting examples of builders include citrates, chelators such as 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-N-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), anthranil Phosphonates suitable for use herein include 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentaerythritol phosphate (DTP), 1-hydroxyethane-1,1-diphosphonic acid (HEDP ... Examples of suitable detergent co-builders include tetrakis(methylene phosphonic acid) (DTMPA, DTPMPA, or DTPMP), nitrilotris(methylene phosphonic acid) (ATMP or NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), and hexamethylenediaminetetrakis(methylene phosphonic acid) (HDTMP). The compositions may also contain 0 to 50% by weight, for example, about 5% to about 30%, of a detergent co-builder. The detergent compositions may contain the co-builder alone or in combination with a builder, such as a zeolite builder.Non-limiting examples of co-builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA) or polyaspartic acid. Further exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and U.S. Pat. No. 5,977,053. In some embodiments, the builder is a non-phosphorus-based 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. In addition, in cases where a preferred builder comprises citrate 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 phosphate-free.

[0158] bleaching ingredients The cleaning composition may contain 0 to 30% by weight of a bleaching system, such as from about 1% to about 20%. Any bleaching system containing ingredients known in the art for use in cleaning detergents may be utilized. Suitable bleaching system ingredients include a source of hydrogen peroxide; a source of peracid; and a bleach catalyst or bleach booster.

[0159] Sources of hydrogen peroxide: Suitable sources of hydrogen peroxide are inorganic persalts, including sodium percarbonate and sodium perborate (usually the monohydrate or tetrahydrate), and alkali metal salts such as hydrogen peroxide-urea (1 / 1).

[0160] Source of peracid: Peracid can be (a) directly incorporated as preformed peracid, or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis), or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, e.g., an ester.

[0161] a) Suitable preformed 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-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic acid, diperoxyisophthalic acid, and diperoxyterephthalic acid; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; and mixtures of the foregoing compounds. It will be understood that the peracids mentioned may in some cases best be added as a suitable salt, for example an alkali metal salt (eg Oxone®) or alkaline earth metal salt.

[0162] b) Suitable bleach activators include those belonging to the classes of esters, amides, imides, nitriles, or anhydrides, as well as their salts, as appropriate. Suitable examples include tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), sodium 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), sodium 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), sodium 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of bleach activators of interest is disclosed in EP 624154, and within that family, acetyltriethyl citrate (ATC) is particularly preferred. ATC or the short-chain triglyceride-like triacetin has the advantage of being environmentally friendly. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are effective bleach activators. Finally, ATC is multifunctional because the citrate released during the perhydrolysis reaction can function as a builder.

[0163] Bleaching catalysts and accelerators The bleaching system may also include bleaching catalysts or accelerators.Some non-limiting examples of bleaching catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), especially manganese complexes with Me3-TACN, such as: The dinuclear manganese complexes are [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2 and [2,2',2"-nitrilotris(ethane-1,2-diylazanilylidene-κN-methanilylidene)triphenolato-κ3O]manganese(III). The bleach catalyst can also include other metal compounds, such as iron or cobalt complexes. Other suitable bleach catalysts include acylhydrazone catalysts, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0323381.

[0164] In some embodiments that include a source of peracid, the source may be of the formula: [ka] (iii) and mixtures thereof, wherein each R1 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 R1 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 R1 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, may be used.

[0165] Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, EP 1 867 708 (vitamin K), and WO 2007 / 087242. Suitable photobleaches can include, for example, sulfonated zinc or aluminum phthalocyanine.

[0166] The selection of a detergent composition may include, in the case of fabric care, considerations of the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning will be performed, and the formulation of the detergent product. The ingredients mentioned below are categorized by general headings according to functionality, but this should not be construed as limiting, as it will be understood by those skilled in the art that ingredients, including the exemplary non-limiting ingredients set forth below, may also include additional functionality.

[0167] Hydrotrope The detergent composition may contain 0 to 10% by weight, e.g., 0 to 5% by weight, e.g., about 0.5 to about 5%, or about 3% to about 5% of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-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.

[0168] polymer The detergent composition may contain 0 to 10% by weight of polymer, e.g., 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 may be utilized. The polymer may function as a co-builder as described above, or may provide anti-redeposition, fabric protection, soil release, dye transfer inhibition, grease cleaning, and / or defoaming properties. Some polymers may have two or more of the above properties and / or two or more of the following motifs: Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, polyaspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene 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 oxide and polypropylene oxide (PEO-PPO), and diquaternium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the aforementioned polymers are also contemplated.

[0169] Fabric color toning agent The detergent compositions of the present invention may also contain fabric hueing agents, such as dyes or pigments, which, when incorporated into the detergent composition, can be deposited on fabrics when the fabrics come into contact with a wash liquor containing the detergent composition, thereby changing the color of the fabric by absorbing / reflecting visible light. Optical brighteners emit at least some visible light. In contrast, fabric hueing agents absorb at least a portion of the visible light spectrum, thereby changing the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include, for example, small molecule dyes selected from the group consisting of dyes classified as Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red according to the Color Index (CI) classification, or mixtures thereof, as described in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276, and EP 1 876 226 (incorporated herein by reference). The detergent composition preferably comprises from about 0.00003% to about 0.2%, from about 0.00008% to about 0.05%, or from about 0.0001% to about 0.04% by weight of the fabric hueing agent. The composition may contain from 0.0001% to about 0.2% by weight of a fabric hueing agent, which may be preferred, particularly when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0170] enzyme The detergent composition may also comprise one or more additional enzymes such as proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, hexosaminidases, oxidases such as laccases and / or peroxidases.

[0171] In general, the properties of the selected enzyme should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.), and the enzyme should be present in an effective amount.

[0172] cellulase Suitable cellulases include those of bacterial or fungal origin, including chemically modified or protein engineered variants. Suitable cellulases include, for example, cellulases derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, and 5,776,757, and WO 89 / 09259, such as fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum.

[0173] Particularly suitable cellulases are alkaline or neutral cellulases, which have color protection advantages. Examples of such cellulases include those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples include cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and WO 99 / 001544.

[0174] Other cellulases include endo-beta-1,4-glucanase enzymes having a sequence that is at least 97% identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 in WO 2002 / 099091, or family 44 xyloglucanases, which have a sequence that is at least 60% identical to positions 40 to 559 of SEQ ID NO:2 in WO 2001 / 062903.

[0175] 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™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0176] Proteases Suitable proteases may be of any origin, preferably bacterial or fungal, and optionally in the form of engineered or chemically modified variants. 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 the S8 family, such as subtilisin. The metalloprotease may be, for example, thermolysin, for example from the M4 family, or another metalloprotease, for example from the M5, M7, or M35 family.

[0177] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subdivisions: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family.

[0178] Although proteases suitable for detergent use can be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases are generally obtained from bacteria, particularly 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, and subtilisin 168, as well as, for example, protease PD138 (described in WO 93 / 18140). Other useful proteases include, for example, those described in WO 01 / 16285 and WO 02 / 16547.

[0179] Examples of trypsin-like proteases include the Fusarium proteases described in WO 94 / 25583 and WO 2005 / 040372, and the chymotrypsin proteases from Cellulomonas, described in WO 2005 / 052161 and WO 2005 / 052146.

[0180] Examples of metalloproteases include the neutral metalloproteases described in WO 2007 / 044993, such as those derived from Bacillus amyloliquefaciens, and the metalloproteases described in WO 2015 / 158723 and WO 2016 / 075078, for example.

[0181] Examples of useful proteases include those described in WO 89 / 06279, WO 92 / 19729, WO 96 / 34946, WO 98 / 20115, WO 98 / 20116, WO 99 / 11768, WO 01 / 44452, WO 03 / 006602, WO 04 / 006603, WO 05 / 006604, WO 06 / 006606, WO 07 / 006608, WO 08 / 006609, WO 09 / 006610, WO 10 / 006611, WO 11 / 006612, WO 12 / 006613, WO 13 / 006614, WO 14 / 006615, WO 15 / 006616, WO 16 / 006617, WO 17 / 006618, WO 18 / 006619, WO 19 ... and protease variants described in WO 2004 / 003186, WO 2004 / 041979, WO 2007 / 006305, WO 2011 / 036263, WO 2014 / 207227, WO 2016 / 087617, and WO 2016 / 174234. Preferred protease variants are, for example: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, A120S, S1 26L, P127Q, S128A, S154D, A156E, G157D, G157P, 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 The protease may comprise one or more mutations selected from the group consisting of T268A, T268B, and R269H, where the position numbers correspond to the positions in Bacillus lentus protease set forth in SEQ ID NO: 1 of WO 2016 / 001449.Protease variants having one or more of these mutations are preferably variants of the Bacillus lentus protease (Savinase®, also known as subtilisin 309) set forth in SEQ ID NO: 1 of WO 2016 / 001449, or variants of the Bacillus amyloliquefaciens protease (BPN') set forth in SEQ ID NO: 2 of WO 2016 / 001449. Such protease variants preferably have at least 80% sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 2 of WO 2016 / 001449.

[0182] Another protease of interest is the alkaline protease from Bacillus lentus DSM 5483, described, for example, in WO 91 / 02792, and variants thereof, described, for example, in WO 92 / 21760, WO 95 / 23221, EP 1921147, EP 1921148, and WO 2016 / 096711.

[0183] Alternatively, the protease may be a variant of the TY145 protease having SEQ ID NO: 1 of WO 2004 / 067737, for example a variant comprising 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 of WO 2004 / 067737, wherein the protease variant has at least 75% but less than 100% sequence identity to SEQ ID NO: 1 of WO 2004 / 067737. TY145 variants of interest are described, for example, in WO 2015 / 014790, WO 2015 / 014803, WO 2015 / 014804, WO 2016 / 097350, WO 2016 / 097352, WO 2016 / 097357, and WO 2016 / 097354.

[0184] Suitable commercially available protease enzymes include Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 160T, Blaze Evity® 170T, Blaze Evity® 180T, Blaze Evity® 190T, Blaze Evity® 200T, Blaze Evity® 210T, Blaze Evity® 220T, Blaze Evity® 230T, Blaze Evity® 240T, Blaze Evity® 250T, Blaze Evity® 260T, Blaze Evity® 270T, Blaze Evity® 280T, Blaze Evity® 290T, Blaze Evity® 300T, Blaze Evity® 310T, Blaze Evity® 320T, Blaze Evity® 330T, Blaze Evity® 340T, Blaze Evity® 350T, Blaze Evity® 360T, Blaze Evity® 370T, Blaze Evity® 380T, Blaze Evity® 390T, Blaze Evity® 400T, Blaze Evity® 410T, Blaze Evity® 420T, Blaze Evity® 430T, Blaze Evity® 440T, Blaze Evity® 450T, Blaze Evity® 460T, Blaze Evity® Products sold under the trade names Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In, Progress® Key and Progress® Excel (Novozymes A / S), Maxatase™, Maxacal™, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, FN4 ex(trademark), Excellase®, Excellenz® P1000, Excellenz® P1250, 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), BLAP (sequence shown in Figure 29 of U.S. Pat. No. 5,352,604) and its variants (Henkel AG), and KAP (Bacillus alkalophilus) from Kao. Examples include those sold under the trade name (B. alkalophilus) subtilisin.

[0185] Lipase and cutinase Suitable lipases and cutinases include those of bacterial or fungal origin. These include chemically modified or engineered mutant enzymes. Examples include those derived from the genus Thermomyces, such as T. lanuginosus (formerly Humicola lanuginosa), as described in EP 258068 and EP 305216. Lipases derived from the genus Humicola, such as H. insolens (formerly named H. lanuginosa), cutinase derived from the genus Pseudomonas (some of which have now been renamed Burkholderia), such as P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (P.Lipases from strains of Pseudomonas wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), cutinase from Magnaporthe grisea (WO 10 / 107560), cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca (WO 11 / 084412), Geobacillus stearothermophilus lipase (WO 11 / 084417), Bacillus Examples include lipases from S. subtilis (WO 11 / 084599), and lipases from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0186] Other examples include lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0187] Preferred commercially available lipase products include Lipolase™, Lipex™; Lipolex™, and Lipoclean™ (Novozymes A / S), Lumafast (originally manufactured by Genencor), and Lipomax (originally manufactured by Gist-Brocades).

[0188] Further examples include lipases, sometimes referred to as acyltransferases or perhydrolases, such as acyltransferases with homology to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782), perhydrolases from the CE 7 family (WO 09 / 67279), and variants of M. smegmatis perhydrolase, in particular the S54V variant used in the product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0189] amylase Suitable amylases that can be used with the DNase of the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin, including chemically modified or engineered variants. Examples of amylases include alpha-amylases obtained from specialized strains of the Bacillus genus, such as Bacillus licheniformis, as described in more detail in British Patent No. 1,296,839.

[0190] Suitable amylases include amylases having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants are set out in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0191] Suitable different amylases include the amylase having SEQ ID NO: 6 in WO 02 / 010355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 is one having deletions at positions 181 and 182 and a substitution at position 193.

[0192] Another suitable amylase is a hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or a variant thereof with 90% sequence identity. Preferred variants of this hybrid alpha-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. The most preferred variant of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 comprises the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It has the following characteristics.

[0193] A further suitable amylase is the amylase having SEQ ID NO: 6 in WO 99 / 019467, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those with substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those with deletions at positions R181 and G182, or H183 and G184.

[0194] Additional amylases that can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 7 of WO 96 / 023873, or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7 are those that have substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476, using SEQ ID NO: 2 of WO 96 / 023873 for numbering. More preferred variants are those having deletions at two positions selected from positions 181, 182, 183, and 184, for example, positions 181 and 182, positions 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those having deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0195] Other amylases that can be used include amylases having SEQ ID NO: 2 in WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 in WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 include those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0196] Further suitable amylases include amylases having SEQ ID NO: 2 of WO 09 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 include those having C-terminal truncations and / or substitutions, deletions or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO: 2 include substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 include substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0197] Another suitable amylase is the alpha-amylase having SEQ ID NO: 12 in WO 01 / 66712, or a variant having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants include those having substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and substitutions R118K, N195F, R320K, and R458K, as well as variants additionally having substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, most preferred are variants additionally having substitutions at all of these positions.

[0198] Other examples include amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087.

[0199] Commercially available amylases include Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (manufactured by Novozymes A / S), and Rapidase™, Purastar™ / Effectenz™, Powerase, and Preferenz S100 (manufactured by Genencor International Inc. / DuPont).

[0200] Hexosaminidase The detergent compositions containing DNase of the present invention may also contain one or more hexosaminidases. The term "hexosaminidase" includes "dispersin" and the abbreviation "Dsp," which refers to EC 3.2.1.-, a polypeptide with hexosaminidase activity that catalyzes the hydrolysis of β-1,6-glycosidic bonds in N-acetylglucosamine polymers found, for example, in biofilms. The term "hexosaminidase" includes polypeptides with N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity.

[0201] Polypeptides having hexosaminidase activity can be obtained from any genus of microorganism, particularly bacteria or fungi. Preferably, the hexosaminidase, e.g., dispersin, is obtained from the genera Terribacillus, Curtobacterium, Aggregatibacter, Haemophilus, or Actinobacillus, preferably Terribacillus. The hexosaminidase can also be a variant of a polypeptide obtained from any of these or other organisms.

[0202] Suitable hexosaminidases include those listed in International Publication Nos. WO 2017186936, WO 2017186937, WO 2017186943, WO 2017207770, WO 2018184873, WO 2019086520, WO 2019086528, WO 2019086530, WO 2019086532, WO 2019086521, and WO 2019086 526 pamphlet, International Publication No. 2020002604 pamphlet, International Publication No. 2020002608 pamphlet, International Publication No. 2020007863 pamphlet, International Publication No. 2020007875 pamphlet, International Publication No. 2020008024 pamphlet, International Publication No. 2020070063 pamphlet, International Publication No. 2020070249 pamphlet, International Publication No. 2020088957 pamphlet, International Publication No. 2020088958 pamphlet, and International Publication No. 2020207944 pamphlet.

[0203] Peroxidase / Oxidase The peroxidase may be included in the enzyme classification EC 1.11.1.7 as described by the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment thereof exhibiting peroxidase activity. Suitable peroxidases include those of plant, bacterial, or fungal origin. Chemically modified or protein-engineered variants are also included. Examples of useful peroxidases include peroxidase from the genus Coprinopsis, such as C. cinerea (EP 179,486), and its variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Peroxidases may also include haloperoxidase enzymes, such as chloroperoxidase and bromoperoxidase, and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (EC 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions. In one embodiment, the haloperoxidase is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium peroxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method, a vanadate-containing haloperoxidase is combined with a chloride ion source.Haloperoxidases have been isolated from a variety of fungi, particularly from the dematiaceous hyphomycetes group, such as the genera Caldariomyces (e.g., C. fumago), Alternaria, Curvularia (e.g., C. verruculosa and C. inaequalis), Drechslera, Ulocladium, and Botrytis. Haloperoxidases have also been isolated from bacteria, such as Pseudomonas (e.g., P. pyrrocinia) and Streptomyces (e.g., S. aureofaciens). In a preferred embodiment, the haloperoxidase is derived from a Curvularia species, in particular Curvularia verruculosa or Curvularia inaequalis, e.g., C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or Drechslera hartlebii as described in WO 01 / 79459, Dendrophiella salina as described in WO 01 / 79458, or salina, Phaeotrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium species as described in WO 01 / 79460.

[0204] Oxidases include any laccase enzyme encompassed by enzyme classification EC 1.10.3.2, or any fragment derived therefrom that exhibits laccase activity, or compounds that exhibit 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).

[0205] Preferred laccase enzymes are those of bacterial origin. The enzymes can be obtained from plants, bacteria, or fungi (including filamentous fungi and yeasts).

[0206] Suitable examples of fungal origin include species of the genus Bacillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., For example, T. villosa and T. versicolor, the genus Rhizoctonia, such as R. solani, the genus Coprinopsis, such as C. cinerea, C. comatus, C. friesii, and C. plicata, atilis, Psathyrella such as P. condoleana, Panaeolus such as P. papilionaceus, Myceliophthora such as M. thermophila, Schytalidium such as S. thermophilum, Polyporus such as P. pinsitus, Phlebia such as P. radiata (WO 92 / 01046), or Coriolus such as C. hirsutus (JP Patent Publication No. 2238885).

[0207] Suitable examples of bacterial origin include laccases inducible from strains of the genus Bacillus.

[0208] Laccases obtained from the genus Coprinopsis or Myceliophthora; laccases obtained from Coprinopsis cinerea as disclosed in WO 97 / 08325; or laccases obtained from Myceliophthora thermophila as disclosed in WO 95 / 33836 are preferred.

[0209] Detergent enzymes can be included in detergent compositions by adding separate additives containing one or more enzymes or by adding combined additives containing these enzymes. The detergent additives of the present invention, i.e., separate additives or combined additives, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, non-shattering granules, liquids, stabilized liquids, and slurries.

[0210] Non-shattering granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials include poly(ethylene oxide) products (polyethylene glycols, PEGs) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed techniques are given in British Patent No. 1,483,591. Liquid enzyme preparations may be stabilized, for example, by adding polyols, such as propylene glycol, sugars or sugar alcohols, lactic acid, or boric acid, according to established methods. The protected enzyme may be prepared according to the method disclosed in EP 238,216.

[0211] Dispersants The detergent composition of the present invention may also contain a dispersant. Powder detergents may contain a dispersant. Suitable water-soluble organic materials include homo- or copolymeric acids or their salts, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series Volume 71, Marcel Dekker, Inc.

[0212] Dye transfer inhibitor The cleaning compositions of the present invention may also contain one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, and polyvinylimidazole, or mixtures thereof. When present in the subject compositions, the dye transfer inhibitors may be present at levels of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0213] Optical brighteners The latent composition may also preferably contain additional ingredients, such as optical brighteners or optical brighteners, that can adjust the color tone of the item being cleaned. When present, the optical brightener is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used optical brighteners belong to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyl-distyryl derivatives. Examples of diaminostilbene-sulfonic acid derivative type fluorescent whitening agents include: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydro) Examples of suitable fluorescent whitening agents include 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and the sodium salt of sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, commercially available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenylstyryl)-disulfonate. Another preferred optical brightener is Parawhite KX, available from Paramount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is disodium 4,4'-bis-(sulfostyryl)-biphenyl, also known as disodium distyrylbiphenyl disulfonate.Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins.

[0214] Suitable fluorescent brightener levels include lower levels of about 0.01, 0.05, about 0.1, or even about 0.2% by weight, to upper levels of 0.5 or even 0.75% by weight.

[0215] Dirt-releasing polymer The detergent composition may also include one or more soil-releasing polymers to aid in the removal of soil from fabrics, such as cotton and polyester-based fabrics, and the removal of hydrophobic soil from polyester-based fabrics. The soil-releasing polymer may be, for example, a nonionic or anionic terephthalate-based polymer, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, or polyester polyamides, as described, for example, in Chapter 7, Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc. Another type of soil-releasing polymer is an amphiphilic alkoxylated grease-cleaning polymer comprising a core structure and multiple alkoxylate groups attached to the core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure, as detailed in WO 2009 / 087523 (herein incorporated by reference). Additionally, random graft copolymers are suitable soil-releasing polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (incorporated herein by reference). Other soil-releasing polymers are substituted polysaccharide structures, particularly substituted cellulosic structures, such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003 / 040279 (both incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

[0216] Anti-redeposition agent The detergent compositions of the present invention may also include one or more anti-redeposition agents, such as carboxymethyl cellulose (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 cellulosic polymers described above under soil release polymers may also function as anti-redeposition agents.

[0217] Rheology Modifiers The detergent compositions of the present invention may also contain one or more rheology modifiers, structurants, or thickeners different from the viscosity reducing agent. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, and polymeric rheology 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 shown in EP 2169040.

[0218] Other suitable adjunct materials include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, disinfectants, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, suds suppressors, solvents, and liquid detergent structurants and / or structural elasticizers.

[0219] Other materials In addition, any detergent ingredient known in the art may be used in the cleaning composition of the present invention. Other optional detergent ingredients include, alone or in combination, anticorrosion agents, anti-shrinkage agents, soil redeposition inhibitors, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric softeners including clay, fillers / processing aids, optical brighteners / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil suspending agents, softeners, foam suppressors, tan inhibitors, and wicking agents. Any ingredient known in the art for use in detergents may be used. The selection of such ingredients is well within the skill of those skilled in the art.

[0220] Formulation of detergent products The detergent composition can be in any convenient form, such as a bar, a homogenous tablet, a tablet having two or more layers, a plain or compressed powder, a granule, a paste, a gel, or a plain, compressed, or concentrated liquid. Other detergent formulation forms include single unit dose forms, such as layered forms and pouches.

[0221] The pouch can be configured as a single compartment or multiple compartments. It can be of any form, shape, and material suitable for holding the composition without releasing it from the pouch, for example, prior to contact with water. The pouch can be made of a water-soluble film that encompasses the contents and can be divided into compartments. Preferred films are polymeric materials, preferably polymers that can be formed into films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, and polymethacrylates, with polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC) being most preferred. Preferably, the level of polymer in the film is, for example, at least about 60%. Preferred average molecular weights are typically from about 20,000 to about 150,000. The membrane can also be a mixture of hydrolytically degradable and water-soluble polymers, such as polylactide and polyvinyl alcohol, and plasticizers, such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch can contain a solid laundry cleaning composition or some of its components and / or a liquid cleaning composition or some of its components separated by a water-soluble film. The compartment for the liquid components can have a different composition from the compartment containing the solids (see, for example, U.S. Patent Application Publication No. 2009 / 0011970A1).

[0222] The detergent ingredients can be physically separated from each other by compartments within a water-soluble pouch or different layers of a tablet to avoid negative storage interactions between the ingredients, and the different dissolution profiles of each of the compartments can result in delayed dissolution of selected ingredients in the wash solution.

[0223] Non-unit-dose liquid or gel detergents may be aqueous, typically containing at least 20% by weight and up to 95% water, e.g., up to about 70%, up to about 65%, up to about 55%, up to about 45%, or up to about 35% water. Concentrated liquid detergents may have a lower water content, e.g., up to about 30% or up to about 20%, e.g., in the range of about 1% to about 20%, such as about 2% to about 15%. Other types of liquids may be included in aqueous liquids or gels, including, but not limited to, alkanols, amines, diols, ethers, and polyols. Water-soluble liquid or gel detergents may contain 0 to 30% organic solvents. Alternatively, liquid or gel detergents may be non-aqueous.

[0224] Liquid detergent compositions can be formulated to have a moderate pH of about 6 to about 10, such as about pH 7, about pH 8, or about pH 9, or can be formulated to have a higher pH of about 10 to about 12, such as about pH 10, about pH 11, or about pH 12.

[0225] Unless otherwise indicated, the term "liquid" as used herein should be understood to encompass any type of liquid detergent composition, e.g., concentrated liquid, gel, or the liquid or gel portion of, e.g., a pouch having one or more compartments.

[0226] Granular detergent formulations Enzymes in the form of granules containing an enzyme-containing core and, optionally, one or more coating agents are commonly used in granular (powdered) detergents. Various methods for preparing the cores are well known in the art, including, for example, a) spray-drying a liquid enzyme-containing solution; b) producing a layered product in which the enzyme is coated as a layer surrounding a preformed inert core particle, e.g., using a fluidized bed apparatus; c) absorbing the enzyme onto and / or into the preformed core; d) extruding an enzyme-containing paste; e) suspending an enzyme-containing powder in molten wax and atomizing it to produce a pelletized product; f) mixer-granulating by adding an enzyme-containing liquid to a dry powder composition of the granulation ingredients; g) reducing the size of the enzyme-containing core by grinding or crushing larger particles, pellets, etc.; and h) fluidized bed granulation. The enzyme-containing cores can be dried, e.g., using a fluidized bed dryer or other known methods for drying granules in the feed or enzyme industry, to a moisture content typically of 0.1 to 10% water by weight.

[0227] The enzyme-containing core is sometimes provided with a coating to improve storage stability and / or reduce dust formation. One type of coating often used on enzyme granules for detergents is a salt coating, typically an inorganic salt coating, which can be applied as a salt solution, for example, using a fluidized bed. Other coating materials that can be used include, for example, polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). The granules can contain two or more coatings, for example, a salt coating followed by an additional coating of a material such as PEG, MHPC, or PVA.

[0228] For further details regarding enzyme granules and their production, see WO 2013 / 007594, as well as, for example, WO 2009 / 092699, EP 1705241, EP 1382668, WO 2007 / 001262, U.S. Pat. No. 6,472,364, WO 2004 / 074419, and WO 2009 / 102854.

[0229] Formulation of enzymes in co-granules DNase can be formulated as granules, for example, as multi-enzyme granules combining one or more enzymes, where each enzyme is present in more granules to ensure a more uniform distribution of the enzymes within the detergent. This also reduces physical separation of the various enzymes due to different particle sizes. A method for producing multi-enzyme multi-granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0230] Another example of formulating enzymes using composite granules is disclosed in WO 2013 / 188331, which relates to a detergent composition comprising: (a) a multi-enzyme composite granule; (b) less than 10 wt. of zeolite (on an anhydrous basis); and (c) less than 10 wt. of phosphate (on an anhydrous basis), wherein the enzyme composite granule comprises 10 to 98 wt. % of a moisture-absorbing component, and the composition additionally comprises 20 to 80 wt. % of a detergent moisture-absorbing component. WO 2013 / 188331 also relates to a method of treating and / or cleaning a surface, preferably a fabric surface, comprising the steps of: (i) contacting the surface with a detergent composition in an aqueous wash liquor as claimed and described herein; and (ii) rinsing and / or drying the surface.

[0231] Liquid formulations The present invention also relates to liquid compositions comprising the DNase variants of the present invention, which may contain enzyme stabilizers (e.g., polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid).

[0232] 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 such as sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

[0233] In another aspect, the liquid formulation comprises 20-80% by weight of polyol. In one embodiment, the liquid formulation comprises 0.001-2% by weight of a preservative.

[0234] In another embodiment, the present invention provides: (A) 0.001 to 25% by weight of the variant of the invention; (B) 20 to 80 wt. % polyol; (C) optionally 0.001 to 2% by weight of a preservative; and (D)Water The present invention relates to a liquid formulation comprising:

[0235] In another embodiment, the present invention provides: (A) 0.001 to 25% by weight of the variant of the invention; (B) 0.001 to 2% by weight of a preservative; (C) optionally 20 to 80 wt. % of a polyol; and (D)Water The present invention relates to a liquid formulation comprising:

[0236] In another embodiment, the liquid formulation comprises one or more formulating agents 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, acetates, and phosphates, preferably 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) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0237] In another embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol. In one embodiment, the liquid formulation comprises 20% to 80% polyol, e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0238] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% by weight of the preservative, e.g., 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% by weight of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% by weight of the preservative, 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0239] In another embodiment, the liquid formulation further comprises one or more additional enzymes, for example, as described above.

[0240] Use of DNase variants The DNase variants of the present invention are suitable for use in cleaning processes, such as laundry cleaning or hard surface cleaning, in particular for laundry. Thus, one aspect of the present invention relates to a method for washing an item, wherein the item is a fabric, the method comprising: a) exposing the item to a wash solution containing a DNase variant of the present invention; b) completing at least one cleaning cycle; and c) a step for rinsing the item; Includes:

[0241] 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.

[0242] The cleaning solution may have a temperature in the range of 5°C to 95°C, or in the range of 10°C to 80°C, or in the range of 10°C to 70°C, or in the range of 10°C to 60°C, or in the range of 10°C to 50°C, or in the range of 15°C to 40°C, or in the range of 20°C to 30°C.

[0243] The concentration of DNase variant enzyme in the wash solution is typically in 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 of enzyme protein.

[0244] When used, items such as T-shirts or sportswear are exposed to bacteria from the user's body and from the remaining environment in which the item is used. This can cause malodor in the item even after it has been washed. Therefore, the present invention also relates to a method for removing or reducing malodor from fabrics. Malodor can be caused by bacteria that produce unpleasant-smelling compounds. One example of such an unpleasant-smelling compound is E-2-nonenal. Malodor can be present in freshly washed fabrics that are still wet, or in freshly washed and dried fabrics. Malodor can also be present in fabrics that have been stored for a period of time after washing. Therefore, the present invention also relates to the use of the DNase variants of the present invention for reducing or removing malodor, such as E-2-nonenal, from wet or dry fabrics.

[0245] In one embodiment, the DNase variants of the present invention have improved malodour removal properties compared to a parent or reference DNase such as SEQ ID NO: 1, where malodour is measured as described in Assay II.

[0246] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0247] Assays and detergent compositions Model detergent A2 (liquid) Ingredients: 12% LAS, 12% AEO, 4% AEOS / SLES, 2% MPG (monopropylene glycol), 3% ethanol, 2% TEA, 3% soap, 0.5% NaOH, 3.9% sodium citrate, 1.5% DTMPA Na7, 0.5% phenoxyethanol, and water to 100% (all percentages are w / w); pH adjusted to 8.4 with NaOH

[0248] Assay I: Test for DNase activity DNase activity can be determined using DNase Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA), which is prepared according to the supplier's instructions. Briefly, 21 g of agar is dissolved in 500 ml of water and then autoclaved at 121°C for 15 minutes. The autoclaved agar is heated to 48°C in a water bath, and 20 ml of the agar is poured into a Petri dish and allowed to solidify overnight at room temperature by incubation. 5 μl of enzyme solution is added to the solidified agar plate, and DNase activity is observed as a colorless zone around the spotted enzyme solution.

[0249] Assay II: Analysis of dough for E-2-nonenal using E-nose. One method for testing fabrics for the presence of malodors is to use E-2-nonenal as a malodor marker, since this compound contributes to laundry malodors. A solution of E-2-nonenal is added to a 5 cm x 5 cm fabric swatch, the swatch is placed in a 20 mL glass vial for gas chromatography (GC) analysis, and the vial is capped. After 20 minutes of incubation at 40°C, a 5 mL headspace of the capped vial is analyzed using a Heracles II Electronic Nose (Alpha MOS, France), a dual-column gas chromatograph with two FIDs, column 1: MXT5 and column 2: MXT1701.

[0250] method General methods of PCR, cloning, ligation of nucleotides, etc. are well known to those skilled in the art and can be found, for example, in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons, (1995); Harwood, CR, and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", DN Glover ed. (1985); "Oligonucleotide Synthesis", MJ Gait ed. (1984); "Nucleic Acid Hybridization", B.D. Hames & S.J. Higgins eds. (1985); "A Practical Guide To Molecular Cloning", B. Perbal, (1984).

[0251] Example 1: Preparation and initial screening of DNase variants Site-directed variants of the DNase of SEQ ID NO: 1 were generated using specific primers containing the desired new mutations. The new codons selected were those with the highest natural abundance for the particular amino acid in Aspergillus oryzae. Transformation substrates were generated using PCR:

[0252] 1) Separate amplification of N- and C-terminal fragments (compared to the mutation site) The N-terminal fragment was amplified using a universal forward primer and a site-specific reverse primer. The C-terminal fragment was amplified using a mutation-specific forward primer and a universal reverse primer. Both universal primers are complementary to sequences essential for homologous integration in the Aspergillus genome.

[0253] 2) Assembly of N- and C-terminal fragments by in vivo recombination in cells. The resulting transformation substrates were transformed into Aspergillus oryzae, and transformants were selected by growth on nitrate as the sole nitrogen source. Three single colonies of each type were picked into microtiter plates and grown in Aspergillus-specific broth at 30°C for 4 days.

[0254] The supernatant was used for initial stability screening. Based on the initial screening, substitutions of interest were combined into new variants containing multiple substitutions, which were purified and subjected to further stability testing as described below.

[0255] Example 2: Purification of DNase variants Purification of Aspergillus oryzae DNase culture supernatant containing multiple substitutions compared to SEQ ID NO:1 was carried out by CaptoMMC as follows: The culture broth was filtered through a Nalgene 0.2 μm filtration unit to remove host cells. The filtered supernatant was applied to a 25 mL CaptoMMC column (Cytiva) equilibrated with 20 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6.0. The supernatant was applied by in-line dilution of 1:3 using 20 mM MES, pH 6.0 buffer as the diluent. The column was washed with 20 mM MES, pH 6.0, and then eluted by a step gradient to 100% 50 mM Tris, 1 M NaCl, pH 9.0, retained for 5 column volumes. Fractions were analyzed by SDS-PAGE and pooled according to purity for use as purified enzyme preparations for further experiments.

[0256] Example 3: Determination of the melting temperature (Tm) of purified DNase variants by nDSF Purified DNase variants were diluted to 0.6 mg / ml with 0.01% Triton X-100. Concentrations were calculated from absorbance at 280 nm. 40 μl of diluted sample was mixed with 40 μl of buffer (0.2 M Hepes, 20 mM MgCl, pH 8.5) or 40 μl of 20% model detergent A2.

[0257] The thermal stability of purified DNase variants was estimated by differential scanning fluorimetry (nanoDSF or nDSF) using a Prometheus NT.48 or NT.Plex instrument (NanoTemper Technologies GmbH, Germany), which measures the change in intrinsic tryptophan and tyrosine fluorescence due to unfolding during a temperature increase (20°C to 95°C, ramp rate 3.3°C / min). Melting temperatures are calculated from the inflection points in the melting curves using the first and second derivatives. Variants were usually tested in triplicate.

[0258] Example 4: Storage stability assay for purified DNase variants The storage stability of purified DNase variants of the invention with multiple substitutions compared to SEQ ID NO: 1 was determined using an accelerated storage stability assay in which the variants were stored for 7 days in a detergent composition at elevated temperatures of 40° C. or 45° C. The remaining DNase activity was determined after various incubation times and the results were compared to those for the wild-type DNase of SEQ ID NO: 1 stored at the same temperatures.

[0259] Purified DNase variants were diluted with 0.01% Triton X-100 to concentrations ranging from 10 ppm to 150 ppm in the final reaction. Enzyme concentrations were calculated from absorbance at 280 nm. 15 μl of diluted DNase sample was mixed with 285 μl of concentrated model detergent A2 and 0.5% protease (Progress Uno 101L, Novozymes A / S) and added to wells of a microtiter plate (detergent plate, Nunc U96 PP 0.5 ml) using a magnetic bar. After mixing, the detergent plate was incubated at 40°C or 45°C in a Biosan PST-100HL thermomixer.

[0260] After various incubation times (e.g., 0, 3, 24, 96, and 168 hours), the remaining DNase activity was measured. Five μl from the detergent plate was mixed with 195 μl of DNA substrate solution (3.3 mg of DNA (Sigma D1626) in 50 mM Tris pH 7, 20 mM MgCl2, 20 mM CaCl2, 1.3 mM EDTA). Viscosity was measured every minute for 30 minutes using pressure sensing during aspiration with a Hamilton Microlab STAR, and activity was calculated from the measured viscosity reduction. Each variant was tested in duplicate at each of two different enzyme concentrations.

[0261] The decline in activity during incubation with detergent was assumed to be exponential. Half-lives (T1 / 2) were found from linear regression of the logarithm (assumed) versus incubation time, and half-life improvement factors (HIF or T1 / 2IF) were calculated as the half-life of the DNase variant compared to the half-life of the reference, in this case DNase SEQ ID NO: 1. Variants assayed at 40°C or 45°C were compared to DNase SEQ ID NO: 1 assayed at the same temperatures. The HIF value for DNase SEQ ID NO: 1 was defined as 1.

[0262] Example 5: Melting Point and Storage Stability Results The results for the melting temperature (Tm) and half-life improving factor (HIF) (determined as described above) of the DNase variants of the invention are shown below in Tables 1 and 2. Table 1 shows data for the melting temperature (Tm) determined by nDSF in both the buffer and model detergent A2 described in Example 3. Table 2 shows data for the half-life improving factor (HIF) compared to the DNase of SEQ ID NO: 1 determined in model detergent A2 at the temperatures indicated as described in Example 4.

[0263] [Table 2]

[0264] [Table 3]

[0265] [Table 4]

[0266] [Table 5]

[0267] [Table 6]

[0268] [Table 7]

[0269] [Table 8]

[0270] The present invention as described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended as illustrations of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure, including definitions, will control.

[0271] The invention is further defined by the following numbered paragraphs:

[0272] 1. A DNase variant comprising at least two substitutions, for example at least three substitutions, at positions selected from the group consisting of 111, 32, 35, 69, 102, 105, and 181, wherein the position numbers are based on the numbering of SEQ ID NO: 1, wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0273] 2. A DNase variant comprising three or more substitutions at positions selected from the group consisting of 26, 32, 35, 65, 67, 69, 98, 102, 105, 111, 115, 150, 157, 159, 161, 172, 181, 182, 185, 187, 192, 206, 208, and 212, wherein the position numbers are based on the numbering of SEQ ID NO: 1, wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0274] 3. A DNase variant comprising three or more substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V and K212E, wherein the position numbers are based on the numbering of SEQ ID NO: 1, wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0275] 4. A DNase variant comprising at least two substitutions, such as at least three substitutions, selected from the group consisting of A111P, D32E, V35I, S69V, Q102E, K105N, and G181N, wherein the position numbers are based on the numbering of SEQ ID NO: 1, and wherein the variant has at least 60% and less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity.

[0276] 5. A variant of any of the preceding paragraphs, wherein the variant contains the substitution A111P.

[0277] 6. The variant of paragraph 5, comprising 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.

[0278] 7. A variant of any of the preceding paragraphs comprising the substitutions D32E+V35I and / or the substitution G181N.

[0279] 8. A variant of any of the preceding paragraphs containing the substitution D32E+V35I+A111P.

[0280] 9. A variant of any of the preceding paragraphs containing the substitutions A111P+G181N.

[0281] 10. A variant of any of the preceding paragraphs containing the substitutions S69V and / or Q102E.

[0282] 11. A variant of any of the preceding paragraphs containing the substitution K105N.

[0283] 12. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0284] 13. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0285] 14. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0286] 15. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0287] 16. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+S69V+Q102E+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0288] 17. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+S69V+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0289] 18. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions S69V+Q102E+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0290] 19. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions S69V+Q102E+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0291] 20. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions S69V+Q102E+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0292] 21. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions Q102E+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0293] 22. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions S69V+Q102E+K105N+A111P+G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0294] 23. The variant of any of the preceding paragraphs, wherein the variant includes the substitutions D32E+V35I+K105N+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E.

[0295] 24. The variant of any of the preceding paragraphs, comprising at least one additional substitution selected from the group consisting of K18S, T19P, D32P, K86E, A101E, A101T, K105D, K105E, K105T, G137R, N146H, K147N, K155E, A172D, K178S, K192A, N214D, and N217A.

[0296] 25. A variant of any of the preceding paragraphs having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0297] 26. A variant of any of the preceding paragraphs, wherein the number of substitutions is 1 to 20, e.g., 1 to 10 or 1 to 5, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

[0298] 27. A variant of any of the preceding paragraphs, consisting of 195 to 230 amino acids, preferably 200 to 225 amino acids, for example 204 to 221 amino acids.

[0299] 28. · S69V+K86E+Q102E+K147N+K155E+A172D+K192A+Q208V; G20C+K30C+D32E+V35I+A111P; · S69V+K86E+Q102E+K147N+K155E+A172D+G181N+K192A+Q208V; · D32E+V35I+N77E+A111P+G181N+S182V; · D32E+E34N+V35I+K86E+A111P+G181N; · S69V+K86E+Q102E+K105T+K147N+K155E+A172D+K192A+Q208V; · S69V+Q102E+Q157E+T159Q+N214D+N217A; · S26H+D32E+V35I+A111P+K155L; · K18S+D32E+V35I+A111P+K185I+K215T; · D32E+V35I+A111P+K185I+K215T; · D32E+V35I+K82G+A111P+G181N+S182V; · D32E+V35I+A111P+K178M+K212T; · K18S+D32E+V35I+A111P+K178S+K212L; · S69V+Q102E+V138A+S144R+Q157E+T159Q+A172E+G181N; · D32E+V35I+A111P+K204L+K215I; · K18F+D32E+V35I+A111P+K185Q; · D32E+V35I+A111P+K147N+K204L; · S69V+K86E+Q102E+K105D+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+S69V+K86E+Q102E+A111P+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P+K147N; · S69V+K86E+Q102E+S115T+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P+N140H; · D32E+V35I+K65Q+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; · D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+K178S+G181N+K192A+Q208V+N217A; · D32E+V35I+A111P; · E6D+A101E+Q150E+G181N+Q208V; · S26H+D32E+V35I+A111P; · K18Q+D32E+V35I+K105E+A111P+K155F; · D32E+V35I+A111P+G199E; · S69V+Q102E+Q157E+T159Q+G181N; · D32E+V35I+K67A+A111P+P175N+S182V; · D32E+V35I+A111P+N214D; · S69V+Q102E+Q157E+T159Q+A172E; · E6D+A101E+Q150E+G181N+Q208V; · D32E+V35I+S69E+A111P; · D32E+V35I+Q85E+A111P; · K18Q+D32E+V35I+A111P; · D32E+V35I+A101E+K105T+A111P+G181N+K212E+Y218E; · D32E+V35I+A111P+G181N+S182V+K185E; · D32E+V35I+S98R+A111P+G181N+S182Y; · D32E+V35I+A101E+Q102E+A111P+G181N; · T19P+D32E+V35I+Q102E+K105E+A111P; · D32E+V35I+D72V+A111P; · S69V+K86E+Q102E+F112W+K147N+K155E+A172D+K192A+Q208V; · S69V+Q102E+A111P+Q157E+T159Q+A172E; · K67A+S69V+Q102E+K147N+Q157E+T159Q+A172E+D197S; · S69V+Q102E+S115T+Q157E+T159Q+A172E; · S69V+K86E+Q102E+G136E+K147N+K155E+A172D+K192A+Q208V; · D32E+V35I+K67A+A111P+K204G; · A101E+A111P+Q150E+Q208V; · D32E+V35I+A101E+K105E+A111P+S182V; · D32E+V35I+A101E+K105E+A111P+S182V; · D32E+V35I+A101T+K105N+A111P+K147E+G181N+S182Y; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+Q102E+A111P; · S26H+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+K67A+K105T+A111P+K178L+K185T; · K67A+K105T+G181N; · T19P+D32E+V35I+A101E+K105T+A111P+K160A+G181N; · D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; · D32E+V35I+Q102E+A111P; · K18S+D32E+V35I+Q102E+K105E+A111P; · D32E+V35I+K65E+A101E+K105T+A111P+N217A; · D32E+V35I+K105T+A111P+G181N+K204W+K215L; · D32E+V35I+K105T+A111P+K178L+G181N+K212T; · S26H+S69V+Q102E+A111P+N146H+Q150E+Q157E+T159Q+A172E; · K18Q+D32E+V35I+K67A+K105T+A111P+K215V; · D32E+V35I+Q85E+A111P; · K18S+T19P+D32E+V35I+A101E+K105T+A111P+G181N; · D32E+V35I+A101E+K105N+A111P; · K18M+D32E+V35I+K67A+K105T+A111P+K178S; · D32E+V35I+K105T+A111P+G181N; · D32E+V35I+K67A+K105T+A111P+K185G+K212M; · D32E+V35I+A101T+K105N+A111P+G181N+S182Y+N214D; · T19P+D32E+V35I+A101E+K105T+A111P+K178S+G181N; · D32E+V35I+K105T+A111P+G181N+K185L+K192V+K204W; · D32E+V35I+S98R+K105N+A111P+K147N+G181N+S182Y; · D32E+V35I+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+K105T+A111P; · D32E+V35I+K53F+K105N+A111P+G181N+S182Y+K212E; · K18S+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+S98R+K105N+A111P+Q158D+G181N+S182Y; · D32E+V35I+K67A+K105T+A111P+K190V+K204G; · N4K+D32E+V35I+S98R+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+K67A+K105T+A111P+K147N+K190N; · D32E+V35I+S98R+K105N+A111P+G181N+S182Y+Q208V; · D32E+V35I+A101E+K105T+A111P+G181N+K212E+N214D; · D32E+V35I+K82T+A101E+K105T+A111P+G181N+K212E; · D32E+V35I+A111P+S115T; · S26H+S69V+Q102E+A111P+K147N+A149S+Q150E+Q157E+T159Q+A172E; · K18S+D32E+V35I+K67A+K105T+A111P+K185L+K192F; · V12L+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+S69E+K105N+A111P+K147E+G181N+S182Y; · K18S+D32E+V35I+A101E+K105T+A111P+G181N+K212E; · D32E+V35I+K65E+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+S98R+K105N+A111P+G181N+S182Y+D189N+N214D; · D32E+V35I+S98R+A101T+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+A111P+Q157E+T159Q+G181N+A206G+Q208V; · D32E+V35I+K67A+S69R+A111P+G181N+K212E; · D32E+V35I+K67A+A111P+G181N+A206G+Q208V; · D32E+V35I+A101E+K105T+A111P+G181N; · D32E+V35I+K67A+A101E+K105D+A111P+N217A; · D32E+V35I+K67A+A111P+Q157E+G181N+Q208V; · D32E+V35I+K67A+A111P+Q157E+A172E+G181N+A206G+Q208V; · S26H+D32E+V35I+K67A+S69R+A111P+G181N+K212E; · D32E+V35I+S69E+S98R+K105N+A111P+G181N+S182Y; · D32E+V35I+K67A+K105N+A111P+G181N+S182Y+K212E; · D32E+V35I+K105D+A111P+S115T; · D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; · D32E+V35I+K65E+K67A+K105N+A111P+G181N+S182Y; · S69V+Q102E+K105N+A111P+S115T+V138M+Q150E+G161R+G181N+V187N+K192I; · D32E+V35I+K67A+S69V+K105T+A111P+Q157E+G181N+A206G+Q208V; · S69V+Q102E+K105N+A111P+S115T+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+Q150E+K160T+G161R+G181N+V187N+K192I; · S69V+D72S+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; · S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+S134H+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · D32P+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+T120H+N146H+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+Q157E+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · S26H+D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; · K65Q+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · S69A+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; · S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+A172D+G181N+V187N+K192I; · D32P+S69A+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+K192I; D32P+S69V+Q102E+K105D+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; and · S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I A variant of any of the preceding paragraphs, comprising a set of substitutions selected from the group consisting of:

[0300] 29. The variant of paragraph 28, comprising or consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, together with one of said sets of substitutions.

[0301] 30. The variant of any of the preceding paragraphs, wherein the variant has improved stability compared to the polypeptide of SEQ ID NO: 1 or to a parent polypeptide having the same amino acid sequence as the variant without said substitution.

[0302] 31. A variant of paragraph 30, wherein the improved stability is selected from the group consisting of detergent stability and thermal stability.

[0303] 32. A detergent composition comprising the DNase variant of any of paragraphs 1 to 31 and at least one detergent adjunct ingredient.

[0304] 33. Use of a DNase variant according to any of paragraphs 1 to 31 or a detergent composition according to paragraph 32 in a cleaning process such as laundry or hard surface cleaning, for example dishwashing.

[0305] 34. A method of cleaning an item, comprising exposing the item to a cleaning solution comprising the DNase variant of any of paragraphs 1 to 31 or the detergent composition of paragraph 32.

[0306] 35. The method of paragraph 34, wherein the item is a surface such as a fabric or hard surface, for example, a dish, or a surface such as a tabletop, a wall or floor, or the interior surface of a machine such as a washing machine or dishwasher.

[0307] 36. A method for washing fabrics comprising: a) exposing a fabric to a wash liquor comprising a DNase variant according to any of paragraphs 1 to 31 or a detergent composition according to paragraph 32; b) completing at least one wash cycle; and c) Rinse the items and A method comprising:

[0308] 37. Granules containing: (a) a core containing any one of the DNase variants of paragraphs 1 to 31 and, if applicable, (b) a coating consisting of one or more layers surrounding the core; Granules containing

[0309] 38. A liquid composition comprising the DNase variant of any one of paragraphs 1 to 31 and an enzyme stabilizer, such as 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.

[0310] 39. A polynucleotide encoding a DNase variant of any of paragraphs 1 to 31, a nucleic acid construct or expression vector comprising the polynucleotide, or a recombinant host cell transformed with the polynucleotide.

[0311] 40. A method for producing a DNase variant, comprising: a. culturing the recombinant host cell of paragraph 39 under conditions suitable for expression of the variant; b. Recovering variants and A method comprising:

Claims

1. 1. A DNase variant comprising at least two substitutions, such as at least three substitutions, selected from the group consisting of A111P, D32E, V351, S69V, Q102E, K105N, and G181N, wherein the position numbers are based on the numbering of SEQ ID NO: 1, wherein the variant has at least 60% and less than 100% sequence identity to a polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the variant has DNase activity.

2. The variant of claim 1, wherein the variant comprises the substitution A111P.

3. 3. The variant of claim 2, further comprising 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.

4. The variant is: D32E+V35I, for example D32E+V35I+A111P; and / or G181N, e.g., A111P+G181N; and / or S69V+Q102E The variant of claim 1, comprising:

5. The variant is: D32E+V35I+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; D32E + V35I + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; D32E + V35I + Q102E + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; D32E + V35I + K105N + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; D32E + V35I + S69V + Q102E + A111P, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; D32E + V35I + S69V + Q102E + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; S69V+Q102E+A111P and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; S69V + Q102E + G181N and, optionally, one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, A111P, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; S69V + Q102E + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, K105N, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; Q102E + K105N + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S69E, S69V, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; S69V + Q102E + K105N + A111P + G181N, and optionally one or more additional substitutions selected from the group consisting of S26H, D32E, V35I, K65E, K67A, S98R, S115T, Q150E, Q157E, T159Q, G161R, A172E, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E; or D32E + V35I + K105N + A111P, and optionally one or more additional substitutions selected from the group consisting of S26H, K65E, K67A, S69E, S69V, S98R, Q102E, S115T, Q150E, Q157E, T159Q, G161R, A172E, G181N, S182Y, S182V, K185E, V187N, K192I, A206G, Q208V, and K212E. The variant of claim 1, comprising:

6. 2. The variant of claim 1, having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

7. ・S69V+K86E+Q102E+K147N+K155E+A172D+K192A+Q208V; ・G20C+K30C+D32E+V35I+A111P; ・S69V+K86E+Q102E+K147N+K155E+A172D+G181N+K192A+Q208V; ・D32E+V35I+N77E+A111P+G181N+S182V; ・D32E+E34N+V35I+K86E+A111P+G181N; ・S69V+K86E+Q102E+K105T+K147N+K155E+A172D+K192A+Q208V; ・S69V+Q102E+Q157E+T159Q+N214D+N217A; ・S26H+D32E+V35I+A111P+K155L; ・K18S+D32E+V35I+A111P+K185I+K215T; ・D32E+V35I+A111P+K185I+K215T; ・D32E+V35I+K82G+A111P+G181N+S182V; ・D32E+V35I+A111P+K178M+K212T; ・K18S+D32E+V35I+A111P+K178S+K212L; ・S69V+Q102E+V138A+S144R+Q157E+T159Q+A172E+G181N; ・D32E+V35I+A111P+K204L+K215I; ・K18F+D32E+V35I+A111P+K185Q; ・D32E+V35I+A111P+K147N+K204L; ・S69V+K86E+Q102E+K105D+K147N+K155E+A172D+K192A+Q208V; ・D32E+V35I+S69V+K86E+Q102E+A111P+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; ・D32E+V35I+A111P+K147N; ・S69V+K86E+Q102E+S115T+K147N+K155E+A172D+K192A+Q208V; ・ D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; ・ D32E+V35I+A111P+N140H; ・ D32E+V35I+K65Q+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+G181N+K192A+Q208V+N217A; ・ D32E+V35I+S69V+K86E+Q102E+A111P+K147N+K155E+T159Q+A172D+K178S+G181N+K192A+Q208V+N217A; ・ D32E+V35I+A111P; ・ S26H+D32E+V35I+A111P; ・ K18Q+D32E+V35I+K105E+A111P+K155F; ・ D32E+V35I+A111P+G199E; ・ S69V+Q102E+Q157E+T159Q+G181N; ・ D32E+V35I+K67A+A111P+P175N+S182V; ・ D32E+V35I+A111P+N214D; ・ S69V+Q102E+Q157E+T159Q+A172E; ・ D32E+V35I+S69E+A111P; ・ D32E+V35I+Q85E+A111P; ・ K18Q+D32E+V35I+A111P; ・ D32E+V35I+A101E+K105T+A111P+G181N+K212E+Y218E; ・ D32E+V35I+A111P+G181N+S182V+K185E; ・ D32E+V35I+S98R+A111P+G181N+S182Y; ・ D32E+V35I+A101E+Q102E+A111P+G181N; ・ T19P+D32E+V35I+Q102E+K105E+A111P; ・ D32E+V35I+D72V+A111P; ・ S69V+K86E+Q102E+F112W+K147N+K155E+A172D+K192A+Q208V; ・ S69V+Q102E+A111P+Q157E+T159Q+A172E; ・ K67A+S69V+Q102E+K147N+Q157E+T159Q+A172E+D197S; ・ S69V+Q102E+S115T+Q157E+T159Q+A172E; ・ S69V+K86E+Q102E+G136E+K147N+K155E+A172D+K192A+Q208V; ・ D32E+V35I+K67A+A111P+K204G; ・ D32E+V35I+A101E+K105E+A111P+S182V; ・ D32E+V35I+A101E+K105E+A111P+S182V; ・ D32E+V35I+A101T+K105N+A111P+K147E+G181N+S182Y; ・ D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; ・ D32E+V35I+Q102E+A111P; ・ S26H+S69V+Q102E+A111P+Q157E+T159Q+A172E; ・ D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; ・ D32E+V35I+K67A+K105T+A111P+K178L+K185T; ・ T19P+D32E+V35I+A101E+K105T+A111P+K160A+G181N; ・ D32E+V35I+S69V+Q102E+A111P+Q157E+T159Q+A172E; ・ D32E+V35I+Q102E+A111P; ・ K18S+D32E+V35I+Q102E+K105E+A111P; ・ D32E+V35I+K65E+A101E+K105T+A111P+N217A; ・ D32E+V35I+K105T+A111P+G181N+K204W+K215L; ・ D32E+V35I+K105T+A111P+K178L+G181N+K212T; ・ S26H+S69V+Q102E+A111P+N146H+Q150E+Q157E+T159Q+A172E; ・ K18Q+D32E+V35I+K67A+K105T+A111P+K215V; ・ D32E+V35I+Q85E+A111P; ・ K18S+T19P+D32E+V35I+A101E+K105T+A111P+G181N; ・ D32E+V35I+A101E+K105N+A111P; ・ K18M+D32E+V35I+K67A+K105T+A111P+K178S; ・ D32E+V35I+K105T+A111P+G181N; ・ D32E+V35I+K67A+K105T+A111P+K185G+K212M; ・ D32E+V35I+A101T+K105N+A111P+G181N+S182Y+N214D; ・ T19P+D32E+V35I+A101E+K105T+A111P+K178S+G181N; ・ D32E+V35I+K105T+A111P+G181N+K185L+K192V+K204W; ・ D32E+V35I+S98R+K105N+A111P+K147N+G181N+S182Y; ・ D32E+V35I+K105N+A111P+G181N+S182Y; ・ D32E+V35I+K67A+K105T+A111P; ・ D32E+V35I+K53F+K105N+A111P+G181N+S182Y+K212E; ・ K18S+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; ・ D32E+V35I+S98R+K105N+A111P+Q158D+G181N+S182Y; ・ D32E+V35I+K67A+K105T+A111P+K190V+K204G; ・ N4K+D32E+V35I+S98R+K105N+A111P+G181N+S182Y+K212E; ・ D32E+V35I+K67A+K105T+A111P+K147N+K190N; ・ D32E+V35I+S98R+K105N+A111P+G181N+S182Y+Q208V; ・ D32E+V35I+A101E+K105T+A111P+G181N+K212E+N214D; ・ D32E+V35I+K82T+A101E+K105T+A111P+G181N+K212E; ・ D32E+V35I+A111P+S115T; ・ S26H+S69V+Q102E+A111P+K147N+A149S+Q150E+Q157E+T159Q+A172E; ・ K18S+D32E+V35I+K67A+K105T+A111P+K185L+K192F; ・ V12L+D32E+V35I+S98R+K105N+A111P+G181N+S182Y; ・ D32E+V35I+S69E+K105N+A111P+K147E+G181N+S182Y; ・ K18S+D32E+V35I+A101E+K105T+A111P+G181N+K212E; ・ D32E+V35I+K65E+K105N+A111P+G181N+S182Y+K212E; ・ D32E+V35I+S98R+K105N+A111P+G181N+S182Y+D189N+N214D; ・ D32E+V35I+S98R+A101T+K105N+A111P+G181N+S182Y; ・ D32E+V35I+K67A+A111P+Q157E+T159Q+G181N+A206G+Q208V; ・ D32E+V35I+K67A+S69R+A111P+G181N+K212E; ・ D32E+V35I+K67A+A111P+G181N+A206G+Q208V; ・ D32E+V35I+A101E+K105T+A111P+G181N; ・ D32E+V35I+K67A+A101E+K105D+A111P+N217A; ・ D32E+V35I+K67A+A111P+Q157E+G181N+Q208V; ・ D32E+V35I+K67A+A111P+Q157E+A172E+G181N+A206G+Q208V; ・ S26H+D32E+V35I+K67A+S69R+A111P+G181N+K212E; ・ D32E+V35I+S69E+S98R+K105N+A111P+G181N+S182Y; ・ D32E+V35I+K67A+K105N+A111P+G181N+S182Y+K212E; ・ D32E+V35I+K105D+A111P+S115T; ・ D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; ・ D32E+V35I+K65E+K67A+K105N+A111P+G181N+S182Y; ・ S69V+Q102E+K105N+A111P+S115T+V138M+Q150E+G161R+G181N+V187N+K192I; ・ D32E+V35I+K67A+S69V+K105T+A111P+Q157E+G181N+A206G+Q208V; ・ S69V+Q102E+K105N+A111P+S115T+G161R+G181N+V187N+K192I; ・ S69V+Q102E+K105N+A111P+S115T+Q150E+K160T+G161R+G181N+V187N+K192I; ・ S69V+D72S+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・ D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A; ・ S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+S134H+Q150E+G161R+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; ・ D32P+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+T120H+N146H+Q150E+G161R+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+Q157E+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; ・ S26H+D32P+S69V+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+V187N+K192I; ・ K65Q+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・ S69A+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・ D32P+S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; ・S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; ・S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I; ・S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+A172D+G181N+V187N+K192I; ・D32P+S69A+Q102E+K105N+A111P+S115T+N146H+Q150E+G161R+G181N+K192I; ・D32P+S69V+Q102E+K105D+A111P+S115T+Q150E+G161R+G181N+V187N+K192I; and ・S69V+Q102E+K105N+A111P+S115T+G137R+Q150E+G161R+G181N+V187N+K192I 2. The variant of claim 1, comprising a set of substitutions selected from the group consisting of:

8. 8. The variant of claim 7, comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, together with one of said sets of substitutions.

9. 2. The variant of claim 1, wherein the variant has improved stability compared to the polypeptide of SEQ ID NO: 1 or a parent polypeptide having the same amino acid sequence as the variant without the substitution, for example, the improved stability is selected from the group consisting of detergent stability and thermal stability.

10. A detergent composition comprising the DNase variant according to any one of claims 1 to 9 and at least one detergent auxiliary ingredient.

11. 11. Use of the detergent composition according to claim 10 in cleaning processes such as laundry or hard surface cleaning, e.g. dishwashing.

12. 11. A method of cleaning an item, the method comprising exposing the item to a wash liquor comprising the detergent composition of claim 10, for example the item being a fabric or hard surface.

13. 1. A method of laundering a fabric comprising: a) exposing the fabric to a wash liquor comprising the detergent composition of claim 10; b) completing at least one cleaning cycle; and optionally c) rinsing said items; and A method comprising:

14. A polynucleotide encoding the variant of any one of claims 1 to 9, a nucleic acid construct or expression vector comprising said polynucleotide, or a recombinant host cell transformed with said polynucleotide.

15. 1. A method for producing a DNase variant, comprising: a) culturing the recombinant host cell of claim 14 under conditions suitable for expression of said variant; b) recovering said variant; A method comprising: