Cleaning compositions containing polypeptide variants

Abstract

A detergent composition comprising a DNase variant and a cleaning adjunct. A method of treating a surface, such as a fabric, by contacting the surface with an aqueous wash liquor having the detergent composition. The composition and method are, among others, for improving the whiteness of the fabric, improving stain removal from the fabric, removing malodors from the fabric, providing anti-wrinkle benefits, anti-redeposition benefits, and / or improving the drying of the fabric.

Description

[Technical Field]

[0001] (Reference to sequence listing) The present specification contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to detergent compositions and cleaning methods comprising specific DNase enzymes with improved properties, such as improved stability in detergents and / or during storage. The compositions are preferably laundry detergent compositions and / or hard surface cleaning compositions, such as dishwashing compositions, suitable for use in hand or automatic washing. The invention also relates to methods for making such detergent compositions and methods for cleaning surfaces by hand and / or in an automatic washing machine, as well as methods for using such compositions. [Background technology]

[0003] Hard surfaces and fabrics are exposed to soils and microorganisms from the environment and personal soils. Microorganisms contained in accumulated soil can promote the formation of soils of microbial origin, such as biofilm soils. For example, in laundry, biofilm-producing bacteria include Acinetobacter, Aeromicrobium, Brevundimonas, Microbacterium, Micrococcus luteus, Pseudomonas, Staphylococcus epidermidis, and Stenotrophomonas. Biofilms contain a matrix of extracellular polymeric substances (EPS): a polymeric mixture generally containing extracellular DNA, proteins, and polysaccharides. These stains are sticky, difficult to remove, and tend to induce further adhesion of other soils. Therefore, household care-related stains are complex and may contain particulates, proteins, starches, oils, and EPS. As a result, stain removal is also complex, and various enzymes, including deoxyribonuclease (DNase) enzymes, can be useful.

[0004] Detergent compositions containing DNase are already known. However, to be useful in household cleaning processes, enzymes such as DNase must be stable in the detergent composition, for example, in the presence of detergent ingredients such as surfactants, builders, and / or bleaches. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides detergent compositions containing DNase enzymes with good stability. [Means for solving the problem]

[0006] The present invention provides a detergent composition comprising a polypeptide having DNase activity and good stability in the detergent composition, and a cleaning adjuvant, wherein the polypeptide is a DNase variant comprising at least one, and preferably two or more, substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S compared to the polypeptide of SEQ ID NO: 1, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1 and has DNase activity.

[0007] The present invention also provides a method of treating a surface, preferably a fabric, the method comprising contacting the surface with an aqueous wash solution comprising a polypeptide having DNase activity, the polypeptide being a DNase variant comprising at least one substitution, and typically two or more substitutions, selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S compared to the polypeptide of SEQ ID NO: 1, the variant comprising a polypeptide having at least 60% sequence identity to SEQ ID NO: 1, and a cleaning adjunct.

[0008] Preferably, the surface is contacted with the aqueous cleaning solution at a temperature of 60° C. or less, or more preferably at a temperature of 40° C. or less or 35° C. or less, and most preferably at a temperature of 30° C. or less or even 30° C. or less, and (iii) rinsing the surface. The compositions and methods herein are particularly useful for treating surfaces, for example, fabrics made from synthetic or natural materials, including blended fabrics such as cotton, wool, silk, polyester, nylon, elastane, or polycotton, and / or hard surfaces such as dishware.

[0009] The present invention also relates to the use of a composition or method as described above for improved cleaning, such as deep cleaning; maintaining or improving the whiteness of fabrics; improved stain removal from surfaces such as fabrics; reducing or eliminating malodors from surfaces such as fabrics; preventing fabrics from wrinkling; improving fabric drying; preventing soil redeposition, preventing and / or reducing surface stickiness, pre-treating stains, and preventing and / or reducing the adhesion of soils to surfaces.

[0010] The present invention also relates to a method of making a detergent composition comprising preparing a polypeptide having DNase activity as described herein and mixing it with a cleaning adjunct.

[0011] array SEQ ID NO: 1 A variant of the polypeptide of SEQ ID NO: 27 obtained from Bacillus sibiricus SEQ ID NO: 2 Mature polypeptide obtained from Bacillus horikoshii SEQ ID NO: 3 Mature polypeptide obtained from Bacillus sp. strain 62520 SEQ ID NO: 4 Mature polypeptide obtained from Bacillus horikoshii SEQ ID NO: 5 Mature polypeptide obtained from Bacillus horikoshii SEQ ID NO: 6 Mature polypeptide obtained from Bacillus sp. strain 16840 SEQ ID NO: 7 Mature polypeptide obtained from Bacillus sp. strain 16840 SEQ ID NO: 8 Mature polypeptide obtained from Bacillus sp. strain 62668 SEQ ID NO: 9 Mature polypeptide obtained from Bacillus sp. strain 13395 SEQ ID NO: 10 Mature polypeptide obtained from Bacillus horneckiae SEQ ID NO: 11 Mature polypeptide obtained from Bacillus sp. 11238 SEQ ID NO: 12 Mature polypeptide obtained from Bacillus sp. strain 62451 SEQ ID NO: 13 Mature polypeptide obtained from Bacillus sp. strain 18318 SEQ ID NO: 14 Mature polypeptide obtained from Bacillus idriensis SEQ ID NO: 15 Mature polypeptide obtained from Bacillus algicola SEQ ID NO: 16 Mature polypeptide obtained from environmental sample J SEQ ID NO: 17 Mature polypeptide obtained from Bacillus vietnamensis SEQ ID NO: 18 Mature polypeptide obtained from Bacillus hwajinpoensis SEQ ID NO: 19 Mature polypeptide obtained from Paenibacillus mucilaginosus SEQ ID NO: 20 Mature polypeptide obtained from Bacillus indicus SEQ ID NO: 21 Mature polypeptide obtained from Bacillus maris flavi SEQ ID NO: 22 Mature polypeptide obtained from Bacillus luciferensis SEQ ID NO: 23 Mature polypeptide obtained from Bacillus maris flavi SEQ ID NO: 24 Mature polypeptide obtained from Bacillus sp. strain SA2-6 SEQ ID NO: 25 Motif [D / M / L][S / T]GYSR[D / N] SEQ ID NO: 26 Motif ASXNRSKG SEQ ID NO: 27 Mature polypeptide obtained from Bacillus sibiricus SEQ ID NO: 28 Protease variant

[0012] definition The term "clade" refers to a group of polypeptides clustered together based on homologous characteristics derived from a common ancestor. A clade of polypeptides can be visualized as a phylogenetic tree, where a clade is a group of polypeptides consisting of a common ancestor and all of its direct descendants. Polypeptides that form a group, e.g., a clade, shown in a phylogenetic tree often share common characteristics and are more closely related than other polypeptides that do not belong to the clade.

[0013] The term "deep cleaning" refers to the disruption or removal of components of organic matter, such as biofilms, such as polysaccharides, proteins, DNA, dirt, or other components present in organic matter.

[0014] The terms "DNase," "DNase variant," or "DNase parent" refer to a polypeptide having DNase activity (i.e., deoxyribonuclease activity) that catalyzes the hydrolytic cleavage of phosphodiester bonds in DNA, thereby degrading DNA. DNases belong to the esterase subgroup (EC number 3.1) of hydrolases. DNases are classified, for example, in EC 3.1.11, EC 3.1.12, EC 3.1.15, EC 3.1.16, EC 3.1.21, EC 3.1.22, EC 3.1.23, EC 3.1.24, and EC 3.1.25, as well as EC 3.1.21.X (where X = 1, 2, 3, 4, 5, 6, 7, 8, or 9). The terms "DNase" and "polypeptide having DNase activity" may be used interchangeably throughout this application. For purposes of the present invention, deoxyribonuclease activity can be determined according to the procedure described in the assay below. The DNase variants of the present invention preferably have at least one improved property compared to the parent DNase. In one embodiment, the DNase variants have improved stability, e.g., improved storage stability in a detergent composition, compared to the parent DNase. Preferably, the DNase variants have a half-life improvement factor (T1 / 2 IF, or HIF) of at least 1.1, more preferably at least 1.2, preferably at least 1.5, more preferably at least 2, e.g., at least 3, after storage in a liquid detergent, as compared to the half-life of a reference DNase, e.g., the DNase of SEQ ID NO: 1 and / or the parent DNase, as determined, e.g., as described in Example 3 herein. The half-life improvement factor of a DNase variant is calculated as the half-life of the DNase variant relative to the half-life of the reference DNase.

[0015] An "effective amount" with respect to an enzyme refers to the amount of enzyme necessary to achieve the enzymatic activity required for a particular application, e.g., in a given detergent composition. Such an effective amount is readily ascertainable by one of ordinary skill in the art and is based on many factors, including the specific enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granule, bar) composition is required. The term "effective amount" of a DNase variant refers to the amount of said DNase variant that achieves a desired level of enzymatic activity, e.g., in a given detergent composition.

[0016] The term "expression" includes any step involved in producing a variant, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0017] The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant, operably linked to additional nucleotides that effect its expression.

[0018] The term "fabric" includes any textile material. Thus, the term is intended to encompass fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, in addition to garments.

[0019] The term "host cell" refers to any cell type that is amenable to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0020] The term "improved properties" refers to characteristics associated with a variant that are improved compared to a parent and / or DNase polypeptide having SEQ ID NO: 1, or compared to a DNase having the same amino acid sequence as said variant but lacking the modifications defined herein at one or more of the above-specified positions. Such improved properties include, but are not limited to, stability, such as detergent stability, e.g., detergent storage stability, and cleaning performance, e.g., deep cleaning effect, which may include de-adhesion effect. The DNase variant preferably has improved stability, in particular improved detergent storage stability, compared to the polypeptide of SEQ ID NO: 1.

[0021] The term "improved DNase activity" is defined herein as a change in DNase activity relative to the activity of a parent DNase, for example, due to increased catalysis of the hydrolytic cleavage of phosphodiester bonds in DNA, such as compared to a DNase variant having SEQ ID NO: 1.

[0022] The term "improved cleaning performance" includes, but is not limited to, the term "deep cleaning effect." Improved performance, e.g., deep cleaning performance, of the DNase variants of the present invention is measured compared to the DNase parent, e.g., the DNase set forth in SEQ ID NO: 1 or SEQ ID NO: 27. Improved performance, e.g., deep cleaning performance, can be expressed as the remission value of the stained swatch. After washing and rinsing, the swatches are laid flat and allowed to air dry overnight at room temperature. All washed swatches are evaluated the same day after washing. Light reflectance evaluation of the swatches is performed using a Macbeth Color Eye 7000 reflectance spectrometer with a very small aperture. Measurements are performed without ultraviolet light in the incident light, and the reflectance at 460 nm is extracted. A positive reaction indicates that the stain has been removed. This may include stains that adhere to the fabric due to, for example, a sticky biofilm layer.

[0023] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. The N-terminus of a mature polypeptide can be determined experimentally based on EDMAN N-terminal sequencing data and Intact MS data. It is known in the art that a host cell 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.

[0024] The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having DNase activity.

[0025] The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that has been isolated from a naturally occurring gene or modified to contain segments of nucleic acid that are not otherwise found in nature, or that is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

[0026] The term "operably linked" refers to a configuration in which control sequences are positioned in appropriate relation to a coding sequence of a polynucleotide so that the control sequences direct the expression of the coding sequence.

[0027] The terms "parent DNase," "DNase parent," or "precursor DNase" may be used interchangeably. In the context of the present invention, a "parent DNase" should be understood as a DNase in which at least one modification is made in the amino acid sequence to generate a DNase variant with an amino acid sequence that is less than 100% identical to the DNase sequence in which the modification is made. Thus, a parent is a DNase that has an identical amino acid sequence compared to the variant but does not have a modification at one or more of the specified positions. The DNase parent may be 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 71%, 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% or 100% identity to the polypeptide set forth in SEQ ID NO:1.

[0028] The term "relevant washing conditions" is used herein to refer to the conditions used in homes in the detergent market segment, in particular washing temperature, time, washing machine, detergent concentration, detergent type, and water hardness.

[0029] The term "stability" includes storage stability and stability during use, e.g., during the washing process, and reflects the stability of the DNase variants of the present invention as a function of time, e.g., how much activity is retained when the DNase variant is maintained in a detergent or detergent solution. Stability is affected by many factors, e.g., pH, temperature, detergent composition, e.g., amount of builder, surfactant, etc. DNase stability can be measured as described in Example 3. The terms "improved stability" or "increased stability" are defined herein as a variant DNase that exhibits increased stability in solution, e.g., a detergent solution, relative to the stability of the parent DNase and / or relative to SEQ ID NO: 1. "Improved stability" and "increased stability" can refer to detergent stability during storage or use ("washing stability").

[0030] The term "variant" refers to a polypeptide that has DNase activity and contains a substitution at one or more positions as defined elsewhere herein. A substitution refers to replacing an amino acid occupying a position with a different amino acid; a deletion refers to removing an amino acid occupying a position; and an insertion refers to adding amino acids, e.g., 1 to 10 amino acids, preferably 1 to 3 amino acids, adjacent to the amino acid occupying a position. Variants of the invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, of the DNase activity of the polypeptide of SEQ ID NO: 1.

[0031] The term "washing solution" refers to an aqueous solution containing a DNase variant and a washing adjuvant as described herein.

[0032] The terms "water hardness" or "hardness" or "dH" or "°dH" as used herein refer to the German scale of hardness, where one degree is defined as 10 milligrams of calcium oxide per liter of water.

[0033] Mutant designation arrangements Unless otherwise indicated, the polypeptide disclosed in SEQ ID NO: 1 is used for the purpose of the present invention to determine the corresponding amino acid residue in another DNase. The amino acid sequence of another DNase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program, preferably version 5.0.0 or higher, of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0034] Identification of corresponding amino acid residues in other DNases can be achieved using, but is not limited to, MUSCLE (multiple sequence comparison by log prediction, version 3.5 or higher; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or higher; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA employing It can be determined by aligning multiple polypeptide sequences using several computer programs, including EMBOSS EMMA using ClustalW (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0035] Other pairwise sequence comparison algorithms may be used when other enzymes diverge from the polypeptide of SEQ ID NO: 1, such that traditional sequence-based comparisons fail to detect these relationships (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615). Greater sensitivity in sequence-based searches can be achieved by using search programs that utilize probabilistic representations (profiles) of polypeptide families to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process, allowing the detection of distant homologs (see Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved when a family or superfamily of polypeptides has more than one representation in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvate potential) as input to neural networks that predict the structural fold of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models of the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

[0036] For proteins of known structure, several tools and resources are available for searching and creating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures; implementations of these algorithms can then be used to query structural databases for the target structure to find potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0037] Because different amino acids may be present at a given position depending on the parent selected for the variant, the amino acid positions are designated as #1, #2, etc. in the definitions below. In describing the variants of the present invention, the following nomenclature is adopted for ease of reference: The generally accepted IUPAC one-letter or three-letter amino acid abbreviations are used.

[0038] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of valine with alanine at position #1 is designated "Val#1Ala" or "V#1A." Multiple mutations are separated by a plus sign ("+") or a comma (,), e.g., "Val#1Ala+Pro#2Gly" or V#1A,P#2G" represents the substitution of valine (V) and proline (P) with alanine (A) and glycine (G) at positions #1 and #2, respectively. When more than one amino acid can be substituted at a given position, these are listed in parentheses, such as [X] or {X}. Thus, if both Trp and Lys can be substituted for the amino acid occupying position #1, this is designated as X#1{W,K}, X#1[W,K], or X#1[W / K] (where X represents the amino acid residue present at that position in the parent DNase, e.g., the DNase set forth in SEQ ID NO: 1 or a DNase with at least 60% identity thereto). In some cases, the variant can also be designated #1{W,K} or X#2P, indicating that the substituted amino acid varies depending on the parent.

[0039] For convenience, SEQ ID NO: 1 is used to number the substitutions and other mutations disclosed herein, and therefore the amino acid at the corresponding position in SEQ ID NO: 1 will be indicated, for example, as T65V. However, it will be clear to those skilled in the art that DNase variants containing T65V are not limited to parent DNases having a threonine at the position corresponding to position 65 in SEQ ID NO: 1. For example, in a parent DNase having an asparagine at position 65, those skilled in the art would change the mutation designated T65V to N65V. If the parent DNase has an alanine at position 65, those skilled in the art would recognize that the parent DNase is unchanged at this position. The same applies to deletions and insertions described below. This may also be indicated using "X," defined as any of the 20 naturally occurring amino acids, as the original amino acid; for example, X65V means that any amino acid residue other than V at position 65 is changed to V.

[0040] Deletions: For amino acid deletions, use the following nomenclature: original amino acid, position, * Thus, the deletion of valine at position #1 is expressed as "Val#1 * " or "V#1 * " Multiple deletions are represented as, for example, "Val#1 * +Pro#2 * " or V#1 * ,P#2 * " and are separated by a plus sign ('+') or a comma.

[0041] Insertion: For example, the insertion of an additional amino acid residue, such as a lysine, after Val#1 can be indicated by Val#1ValLys or V#1VK. Alternatively, the insertion of an additional amino acid residue, such as a lysine, after V#1 can be indicated by * For example, when more than one amino acid residue, such as Lys and Gly, is inserted after #1, this can be represented as Val#1ValLysGly or V#1VKG. In such cases, the inserted amino acid residue may be numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue, in this example: * #1aK * #1bG.

[0042] Multiple modifications: Variants containing multiple modifications are separated by a plus sign ("+") or a comma (,), e.g., "Val#1Trp+Pro#2Gly" or "V#1W,P#2G" or "V#1W+P#2G" represent substitutions of tryptophan and glycine for valine and proline at positions #1 and #2, respectively, as described above.

[0043] Different modifications: When different modifications can be introduced at a position, they may be separated by a comma, for example, "Val#1Trp,Lys" or V#1W,K represents the substitution of valine with tryptophan or lysine at position #1. Thus, "Val#1Trp,Lys+Pro#2Asp" represents the following variants: "Val#1Trp+Pro#2Asp", "Val#1Lys+Pro#2Asp" or V#1W,K+P#2D.

[0044] Different alterations may also be indicated using the nomenclature [IV] or [I / V], meaning that the amino acid at this position may be isoleucine (Ile, I) or valine (Val, V). Similarly, the nomenclature [LVI] and [L / V / I] means that the amino acid at this position may be leucine (Leu, L), valine (Val, V), or isoleucine (Ile, I), as well as other combinations described herein. For example, T65I / V refers to the substitution of T at position 65 with either I or V. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention provides a detergent composition comprising a DNase variant and a cleaning adjunct, wherein the DNase variant comprises at least one substitution selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S when compared to the polypeptide of SEQ ID NO: 1, and the variant has at least 60% sequence identity to SEQ ID NO: 1, such as at least 65%, at least 70%, at least 75%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the DNase variant comprises at least one, preferably at least two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S.

[0046] The detergent composition of the present invention is for laundry cleaning and / or treatment and / or hard surface cleaning or treatment, particularly for dishwashing. The composition of the present invention may be a solid (e.g., bar, tablet, powder, granule), liquid, gel and / or paste, or may be in the form of a sheet. A preferred form is a unit dose form, which may be a tablet, a sheet, or a water-soluble pouch, preferably containing a liquid and / or solid composition. A multi-compartment pouch is preferred. A preferred unit dose composition is preferably a multi-compartment unit dose, and contains a solid (preferably in powder form), or a liquid, or a combination thereof. Particularly preferred is a unit dose form, in the form of a pouch, optionally a multi-compartment pouch, containing a liquid laundry detergent encapsulated in a water-soluble material.

[0047] DNase variants In one aspect the DNase variant comprises one or preferably two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D and L181S when compared to the polypeptide of SEQ ID NO: 1, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, such as at least 70%, at least 75%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90% or at least 95% sequence identity to SEQ ID NO: 1 and has DNase activity.

[0048] DNase variants may contain two, three, four, five or more of the above substitutions, typically two, three, four or five substitutions, and may optionally contain one or more further substitutions as described herein.

[0049] In one embodiment, the DNase variant comprises the substitutions N61D+T65I, and optionally one or more additional substitutions described herein.

[0050] In one embodiment, the DNase variant comprises the substitutions N61D+T65V, and optionally one or more additional substitutions described herein.

[0051] In one embodiment, the DNase variant comprises the substitutions N61D+S82R, and optionally one or more additional substitutions described herein.

[0052] In one embodiment, the DNase variant comprises the substitutions N61D+K107Q, and optionally one or more additional substitutions described herein.

[0053] In one embodiment, the DNase variant comprises the substitutions N61D+T127S, and optionally one or more additional substitutions described herein.

[0054] In one embodiment, the DNase variant comprises the substitutions N61D+T127V, and optionally one or more additional substitutions described herein.

[0055] In one embodiment, the DNase variant comprises the substitutions N61D+G149N, and optionally one or more additional substitutions described herein.

[0056] In one embodiment, the DNase variant comprises the substitutions N61D+S164D, and optionally one or more additional substitutions described herein.

[0057] In one embodiment, the DNase variant comprises the substitutions N61D+L181S, and optionally one or more additional substitutions described herein.

[0058] In one embodiment, the DNase variant comprises the substitutions T65I+S82R, and optionally one or more additional substitutions described herein.

[0059] In one embodiment, the DNase variant comprises the substitutions T65I+K107Q, and optionally one or more additional substitutions described herein.

[0060] In one embodiment, the DNase variant comprises the substitutions T65I+T127S, and optionally one or more additional substitutions described herein.

[0061] In one embodiment, the DNase variant comprises the substitutions T65I+T127V, and optionally one or more additional substitutions described herein.

[0062] In one embodiment, the DNase variant comprises the substitutions T65I+G149N, and optionally one or more additional substitutions described herein.

[0063] In one embodiment, the DNase variant comprises the substitutions T65I+S164D, and optionally one or more additional substitutions described herein.

[0064] In one embodiment, the DNase variant comprises the substitutions T65I+L181S, and optionally one or more additional substitutions described herein.

[0065] In one embodiment, the DNase variant comprises the substitutions T65V+S82R, and optionally one or more additional substitutions described herein.

[0066] In one embodiment, the DNase variant comprises the substitutions T65V+K107Q, and optionally one or more additional substitutions described herein.

[0067] In one embodiment, the DNase variant comprises the substitutions T65V+T127S, and optionally one or more additional substitutions described herein.

[0068] In one embodiment, the DNase variant comprises the substitutions T65V+T127V, and optionally one or more additional substitutions described herein.

[0069] In one embodiment, the DNase variant comprises the substitutions T65V+G149N, and optionally one or more additional substitutions described herein.

[0070] In one embodiment, the DNase variant comprises the substitutions T65V+S164D, and optionally one or more additional substitutions described herein.

[0071] In one embodiment the DNase variant comprises the substitutions T65V+L181S, and optionally one or more additional substitutions as described herein.

[0072] In one embodiment, the DNase variant comprises the substitutions S82R+K107Q, and optionally one or more additional substitutions described herein.

[0073] In one embodiment, the DNase variant comprises the substitutions S82R+T127S, and optionally one or more additional substitutions described herein.

[0074] In one embodiment, the DNase variant comprises the substitutions S82R+T127V, and optionally one or more additional substitutions described herein.

[0075] In one embodiment, the DNase variant comprises the substitutions S82R+G149N, and optionally one or more additional substitutions described herein.

[0076] In one embodiment, the DNase variant comprises the substitutions S82R+S164D, and optionally one or more additional substitutions described herein.

[0077] In one embodiment, the DNase variant comprises the substitutions S82R+L181S, and optionally one or more additional substitutions described herein.

[0078] In one embodiment, the DNase variant comprises the substitutions K107Q+T127S, and optionally one or more additional substitutions described herein.

[0079] In one embodiment, the DNase variant comprises the substitutions K107Q+T127V, and optionally one or more additional substitutions described herein.

[0080] In one embodiment, the DNase variant comprises the substitutions K107Q+G149N, and optionally one or more additional substitutions described herein.

[0081] In one embodiment, the DNase variant comprises the substitutions K107Q+S164D, and optionally one or more additional substitutions described herein.

[0082] In one embodiment, the DNase variant comprises the substitutions K107Q+L181S, and optionally one or more additional substitutions described herein.

[0083] In one embodiment the DNase variant comprises the substitutions T127S+G149N and optionally one or more additional substitutions as described herein.

[0084] In one embodiment, the DNase variant comprises the substitutions T127S+S164D, and optionally one or more additional substitutions described herein.

[0085] In one embodiment, the DNase variant comprises the substitutions T127S+L181S, and optionally one or more additional substitutions described herein.

[0086] In one embodiment the DNase variant comprises the substitutions T127V+G149N and optionally one or more additional substitutions as described herein.

[0087] In one embodiment, the DNase variant comprises the substitutions T127V+S164D, and optionally one or more additional substitutions described herein.

[0088] In one embodiment the DNase variant comprises the substitutions T127V+L181S, and optionally one or more additional substitutions as described herein.

[0089] In one embodiment, the DNase variant comprises the substitutions G149N+S164D, and optionally one or more additional substitutions described herein.

[0090] In one embodiment, the DNase variant comprises the substitutions G149N+L181S, and optionally one or more additional substitutions described herein.

[0091] In one embodiment, the DNase variant comprises the substitutions S164D+L181S, and optionally one or more additional substitutions described herein.

[0092] In a preferred embodiment, the DNase variant comprises the substitution T65I or T65V together with at least two of the substitutions N61D, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S.

[0093] In another preferred embodiment the DNase variant comprises the substitution N61D together with at least two of the substitutions T65I / V, S82R, K107Q, T127S / V, G149N, S164D and L181S, such as two, three or four of the above substitutions. In a preferred embodiment the variant comprises N61D together with at least two of the substitutions T65I / V, S82R, K107Q, T127S and S164D, such as two, three or four of the above substitutions.

[0094] In another preferred embodiment the DNase variant comprises at least two of the substitutions N61D, T65I, T65V, K107Q, T127S, T127V, G149N, S164D and L181S, such as two, three or four of the above substitutions together with the substitution S82R.

[0095] In another preferred embodiment the DNase variant comprises the substitution K107Q together with at least two of the substitutions N61D, T65I, T65V, S82R, T127S, T127V, G149N, S164D and L181S, such as two, three or four of the above substitutions.

[0096] In another preferred embodiment the DNase variant comprises the substitution T127S together with at least two of the substitutions N61D, T65I, T65V, S82R, K107Q, G149N, S164D and L181S, such as two, three or four of the above substitutions.

[0097] In another preferred embodiment, the DNase variant comprises the substitution G149N together with at least one of the substitutions N61D, T65I, T65V, S82R, K107Q, T127S, T127V, S164D, and L181S, such as two, three, or four of the above substitutions.

[0098] In another preferred embodiment, the DNase variant comprises the substitution S164D together with at least one of the substitutions N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, and L181S, such as two, three, or four of the above substitutions.

[0099] In some embodiments, the variant may further comprise at least one substitution selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W, for example one, two, or more, typically one, two, or three of the above substitutions.

[0100] Thus, the present invention also relates to a polypeptide having at least one substitution, such as two or more substitutions, selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S, as compared to the polypeptide of SEQ ID NO: 1, and at least one substitution, such as two or more substitutions, selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T , D116S, D116W, T171W, L181T, and L181W, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity, and has DNase activity.

[0101] The DNase variant preferably comprises: ● K21L+Q48D+T65I+S82R+K107Q+T127S, ● Q14R+K21L+Q48D+T65I+T127S, ● Q48D+T65I+S82R+T127S+S164D, ● N61D+T65I+K107Q+T127S+S164D, ● Q48D+T65I+S82R+K107Q+T127S, ● Q14R+N61D+T65I+S82R+K107Q, ● N61D+S68L+G149N, ● Q14R+N61D+T65I+S82R+T127S+S164D, ● K21L+Q48D+T65I+S82R+K107Q, ● Q14R+T65I+K107Q+T127S, ● N61D+T65I+S82R+T127S+S164D, ● K21L+N61D+T65I+S82R+K107Q+T127S, ● T65V+T127V+G149N, ● T65I+K107Q+T127S+S164D, ● N61D+T65I+S82R+K107Q, ● Q14R+K21L+N61D+T65I+S82R, ● Q14R+K21L+N61D+T65I+S82R+K107Q, ● Q14R+K21L+N61D+T65I+T127S, ● N61D+T65I+S82R+K107Q+T127S+S164D, ● Q14R+K21L+T65I+K107Q+T127S, ● N61D+T65I+K107Q+T127S, ● T65I+S82R+K107Q+S164D, ● K21L+N61D+T65I+S82R, ● K21L+N61D+T65I+T127S, ● N61D+T65I+S82R+S164D、 ● K21L+N61D+T65I+S82R+K107Q、 ● S68L+S106A+G149N、 ● N61D+T65I+T127S+S164D、 ● Q14R+K21L+N61D+T65I、 ● Q14R+K21L+T65I+T127S、 ● T65V+G149N、 ● T65V+R109T+T127V、 ● Q14R+K21L+T65I+K107Q、 ● K21L+T65I+S82R+K107Q、 ● K21L+T65I+K107Q+T127S、 ● N61D+S68L+S102Y+G149N+S164D+L181T、 ● N61D+S68L+S106A+G149N+S164D、 ● T65V+R109T+G149N、 ● T65V+T127V+T171W、 ● T65V+T127V+L181S、 ● T65I+S164D+L181W、 ● N61D+S66Y+S102Y+S164D、 ● N61D+S66Y+S164D、 ● N61D+T65V+S164D、 ● Q14W+N61D+T65I、 ● Q14W+N61D+T65I+S164D、 ● Q14W+N61D+T65I+S164D+L181W、 ● T65I+D116W+S164D+L181W、 ● T65I+D116W+S164D、 ● Q14W+T65I+S164D、 ● R109T+G149N、 ● G149N+T171W、 ● G149N、 ● S164D、 ● P25S+L33K+D56I+T65V+Y77T+T127V+L181S、 ● P25S+L33K+T65V+Y77T+T127V+L181S、 ● P25S+L33K+D56I+T65V+Y77T+R109Q+T127V+L181S、 ● P25S+L33K+D56I+T65V+Y77T+D116S+T127V+L181S、 ● D56L+T65V+T127V、 ● D56L+T65V+T127V+T171W、 ● T65V+G149N+T171W、 ● Q48D+T65I+K107Q+T127S+S164D、 ● T65V+Y77T+T127V、 ● T65V+R109T+T127V+G149N、 ● T65V+R109T+T171W、 ● T65V+R109T+G149N+T171W、 ● P25S+D56I+T65V+Y77T+T127V+L181S、 ● Q14R+K21L+T65I+K107Q+T127S、 ● N61D+S68L+S102Y、 ● S66Y+T127V+L181S、 ● N61D+T65I+S82R+K107Q+L181D、 ● T65V+G149N+L181E、 ● T65V+T127V+G149N+Y182D、 ● Q48D+N61D+T65I+S82R+K107Q、 ● Q48D+T65V+G149N、 ● N61D+T65I+S82R+T127S+S164D+Y182D、 ● N61D+T65I+S82R+T127S+S164D+L181D、 ● Q48D+N61D+T65I+K107Q+T127S+S164D、 ● N61D+T65I+S82R+T127S+S164D+Y182N、 ● T65I+K107Q+T127S+S164D+Y182D、 ● N61D+T65I+K107Q+T127S+S164D+L181E、 ● N61D+T65I+K107Q+T127S+S164D+L181D、 ● T65I+K107Q+T127S+S164D+L181T、 ● T65I+K107Q+T127S+S164D+L181E、 ● N61D+T65I+K107Q+T127S+S164D+Y182D、 ● T65I+K107Q+T127S+S164D+Y182N、 ● N61D+T65I+K107Q+T127S+S164D+L181Q、 ● N61D+T65V+S164D+Y182N、 ● N61D+T65I+K107Q+T127S+S164D+L181T、 ● T65I+K107Q+T127S+S164D+L181D、 ● T65I+K107Q+T127S+S164D+L181Q、 ● N61D+T65V+T127S+S164D、 ● Q48D+N61D+T65V+S164D、 ● K21L+N61D+T65I+K107Q+T127S、 ● Q14W+N61D+T65I+R109T+G149N+S164D+L181W、 ● K21L+N61D+T65I+K107Q、 ● Q14W+N61D+T65I+R109T+G149N、 ● Q14W+T65V+R109T+G149N+W154I+L181W、 ● Q14W+T65V+R109T+G149N+W154I+S164D、 ● Q14W+N61D+T65V+R109T+G149N+L181W、 ● Q14W+T65I+R109T+D116W+G149N+S164D+L181W、 ● T65V+R109T+T127V+T171W、 ● R109T+T127V+T171W、 ● N61D+T65I+S82R+T127S+S164D+T171E+D175G+L181S、 ● T65V+Y77R+G149N、 ● T65V+Y77H+G149N、 ● T65V+L76K+G149N、 ● T65V+L76R+G149N、 ● T19E+P25S+L33K+D56I+T65V+Y77T+T127V+L181S、 ● T65I+L181S、 ● S66L+G149N、 ● T65V+Y77T+G149N+L181S、 ● T65V+Y77T+G149N、 ● D56N+T65V+Y77T+G149N、 ● D56N+T65V+L76H+G149N、 ● D56Q+T65V+L76H+G149N、 ● S68Q+G149N+L181S、 ● G149N+L181S、 ● S66L+K107E+G149N+Q166D+L181S、 ● T65I+G149N+Q166D、 ● T65I+G149N+Q166D+Y182G、 ● T65I+G149N+Q166D+L181S、 ● T65I+G149N+L181S、 ● T65I+Q166D+Y182G、 ● T65I+Y182G、 ● S68Q+G149N+Q166D+L181S、 ● T65V+G149N+L181T、 ● T19I+S68Q+G149N+Y182G、 ● T65I+K107E+G149N+Q166D+Y182G, ● G149N+Q166D+Y182G, ● T65I+K107E+G149N, ● S68Q+G149N+Q166D, ● G149N+Y182G, ● G149N+Q166D+L181S, ● S68Q+K107E+G149N+Y182G, ● K107E+G149N+Y182G, ● T19E+T65I+K107Q+T127S+S164D, and - It comprises a set of substitutions selected from the group consisting of: N61D+T65I+K107Q+T127S+S164D+Y182N.

[0102] Preferably, the DNase variant may comprise or consist of SEQ ID NO: 1 with one of the sets of substitutions listed above.

[0103] Preferably, the DNase variant is ● T65V+T127V+L181S, ● N61D+T65I+S82R+K107Q, ● T65V+G149N, ● N61D+T65I+K107Q+T127S+S164D, ● N61D+T65I+T127S+S164D, ● N61D+T65V+S164D, ● T65V+T127V+G149N, ● N61D+T65I+S82R+T127S+S164D, and - It comprises a set of substitutions selected from the group consisting of: T65I+K107Q+T127S+S164D.

[0104] In a further preferred embodiment, the DNase variant may comprise or consist of SEQ ID NO: 1 with one of the preferred sets of substitutions listed above.

[0105] Preferably, the DNase variant comprises at least five, such as at least ten, or at least fifteen of the indicated amino acid residues at the following positions: I at position 1, Y at position 13, P at position 22, P at position 25, L at position 27, P at position 39, G at position 42, W at position 57, V at position 59, L at position 76, Y at position 77, R at position 109, D at position 116, P at position 144, H at position 147, L at position 167, D at position 175, L at position 181.

[0106] DNase variants may, for example, contain 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the indicated amino acid residues at the following positions: I at position 1, Y at position 13, P at position 22, P at position 25, L at position 27, P at position 39, G at position 42, W at position 57, V at position 59, L at position 76, Y at position 77, R at position 109, D at position 116, P at position 144, H at position 147, L at position 167, D at position 175, and L at position 181.

[0107] The DNase variant preferably comprises one or more substitutions, such as two, three, four or more substitutions selected from the group consisting of I1T, Y13S, P22T, P25S, L27S, P39S, G42S, W57S, V59S, L76V, Y77T, R109Q, D116S, P144S, H147A, L167S and D175G in addition to the substitutions disclosed elsewhere herein.

[0108] DNase variants preferably contain additional modifications, typically substitutions, at one or more positions of SEQ ID NO: 1 selected from the group consisting of positions 14, 21, 25, 33, 48, 56, 66, 68, 77, 102, 106, 109, 116, 171 and 181. Preferred substitutions at these positions include one or more substitutions selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T and L181W.

[0109] In one aspect, the present invention provides a polypeptide comprising a nucleotide sequence selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S, as compared to the polypeptide of SEQ ID NO: 1, and a nucleotide sequence selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q , R109T, D116S, D116W, T171W, L181T, and L181W, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, e.g., at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity, and has DNase activity.

[0110] In one aspect the invention provides a DNase variant comprising the substitution G149N and / or S164D compared to the polypeptide of SEQ ID NO: 1, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, such as at least 70%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity, and has DNase activity.

[0111] In one embodiment of this aspect, the DNase variant comprises the substitution G149N, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, for example at least 70%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity, and has DNase activity. The variant may further comprise one or more additional substitutions, for example, (i) one or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, S164D, and L181S, and / or (ii) one or more substitutions selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W. The DNase variant may further comprise one or more substitutions selected from the group consisting of I1T, Y13S, P22T, P25S, L27S, P39S, G42S, W57S, V59S, L76V, Y77T, R109Q, D116S, P144S, H147A, L167S, D175G, and L181S.

[0112] In another embodiment of this aspect, the DNase variant comprises the substitution S164D, wherein the variant has at least 60% sequence identity to SEQ ID NO: 1, e.g., at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity, and has DNase activity. The variant may further comprise one or more additional substitutions, for example, (i) one or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, and L181S, and / or (ii) one or more substitutions selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W. The DNase variant may further comprise one or more substitutions selected from the group consisting of I1T, Y13S, P22T, P25S, L27S, P39S, G42S, W57S, V59S, L76V, Y77T, R109Q, D116S, P144S, H147A, L167S, D175G, and L181S.

[0113] In a further aspect, the present invention provides a polypeptide having DNase activity comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0114] As noted above, the DNase variant has at least 60% sequence identity to SEQ ID NO: 1, for example at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO: 1. The DNase variant may have at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 1.

[0115] Thus, DNase variants may comprise more than two modifications, typically substitutions, such as 3, 4, 5, 6, 7, 8, 9 or 10 modifications compared to SEQ ID NO: 1. In other embodiments, DNase variants may comprise more than 10 modifications, typically substitutions, for example up to 15 or up to 20 modifications, compared to SEQ ID NO: 1.

[0116] Preferably, the DNase variants have improved properties, which may be, for example, improved stability in detergents, improved stability during washing, and / or improved thermal stability. Preferably, the DNase variants have improved stability, detergent stability, in particular improved detergent storage stability. Furthermore, the variants preferably have substantially maintained, or more preferably improved, relative cleaning performance compared to a reference DNase, which may be, for example, the DNase of SEQ ID NO: 1 or SEQ ID NO: 27.

[0117] Variants may contain additional mutations to those listed above, which preferably do not significantly alter the improved properties of the variant DNase and may, for example, be conservative substitutions.

[0118] Examples of conservative substitutions are those within the group 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 change the specific activity are known in the art and are described, for example, in H. Neurath and R.L. Hill, 1979, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0119] Essential amino acids in a polypeptide can be identified by procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for DNase activity to identify amino acid residues important for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of amino acids at putative contact sites. 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.

[0120] Parent DNase Preferably, the DNase is selected from any of the enzyme classes EC 3.1.11, EC 3.1.12, EC 3.1.15, EC 3.1.16, EC 3.1.21, EC 3.1.22, EC 3.1.23, EC 3.1.24, and EC 3.1.25.

[0121] Preferably, the DNase parent is obtained from a microorganism and the DNase is a microbial enzyme. The DNase is preferably of fungal or bacterial origin.

[0122] The DNase parent is preferably obtained from the genus Bacillus, such as Bacillus sibii, Bacillus strain 62451, Bacillus horikoshii, Bacillus strain 16840, Bacillus strain 62668, Bacillus strain 13395, Bacillus hornekiae, Bacillus strain 11238, Bacillus idriensis, Bacillus strain 62520, Bacillus argicola, Bacillus vietnamensis, Bacillus huajingpoensis, Bacillus indicus, Bacillus marisflavi, Bacillus luciferensis, Bacillus strain SA2-6, etc.

[0123] The DNase parent is preferably a DNase containing the conserved motif [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26), and belongs to a group of DNases within the GYS clade that share similar structural and functional properties. See, for example, WO 2017 / 060475. DNases of the GYS clade are preferably obtained from the genus Bacillus.

[0124] In one embodiment, the variant is a variant of a DNase parent of the GYS-clade having DNase activity, optionally wherein the parent comprises one or both motifs [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 25), ASXNRSKG (SEQ ID NO: 26), or the parent is a variant of a DNase polypeptide comprising one or both of these motifs, for example a variant of a DNase polypeptide obtained from a naturally occurring microorganism, wherein the polypeptide is a) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 1; b) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO:2; c) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 3; d) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO:4; e) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 5; f) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 6; g) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 7; h) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 8; i) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 9; j) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 10; k) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 11; l) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 12; m) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 13; n) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 14; o) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 15; p) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 16; q) a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 17; r) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 18; s) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 19; t) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 20; u) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 21; v) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 22; w) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 23; x) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 24; y) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 27.

[0125] Polypeptides with DNase activity and containing motifs of the GYS clade have particularly good deep cleaning properties, and compositions containing such DNases are therefore particularly effective in removing or reducing organic matter, such as soils of microbial origin, from items such as textiles or hard surfaces.

[0126] In preferred embodiments, the parent has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide set forth in SEQ ID NO: 1 which has DNase activity.

[0127] In some embodiments, the parent comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0128] Preparation of variants The present invention also relates to a method for making a detergent composition comprising obtaining a DNase variant having at least one improved property compared to the parent DNase, e.g., compared to the polypeptide set forth in SEQ ID NO: 1, and mixing with a cleaning adjunct.

[0129] Thus, this aspect relates to a method of making a detergent composition, the method comprising: (i) a) obtaining a DNase variant, comprising introducing into a parent DNase having at least 60% sequence identity to SEQ ID NO: 1 two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S (numbering based on SEQ ID NO: 1); (ii) recovering the variant, wherein the variant has DNase activity; (iii) mixing with a cleaning adjunct.

[0130] It will be appreciated that the method for obtaining a DNase variant may further comprise the introduction of any of the other substitutions or combinations of substitutions described above. For example, the method for obtaining a DNase variant may further comprise the introduction of at least one substitution selected from the group consisting of Q14R, Q14W, K21L, P25S, L33K, Q48D, D56I, D56L, S66Y, S68L, Y77T, S102Y, S106A, R109Q, R109T, D116S, D116W, T171W, L181T, and L181W. The method may also include the introduction of at least one substitution selected from the group consisting of I1T, Y13S, P22T, P25S, L27S, P39S, G42S, W57S, V59S, L76V, Y77T, R109Q, D116S, P144S, H147A, L167S, and D175G.

[0131] In a preferred embodiment, the parent DNase has at least 80% sequence identity to the polypeptide of SEQ ID NO: 1, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1. In another aspect, the parent DNase has the amino acid sequence of SEQ ID NO: 1.

[0132] In other embodiments, the variant may be based on a parent DNase selected from those mentioned above, i.e. a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% identity, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to any of these.

[0133] Variants can be prepared by procedures well known in the art, such as those described below.

[0134] Site-directed mutagenesis is a technique for introducing mutations at one or more defined sites in a parent encoding polynucleotide. Site-directed mutagenesis can be performed in vitro by PCR, involving the use of oligonucleotide primers containing the desired mutations. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves cleaving a site in a plasmid containing a parent encoding polynucleotide with a restriction enzyme, followed by ligation of an oligonucleotide containing the mutation in the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the cohesive ends of the plasmid and the insert to be ligated together. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955 and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0135] Site-directed mutagenesis can also be performed in vivo by methods known in the art (see, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16).

[0136] Synthetic gene construction involves the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using several techniques, such as the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), in which oligonucleotides are synthesized and assembled on a photoprogrammable microfluidic chip, and similar techniques.

[0137] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; Ibid. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0138] Mutagenesis / shuffling methods may be combined with high-throughput automated screening methods to detect the activity of cloned mutant polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutant DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0139] Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction is exemplified by processes that utilize synthesized polynucleotide fragments combined with PCR technology. In this manner, defined regions of a gene can be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, and still other regions may be subjected to error-prone or non-error-prone PCR amplification. Subsequent polynucleotide sequences may then be shuffled.

[0140] Nucleic Acid Constructs Disclosed herein are 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.

[0141] Polynucleotides can be manipulated in a variety of ways to provide for expression of a polypeptide. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0142] The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention. A promoter contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in a host cell, including mutated, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0143] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in bacterial host cells include the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13:97-107), the E. coli lac operon, and the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agarase gene (dagA), and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria" by Gilbert et al., 1980, Scientific American 242:74-94, and by Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0144] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in filamentous fungal host cells include promoters for Bacillus nidulans acetamidase, Bacillus niger neutral alpha-amylase, Bacillus niger acid-stable alpha-amylase, Bacillus niger or Bacillus awamori glucoamylase (glaA), Bacillus sibirica TAKA amylase, Bacillus sibirica alkaline protease, Bacillus sibirica phosphate isomerase, Fusarium oxysporum trypsin-like protease, and Bacillus niger trypsin-like protease. Protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum dahlia (WO 00 / 56900), Fusarium venenatum mucin (WO 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase promoters derived from Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor, and the NA2-tpi promoter (non-translated leader and modified promoters derived from the Bacillus neutral alpha-amylase gene in which the non-translated leader has been replaced with the non-translated leader derived from the Bacillus triose phosphate isomerase gene, including, but not limited to, modified promoters derived from the Bacillus niger neutral alpha-amylase gene in which the non-translated leader has been replaced with the non-translated leader derived from the Bacillus nidulans or Bacillus sibitriose phosphate isomerase gene, as well as mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0145] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0146] The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that functions in the host cell may be used in the present invention.

[0147] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB). Other terminators can be obtained from the genes for Bacillus nidulans acetamidase, Bacillus nidulans anthranilate synthase, Bacillus niger glucoamylase, Bacillus niger alpha-glucosidase, or Bacillus sibirica TAKA amylase.

[0148] Preferred terminators for filamentous fungal host cells include Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei β-glucosidase ... The genes for lobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor are obtained.

[0149] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0150] The regulatory sequence may also be an mRNA stabilizing region downstream of the gene's promoter and upstream of the coding sequence, which increases expression of the gene. Examples of suitable mRNA stabilizing regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0151] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0152] Preferred leaders for filamentous fungal host cells are obtained from the genes for Bacillus sibiricus TAKA amylase and Bacillus nidulans triose phosphate isomerase.

[0153] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0154] The control sequence may also be a polyadenylation sequence, which is operably linked to the 3' end of the polynucleotide and, when transcribed, is recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0155] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Bacillus nidulans anthranilate synthase, Bacillus niger glucoamylase, Bacillus niger alpha-glucosidase, Bacillus sibirica TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0156] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0157] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide, directing the polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence that is naturally linked in translation reading frame with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the native signal peptide coding sequence may simply be replaced with a foreign signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs an expressed polypeptide into the secretory pathway of a host cell may be used.

[0158] Signal peptide coding sequences useful in bacterial host cells include those obtained from the genes encoding Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0159] Signal peptide coding sequences effective in filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0160] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0161] The control sequence may also be a propeptide coding sequence encoding a propeptide located at the N-terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes a zymogen). The polypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophysora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0162] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0163] It may also be desirable to add regulatory sequences that regulate polypeptide expression relative to the growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which amplifies in the presence of methotrexate, and the metallothionein gene, which amplifies with heavy metals. In these cases, the polynucleotide encoding the polypeptide is operably linked to the regulatory sequence.

[0164] Expression vector The present application also discloses recombinant expression vectors containing polynucleotides encoding the DNase variants of the present invention, promoters, and transcriptional and translational stop signals. Various nucleotides and control sequences may be joined together to generate recombinant expression vectors, which may contain one or more convenient restriction enzyme sites to allow for insertion or substitution of a polynucleotide encoding a polypeptide at such sites. Alternatively, polynucleotides can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a vector suitable for expression. In creating an expression vector, a coding sequence is positioned within the vector such that the coding sequence is operably linked to suitable control sequences for expression. In a further embodiment, the codons of the polynucleotide sequence are modified by nucleotide substitution to correspond to the codon usage of the host organism in which the polypeptide of the present invention is intended to be produced. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and capable of expressing a polynucleotide. The choice of vector typically depends on the compatibility of the vector with the host cell into which it will be introduced. The vector may be a linear or closed circular plasmid.

[0165] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means for ensuring autonomous replication. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, may be used that together contain the entire DNA or transposon to be introduced into the genome of the host cell.

[0166] Vectors preferably contain one or more selectable markers which permit easy selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0167] Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinolothrixin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and their equivalents. Preferred for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Preferred for use in Trichoderma cells are the adeA, adeB, amdS, hph, and pyrG genes.

[0168] The selectable marker may be a double selectable marker system as described in WO 2010 / 039889. In one aspect, the double selectable marker is the hph-tk double selectable marker system.

[0169] The vector preferably contains elements that allow the vector to integrate into the genome of a host cell or to replicate autonomously within the cell independent of the genome. For integration into the genome of a host cell, the vector may rely on a polynucleotide sequence encoding a polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into the genome of the host cell at a precise location in a chromosome by homologous recombination. To increase the likelihood of integration at a precise location, the integration element must contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, with high sequence identity to the corresponding target sequence to increase the likelihood of homologous recombination. The integration element may be any sequence homologous to the target sequence in the genome of the host cell. Furthermore, the integration element may be a non-coding or coding polynucleotide. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0170] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the intended host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0171] Examples of bacterial origins of replication are the replication origins of the plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0172] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0173] Examples of replication origins useful in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175, WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be carried out according to the methods disclosed in WO 00 / 24883.

[0174] More than one copy of a polynucleotide of the invention can also be inserted into a host cell to increase production of the polypeptide. Increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including an amplifiable selectable marker gene with the polynucleotide, such that cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selectable agent.

[0175] The procedures used to ligate the above elements to construct the recombinant expression vectors of the present invention are well known to those of skill in the art (see, eg, Sambrook et al., 1989, supra).

[0176] host cell The present specification discloses recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector containing the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described above. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will largely depend on the gene encoding the polypeptide and its origin.

[0177] The host cell may be any cell useful in the recombinant production of the polypeptides of the invention, eg, prokaryotic or eukaryotic.

[0178] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Iriobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0179] Bacterial host cells include Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus erythrocytes ... The cell may be any Bacillus cell, including, but not limited to, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0180] Alternatively, the bacterial host cell may be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0181] Alternatively, the bacterial host cell may be any Streptomyces cell, including, but not limited to, cells of Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans.

[0182] Introduction of DNA into Bacillus cells can be carried out by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli cells can be carried out by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be carried out by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be carried out by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells can be carried out by natural competent transformation (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), electroportation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0183] The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0184] The host cell may be a fungal cell. "Fungi," as used herein, includes the Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0185] The fungal host cell may be a yeast cell. "Yeast," as used herein, includes ascospore-forming yeasts (Endomycetales), basidiospore-forming yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Because the classification of yeasts may change in the future, for purposes of the present invention, yeasts shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0186] Yeast host cells include cells of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica. lipolytica cells.

[0187] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Euglena and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0188] The filamentous fungal host cell can be a cell of the genus Acremonium, Bacillus, Aureobasidium, Bialecandera, Seripoliopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophysora, Neocallimastyx, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllium, Talaromyces, Cellmoascus, Chelabia, Tolypocladium, Trametes, or Trichoderma.

[0189] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, and Ceriporiopsis subbulfa. subrufa), Ceriporiopsis subvermispora, Chrysosporium inopus, Chrysosporium keratinophyllum, Chrysosporium lacnowense, Chrysosporium meldarium, Chrysosporium pannicola, Chrysosporium queenstrandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioides, Fusarium cerealis, Fusarium cloquewellens, Fusarium curmorum, Fusarium graminearum, Fusarium gramineum, Fusarium heterosporum Fusarium negundii, Fusarium oxysporum, Fusarium reticulantum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium, Fusariumus porotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothesioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophysora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngiiThe cell may be a cell of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0190] Fungal cells can be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a process known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163 and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0191] Production method The present invention also relates to a method of producing a detergent composition comprising a DNase variant, the method comprising (a) culturing a recombinant host cell as described above under conditions conducive to production of the DNase variant, and optionally (b) recovering the DNase variant and combining the DNase variant with a cleaning adjunct.

[0192] Cleaning Aids The composition includes a cleaning adjuvant. Typically, the cleaning adjuvant is present in the composition in an amount of 1 to 98.9% by weight, more typically 5 to 80% by weight of the cleaning adjuvant. Suitable cleaning adjuvants include surfactants, builders, bleaching agents, colorants, chelating agents, dye transfer agents, deposition aids, dispersants, additional enzymes and enzyme stabilizers, catalytic materials, optical brighteners, photoactivators, fluorescent agents, fabric hueing agents (shading dyes), fabric conditioners, preformed peracids, polymeric dispersants, mud stain removers / anti-redeposition agents, filler salts, hydrotropes, brighteners, suds suppressors, structural stretchers, fabric softeners, preservatives, antioxidants, shrinkage inhibitors, disinfectants, sanitizers, discoloration inhibitors, corrosion inhibitors, alkalinity sources, solubilizers, carriers, processing aids, pigments, dyes, fragrances, pH adjusters, encapsulating agents, polymers, and mixtures thereof.For example, these include bleach components such as bleach activators, bleach accelerators such as imine bleach accelerators, bleach catalysts, hydrogen peroxide, hydrogen peroxide sources such as percarbonates and / or perborates, particularly percarbonates coated with materials such as carbonates and / or sulfates, silicates, borosilicates, and any mixtures thereof, preformed peracids, including encapsulated preformed peracids, transition metal catalysts; foam suppressors or foam suppressor systems, such as silicone-based foam suppressors and / or fatty acid-based foam suppressors, clays, silicones, and / or quaternary ammonium compounds. fabric softeners such as polyesters, flocculants such as polyethylene oxide, dye transfer inhibitors such as polyvinylpyrrolidone, poly 4-vinylpyridine N-oxide, and / or copolymers of vinylpyrrolidone and vinylimidazole, fabric integrity components such as oligomers produced by condensation of imidazole with epichlorohydrin, soil dispersants and soil anti-redeposition aids such as alkoxylated polyamines and ethoxylated ethyleneimine polymers, anti-redeposition components such as polyesters, carboxylate polymers, e.g., maleimide polymers, the like; a carboxylic acid polymer, or a copolymer of maleic acid and acrylic acid; a perfume, for example, a perfume microcapsule, a starch-encapsulated accord, a perfume propellant, a soap ring, a cosmetic particle, a filler such as sodium aesthetic dye sulfate and / or citrus fiber (although it may be preferred that the composition be substantially free of fillers); a silicate such as sodium silicate, including 1.6R and 2.0R sodium silicate, or sodium metasilicate; a copolyester of a dicarboxylic acid and a diol; a cellulosic polymer such as methylcellulose, carboxymethylcellulose, hydroxyethoxycellulose, or other alkyl or alkylalkoxycellulose; a solvent such as 1,2 propanediol, monoethanolamine, diethylene glycol, ethanol, and any mixtures thereof; a hydrotrope, for example, sodium cumene sulfonate, sodium xylene sulfonate, sodium toluene sulfonate, and any mixtures thereof; an organic acid and its salt, for example, citric acid / citrate; and any combinations thereof.

[0193] Preferably, the composition contains a surfactant. Preferably, the composition contains an anionic surfactant. Preferred anionic surfactants are sulfonate and sulfate surfactants, preferably alkylbenzene sulfonates and / or (optionally alkoxylated) alkyl sulfates. Particularly preferred anionic surfactants include linear alkylbenzene sulfonates (LAS). Preferred alkyl sulfates include alkyl ether sulfates, particularly C-9-15 alcohol ether sulfates, especially those having an average degree of ethoxylation of 0.5-7, preferably 1-5, C-8-C-16 ester sulfates, and C-10-C-14 ester sulfates such as monododecyl ester sulfate. In preferred compositions, the anionic surfactant comprises alkylbenzene sulfonate and, optionally, an optionally ethoxylated alkyl sulfate, preferably having an ethoxylation degree of 0-7, more preferably 0.5-3. 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, alcohol ethoxy sulfates or aliphatic Also suitable anionic surfactants are 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.

[0194] The anionic surfactant is preferably added to the detergent composition in the form of a salt. Preferred cations are alkali metal ions such as sodium and potassium. However, the salt form of the anionic surfactant may be formed in situ by neutralizing the acid form of the surfactant with an alkali, such as sodium hydroxide, or an amine, such as mono-, di-, or triethanolamine. The composition preferably contains 1 to 60%, or 1 to 50%, or 2%, or 5 to 40% by weight of the composition of anionic surfactant. The surfactant preferably comprises a surfactant system containing an anionic surfactant and one or more additional surfactants, which may be nonionic, including semi-polar and / or cationic and / or zwitterionic and / or amphoteric and / or semi-polar nonionic and / or mixtures thereof.

[0195] The compositions of the present invention preferably comprise a cleaning adjunct comprising surfactants, the surfactants comprising anionic and nonionic surfactants, preferably in a weight ratio of anionic to nonionic from 30:1 to 1:2, preferably from 20:1 to 2:3 or 1:1.

[0196] Suitable nonionic surfactants include alcohol ethoxylates (AE), 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. Alcohol ethoxylates are particularly preferred, preferably having a C9-18 or preferably C12-15 alkyl chain, and preferably having an average degree of ethoxylation of 3 to 9, more preferably 3 to 7. Commercially available non-ionic surfactant washes include Plurafac™, Lutensol™, and Pluronic™ from BASF, the Dehypon™ series from Cognis, and the Genapol™ series from Clariant.

[0197] The detergent composition preferably comprises from 0.5% to about 40% by weight of a nonionic surfactant, preferably from 1 to 30% by weight of the composition.

[0198] The composition may be such that the cleaning adjunct comprises one or more selected from the group consisting of: (i) perfume microcapsules, (ii) fabric hueing agents, (iii) proteases, (iv) amphiphilic cleaning polymers, (v) lipases, or (vi) mixtures thereof.

[0199] The detergent composition preferably comprises one or more additional enzymes selected from the group consisting of aminopeptidase, amylase, arabinose, alginate lyase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, galactanase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hexosaminidase, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzymes, ribonuclease, transglutaminase, xylanase, xanthan lyase, xanthanase, endo-β-1,3-glucanase, and mixtures thereof. Preferably, the cleaning composition comprises additional enzymes selected from amylases, additional nucleases such as further DNases and RNases and mixtures thereof, hexosaminidases, mannanases, xanthan lyases, xanthanases, amylases and mixtures thereof.

[0200] Preferably, the composition comprises an additional enzyme selected from xanthan lyase, xanthanase, mannanase, hexosaminidase, and mixtures thereof, with mannanase being particularly preferred.

[0201] The additional enzyme may be derived from, for example, Aspergillus species, such as Aspergillus aculetus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium species, such as Fusarium bactridioides, Fusarium cerealis, Fusarium cloquewellens, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negunzi, or Fusarium oxysporum. The fungus may be produced by a microorganism belonging to the genus Humicola, such as Humicola insolens or Humicola lanuginosa; or a microorganism belonging to the genus Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0202] Preferably, the composition comprises a protease or a mixture of two or more proteases, a lipase or a mixture of two or more lipases, a peroxidase or a mixture of two or more peroxidases, one or more starch-degrading enzymes, such as α-amylase, glucoamylase, maltogenic amylase, and / or cellulase or a mixture thereof.

[0203] Generally, the properties of the selected enzyme should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount. Preferably, the products of the present invention contain at least 0.01 mg, preferably from about 0.05 to about 10 mg, more preferably from about 0.1 to about 6 mg, and especially from about 0.2 to about 5 mg of additional active enzyme per gram of composition.

[0204] Proteases: The compositions of the present invention may include one or more proteases. A mixture of two or more proteases can contribute to enhanced cleaning over a wider temperature, cycle duration, and / or substrate range, and / or can provide superior shine benefits, especially when used in conjunction with anti-redeposition agents and / or sulfonated polymers.

[0205] Suitable proteases for use in combination with the variant proteases of the present invention include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisin (EC 3.4.21.62). Suitable proteases include those of animal, plant, or microbial origin. In one aspect, such suitable proteases may be of microbial origin. Suitable proteases include chemically or genetically modified variants of the aforementioned suitable proteases. In one aspect, suitable proteases may be serine proteases, such as alkaline microbial proteases and / or trypsin-type proteases. Examples of suitable neutral or alkaline proteases include: Subtilisins (EC 3.4.21.62), in particular WO 2004067737, WO 2015091989, WO 2015091990, WO 2015024739, WO 2015143360, U.S. Pat. Nos. 6,312,936(B1), 5,679,630, and 4,760,025, German Patent Publication No. 102006022216(A1), WO 2015089447, WO 2015089441, WO 2016066756, WO 2016066757, WO 2016069557, and WO 201606956 Bacillus species, such as B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. gibsonii, B. akibaii, Bacillus clausii, and B. clarkii, as described in Patent Nos. 3, 2016069569, 2016174234, 2017 / 089093, and 2020 / 156419. Specifically, mutations S9R, A15T, V66A, A188P, V199I, N212D, Q239R, N255D, X9E, X200L, X256E, X9R, X19L, X60D (Savinase numbering system); Subtilisins derived from B. pumilus, such as those described in German Patent Publication No. 102006022224(A1), International Publication Nos. 2020 / 221578, 2020 / 221579 and 2020 / 221580, for example variants comprising an amino acid substitution at at least one of positions 9, 130, 133, 144, 252 and 271 (BPN' numbering system); Trypsin- or chymotrypsin-type proteases such as trypsin (e.g., of porcine or bovine origin), including the Fusarium proteases described in WO 89 / 06270, and the chymotrypsin proteases from Cellulomonas described in WO 05 / 052161 and WO 05 / 052146. Metalloproteases, in particular Bacillus amyloliquefaciens, as described in WO 07 / 044993(A2), amyloliquefaciens, those derived from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus, or Streptomyces species as described in WO 2014194032, WO 2014194054, and WO 2014194117, those derived from Kluyvera aluminosa as described in WO 2015193488, and those derived from Streptomyces and Lysobacter as described in WO 2016075078, A protease having at least 90% identity to the subtilase from Bacillus sp. TY145, NCIMB 40339, described in WO 92 / 17577 (Novozymes A / S), including variants of this Bacillus sp. TY145 subtilase described in WO 2015024739 and WO 2016066757.

[0206] Particularly preferred additional proteases for the composition exhibit at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, and especially 100% identity to the wild-type enzyme from Bacillus lentus and include the following positions: S9R, A15T, V68A, N76D, N87S, S99D, S99SD, S99A, S101G, S101M, S103A, V104N / I, G118V, G118R, S128L, P12 and / or M222S (using the BPN' numbering system and amino acid abbreviations exemplified in WO 00 / 37627, incorporated herein by reference).

[0207] Most preferably, the additional protease is selected from the group of proteases that comprise the following mutations (BPN numbering system) relative to either PB92 wild type (SEQ ID NO: 2 of WO 08 / 010925) or subtilisin 309 wild type (sequence according to the PB92 backbone but containing the natural mutation N87S): (i) G118V + S128L + P129Q + S130A (ii)S101M+G118V+S128L+P129Q+S130A (iii)N76D+N87R+G118R+S128L+P129Q+S130A+S188D+N248R (iv)N76D+N87R+G118R+S128L+P129Q+S130A+S188D+V244R (v)N76D+N87R+G118R+S128L+P129Q+S130A (vi) V68A+N87S+S101G+V104N (vii)S99AD (viii)S9R+A15T+V68A+N218D+Q245R

[0208] Suitable additional commercially available protease enzymes include: Suitable commercially available additional protease enzymes include those sold by Novozymes under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Liquanase®, Evity®, Savinase® Evity®, Ovozyme®, Neutrase®, Everlase®, Coronase®, Blaze®, Blaze Ultra®, Blaze® Evity®, Blaze® Exceed, Blaze® Pro, Esperase®, Progress® Uno, Progress® Excel, Progress® Key, Ronozyme®, Vinzon®, and Het Ultra®. Sold by A / S (Denmark); those sold by DuPont under the trade names Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®, Ultimase® and Purafect OXP®; those sold by Solvay Enzymes under the trade names Opticlean® and Optimase®; those available from Henkel / Kemira, namely BLAP (the sequence of which is shown in Figure 29 of US Patent No. 5,352,604 and which has the mutations S99D+S101R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I) and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D); optionally containing the further mutations 101E or 101D; KAP (Bacillus alcaliphilus subtilisin with mutations A230V+S256G+S259N) manufactured by Kao; and Lavergy®, Lavergy® Pro, and Lavergy® C Bright manufactured by BASF.

[0209] Commercially available proteases selected from the group consisting of Properase®, Blaze®, Ultimase®, Everlase®, Savinase®, Excellase®, Blaze Ultra®, BLAP and BLAP variants are particularly preferred for use herein in combination with the variant proteases of the invention.

[0210] Preferred concentrations of protease in the products of the present invention include about 0.05 to about 10 mg, more preferably about 0.5 to about 7 mg, and especially about 1 to about 6 mg of active protease per gram of composition.

[0211] Lipase: The composition preferably comprises a lipase. The presence of oils and / or fats can further increase the recovery of stains containing mannans and other polysaccharides. Therefore, the presence of lipase in the enzyme package can further improve the removal of such stains. Suitable lipases include those of bacterial, fungal or synthetic origin, including chemically modified or genetically engineered variants of the protein. Examples of useful lipases include lipases from the genus Humicola (also known as Thermomyces), such as H. lanuginosa (T. lanuginosus) or H. insolens; lipases from the genus Pseudomonas, such as P. alcaligenes or P. pseudoalcaligenes, P. cepacia, P. stutzeri, P. fluorescens, Pseudomonas sp. SD705, and P. wisconsinensis; lipases from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus, or B. pumilus.

[0212] The lipase may be a "first cycle lipase" as described in U.S. Patent No. 6,939,702 (B1) and U.S. Patent Application Publication No. 2009 / 0217464. In one aspect, the lipase is a first wash lipase, preferably a variant of the wild-type lipase from Thermomyces lanuginosus, containing the T231R and N233R mutations. The wild-type sequence is Swiss-Prot Accession No. Swiss-Prot O59952 (269 amino acids (amino acids 23-291) from Thermomyces lanuginosus (Humicola lanuginosa)). Preferred lipases include those sold under the tradenames Lipex®, Lipolex®, and Lipoclean®.

[0213] Other suitable lipases include, for example, Liprl 139 as described in WO 2013 / 171241, TfuLip2 as described in, for example, WO 2011 / 084412 and WO 2013 / 033318, Pseudomonas stutzeri lipase as described in, for example, WO 2018228880, Microbulbifer thermotolerans lipase as described in, for example, WO 2018228881, Sulfobacillus acidocaldarius lipase as described in, for example, EP 3299457, acidocaldarius lipases, such as LIP062 lipase as described in WO2018209026, PinLip lipase as described in WO2017036901, and Absidia lipases as described in WO2017005798.

[0214] Suitable lipases are variants of SEQ ID NO: 5, including: (a) Substitution T231R (b) Substitution N233R or N233C (c) at least three additional substitutions selected from E1C, D27R, N33Q, G38A, F51V, G91Q, D96E, K98L, K98I, D111A, G163K, H198S, E210Q, Y220F, D254S, I255A, and P256T; wherein the positions correspond to those of SEQ ID NO: 5, and the lipase variant has at least 90% but less than 100% sequence identity with a polypeptide having the amino acid sequence of SEQ ID NO: 5, and the variant has lipase activity.

[0215] One preferred lipase is a variant of SEQ ID NO:5 containing the following substitutions: T231R, N233R, D27R, G38A, D96E, D111A, G163K, D254S, and P256T. One preferred lipase is a variant of SEQ ID NO:5 containing the following substitutions: T231R, N233R, N33Q, G91Q, E210Q, I255A.

[0216] Suitable lipases are commercially available from Novozymes, for example as Lipex Evity 100L, Lipex Evity 200L (both liquid ingredients) and Lipex Evity 105T (granules), which have a different structure to the products Lipex 100L, Lipex 100T and Lipex Evity 100T, which are outside the scope of the present invention.

[0217] Cellulase: Suitable cellulases include cellulases of bacterial or fungal origin, including chemically modified or genetically engineered variants of proteins. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Patent Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and 5,691,178.

[0218] In one aspect, preferred enzymes include endoglucanases from microorganisms exhibiting endo-beta-1,4-glucanase activity (EC 3.2.1.4), preferably selected from the group comprising: (a) a bacterial polypeptide endogenous to a member of the genus Bacillus having a sequence that is at least 90%, 94%, 97%, or even 99% identical to the amino acid sequence of SEQ ID NO:2 in U.S. Patent No. 7,141,403(B2), with preferred substitutions including one or more positions corresponding to positions 292, 274, 266, 265, 255, 246, 237, 224, and 221 of the mature polypeptide of SEQ ID NO:2, wherein the variant has cellulase activity; (b) a glycosyl hydrolase having enzymatic activity on both xyloglucan and amorphous cellulose substrates, the glycosyl hydrolase being selected from GH family 5, 7, 12, 16, 44, or 74; (c) glycosyl hydrolases having a sequence that is at least 90%, 94%, 97%, and even 99% identical to the amino acid sequence of SEQ ID NO: 3 of WO 09 / 148983; (d) A variant exhibiting at least 70% identity to SEQ ID NO: 5 in WO2017106676. Preferred substitutions include one or more positions corresponding to positions 4, 20, 23, 29, 32, 36, 44, 51, 77, 80, 87, 90, 97, 98, 99, 102, 112, 116, 135, 136, 142, 153, 154, 157, 161, 163, 192, 194, 204, 208, 210, 212, 216, 217, 221, 222, 225, 227, and 232; (e) and mixtures thereof.

[0219] Suitable endoglucanases are sold under the trade names Celluclean® and Whitezyme® (Novozymes A / S, Bagsvaerd, Denmark). Examples include Celluclean® 5000L, Celluclean® Classic 400L, Celluclean® Classic 700T, Celluclean® 4500T, Whitezyme® 1.5T, and Whitezyme® 2.0L.

[0220] Other commercially available cellulases include Celluzyme®, Carezyme®, Carezyme® Premium (Novozymes A / S), Clazinase®, Puradax HA®, Revitalenz® 1000, Revitalenz® 2000 (Genencor International Inc.), KAC-500(B)® (Kao Corporation), Biotouch® FCL, Biotouch® DCL, Biotouch® DCC, Biotouch® NCD, Biotouch® FCC, Biotouch® FLX1 (AB Enzymes).

[0221] Suitable glucanases include endo-β-1,3-glucanases, preferably from EC class 3.2.1.39, preferably obtained from a microorganism of the genus Paenibacillus, Zobellia galactanivorans, Thermotoga petrophila, or Trichoderma sp., preferably Paenibacillus or Zobellia galactanivorans, most preferably Paenibacillus.

[0222] Amylase: Preferably, the compositions of the present invention comprise an amylase. Suitable α-amylases include those of bacterial or fungal origin, including chemically or genetically modified variants. Preferred alkaline α-amylases are derived from Bacillus species, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus species, such as Bacillus species NCBI 12289, NCBI 12512, NCBI 12513, DSM 9375 (U.S. Pat. No. 7,153,818), DSM 12368, DSM Z No. 12649, KSM AP1378 (WO 97 / 00324), KSM K36, or KSM K38 (EP 1,022,334). Preferred amylases include: (a) Variants described in U.S. Pat. No. 5,856,164 and WO 99 / 23211, WO 96 / 23873, WO 00 / 60060, WO 06 / 002643, and WO 2017 / 192657, in particular variants having one or more substitutions at the following positions relative to the AA560 enzyme described as SEQ ID NO: 12 in WO 06 / 002643: 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 202, 214, 231, 246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484, preferably D183 * and G184 * Variants that also contain deletions. (b) SEQ ID NO: 4 in WO 06 / 002643, the wild-type enzyme from Bacillus sp. SP722, in particular variants in which positions 183 and 184 are deleted, and variants as described in WO 00 / 60060, WO 2011 / 100410 and WO 2013 / 003659, in particular variants which have one or more substitutions at the following positions relative to SEQ ID NO: 4 in WO 06 / 002643, which are incorporated herein by reference: 51, 52, 54, 109, 304, 140, 189, 134, 195, 206, 243, 260, 262, 284, 347, 439, 469, 476, and 477. (c) Variants exhibiting at least 90% identity to the wild-type enzyme from Bacillus sp. 707 (SEQ ID NO: 7 in U.S. Pat. No. 6,093,562), particularly those containing one or more of the following mutations: M202, M208, S255, R172, and / or M261. Preferably, the amylase contains one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, and / or R172Q. Particularly preferred are those containing the M202L or M202T mutation. Additional relevant mutations / deletions based on the SP707 backbone are W48, A51, V103, V104, A113, R118, N125, V131, T132, E134, T136, E138, R142, S154, V165, R182, G182, H183, E190, D192, T193, I206, M208, D209, E212, V213, V214, N215, L217, R218, N219 Includes 9, V222, T225, T227, G229, I235, K242, Y243, S244, F245, T246, I250, S255, A256, H286, V291, T316, V317, V318, N417, T418, A419, H420, P421, I428, M429, F440, R443, N444, K445, Q448, S451, A465, N470, S472. (d) a variant described in WO 09 / 149130, preferably SEQ ID NO: 1 or SEQ ID NO: 2 in WO 09 / 149130, which shows at least 90% identity to the wild-type enzyme from Geobacillus Stearophermophilus, or a truncated version thereof. (e) a variant described in WO 10 / 115021, in particular SEQ ID NO: 2 in WO 10 / 115021, which exhibits at least 75%, or at least 85%, or at least 90%, or at least 95% identity with the alpha-amylase from Bacillus sp. TS-23. (f) Variants exhibiting at least 89% identity to SEQ ID NO: 1 in WO2016091688, in particular those containing a deletion at positions H183+G184 and further containing one or more mutations at positions 405, 421, 422, and / or 428. (g) Variants described in WO2014099523, in particular those exhibiting at least 60% amino acid sequence identity with "PcuAmyl α-amylase" from Paenibacillus curdlanolyticus YK9 (SEQ ID NO: 3 in WO2014099523). (h) Variants described in WO 2014099523, in particular "CspAmy2 amylase" from Cytophaga sp. (showing at least 60% amino acid sequence identity with SEQ ID NOs: 1 and 6 in WO 2014164777, in particular those containing one or more of the following deletions and / or mutations based on SEQ ID NO: 1 in WO 2014164777: R178 * , G179 * , T38N, N88H, N126Y, T129I, N134M, F153W, L171R, T180D, E187P, I203Y, G476K, G477E, Y303D. (i) A variant showing at least 85% identity with Bacillus subtilis (SEQ ID NO: 1 in WO 2009149271). (j) A variant showing at least 90% identity with the wild-type amylase derived from Bacillus sp. KSM-K38 under accession number AB051102. (k) variants described in WO 2016180748, in particular those exhibiting at least 80% identity with the mature amino acid sequence of AAI10 from Bacillus sp. as set forth in SEQ ID NO: 7 in WO 2016180748; those exhibiting at least 80% identity with the mature amino acid sequence of Alicyclobacillus sp. amylase as set forth in SEQ ID NO: 8 in WO 2016180748, and those exhibiting at least 80% identity with the mature amino acid sequence of SEQ ID NO: 13 in WO 2016180748, in particular those having the following mutations: * , N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G184T, N195F, V206L, K391A, P473R, G476K. (l) Variants described in WO2018060216, in particular those exhibiting at least 70% identity to the mature amino acid sequence of SEQ ID NO: 4 in WO2018060216, fusion molecules of Bacillus amyloliquefaciens and Bacillus licheniformis. In particular, those containing one or more substitutions at positions H1, N54, V56, K72, G109, F113, R116, T134, W140, W159, W167, Q169, Q172, L173, A174, R181, G182, D183, G184, W189, E194, N195, V206, G255, N260, F262, A265, W284, F289, S304, G305, W347, K391, Q395, W439, W469, R444, F473, G476, and G477.

[0223] A preferred amylase is a genetically engineered enzyme in which one or more amino acids susceptible to oxidation are replaced with amino acids less susceptible to oxidation. In particular, methionine residues are preferably replaced with any other amino acid. In particular, the methionine residue most susceptible to oxidation is preferably replaced. Preferably, the methionine at position 202 in SEQ ID NO: 11 is replaced. Preferably, the methionine at this position is replaced with threonine or leucine, preferably leucine.

[0224] Suitable commercially available alpha-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL®, ATLANTIC®, ACHIEVE ALPHA®, AMPLIFY® PRIME, INTENSA®, and BAN® (Novozymes A / S, Bagsvaerd, Denmark), KEMZYM® AT 9000 (Biozym Biotech Trading GmbH, Wehlistrasse 27b A-1200 Wien, Austria), RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE®, and PREFERENZ. Examples include the S (registered trademark) series (including PREFERENZ S1000 (registered trademark) and PREFERENZ S2000 (registered trademark)), PURASTAR OXAM (registered trademark) (DuPont, Palo Alto, California), and KAM (registered trademark) (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku, Tokyo 103-8210, Japan).

[0225] Preferably, the composition contains at least 0.01 mg, preferably about 0.05 to about 10, more preferably about 0.1 to about 6, especially about 0.2 to about 5 mg of active amylase per gram of composition.

[0226] Peroxidases / Oxidases: Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin, including chemically modified or genetically engineered variants of the protein. Examples of useful peroxidases include peroxidases from the genus Coprinus, such as those from C. cinereus and variants thereof, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0227] Commercially available peroxidases include GUARDZYME® (Novozymes A / S).

[0228] Pectate lyases: Suitable pectate lyases include those sold under the trade names Pectawash®, Pectaway®, X-Pect® (all Novozymes A / S, Bagsvaerd, Denmark) and Preferenz® F1000 (DuPont Industrial Biosciences).

[0229] Mannanase. The composition preferably contains one or more additional mannanase enzymes. As used herein, the term "mannanase" or "galactomannanase" refers to a mannanase enzyme, defined in accordance with what is known in the art as mannan endo-1,4-beta-mannosidase, also known as beta-mannanase and endo-1,4-mannanase, that catalyzes the hydrolysis of 1,4-beta-D-mannosidic linkages in mannans, galactomannans, glucomannans, and galactoglucomannans. Mannanases are classified as EC 3.2.1.78 according to enzyme nomenclature and belong to glycosyl hydrolase families 5, 26, and 113. Many suitable mannanases belong to glycosyl hydrolase family 5. Commercially available mannanases include those sold under the trade name Mannaway® (Novozymes A / S), such as Mannaway® 200L and Mannaway Evity 4.0T. Other commercially available mannanases include Effectene® M1000, Mannastar® 375, Prefienz M100, and Purabrite® (all DuPont Industrial Biosciences) and Biotouch M7 (AB enzyme). Other suitable mannanases belong to glycosyl hydrolase family 26, such as those described in WO 2018 / 191135, WO 2015040159, WO 2017 / 021515, WO 2017 / 021516, WO 2017 / 021517, and WO 2019 / 081515. Suitable mixtures of mannanases include the combination of glycosyl hydrolase family 5 mannanase and glycosyl hydrolase family 26 mannanase described in WO 2019 / 081515.

[0230] Xanthan gum-degrading enzymes: The compositions may contain one or more xanthan gum-degrading enzymes. Suitable enzymes for degrading xanthan gum-based stains include xanthan endoglucanases, optionally in combination with xanthan lyases. As used herein, the term "xanthan endoglucanase" refers to an enzyme that exhibits endo-β-1,4-glucanase activity that, together with a suitable xanthan lyase enzyme, can catalyze the hydrolysis of the 1,4-linked β-D-glucose polymer backbone of xanthan gum. Suitable xanthan endoglucanases are described in WO 2013 / 167581, WO 2015 / 181299, WO 2015 / 181292, WO 2017 / 046232, WO 2017 / 046260, WO 2018 / 37062, WO 2018 / 37065, WO 2019 / 038059, and WO 2019 / 162000. As used herein, the term "xanthan lyase" refers to an enzyme that cleaves the β-D-mannosyl-β-D-1,4-glucuronosyl bond in xanthan gum. Such enzymes belong to the EC 4.2.2.12 class. Suitable xanthan gels are described in WO 2015 / 001017, WO 2018 / 037061, WO 2018 / 37064, WO 2019 / 038060, WO 2019 / 162000, and WO 2019 / 038057.

[0231] RNases: Suitable RNases include wild-type and variants defined by SEQ ID NOs: 3, 6, 9, 12, 15, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 72, and 73 in WO2018178061 (Novozymes), which are incorporated herein by reference.

[0232] Hexosaminidase: The composition may contain one or more hexosaminidases. The term "hexosaminidase" includes "dispersant" and the abbreviation "Dsp," meaning a polypeptide (EC 3.2.1) having hexosaminidase activity that catalyzes the hydrolysis of β-1,6-glycosidic bonds in N-acetyl-glucosamine polymers found in soils of microbial origin. The term "hexosaminidase" includes polypeptides having N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity. Hexosaminidase activity can be determined according to Assay II described in WO 2018 / 184873. Suitable hexosaminidases include those described in International Publication Nos. 2017186936, 2017186937, 2017186943, 2017207770, 2018184873, 2019086520, 2019086528, 2019086530, 2019086532, 2019086521, and 2019 086526, 2020002604, 2020002608, 2020007863, 2020007875, 2020008024, 2020070063, 2020070249, 2020088957, 2020088958, and 2020207944. Variants of the Terribacillus saccharophilus hexosaminidase defined by SEQ ID NO: 1 in WO 2020207944 may be preferred, particularly variants with improved thermostability disclosed therein.

[0233] Galactanase: The composition may include a galactanase, i.e., an extracellular polymer-degrading enzyme, including an endo-beta-1,6-galactanase enzyme. The term "endo-beta-1,6-galactanase" or "polypeptide having endo-beta-1,6-galactanase activity" refers to endo-beta-1,6-galactanase activity (EC 3.2.1.164) from glycoside hydrolase family 30 that catalyzes the hydrolytic cleavage of 1,6-3-D-galactooligosaccharides with a degree of polymerization (DP) greater than 3 and their acidic derivatives bearing a 4-O-methyl glucosyluronate or glucosyluronate group at the non-reducing end. For purposes of this disclosure, endo-beta-1,6-galactanase activity is determined in Assay I according to the procedure described in WO2015185689. Suitable examples from classification EC3.2.1.164 are described in WO2015185689, e.g., mature polypeptide SEQ ID NO:2.

[0234] The additional enzymes may be included in the detergent composition by adding a separate enzyme additive containing the additional enzymes or a mixed enzyme additive containing two, some, or all of the additional enzymes. Such enzyme additives may be in the form of granules, liquids, or slurries, and preferably further comprise an enzyme stabilizer.

[0235] Preferably, the or each additional enzyme is present in an amount of at least 0.0001% to about 0.1% by weight of pure active enzyme protein, such as from about 0.0001% to about 0.01%, from about 0.001% to about 0.01%, or from about 0.001% to about 0.01% by weight, based on the weight of the composition.

[0236] Fabric hueing agents. The composition may include a fabric hueing agent (sometimes referred to as a tinting agent, bluing agent, or whitening agent / dye). Typically, the hueing agent imparts a blue or purple hue to the fabric. Hueing agents can be used either alone or in combination to create a particular hue and / or tint different types of fabric. This can be achieved, for example, by mixing a red and a green-blue dye to produce a blue or purple hue. The hueing agent may be selected from any known chemical class of dyes, including, but not limited to, acridines, anthraquinones (including polycyclic quinones), azines, azos including premetallized azos (e.g., monoazos, diazos, trisazos, tetrakisazos, polyazos), benzodifurans and benzodifuranones, carotenoids, coumarins, cyanines, diazahemicyanines, diphenylmethanes, formazans, hemicyanines, indigoids, methanes, naphthalimides, naphthoquinones, nitro and nitroso, oxazines, phthalocyanines, pyrazoles, stilbenes, styryls, triarylmethanes, triphenylmethanes, xanthenes, and mixtures thereof. Azo dyes, particularly monoazo or bis-azo dyes, triarylmethane dyes, and anthraquinone dyes are preferred.

[0237] Suitable fabric hueing agents include dyes, dye-clay conjugates, and organic and inorganic pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes classified in the Color Index (CI) classification of direct dyes, basic dyes, reactive dyes, or hydrolysis reactive dyes, solvent dyes, or disperse dyes. Examples of suitable small molecule dyes include, for example, the Color Index (Society of Dyers and Colorists). Colourists, Bradford, UK) Direct violet dyes such as 9, 35, 48, 51, 66, and 99; direct blue dyes such as 1, 71, 80, and 279; acid red dyes such as 17, 73, 52, 88, and 150; acid violet dyes such as 15, 17, 24, 43, 49, 50, and 51; acid blue dyes such as 15, 17, 25, 29, 40, 45, 75, 80, 83, 90, and 1 and small molecule dyes selected from the group consisting of: acid black dye 1, such as 13; basic violet dyes 1, 3, 4, 10, and 35; basic blue dyes 3, 16, 22, 47, 66, 75, and 159; disperse or solvent dyes such as those described in European Patent No. 1794275 or 1794276; or the dyes disclosed in U.S. Patent No. 7,208,459 B2; and mixtures thereof.

[0238] Preferred are polymeric dyes, including polymers containing covalently attached (sometimes referred to as conjugated) chromogens (dye-polymer conjugates), such as polymers having chromogens copolymerized into the backbone of the polymer, and mixtures thereof, including those described in WO 2011 / 98355, WO 2011 / 47987, U.S. Patent Application Publication No. 2012 / 090102, WO 2010 / 145887, WO 2006 / 055787, and WO 2010 / 142503.

[0239] Preferred polymeric dyes include alkoxylated, preferably ethoxylated azo or anthraquinone, triarylmethane dyes. Particularly preferred are polymeric dyes selected from the group consisting of ethoxylated thiophene azo dyes, such as the fabric substantive colorant sold under the name Liquitint® (Milliken, Spartanburg, South Carolina, USA), and dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of polymers containing a moiety selected from the group consisting of hydroxyl moieties, primary amine moieties, secondary amine moieties, thiol moieties, and mixtures thereof. Suitable polymeric dyes include polymeric dyes selected from the group consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC) covalently bonded to a reactive blue, reactive violet, or reactive red dye, such as CMC conjugated with CI Reactive Blue 19 sold under the trade name AZO-CM-CELLULOSE, product code S-ACMC by Megazyme (Wicklow, Ireland), alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophene polymeric colorants, and mixtures thereof.

[0240] Preferred hueing dyes include the alkoxylated thiophene azo brighteners found in U.S. Patent Application Publication No. 2008 / 0177090, and optionally can be anionic, such as those selected from Examples 1-42 in Table 5 of WO 2011 / 011799. Other preferred dyes are disclosed in U.S. Patent No. 8,138,222.

[0241] Suitable pigments include pigments selected from the group consisting of Ultramarine Blue (CI Pigment Blue 29), Ultramarine Violet (CI Pigment Violet 15), and mixtures thereof. Pigments and / or dyes may also be added to add color for cosmetic reasons. Preferred are organic blue, purple, and / or green pigments.

[0242] Builders: The detergent composition may further contain builders such as builders based on carbonates, bicarbonates or silicates, which may be zeolites such as zeolite A, zeolite MAP (Maximum Aluminum type P). Laundry-safe zeolites are preferably of the formula Na 12 (AlO2) 12 (SiO2) 12 ·27H2O, with particle sizes typically ranging from 1 to 10 μm for zeolite A and 0.7 to 2 μm for zeolite MAP. Another builder is the strongly alkaline sodium metasilicate (Na2SiO3·nH2O or Na2SiO5·nH2O), preferably used in dishwashing. In preferred embodiments, the amount of detergent builder may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50%, or may be less than 80% or less than 65%. In dishwashing detergents, the builder concentration is typically 40 to 65%, particularly 50 to 65%, or even 75 to 90%.

[0243] Encapsulated Agent: The composition may comprise an encapsulated benefit agent, comprising a core and a shell having an inner and outer surface, the shell encapsulating the core. The core may comprise a material selected from the group consisting of fragrances; whitening agents; dyes, insect repellents; silicones; waxes; fragrances; vitamins; fabric softeners; skin care agents, and in one embodiment, paraffin; enzymes; antimicrobial agents; bleaching agents; sensates; and mixtures thereof. The shell may comprise a material selected from the group consisting of polyethylene; polyamide, polystyrene; polyisoprene; polycarbonate; polyester; polyacrylate; aminoplast (in one aspect, the aminoplast may comprise polyurea, polyurethane, and / or polyureaurethane, and in one aspect, the polyurea may comprise polyoxymethylene urea and / or melamine formaldehyde); polyolefin; polysaccharide (in one aspect, the polysaccharide may comprise alginate and / or chitosan); gelatin; shellac; epoxy resin; vinyl polymer; water-insoluble inorganic material; silicone; and mixtures thereof. A preferred encapsulant comprises a core containing a perfume. Such an encapsulant is a perfume microcapsule.

[0244] Enzyme Stabilizers: The compositions may include an enzyme stabilizer. Suitable enzyme stabilizers may be selected from the group consisting of: (a) inorganic salts selected from the group consisting of calcium salts, magnesium salts, and mixtures thereof; (b) carbohydrates selected from the group consisting of oligosaccharides, polysaccharides, and mixtures thereof, and sugars or sugar alcohols; (c) phenylboronic acid and derivatives thereof, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid, or peptide aldehydes, such as di-, tri-, or tetrapeptide aldehydes or aldehyde analogs (any of the forms B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), and B0 is a single amino acid residue (preferably an optionally substituted aliphatic or aromatic side chain). ; B1 consists of one or more amino acid residues (preferably 1, 2 or 3), optionally including an N-terminal protecting group], or as described in WO 09118375, WO 98 / 13459); (d) mass-efficient reversible protease inhibitors selected from the group consisting of: (e) polyols, such as propylene glycol or glycerol 1-2 propanediol; (f) calcium formate and / or sodium formate; (g) protein-type protease inhibitors such as RASI, BASI, WASI (rice, barley and wheat bifunctional α-amylase / subtilisin inhibitor) or Cl2 or SSI, and (h) any combination thereof.

[0245] Structuring Agents: In one aspect, the composition may include a structuring agent selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0246] Polymers: The composition preferably comprises one or more polymers. Preferred examples include carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers, and amphiphilic polymers, and mixtures thereof.

[0247] Amphiphilic cleaning polymer: Preferably, the amphiphilic cleaning polymer has the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + Compounds having the formula -(CH3)-bis((C2H5O)(C2H4O)n) where n=20-30 and x=3-8, or sulfated or sulfonated variants thereof.

[0248] The amphiphilic alkoxylated grease cleaning polymer of the present invention refers to any alkoxylated polymer that has a balance of hydrophilic and hydrophobic properties to remove grease particles from fabrics and surfaces. Specific embodiments of the amphiphilic alkoxylated grease cleaning polymer of the present invention comprise a core structure and a plurality of alkoxylate groups attached to the core structure. These may comprise alkoxylated polyalkyleneimines, preferably having an inner polyethylene oxide block and an outer polypropylene oxide block.

[0249] The core structure may comprise a polyalkyleneimine structure comprising repeat units of formulae (I), (II), (III), and (IV) in condensed form:

[0250] [ka] where # in each case indicates a bond between a nitrogen atom and a group A of two adjacent repeating units of formula (I), (II), (III) or (IV). 1indicates half of the binding between the free binding sites of * indicates in each case half of a bond with one of the alkoxylate groups; A 1 are independently selected from straight-chain or branched C2-C6-alkylene; the polyalkyleneimine structure consists of 1 repeat units of formula (I), x repeat units of formula (II), y repeat units of formula (III), and y+1 repeat units of formula (IV), where x and y in each instance have values ​​ranging from 0 to about 150, and the average weight average molecular weight Mw of the polyalkyleneimine core structure has a value ranging from about 60 to about 10,000 g / mol.

[0251] Alternatively, the core structure comprises a polyalkanolamine structure that is a condensation product of at least one compound selected from N-(hydroxyalkyl)amines of formula (Ia) and / or (Ib):

[0252] [ka] wherein A is independently selected from C1-C6-alkylene; 1 , R 1* , R 2 , R 2* , R 3 , R 3* , R 4 , R 4* , R 5 and R 5* are independently selected from hydrogen, alkyl, cycloalkyl, or aryl, where the alkyl, cycloalkyl, or aryl radicals may be optionally substituted; R 6 is selected from hydrogen, alkyl, cycloalkyl, or aryl, where the alkyl, cycloalkyl, or aryl radicals may be optionally substituted.

[0253] The multiple alkyleneoxy groups attached to the core structure are independently selected from alkyleneoxy units of formula (V):

[0254] [ka] (In the ceremony * represents in each case half of the bond to the nitrogen atom of a repeating unit of formula (I), (II), or (IV), and A 2 is in each occurrence independently selected from 1,2-propylene, 1,2-butylene, and 1,2-isobutylene; A 3 is 1,2-propylene; R in each occurrence is independently selected from hydrogen and C1-C4-alkyl; m has an average value ranging from 0 to about 2; n has an average value ranging from about 20 to about 50; and p has an average value ranging from about 10 to about 50.

[0255] Carboxylate polymer: The composition may preferably include one or more carboxylate polymers, such as a maleate / acrylate random copolymer or a polyacrylate homopolymer. In one embodiment, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of 4,000 Da to 9,000 Da or 6,000 Da to 9,000 Da.

[0256] Soil Release Polymers: The composition preferably also comprises one or more soil release polymers. Preferred are polymers having a structure defined by one of the following structures (I), (II), or (III): (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e (III)-[(OCHR 5 -CHR 6 ) c -OR 7 ] f (In the formula, a, b, and c are 1 to 200; d, e, and f are 1 to 50; Ar is 1,4-substituted phenylene; sAr is 1,3-substituted phenylene substituted at the 5-position with SO3Me; Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, mono-, di-, tri-, or tetra-alkylammonium (the alkyl groups are C1-C 18 Alkyl or C2-C 10 hydroxyalkyl), or mixtures thereof; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently H or C1 to C 18 n- or iso-alkyl; R 7 is a straight chain or branched C1-C 18 Alkyl, or straight or branched C2-C 30 Alkenyl or cycloalkyl groups having 5 to 9 carbon atoms, or C8 to C 30 Aryl group or C6-C 30 It is an arylalkyl group.

[0257] Suitable soil release polymers are polyester soil release polymers such as Repel-o-tex polymers, including Repel-o-tex SF, SF-2, and SRP6, supplied by Rhodia. Other suitable soil release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, supplied by Clariant. Other suitable soil release polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0258] Cellulosic polymer: The composition may include one or more cellulosic polymers, preferably selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, and alkyl carboxyalkyl celluloses. In one embodiment, the cellulosic polymer is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methylcarboxymethyl cellulose, and mixtures thereof. In one embodiment, the carboxymethyl cellulose has a degree of carboxymethyl substitution of 0.5 to 0.9 and a molecular weight of 100,000 Da to 300,000 Da.

[0259] Bleaching System: The compositions may contain a bleaching system comprising a source of HO, such as a perborate or percarbonate, which may be combined with a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise, for example, an amide, imide, or sulfone-type peroxyacid. Generally, when a bleaching agent is used, the compositions of the present invention may comprise from about 0.1% to about 30%, or even from about 0.1% to about 25%, by weight of the subject cleaning composition, of bleaching agent.

[0260] Chelating Agents: The compositions preferably include a chelating agent, preferably in an amount of from 0.005% to about 15%, or even from about 3.0% to about 10% chelating agent by weight of the composition. Suitable chelating agents include copper, iron, and / or manganese chelating agents, and mixtures thereof. Preferred chelating agents (complexing agents) include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), iminodisuccinic acid (IDS), carboxymethylinulin, and derivatives thereof and mixtures thereof. Preferred chelating agents are selected from the group consisting of methyl-glycine-diacetic acid (MGDA), its salts and derivatives, glutamic acid-N,N-diacetic acid (GLDA), its salts and derivatives, iminodisuccinic acid (IDS), its salts and derivatives, carboxymethyl inulin, its salts and derivatives, and mixtures thereof. MGDA and its salts are particularly preferred, especially including the trisodium salt of MGDA.

[0261] The compositions may also contain other conventional detergent ingredients such as, for example, viscosity enhancing agents including fabric conditioners, suds boosters, suds suppressors, corrosion inhibitors, soil settling agents, soil redeposition inhibitors, dyes, disinfectants, optical brighteners, hydrotropes, anti-fog agents, organic solvents such as ethanol, or fragrances.

[0262] How to use The present invention also provides a method of treating a fabric, which method comprises, in a contacting step, contacting the fabric with an aqueous wash liquor comprising a DNase variant as described herein and a cleaning adjunct, preferably in an amount of from 0.01 ppm to 10 ppm, preferably from 0.1 ppm to 1 ppm.

[0263] Preferred cleaning adjuvants contain anionic surfactants, preferably in an amount of 0.05 to 50 g / l, more preferably 0.2 g / l to 5 g / l or 0.5 g / l to 3 g / l.

[0264] The aqueous washing liquid can be formed by adding the above-described detergent composition to water, for example in a washing machine or hand washing process. The concentration of the detergent composition is typically 500 ppm to 15,000 ppm, preferably 1,000 to 10,000 ppm, preferably 1,000 to 5,000 ppm.

[0265] Alternatively, the aqueous wash liquor may be formed by adding the DNase variant and the wash adjunct as separate components to water to form the wash liquor. The fabrics may then optionally be washed, and / or rinsed, and / or dried.

[0266] It may be preferable to use mechanical agitation in the contacting step or a subsequent step to facilitate cleaning and removal of the decomposed soil by-products from the fabrics. The pH of the wash liquor is preferably about 7 or about 8 to about 10.5. The wash liquor preferably has a temperature of about 5°C to about 40°C, or preferably 10 to 30°C or less than 30°C. The water to fabric ratio is typically about 1:1 to about 30:1.

[0267] The pH of the wash solution is typically in the range of about 5.5 to about 10, more typically in the range of 7 to 9, for example, in the range of about 7 to about 8.5 or about 7 to about 8.

[0268] The concentration of DNase variant and any additional enzymes in the wash solution is typically in the range of 0.00001 ppm to 10 ppm of enzyme protein, 0.00002 ppm to 10 ppm, 0.0001 ppm to 10 ppm, 0.0002 ppm to 10 ppm, 0.001 ppm to 10 ppm, 0.002 ppm to 10 ppm, 0.01 ppm to 10 ppm, 0.02 ppm to 10 ppm, 0.1 ppm to 10 ppm, 0.2 ppm to 10 ppm, or 0.5 ppm to 5 ppm. [Example]

[0269] Materials and Methods Assay I: Determination of DNase activity DNase activity can be determined using the DNaseAlert™ kit (IDT, Intergrated DNA Technologies, Belgium) according to the supplier's manual. For an example of this assay, see Example 2 below.

[0270] Assay II: Storage stability assay for purified DNase variants The storage stability of purified DNase variants in detergent compositions, expressed as half-life improvement factors relative to a reference DNase, can be determined, for example, as described in Example 3 below.

[0271] Example 1: Construction and screening of variants Site-directed variants of the DNase of SEQ ID NO: 1 containing specific substitutions were constructed. Mutants were generated by conventional DNA fragment cloning (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR with appropriately designed mutagenic oligonucleotides to introduce the desired mutations into the resulting sequence.

[0272] Mutagenic oligos corresponding to the DNA sequences flanking the desired mutation sites, separated by DNA base pairs defining the insertion / deletion / substitution, were designed and purchased from oligo vendors such as Life Technologies. To test DNase variants, DNA containing the variants was integrated into competent Bacillus subtilis strains by homologous recombination. Transformants were grown for 1 hour in antibiotic-free TB-Gly growth medium and then overnight in TB-Gly medium containing 3 μg / ml chloramphenicol. After adding glycerol to 25% v / v, they were stored at -80°C. Replicates were grown for 3–4 days for DNase production in broth supplemented with chloramphenicol and trace elements: 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, and 2 μM CuCl2.

[0273] Example 2: Stability screening of DNase variants in supernatant After growth, each supernatant sample was split into two identical samples by transferring 20 μl of supernatant to two 96-well standard microtiter plates, each containing 170 μl of TidePods® 3-in-1 detergent supplemented with 1% (v / v) liquid laundry protease, Progress® Uno 101 L (Novozymes A / S), and 0.3% (w / v) sodium bisulfite. After shaking at 7500 rpm for 20 minutes on a microtiter plate shaker, one microtiter plate was incubated at 10°C for 3 days (reference condition), and the other microtiter plate was incubated at 50°C in an incubator for 3 days (stress condition). After 3 days, both sample sets (reference and stress conditions) were diluted 20-fold with dilution buffer (50 mM Tris, HCl, 0.01% Tween® 20, pH 7.5) and then diluted with DNAseAlert™ substrate solution (Integrated DNA Technologies / Belgium, part number 11-04-02-04. Ten microliters of 20-fold diluted reference and stressed DNase samples were transferred to a new 384-well microtiter plate, and 40 μL of DNAseAlert™ assay solution was added (50 mM TrisHCl, pH 7.5, 5 mM MnCl2, 0.01% Tween® 20, 10 μM DNAseAlert™ substrate). Fluorescence (excitation 536 nm and emission 556 nm) was read every 90 seconds for a total of 30 minutes. From the rate curves, the slopes of the reference sample (activity under reference conditions) and the corresponding stressed sample (activity under stress conditions) were determined by linear regression. The residual activity (RA) of each DNase variant and the reference DNase (SEQ ID NO: 1) was calculated as slope(stressed sample) / slope(reference sample). RA was used to calculate half-life, and the half-life improvement factor was calculated as the stress time (min). *The half-life was calculated as ln(0.5) / ln(RA). The calculated half-lives for the variant and reference were used to calculate the half-life improvement factor as the ratio between the half-life of the variant and the half-life of the reference. The half-life improvement factor relative to the reference is, by definition, 1.0, and variants with a half-life improvement factor greater than the reference have improved stability under the test conditions.

[0274] Single mutation variants showing improved stability in the supernatant screen were used for the construction and screening of combinatorial variants containing two or more individual mutations.

[0275] Transformations of these improved single-mutation hits were streaked to single colonies, sequenced, and grown overnight in TB-Gly broth containing 3 μg / ml chloramphenicol. After growth in shake flasks in 100 ml of PS-1 growth medium containing trace elements at 30°C for 4 days, the enzyme variants were purified and their stability was tested in two different liquid detergents as described below.

[0276] Single mutations that confer improved stability were combined in combinatorial variants containing two or more mutations compared to SEQ ID NO: 1. These were streaked to single colonies immediately after transformation, then screened and sequenced as described above. The improved combinatorial variants were then grown, purified, and tested for storage stability in two different liquid detergents as described below.

[0277] For variants subsequently tested in purified form, stability data are presented in Tables 2 and 3 of Example 3. Table 1A presents stability data for several additional variants tested as supernatants in concentrated liquid detergent (Tide Pods® 3-in-1) stored at 55°C.

[0278] Other DNase variants were tested in a similar manner as above, but incubated in 10% Ariel Essential detergent with 1% protease (SEQ ID NO: 28) at a temperature of 58° C. The test results are shown in Table 1B below.

[0279] [Table 1]

[0280] [Table 2]

[0281] Example 3: Storage stability assay of purified DNase variants After purification (a two-step procedure using a PPA HyperCel column and an SP-Sepharose® FF column), the purified DNase variants were diluted to 0.2 and 0.1 mg / ml with 0.01% Triton X-100, and concentrations were calculated using absorbance at 280 nm. For each enzyme variant, two wells at high concentration (0.2 mg / ml) and two wells at low concentration (0.1 mg / ml) were tested. 15 μl of diluted DNase sample was mixed in a well of a microtiter plate ("detergent plate," Nunc U 96 PP 0.5 ml) with 285 μl of concentrated detergent (1) Tide Pod® 3-in-1, green compartment supplemented with 0.3% sodium bisulfite and 1% liquid laundry protease Progress® Uno 101 L (Novozymes A / S) or Tide Original HE Heavy-Duty Liquid supplemented with 1% Progress® Uno 101 L using a magnetic bar. After mixing, the detergent plate was incubated at 50°C in a Biosan PST-100HL thermomixer.

[0282] After various incubation times (e.g., 0, 2, 24, and 72 hours), residual DNase activity was measured. Five μl from the detergent plate was mixed with 195 μl of DNA substrate solution (3.3 mg DNA (Sigma D1626) in 50 mM Tris pH 7, 5 mM MgCl2, 5 mM CaCl2, 1.3 mM EDTA). Viscosity was measured once per minute for 30 minutes using pressure sensing during aspiration on a Hamilton Microlab STAR. Activity was calculated from the measured viscosity reduction.

[0283] The decrease in activity during incubation with detergent is assumed to be exponential. The half-life (T) was found from linear regression of Log(activity) versus incubation time, and the half-life improvement factor (HIF) was calculated as the half-life of the DNase variant relative to the half-life of the reference DNase.

[0284] The half-life improvement factors for the DNase variants are shown below in Tables 2 and 3. Table 2 shows the results for variants tested in concentrated liquid detergent Tide Pods® 3-in-1, and Table 3 shows the results for variants tested in heavy-duty liquid detergent Tide Original HE.

[0285] [Table 3-1]

[0286] [Table 3-2]

[0287] [Table 4-1]

[0288] [Table 4-2]

[0289] Detergent Example Examples 1-6. Granular laundry detergent compositions designed for hand washing or top loading washing machines.

[0290] [Table 5]

[0291] Examples 7-13. Granular laundry detergent compositions designed for front-loading automatic washing machines.

[0292] [Table 6] * DNase is given as mg of active enzyme per 100 g of detergent.

[0293] Examples 14 to 23. Heavy-duty liquid laundry detergent compositions

[0294] [Table 7-1]

[0295] [Table 7-2]

[0296] [Table 8]

[0297] Examples 24-30. Unit dose laundry detergent compositions. Such unit dose formulations may contain one or more compartments.

[0298] [Table 9]

[0299] Example 31. Multi-compartment unit dose composition Multi-compartment unit dose laundry detergent formulations of the present invention are provided below. In these examples, the unit doses have three compartments, but similar compositions can be made with two, four, or five compartments. The film used to enclose the compartments is polyvinyl alcohol.

[0300] [Table 10]

[0301] [Table 11]

[0302] Examples 32 to 35. Fabric softener compositions of the present invention

[0303] [Table 12]

[0304] Ingredients and Notes for the Compositions of Examples 1 to 33 Linear alkylbenzene sulfonates with an average aliphatic carbon chain length of C11 to C18 C12-18 Dimethylhydroxyethylammonium Chloride AE3S is a C12-15 alkyl ethoxy (3) sulfate. AE7 is a C12-15 alcohol ethoxylate with an average degree of ethoxylation of 7. AE9 is a C12-16 alcohol ethoxylate with an average degree of ethoxylation of 9. HSAS is a mid-chain branched primary alkyl sulfate having a carbon chain length of about 16-17, as disclosed in US Pat. Nos. 6,020,303 and 6,060,443. Polyacrylate MW4500 is supplied by BASF. The carboxymethylcellulose is Finnfix® V supplied by CP Kelco (Arnhem, Netherlands). CHEC is a cationically modified hydroxyethyl cellulose polymer. An example of a phosphonate chelating agent is diethylenetetraaminepentaacetic acid (DTPA) hydroxyethane diphosphonate (HEDP). Savinase®, Natalase®, Stainzyme®, Lipex®, Celluclean™, Mannaway®, and Whitezyme® are all products of Novozymes (Bagsvaerd, Denmark). Purafect® and Purafect Prime® are products of Genencor International (Palo Alto, California, USA). Optical Brightener 1 is Tinopal® AMS, Optical Brightener 2 is Tinopal® CBS-X, Direct Violet 9 is Pergasol® Violet BN-Z, and NOBS is sodium nonanoyloxybenzenesulfonate. TAED is tetraacetylethylenediamine. S-ACMC is carboxymethylcellulose conjugated with CI Reactive Blue 19, trade name AZO-CM-CELLULOSE. The soil release agent is Repel-o-tex® PF. The acrylic acid / maleic acid copolymer has a molecular weight of 70,000 and an acrylate:maleate ratio of 70:30; EDDS is the sodium salt of ethylenediamine-N,N'-disuccinic acid, (S,S) isomer, a suds suppressor aggregate supplied by Dow Corning (Midland, Michigan, USA). HSAS is a medium-chain branched alkyl sulfate. Liquitint® Violet CT is a polymeric color pigment supplied by Milliken (Spartanburg, South Carolina, USA). The polyethoxylated azothiophene dye is Violet DD™ polymeric hue dye supplied by Milliken (Spartanburg, South Carolina, USA). 1 Random graft copolymers are polyvinyl acetate-grafted polyethylene oxide copolymers having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is about 40-60, and there is no more than one grafting point per 50 ethylene oxide units. 2 Polyethyleneimine (MW=600) with 20 ethoxylate groups per —NH. 3 The amphiphilic alkoxylated polymer is polyethyleneimine (MW600) prepared from a polymer derivatized to contain 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH. 4 Amylase is given as mg of active enzyme per 100g of detergent. 5 DNase in all these examples is given as mg of active enzyme per 100 g of detergent. DNase may contain trace amounts of superoxide dismutase impurity. a Proxel GXL, a 20% aqueous dipropylene glycol solution of 1,2-benzisothiazolin-3-one supplied by Lonza. bN,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride fatty acid ester. The iodine value of the parent fatty acid of this material is 18-22. The material obtained from Evonik contains impurities in the form of free fatty acids, the monoester form of N,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride fatty acid ester, and fatty acid esters of N,N-bis(hydroxyethyl)-N-methylamine. c MP10®, supplied by Dow Corning, 8% active d Expressed as 100% encapsulated perfume oil as described in U.S. Pat. No. 8,765,659 e Rheovis® CDE, a cationic polymeric thickener supplied by BASF f N,N-dimethyloctanamide and N,N-dimethyldecanamide in an approximately 55:45 weight ratio, Steposol® M-8-10 from Stepan Company

[0305] Dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise indicated, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm."

Claims

1. 1. A detergent composition comprising a DNase variant having DNase activity, wherein the DNase variant comprises two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S compared to the polypeptide of SEQ ID NO: 1, and a cleaning adjunct, The DNase variant is ● K21L+Q48D+T65I+S82R+K107Q+T127S, ● Q14R+K21L+Q48D+T65I+T127S, ● Q48D+T65I+S82R+T127S+S164D, ● N61D+T65I+K107Q+T127S+S164D, ● Q48D+T65I+S82R+K107Q+T127S, ● Q14R+N61D+T65I+S82R+K107Q, ● N61D+S68L+G149N, ● Q14R+N61D+T65I+S82R+T127S+S164D, ● K21L+Q48D+T65I+S82R+K107Q, ● Q14R+T65I+K107Q+T127S, ● N61D+T65I+S82R+T127S+S164D, ● K21L+N61D+T65I+S82R+K107Q+T127S, ● T65V+T127V+G149N, ● T65I+K107Q+T127S+S164D, ● N61D+T65I+S82R+K107Q, ● Q14R+K21L+N61D+T65I+S82R, ● Q14R+K21L+N61D+T65I+S82R+K107Q, ● Q14R+K21L+N61D+T65I+T127S, ● N61D+T65I+S82R+K107Q+T127S+S164D, ● Q14R+K21L+T65I+K107Q+T127S, ● N61D+T65I+K107Q+T127S, ● T65I+S82R+K107Q+S164D, ● K21L+N61D+T65I+S82R, ● K21L+N61D+T65I+T127S, ● N61D+T65I+S82R+S164D, ● K21L+N61D+T65I+S82R+K107Q, ● S68L+S106A+G149N, ● N61D+T65I+T127S+S164D, ● Q14R+K21L+N61D+T65I, ● Q14R+K21L+T65I+T127S, ● T65V+G149N, ● T65V+R109T+T127V, ● Q14R+K21L+T65I+K107Q, ● K21L+T65I+S82R+K107Q, ● K21L+T65I+K107Q+T127S, ● N61D+S68L+S102Y+G149N+S164D+L181T, ● N61D+S68L+S106A+G149N+S164D, ● T65V+R109T+G149N, ● T65V+T127V+T171W, ●N61D+T65I+S82R+T127S+S164D+T171E+D175G+L181S, ● T65V+Y77R+G149N, ● T65V+Y77H+G149N, ● T65V+L76K+G149N, ● T65V+L76R+G149N, - A detergent composition comprising a set of substitutions selected from the group consisting of T19E + P25S + L33K + D56I + T65V + Y77T + T127V + L181S, wherein the DNase variant comprises or consists of a polypeptide of SEQ ID NO: 1 having one of the sets of substitutions.

2. The DNase variant is ●N61D+T65I+S82R+K107Q, ● T65V+G149N, ● N61D+T65I+K107Q+T127S+S164D, ● N61D+T65I+T127S+S164D, ● T65V + T127V + G149N, and 2. The detergent composition of claim 1, comprising a set of substitutions selected from the group consisting of: N61D + T65I + S82R + T127S + S164D.

3. 3. The detergent composition of claim 1, comprising an enzyme selected from amylase, hexosaminidase, mannanase, xanthan lyase, xanthanase, and mixtures thereof.

4. 1. A method for making a detergent composition, comprising: (i) obtaining a DNase variant by introducing two or more substitutions selected from the group consisting of N61D, T65I, T65V, S82R, K107Q, T127S, T127V, G149N, S164D, and L181S (numbering based on SEQ ID NO: 1) into a parent DNase, and recovering said variant, wherein said variant has DNase activity; and (ii) mixing said DNase variant with a cleaning adjunct; The DNase variant is ● K21L+Q48D+T65I+S82R+K107Q+T127S, ● Q14R+K21L+Q48D+T65I+T127S、 ● Q48D+T65I+S82R+T127S+S164D、 ● N61D+T65I+K107Q+T127S+S164D、 ● Q48D+T65I+S82R+K107Q+T127S、 ● Q14R+N61D+T65I+S82R+K107Q、 ● N61D+S68L+G149N、 ● Q14R+N61D+T65I+S82R+T127S+S164D、 ● K21L+Q48D+T65I+S82R+K107Q、 ● Q14R+T65I+K107Q+T127S、 ● N61D+T65I+S82R+T127S+S164D、 ● K21L+N61D+T65I+S82R+K107Q+T127S、 ● T65V+T127V+G149N、 ● T65I+K107Q+T127S+S164D、 ● N61D+T65I+S82R+K107Q、 ● Q14R+K21L+N61D+T65I+S82R、 ● Q14R+K21L+N61D+T65I+S82R+K107Q、 ● Q14R+K21L+N61D+T65I+T127S、 ● N61D+T65I+S82R+K107Q+T127S+S164D、 ● Q14R+K21L+T65I+K107Q+T127S、 ● N61D+T65I+K107Q+T127S、 ● T65I+S82R+K107Q+S164D、 ● K21L+N61D+T65I+S82R、 ● K21L+N61D+T65I+T127S、 ● N61D+T65I+S82R+S164D、 ● K21L+N61D+T65I+S82R+K107Q、 ● S68L+S106A+G149N、 ● N61D+T65I+T127S+S164D、 ● Q14R+K21L+N61D+T65I、 ● Q14R+K21L+T65I+T127S、 ● T65V+G149N、 ● T65V+R109T+T127V、 ● Q14R+K21L+T65I+K107Q、 ● K21L+T65I+S82R+K107Q、 ● K21L+T65I+K107Q+T127S、 ● N61D+S68L+S102Y+G149N+S164D+L181T、 ● N61D+S68L+S106A+G149N+S164D、 ● T65V+R109T+G149N、 ● T65V+T127V+T171W、 ●N61D+T65I+S82R+T127S+S164D+T171E+D175G+L181S, ● T65V+Y77R+G149N, ● T65V+Y77H+G149N, ● T65V+L76K+G149N, ● T65V+L76R+G149N, - A method comprising a set of substitutions selected from the group consisting of T19E + P25S + L33K + D56I + T65V + Y77T + T127V + L181S, wherein the DNase variant comprises or consists of a polypeptide of SEQ ID NO: 1 having one of the sets of substitutions.

5. Use of the detergent composition according to any one of claims 1 to 3 in a cleaning process such as laundry or hard surface cleaning.

6. A method for treating a surface, the method comprising contacting the surface with an aqueous cleaning solution comprising a DNase variant having DNase activity according to any one of claims 1 to 3 and a cleaning adjuvant.

7. The method of claim 6, wherein the anionic surfactant is present in the cleaning solution in an amount of 0.2 g / l to 3 g / l.

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