Liquid cleaning composition comprising protease variants

Abstract

The present invention relates to compositions comprising protease variants suitable for use in cleaning or detergent compositions, such as laundry detergent compositions and dishwashing compositions, including automatic dishwashing compositions.

Description

[0001] References to sequence lists

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

[0003] Invention Field

[0004] This invention relates to compositions comprising protease variants suitable for use in, for example, high-pH cleaning or detergent compositions, such as laundry detergent compositions and dishwashing compositions, including automatic dishwashing compositions.

[0005] Related fields description

[0006] Enzymes have been used in detergent formulations for decades in the detergent industry. Enzymes used in such formulations include amylases, cellulases, lipases, mannosidases, and proteases along with other enzymes, or mixtures thereof. Commercially most important enzymes are proteases. Many proteases traditionally used in cleaning processes are alkaline proteases, typically with an optimal pH of around 9, and are therefore suitable for fairly alkaline detergents. However, some specific liquid detergents have pH values ​​above 10, and these detergents were not previously considered suitable for enzymes due to the very harsh conditions and the resulting low storage stability of enzymes in them. Furthermore, enzymes tend to be more unstable in liquid detergents, especially when the water content is high.

[0007] Many useful protease variants have been described, many of which exhibit improved activity, stability, and solubility in various detergents.

[0008] However, these variations are not suitable for detergents with very high pH levels, because the alkaline environment of these detergents will cause the enzymes to break down after a very short time and thus reduce their activity. Invention Overview

[0010] This invention relates to protease variants that are stable under alkaline conditions and suitable for cleaning high-pH liquids, and detergent compositions including such variants.

[0011] The present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising at least 0.01 wt% of a protease, wherein the protease is a variant of a parent protease, and wherein the protease variant has an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C.

[0012] In another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising: (a) at least 0.01 wt% of a protease, wherein the protease is a variant of a parent protease, and wherein the protease variant has an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C; and (b) optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor.

[0013] In another embodiment, the present invention relates to a liquid cleaning composition comprising: (a) water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; (b) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C; and (c) optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor.

[0014] The present invention also relates to a liquid cleaning composition comprising:

[0015] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0016] (b) Water ranging from 40% to 95% wt, preferably from about 70% to about 90% wt;

[0017] (c) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or alkali metal hydroxide or a mixture thereof, to provide a pH from about 11 to 13.5; and

[0018] (d) Protease variants ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt.

[0019] In another embodiment, the invention further relates to a liquid cleaning composition comprising:

[0020] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0021] (b) a protease variant ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt; and

[0022] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0023] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0024] (e) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0025] In the embodiments described, (c1) and (c2) can be used as substitutes for each other, or they can be used in combination.

[0026] definition

[0027] The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0028] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0029] The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0030] The term "control sequence" refers to the nucleic acid sequence required for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, promoters, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the variant.

[0031] The term "detergent component" is defined herein as referring to the type of chemicals that can be used in a cleaning composition, such as in a detergent composition. Examples of detergent components are surfactants, water-soluble solvents, builders, co-builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric toners, fabric conditioning agents, foaming agents, defoaming agents, dispersants, dye transfer inhibitors, optical brighteners, fragrances, optical brighteners, bactericides, fungicides, soil suspending agents, soil-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. The detergent composition may include one or more detergent components of any type.

[0032] The term "cleaning composition" includes "detergent composition" and, unless otherwise specified, includes all forms of detergent compositions such as sticks, uniform tablets, tablets having two or more layers, pouches having one or more chambers, regular or compressed powders, gels, granules, liquids (e.g., regular, compressed, or concentrated liquids), pastes, powders, sprays, or tablet compositions, including heavy-duty liquids (HDLs), fine fabric liquid detergents, liquid and / or solid laundry detergents, and fine fabric detergents; hard surface cleaning formulations for, for example, glass, wood, ceramic and metal countertops and windows; carpet cleaners; stove cleaners; and fabric fresheners. Fabric softeners; pre-spotters for textiles and clothing, along with dishwashing detergents such as hand-wash dishwashing detergents, light-stain dishwashing detergents, and machine-wash dishwashing detergents; general-purpose or heavy-duty cleaners, general-purpose cleaners in liquid, gel, or paste form; liquid cleaners and disinfectants, including antibacterial hand-wash types, cleaning sticks, mouthwashes, denture cleaners, car or carpet shampoos, and bathroom cleaners; hair shampoos and hair rinses; shower gels and bubble baths; metal cleaners; along with cleaning aids such as bleach additives and "stain-sticks" or pre-treatment types. The bag can be configured as a single or multiple compartments. It can have any form, shape, and material suitable for preserving the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane encapsulating an inner volume. The inner volume can be divided into compartments containing the bag. The preferred membrane is a polymeric material forming a membrane or sheet, preferably a polymer. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl methacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a blend of hydrolyzable and water-soluble polymers, such as polylactic acid and polyvinyl alcohol (known under Trade Reference M8630, such as those sold by MonoSol LLC, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. A chamber for a liquid component may differ in composition from a chamber that includes a solid component: US 2009 / 0011970 A1.

[0033] Detergent components (or parts) can be physically separated from each other by compartments in different layers of a water-soluble bag or tablet. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the washing solution can cause delayed dissolution of selected components.

[0034] The cleaning compositions described herein (e.g., liquid cleaning compositions) are suitable for cleaning processes such as laundry or hard surface cleaning, including dishwashing and industrial cleaning. Exemplary laundry processes may be selected from the group consisting of: residential laundry processes, industrial laundry processes, and public institution laundry processes.

[0035] In addition to containing the protease variants of the present invention, the detergent formulation may also contain one or more other enzymes (such as hydrolases, amylases, catalases, cellulases (e.g., endoglucanases), keratinases, halogenated peroxidases, lipases, mannanases, pectinases, xyloglucanases, pectinases, pectin lyases, peroxidases, proteases, DNases, xanthan gumases, and xyloglucanases, or any mixture thereof), and / or components such as surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0036] The term "dishwashing" refers to all forms of dishwashing, such as manual or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of earthenware, such as plates, cups, glasses, and bowls; all forms of tableware, such as spoons, knives, and forks; and serving utensils, including ceramic, plastic (e.g., melamine), metal, porcelain, glass, and acrylic.

[0037] The term "dishwashing composition" refers to all forms of compositions used for cleaning hard surfaces. This invention is not limited to any specific type of dishwashing composition or any specific detergent.

[0038] The term “expression” includes any step involved in the production of a variant, including (but not limited to) transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0039] The term "expression vector" refers to a straight or circular DNA molecule that includes a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0040] The term "hard surface cleaning" is defined here as cleaning hard surfaces, which can include floors, tables, walls, roofs, etc., along with surfaces of hard objects such as cars (car washing) and tableware (dishwashing). Dishwashing includes, but is not limited to, cleaning plates, cups, glasses, bowls, and utensils (such as spoons, knives, and forks), serving utensils, ceramics, plastics (such as melamine), metals, porcelain, glass, and acrylics.

[0041] The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors including the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0042] The term "improved properties" refers to characteristics associated with a protease variant that are improved relative to the parent protease. Such improved properties include, but are not limited to, washing performance, protease activity, thermal activity profile, thermal stability, pH activity profile, pH stability, substrate / cofactor specificity, improved surface properties, substrate specificity, product specificity, increased stability, improved stability under storage conditions, and chemical stability.

[0043] The term "stability" includes both storage stability and stability during use, such as during washing, and reflects the stability of the protease variant according to the invention as a function of time, such as how much activity it retains when the protease variant is placed in solution, particularly in detergent solution. This stability is affected by many factors, such as pH, temperature, detergent composition, such as builders, amount of surfactant, etc. Protease stability can be measured using the assay described in Example 3. The term "improved stability" or "increased stability" is defined herein as the increased stability of a variant protease relative to its parent protease (e.g., relative to SEQ ID NO:2) as shown in solution.

[0044] In one specific aspect of the invention, the improved stability is the improved stability in liquid detergents having a pH of 10 or higher. The term "detergent stability" or "improved detergent stability" specifically refers to the improved stability of protease activity when the protease variant of the invention is incorporated into a liquid detergent formulation having a pH of 10 or higher and then stored at temperatures between 15°C and 50°C (e.g., 20°C, 30°C, or 40°C).

[0045] The term "improved washing performance" is defined herein as the washing performance of the protease variant according to the invention relative to the parent protease, exhibiting improved washing performance, for example, through increased detergency. The term "washing performance" includes washing performance in laundry, but also includes, for example, washing performance in dishwashing. Washing performance can be quantified as described herein under the definition of "washing performance".

[0046] The term "isolated" means a substance that does not exist in nature in a form or environment. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or all of the naturally occurring components associated with it; (3) any substance that is artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0047] The term "laundry" encompasses both household and industrial laundry and refers to the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The laundry process can be performed, for example, using a household or industrial washing machine or manually.

[0048] 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, autocatalytic activation, etc. In one aspect, a mature polypeptide is amino acids 1 to 269 of SEQ ID NO:2 and amino acids 1 to 275 of SEQ ID NO:1. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide.

[0049] The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide with protease activity.

[0050] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, modified in a way that does not normally exist in nature to contain segments of nucleic acid, or synthesized, and that the nucleic acid molecule includes one or more control sequences.

[0051] The term "operably linked" refers to a configuration in which a control sequence is placed in the appropriate position relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0052] The term "parent" refers to a protease to which modifications are made to produce the enzyme variant of the present invention. It should be understood that "having the same amino acid sequence" in the context refers to 100% sequence identity. In one specific embodiment, the parent is a protease having at least 60% identity with the polypeptide of SEQ ID NO:1 or 2, such as at least 65%, at least 70%, 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.

[0053] The term “protease” is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to EC 3.4 (including each of its 13 subclasses). EC numbers refer to the 1992 enzyme nomenclature published by NC-IUBMB Academic Press in San Diego, California, including supplements 1-5 of the European Journal of Biochemistry 1223:1-5 (1994); European Journal of Biochemistry 232:1-6 (1995); European Journal of Biochemistry 237:1-5 (1996); European Journal of Biochemistry 250:1-6 (1997); and European Journal of Biochemistry 264:610-650 (1999). The most widely used proteases in the detergent industry, such as those used for laundry and dishwashing, are serine proteases or serine peptidases. These are subgroups of proteases characterized by the presence of a serine residue at their active site, which forms a covalent adduct with the substrate. Additionally, subtilisinases (and serine proteases) are characterized by having two additional active site amino acid residues besides serine: histidine and aspartic acid. Subtilisinases refer to the serine protease subgroup according to Siezen et al., 1991, *Protein Engineering* 4:719-737 and Siezen et al., 1997, *Protein Science* 6:501-523. Subtilisinases can be divided into six subfamilies: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanoxanthionein family, the Kexin family, and the Pyrolysin family. The term "protease activity" refers to proteolytic activity (EC 3.4). Proteases that can be used in detergents are primarily endopeptidases (EC 3.4.21). Several types of protease activity exist: three main types are: trypsin-like, where cleavage of the amide substrate occurs at P1 after Arg or Lys; chymotrypsin-like, where cleavage occurs at P1, after one of the hydrophobic amino acids; and elastase-like, where cleavage occurs after Ala at P1. For the purposes of this invention, protease activity is determined according to the Suc-AAPF-pNA activity assay as described in the Materials and Methods section below. In one aspect, the protease variants of this invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mature polypeptide of the parent enzyme.In one specific aspect, the protease variant of the present invention has at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the enzymatic activity of the polypeptide of SEQ ID NO:2.

[0054] The term "protease activity" refers to proteolytic activity (EC 3.4). The protease of the present invention is an endopeptidase (EC 3.4.21). Several types of protease activity exist: three main types are: trypsin-like, wherein cleavage of the amide substrate occurs at P1 after Arg or Lys; chymotrypsin-like, wherein cleavage occurs at P1, after one of the hydrophobic amino acids; and elastase-like, wherein cleavage occurs after Ala at P1. For the purposes of the present invention, protease activity is determined according to the procedures described in the following "Materials and Methods". These protease variants of the present invention preferably have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 100% protease activity of the polypeptide of SEQ ID NO:2.

[0055] The term "residual activity" in this context refers to the protease activity remaining or residual after storage, particularly after storage in liquid detergents. When measuring residual activity after the protease has been added to the detergent, the activity of the protease is measured at t1. Then, after a certain period of storage, typically several minutes or weeks, the residual activity is measured at t2. The protease variant of the present invention preferably exhibits residual activity of at least 10%, at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the protease activity when measured at t = 4 hours or longer before storage in a high-pH liquid detergent at 30°C (at t1). In this context, a high-pH liquid detergent is a liquid detergent with a pH of 10 or higher, and in this context, a very high-pH liquid detergent is a liquid detergent with a pH of 12 or higher. The protease variants according to the invention are preferably more stable, i.e., exhibiting higher residual activity after storage in high-pH liquid detergents (pH = 10 or higher) compared to the parental protease (e.g., the protease having SEQ ID NO 2), and / or the protease variants according to the invention are more stable, i.e., exhibiting higher residual activity after storage in liquid detergents (i.e., liquid detergents comprising 40% or more water) compared to the parental protease (e.g., the protease having SEQ ID NO 2). Preferably, the residual activity of the protease variants of the invention, when measured after storage at 30°C for 4 hours in a liquid detergent having pH 10 and / or a liquid detergent having at least 40% water, is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, such as at least 50%, compared to the residual activity of the parental protease, for example, compared to SEQ ID NO 2. As described in Example 3.

[0056] Specifically, the relative stability of the protease variants according to the invention and their corresponding parent proteases differs at high pH, ​​above or at pH 10 from their relative stability at neutral pH, above or at pH 7.

[0057] The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter “sequence consistency”. For the purposes of this invention, the sequence consistency between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453), as performed in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16:276-277), preferably version 5.0.0 or later. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The Needle output, marked as "Longest Consistency" (obtained using the -nobrief option), is used as the percentage consistency and is calculated as follows:

[0058] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0059] For the purposes of this invention, the Niedle-Onsch algorithm (Niedleman and Onsch, 1970, see above), implemented in the Niedle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Niedle output labeled “Longest Consistency” (obtained using the -nobrief option) is used as the percentage consistency and is calculated as follows:

[0060] (Consistent deoxyribonucleotides x 100) / (Alignment length - total number of vacancies in the alignment)

[0061] The different strict conditions are defined as follows.

[0062] The term "substantially pure variant" means a formulation containing, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polypeptide materials, which are naturally or recombinantly related to it. Preferably, the variant is at least 92% pure by weight of the total polypeptide material present in the formulation, for example, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and 100% pure. These variants of the invention are preferably present in a substantially pure form. This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0063] The term "substantially pure polynucleotide" refers to a polynucleotide formulation free of other external or unwanted nucleotides and in a form suitable for use in a genetically engineered peptide production system. Thus, a substantially pure polynucleotide comprises, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polynucleotides naturally or recombinantly associated with that polynucleotide. However, substantially pure polynucleotides may include naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Preferably, the substantially pure polynucleotide is at least 90% pure by weight, for example, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. These polynucleotides of the present invention are preferably present in a substantially pure form.

[0064] The term "textiles" means any textile material including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, and synthetic materials, together with fabrics made from these materials, such as clothing, garments, and other articles. When the terms fabric or garment are used, the term textiles in a broader sense is also intended to be included.

[0065] The term "variant" refers to a polypeptide having protease activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at three or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced with a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that one or more amino acids (e.g., 1, 2, 3, 4, or 5 amino acids) are added adjacent to and immediately following an amino acid occupying a position. The term protease variant can also refer to a variant of the subtilisin parent protease, i.e., a protease variant is a subtilisin protease that includes alterations (i.e., substitutions, insertions, and / or deletions) at three or more (e.g., several) positions compared to the parent protease.

[0066] The term "washing performance" is used to describe the ability of an enzyme to remove stains present on an object to be cleaned during processes such as washing (e.g., laundry or hard surface cleaning). Improvements in washing performance can be quantified by calculating a so-called intensity value (Int) as defined in AMSA assays.

[0067] The term "wild-type protease" refers to a protease expressed by a naturally occurring organism (such as bacteria, archaea, yeast, fungi, plants, or animals found in nature). An example of a wild-type subtilisin is the subtilisin Savinase, which consists of amino acids 1 to 269 of SEQ ID NO:2.

[0068] Variant Naming Rules

[0069] For the purposes of this invention, the amino acid residues corresponding to another protease were determined using the sequence of amino acids 1-275 of SEQ ID NO:1 (Siezen et al., 1991, Protein Engineering 4:719-737). The amino acid sequence of the other protease was compared with the mature polypeptide disclosed in SEQ ID NO:1, and based on the comparison, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, Journal of Molecular Biology 48:443-453) was performed as in the Nieder program of the EMBOSS software package (EMBOSS: European Open Package for Molecular Biology, Rice et al., 2000, Trends in Genetics 16:276-277), preferably version 5.0.0 or later, to determine the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:1. The parameters used are an open space penalty of 10, an extended space penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) replacement matrix.

[0070] The identification of the corresponding amino acid residues in another protease can be determined by comparing multiple polypeptide sequences using several computer programs with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797); MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Kato et al., 2005, Nucleic Acids Research 33:511-518; Kato and Toh, 2007, Bioinformatics 23:372-374; Kato et al., 2009, Methods in Molecular Biology). Biology 537:39-64; Kato and Asato, 2010, Bioinformatics 26:1899-1900; and EMBOSS EMMA using ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22:4673-4680).

[0071] When other enzymes deviate from the mature polypeptide of SEQ ID NO:1, making traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, J.Mol.Biol. 295:613-615), other pairwise sequence comparison algorithms can be applied. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (profiles) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J.Mol.Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J.Mol.Biol. 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0072] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures. Furthermore, implementations of these algorithms can be used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0073] In the description of variations of the invention, the following nomenclature is used for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are adopted.

[0074] replace For amino acid substitutions, the following nomenclature is used: initial amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) with phenylalanine (F). The "X" before a position indicates that any original amino acid at that position can be substituted.

[0075] Missing For amino acid deletions, use the following nomenclature: initial amino acid, position, * Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195". * +Ser411 * "or "G195 * +S411 * ".

[0076] insertThe insertion of additional amino acid residues, such as the insertion of lysine after G195, can be represented as Gly195GlyLys or G195GK. Alternatively, the insertion of additional amino acid residues, such as the insertion of lysine after G195, can be represented as: * 195aK. When more than one amino acid residue is inserted, such as inserting Lys and Ala after G195, it can be represented as: Gly195GlyLysAla or G195GKA. In such cases, the inserted amino acid residues can also be numbered by adding a lowercase letter to the position number of the amino acid residue preceding the inserted amino acid residue, as in this example: * 195aK * 195bA. In the above example, sequences 194 to 196 are therefore:

[0077]

[0078] When substitution and insertion occur in the same location, this can be represented as S99SD+S99A, or simply S99AD. The same modification can also be represented as S99A+ * 99aD.

[0079] When an amino acid residue identical to the existing amino acid residue is inserted, degeneracy clearly occurs in the nomenclature. If, for example, glycine is inserted after glycine in the above example, it is represented as G195GG or *195GaG. The same practical change can also be represented simply as A194AG or... * 194aG, from:

[0080]

[0081] Such cases are obvious to those skilled in the art, and therefore the expression G195GG and the corresponding expression for this type of insertion are intended to include such equivalent degenerate expressions.

[0082] Multiple changes Variations involving multiple changes are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent arginine and glycine at positions 170 and 195 being replaced by tyrosine and glutamic acid, respectively. Alternatively, spaces or commas can be used to separate multiple changes, such as A170Y G195E or A170Y, G195E.

[0083] Different changesWhen different changes can be introduced at a single position, these changes are separated by a comma. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0084] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0085] Alternatively, different changes or optional substitutions may be indicated by parentheses, such as Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or simply R170[Y, G] or R170{Y, G}. Invention Details

[0087] Alkaline liquid detergents with high pH are widely used in cleaning applications such as laundry and dishwashing. Especially in North America, consumers frequently use liquid detergents with elevated pH levels. High-pH cleaning compositions are also used in industrial cleaning processes.

[0088] Alkaline detergents are liquids with detergent properties. The pH of such detergents typically ranges from 9 to 12.5. High-pH detergents typically include components such as surfactants, builders, and bleaching agents, and in addition, they may contain significant amounts of water and alkalis such as NaOH, TSP (trisodium phosphate), ammonia, sodium carbonate, and potassium hydroxide (KOH), which are typically added in amounts corresponding to 0.1% to 30% by weight.

[0089] Examples of commercially available high-pH detergents include, but are not limited to, Arm & Hammer (Church & Dwight), Surf Sparkling Ocean (Unilever), Clean Burst, SUN Triple Clean Tropical Breeze (SUN product), Purex Free and Clear (Henkel), and Xtra Tropical Passion (Church & Dwight).

[0090] Adding enzymes to detergents is highly advantageous because their specific activity effectively removes particular stains from surfaces such as fabrics and tableware. However, the difficulty in maintaining acceptable enzyme stability in high-pH liquid detergents has prevented the inclusion of enzymes in these detergents for many years.

[0091] This invention relates to high-pH liquid cleaning compositions comprising alkaline-stable protease variants suitable for such compositions.

[0092] One aspect of the present invention relates to a liquid cleaning composition comprising:

[0093] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0094] (b) Water ranging from 40% to 95% wt, preferably from about 70% to about 90% wt;

[0095] (c) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or alkali metal hydroxide or a mixture thereof, to provide a pH from about 11 to 13.5; and

[0096] (d) Protease variants ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt.

[0097] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0098] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0099] (b) a protease variant ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt; and

[0100] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0101] (c2) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH from about 7.5 to 13.5, preferably from about 10 to 13.5;

[0102] (d) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0103] The compositions of the present invention preferably comprise an alkaline buffer system to provide a pH of at least about 7.5, at least about 8, at least about 9, and preferably a pH of 10 or higher. Preferably, the pH is from about 9 to about 13. To achieve a high pH, ​​it is necessary to include an alkali metal hydroxide, particularly sodium hydroxide or potassium hydroxide, typically in an amount of 0.1% to about 30% (wt%), and preferably 1.0% to 2.5%, or higher, of a suitable alkali metal silicate, such as a metal silicate, depending on the pH required for the product.

[0104] The compositions of the present invention preferably contain 20% to 95% wt, more preferably 40% to 95% wt, and more preferably from about 70% to about 90% wt of water. Preferably from about 75% to 80% wt of water, more preferably from about 80% to 85% wt of water, more preferably from about 70% to 75% wt of water, or more preferably from about 75% to 80% wt of water. Preferably, the compositions of the present invention comprise 10% wt or more of water, such as at least 20% wt, at least 25% wt, at least 30% wt, at least 35% wt, at least 40% wt, at least 45% wt, at least 50% wt, at least 55% wt, at least 60% wt, at least 65% wt, at least 70% wt, at least 75% wt, at least 80% wt, at least 85% wt, at least 90% wt, or at least 95% wt or more, but less than 100% wt of water.

[0105] These protease variants can be added to the high pH cleaning composition according to the invention in amounts corresponding to 0.01-200 mg of enzyme protein per liter of washing solution, preferably 0.05-50 mg of enzyme protein per liter of washing solution, and particularly 0.1-10 mg of enzyme protein per liter of washing solution.

[0106] Compositions intended for use in laundry detergents may, for example, include 0.0001%-10%, such as 0.001%-7%, such as 0.1%-5% of the protease variant according to the invention by weight of the composition.

[0107] Compositions intended for use in automatic dishwashers (ADW) may, for example, contain 0.0001%-50%, 0.001%-30%, 0.01%-20%, or 0.5%-15% of enzyme protein by weight of the composition.

[0108] The present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising at least 0.01 wt% of a protease, wherein the protease is a variant of a parent protease, and wherein the protease variant has an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C.

[0109] The protease variant added to the cleaning compositions according to the invention exhibits an improved half-life, enabling storage and use of the protease in high-pH (pH>10.0) liquid cleaning compositions such as detergents. Furthermore, the protease variant exhibits significant cleaning performance in high-pH liquids, thus opening up the possibility of improving the cleaning performance of high-pH liquid detergents and / or reducing the concentration of detergent present in these detergent formulations used to achieve the same cleaning performance.

[0110] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of 10 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, wherein the variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO 1). In another embodiment, the present invention provides a liquid cleaning composition having a pH of 10 or higher and comprising a protease, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, wherein the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO:1), preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO:1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 of BPN' (SEQ ID NO:1).

[0111] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of from about 7 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO:1). In another embodiment, the present invention relates to a liquid cleaning composition comprising a protease having a pH of from about 7 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1), preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 of BPN' (SEQ ID NO: 1).

[0112] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH from about 8 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO:1). In another embodiment, the present invention relates to a liquid cleaning composition comprising a protease having a pH from about 8 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1), preferably, the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1).

[0113] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 9 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO:1). In another embodiment, the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 9 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1), preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 of BPN' (SEQ ID NO: 1).

[0114] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 10 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO:1). In another embodiment, the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 10 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1), preferably, the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1).

[0115] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 11 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO 1). In another embodiment, the present invention relates to a liquid cleaning composition comprising a protease having a pH of from about 11 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1), preferably, the variant comprises substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1).

[0116] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH from about 7 to about 13 or higher, wherein the protease is a variant protease having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D. In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 7 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1), preferably, said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1). IDNO:1) replaces one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D.

[0117] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH from about 8 to about 13 or higher, wherein the protease is a variant protease having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D. In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 8 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1), preferably, said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1). IDNO:1) replaces one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D.

[0118] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH from about 9 to about 13 or higher, wherein the protease is a variant protease having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D. In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 9 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1), preferably, said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1). IDNO:1) replaces one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D.

[0119] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 10 to about 13 or higher, wherein the protease is a variant protease having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D. In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 10 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1), preferably, said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1). NO:1) is a substitution for one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D.

[0120] One embodiment of the present invention relates to a liquid cleaning composition comprising a protease having a pH of about 11 to about 13 or higher, wherein the protease is a variant protease having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D. In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 11 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1), preferably, said variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO:1). NO:1) is a substitution for one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D.

[0121] In some preferred embodiments, the protease is selected from the group consisting of the following items:

[0122] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0123] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0124] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0125] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0126] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0127] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0128] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0129] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0130] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0131] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0132] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0133] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0134] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0135] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0136] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0137] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0138] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0139] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0140] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0141] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0142] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0143] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0144] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0145] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0146] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0147] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0148] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0149] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0150] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0151] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0152] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0153] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0154] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0155] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0156] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0157] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0158] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0159] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0160] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0161] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0162] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0163] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0164] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0165] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0166] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0167] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0168] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0169] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0170] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0171] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0172] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0173] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0174] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0175] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0176] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0177] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0178] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0179] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0180] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0181] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0182] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0183] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0184] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0185] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[0186] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E。

[0187] In some preferred embodiments, the cleaning compositions provided herein are formulated to have a pH of about 7.5 to about 13.5, or about 8.0 to about 13.5, about 8.5 to about 13.5, about 9.0 to 13.5, or in alternative embodiments, even from about 9.5 to about 13.5, such as from about 10 to about 13.5, from about 10 to about 13, from about 10 to about 12.5, from about 10 to about 12, from about 10 to about 11.5, from about 10 to about 11, or from about 10 to about 10.5. In some preferred embodiments, the liquid cleaning compositions are formulated to have a pH of about 10 to about 13.5. In other preferred embodiments, the liquid cleaning compositions are formulated to have a pH of about 12 to 13.5.

[0188] One aspect of the present invention relates to a liquid cleaning composition comprising:

[0189] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0190] (b) Water ranging from 40% to 95% wt, preferably from about 70% to about 90% wt;

[0191] (c) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or alkali metal hydroxide or a mixture thereof, to provide a pH from about 11 to 13.5; and

[0192] (d) From 0.001% to 10% wt, preferably 0.1% to 5% wt, of a protease variant, wherein the variant comprises substitutions at one or more sites selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN' (SEQ ID NO:1).

[0193] Another aspect of the present invention relates to a liquid cleaning composition comprising:

[0194] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0195] (b) a protease variant from 0.001% to 10% wt, preferably 0.1% to 5% wt, wherein the variant comprises substitutions at one or more sites selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1); and

[0196] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0197] (c2) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 8 to 13.5, preferably a pH of about 10 to 13.5;

[0198] (d) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0199] In a preferred embodiment, the protease variant includes substituted X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D. Therefore, one embodiment of the present invention relates to a liquid cleaning composition comprising:

[0200] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0201] (b) Water ranging from 40% to 95% wt, preferably from about 70% to about 90% wt;

[0202] (c) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or alkali metal hydroxide or a mixture thereof, to provide a pH from about 11 to 13.5; and

[0203] (d) A protease variant ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt, wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO). 1) The variants replacing X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D, preferably, include variants corresponding to BPN' (SEQ ID NO). 1) Substitution of X205I and X209W, more preferably, the variant includes substitution of X205I and X209W corresponding to BPN' (SEQ ID NO 1) and substitution of X205I and X209W corresponding to BPN' (SEQ ID NO 1). 1) Substitution of one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D.

[0204] Another embodiment of the present invention relates to a liquid cleaning composition comprising:

[0205] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0206] (b) a protease variant from 0.001% to 10% wt, preferably 0.1% to 5% wt, wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D, preferably, the variants include those corresponding to BPN' (SEQ ID). Substitution of X205I and X209W corresponding to BPN' (SEQ ID NO:1), further preferably, the variant includes substitution of X205I and X209W corresponding to BPN' (SEQ ID NO:1) and substitution of X205I and X209W corresponding to BPN' (SEQ ID NO:1). NO:1) substituted for one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D; and

[0207] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0208] (c2) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH from about 8 to 13.5, preferably from about 10 to 13.5;

[0209] (d) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0210] In some preferred embodiments, the protease is selected from the group consisting of the following items:

[0211] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0212] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0213] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0214] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0215] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0216] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0217] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0218] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0219] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0220] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0221] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0222] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0223] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0224] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0225] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0226] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0227] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0228] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0229] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0230] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0231] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0232] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0233] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0234] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0235] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0236] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0237] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0238] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0239] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0240] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0241] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0242] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0243] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0244] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0245] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0246] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0247] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0248] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0249] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0250] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0251] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0252] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0253] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0254] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0255] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0256] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0257] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0258] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0259] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0260] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0261] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0262] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0263] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0264] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0265] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0266] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0267] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0268] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0269] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0270] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0271] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0272] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0273] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0274] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[0275] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E。

[0276] When compared to parental proteases such as those having SEQ ID NO:2, the protease variants of the present invention exhibit improved stability in liquid cleaning compositions at pH 10 or higher. This makes these variants suitable for high-pH liquid detergents, such as for laundry or dishwashing.

[0277] In one embodiment, the protease variant exhibits improved stability, particularly improved storage stability, in high-pH liquid cleaning compositions compared to the parent protease. In a preferred embodiment, the protease variant exhibits improved stability, particularly improved storage stability, and equivalent or improved washing performance compared to the parent protease.

[0278] In one embodiment, the protease variant exhibits improved stability and equivalent or improved washing performance in high-pH liquid cleaning compositions compared to the parent protease, wherein storage stability can be measured using an accelerated storage stability assay and washing performance can be measured using an automated mechanical stress assay (AMSA).

[0279] In one embodiment, the protease variant has at least 60% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 70% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 75% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 80% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 85% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 90% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 93% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 95% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 96% but less than 100% sequence identity with the parent protease. In one embodiment, the protease variant has at least 97% but less than 100% sequence identity with the parent protease. In another embodiment, the protease variant has at least 98% but less than 100% sequence identity with the parent protease.

[0280] In one embodiment, the variant has an amino acid sequence that is at least 60% identical to SEQ ID NO:1, for example, having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0281] In another embodiment, the variant has an amino acid sequence that is at least 60% identical to SEQ ID NO:2, for example, having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0282] On one hand, the total number of changes in the parent protease is between 3 and 30, preferably between 3 and 20, more preferably between 3 and 15, even more preferably between 3 and 10, and most preferably between 3 and 8 changes. On the other hand, the total number of changes in the parent protease is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 changes.

[0283] The parental or precursor protease can be any protease, or even more preferably any subtilisin as defined below. The parental protease is preferably a protease having at least 60% identity with SEQ ID NO:2, but the parent can be any serine protease. Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds and have an essential serine residue at their active site (White, Handler, and Smith, 1973, "Principles of Biochemistry," 5th ed., McGraw-Hill Book Company, New York, pp. 271-272). Bacterial serine proteases have a molecular weight range of 20,000 to 45,000 Daltons. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsins, which are also serine proteases. Even more preferred parents are alkaline proteases, which encompass a subgroup that reflects the high optimum pH of some serine proteases from pH 9.0 to 11.0 (see Priest (1977) Bacteriological Rev. 41:711-753 for a review).

[0284] The parental protease could be a substantia nigra, a subgroup of serine proteases proposed by Siezen et al. (1991), Protein Engineering 4:719-737 and Siezen et al. (1997), Protein Science 6:501-523. They were defined by homology analysis of more than 170 amino acid sequences of serine proteases previously known as substantia nigra-like proteases. Substantia nigra proteases were previously generally defined as serine proteases produced by Gram-positive bacteria or fungi, but are now, according to Siezen et al., a subgroup of substantia nigra proteases. A wide variety of substantia nigra proteases have been identified, and the amino acid sequences of many have been determined. For a more detailed description of such substantia nigra proteases and their amino acid sequences, see Siezen et al. (1997). Substantiates are a subgroup of substantiates, as described above, that are serine proteases from the S8 family, particularly from the S8A subfamily, as defined in the MEROPS database (http: / / merops.sanger.ac.uk / cgi-bin / famsum?family=S8). Examples of substantiates are substantiates BPN' (SEQ ID NO:1) and substantiates 309 (SEQ ID NO:2), which have MEROPS numbers S08.034 and S08.003, respectively. Parental proteases can also be substantiates isolated from natural sources, wherein they have been subsequently modified (e.g., one or more substitutions, substitutions, deletions, and / or insertions of one or more amino acid side chains) while retaining the characteristics of substantiates. In addition, the parental protease can be a subtilis enzyme prepared by DNA shuffling technology, as described by Ness et al., 1999, Nature Biotechnology, 17:893-896.

[0285] Alternatively, the term "parental protease" can be referred to as "precursor protease" and is used to describe the initiating protease in which mutations are performed to obtain the variant of the present invention. This parental protease preferably belongs to the substantia nigra protease subgroup.

[0286] A subgroup of subtilisinases, I-S1 or “true” subtilisin, including “standard” subtilisinases such as subtilisin 168 (BSS168), subtilisin BPN', and Carlsberg subtilisin (subtilisin Carlsberg) Novozymes), and BPN' is a subtilisin DY (BSSDY) derived from Bacillus amyloliquefaciens. BPN' has the amino acid sequence of SEQ ID NO:1. Siezen et al. (see above) identified another subgroup of subtilisinases, I-S2 or highly alkaline subtilisinases. Subgroup I-S2 proteases are described as highly alkaline subtilisinases and include enzymes such as: BPN' PB92 (BAALKP) Genencor International, Inc.), Bacillus subtilis protease 147 (BLS147) Novozymes, alkaline elastase YaB (BSEYAB), and subtilisin 309 with the amino acid sequence SEQ ID NO 2 ( Novozymes).

[0287] The homology between two amino acid sequences, described by the parameter "identity" for the purposes of this invention, is determined using the Niedermann-Onsch algorithm as described above. The results from the program calculate the "percentage identity" between the two sequences in addition to amino acid alignment.

[0288] Based on this description, it will be fairly straightforward for those skilled in the art to identify suitable homologous subtilisases that can be modified according to the present invention.

[0289] The parent protease may be a polypeptide having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:1, and possessing protease activity. In one aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:1 by up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0290] The parent protease may be a polypeptide having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:2, and possessing protease activity. In one aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:1 by up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0291] The parent protease can be a hybrid polypeptide in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0292] This parental protease can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0293] The parent can be a bacterial protease. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. Tomyces protease; or a Gram-negative bacterial polypeptide, such as Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease.

[0294] On one hand, the parent is a protease of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus scoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

[0295] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Dutch Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Agricultural Research Culture Collection (NRRL) of the United States.

[0296] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural habitat are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0297] The high pH-stable protease variant according to the present invention can be prepared by a method comprising the following steps:

[0298] (a) Introducing substitutes for X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D into the parent protease, wherein these positions correspond to SEQ ID The position of NO:1, and

[0299] (b) Recycle the variant.

[0300] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0301] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0302] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0303] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, US 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0304] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0305] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques involving the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0306] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or truncation, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409, WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0307] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0308] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments in conjunction with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenic primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0309] In addition to the protease according to the invention, the liquid cleaning composition according to the invention also includes other components, such as the non-limiting components listed below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or extent of the stain, the temperature at which cleaning is performed, and the formulation of the detergent product.

[0310] In one specific embodiment, the high-pH liquid cleaning composition includes a protease variant of the present invention and one or more detergent components, such as surfactants, water-soluble solvents, builders, co-builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric toners, fabric conditioning agents, foaming agents, defoaming agents, dispersants, dye transfer inhibitors, fluorescent brighteners, fragrances, optical brighteners, bactericides, fungicides, dirt suspending agents, dirt-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0311] The protease variants of the clean compositions of the present invention can be stabilized using conventional stabilizers and protease inhibitors, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, various salts (e.g., NaCl, KCl), lactic acid, formic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters; or phenyl boric acid derivatives such as 4-formylphenylboronic acid (e.g., as in WO 96 / 41859)), or peptide aldehydes (e.g., dipeptide aldehydes, tripeptide aldehydes, or tetrapeptide aldehydes or aldehyde analogs) (or having the form B1-B0-R, wherein R is H, CH3, CX3, CHX2, or CH2X (X = halogen), B0 is a single amino acid residue (preferably having an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two, or three), optionally including an N-terminal protecting group (e.g., as in WO 2009 / 118375, WO 2013 / 004635), or as described in WO (as in WO 2005 / 105826, WO 2009 / 118375, WO 98 / 13459) or protein-type protease inhibitors such as RASI, BASI, WASI (bifunctional α-amylase / subtilisin inhibitors of rice, barley and wheat) (e.g., as in WO 2009 / 095425) or CI2 or SSI.

[0312] Furthermore, the use of peptidaldehydes for stabilizing certain proteases in liquid detergents has been disclosed in PCT publications WO 94 / 04651 and WO 98 / 13460. More specifically, WO 94 / 04651 discloses the use of peptidaldehydes Phe-Gly-Ala-PheH and Phe-Gly-Ala-LeuH for stabilizing Bacillus subtilis protease-type proteases. For stabilizing chymotrypsin-type proteases, WO 94 / 04651 discloses Leu-Leu-TyrH as a suitable peptidaldehyde. Additionally, WO 94 / 04651 suggests using methyl carbamate or methylurea as the N-terminal protecting group of the peptidaldehyde. WO 98 / 13460 discloses the use of peptidase inhibitors, peptide aldehydes, or trifluoromethyl ketones, wherein the peptide chain contains 2-5 amino acids, and the aldehyde / trifluoromethyl ketone is derived from the amino acids alanine, valine, isoleucine, leucine, phenylglycine, phenylalanine, or homophenylalanine, and wherein the N-terminal protecting group is preferably sulphonamide or amide phosphate. As an example, CH3SO2Phe-Gly-Ala-LeuH can be used.

[0313] In another embodiment, a compound having general formula I:

[0314]

[0315] R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, where X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine; it can be used to stabilize and / or inhibit the protease variants of the present invention.

[0316] In another embodiment, the protease inhibitor is a peptide compound having the chemical formula B2-B1-B0-R, wherein:

[0317] R is hydrogen, CH3, CX3, CHX2 or CH2X, where X is a halogen atom;

[0318] B0 is a phenylalanine residue with an OH substitution at the para and / or meta positions;

[0319] B1 is a single amino acid residue; and

[0320] B2 consists of one or more amino acid residues, optionally including an N-terminal protecting group.

[0321] The protease inhibitor having the chemical formula B2-B1-B0-R as described above can be used as an optional protease inhibitor in the sense of this invention (e.g., in combination with or as part of any embodiment of the invention as disclosed herein).

[0322] Therefore, in another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising:

[0323] (a) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence that is at least 60% sequence identical to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C.

[0324] (b) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0325] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0326] (a) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0327] (b) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence that is at least 60% sequence identical to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C.

[0328] (c) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, wherein B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0329] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0330] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0331] (b) a protease variant ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt; and

[0332] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0333] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0334] (d) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0335] In the embodiments described, (c1) and (c2) can be used as substitutes for each other or they can be used in combination.

[0336] Therefore, in another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising:

[0337] (a) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence that is at least 60% sequence identical to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant includes substitutions at one or more positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN' (SEQ ID NO 1);

[0338] (b) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0339] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0340] (a) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0341] (b) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence that is at least 60% sequence identical to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant includes substitutions at one or more positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN' (SEQ ID NO 1);

[0342] (c) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, wherein B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0343] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0344] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0345] (b) a protease variant from 0.001% to 10% wt, preferably 0.1% to 5% wt, preferably comprising substitutions at one or more sites selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1); and

[0346] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0347] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0348] (d) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0349] In the embodiments described, (c1) and (c2) can be used as substitutes for each other or they can be used in combination.

[0350] Therefore, in another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising:

[0351] (a) At least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant includes substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO:1), more preferably, the variant includes substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO:1) and substitutions at one or more of the following positions selected from those corresponding to BPN' (SEQ ID NO:2). The positions of NO:1) are 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262;

[0352] (b) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0353] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0354] (a) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0355] (b) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant includes substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO:1), more preferably, the variant includes substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO:1) and substitutions at one or more of the following positions selected from those corresponding to BPN' (SEQ ID NO:1). 1) The positions of 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262;

[0356] (c) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, wherein B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0357] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0358] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0359] (b) a protease variant from 0.001% to 10% wt, preferably 0.1% to 5% wt, preferably comprising substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1), more preferably comprising substitutions at positions 205 and 209 corresponding to BPN' (SEQ ID NO: 1) and substitutions at one or more of the following positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1); and

[0360] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0361] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0362] (d) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0363] In the embodiments described, (c1) and (c2) can be used as substitutes for each other or they can be used in combination.

[0364] Therefore, in another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising:

[0365] (a) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO:2). 1) Replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D;

[0366] (b) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0367] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0368] (d) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0369] (e) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO:2). 1) Replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D;

[0370] (f) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having the general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, wherein B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0371] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0372] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0373] (b) a protease variant from 0.001% to 10% wt, preferably 0.1% to 5% wt, preferably, said variant comprising one or more substitutions corresponding to BPN' (SEQ ID NO). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D; and

[0374] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0375] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0376] (d) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably, the protease inhibitor is a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0377] In the embodiments described, (c1) and (c2) can be used as substitutes for each other or they can be used in combination.

[0378] Therefore, in another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or higher, the composition comprising:

[0379] (a) At least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1), more preferably, the variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1). 1) Substitution of one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D;

[0380] (b) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0381] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0382] (a) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0383] (b) At least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent at pH 10 at 40°C, preferably, the variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1), more preferably, the variant comprises substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1) and substitutions corresponding to X205I and X209W of BPN' (SEQ ID NO 1). 1) Substitution of one or more of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D;

[0384] (c) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, wherein B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0385] In another embodiment, the present invention relates to a liquid cleaning composition comprising:

[0386] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0387] (b) From 0.001% to 10% wt, preferably 0.1% to 5% wt, of a protease variant, preferably comprising substitutions of X205I and X209W corresponding to BPN' (SEQ ID NO: 1), more preferably, the variant comprising substitutions of X205I and X209W corresponding to BPN' (SEQ ID NO: 1) and substitutions of X205I and X209W corresponding to BPN' (SEQ ID NO: 1). 1) Substitution of X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E, and X262D; and

[0388] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0389] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 11 to 13.5, preferably a pH of about 12 to 13.5;

[0390] (d) Optionally, a protease inhibitor, preferably a peptidaldehyde protease inhibitor, more preferably a compound having general formula I, wherein R is selected from the group consisting of: hydrogen, CH3, CX3, CHX2, CH2X, wherein X is a halogen atom, B1 is a single amino acid residue, and B2 is one or more amino acid residues, B2 optionally including an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0391] In the embodiments described, (c1) and (c2) can be used as substitutes for each other or they can be used in combination.

[0392] In some preferred embodiments, the protease is selected from the group consisting of the following items:

[0393] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0394] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0395] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0396] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0397] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0398] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0399] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0400] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0401] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0402] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0403] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0404] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0405] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0406] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0407] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0408] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0409] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0410] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0411] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0412] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0413] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0414] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0415] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0416] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0417] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0418] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0419] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0420] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0421] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0422] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0423] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0424] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0425] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0426] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0427] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0428] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0429] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0430] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0431] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0432] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0433] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0434] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0435] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0436] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0437] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0438] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0439] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0440] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0441] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0442] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0443] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0444] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0445] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0446] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0447] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0448] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0449] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0450] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0451] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0452] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0453] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0454] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0455] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0456] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[0457] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E。

[0458] The composition can be formulated as described, for example, in WO 92 / 19709, WO 92 / 19708 and US 6,472,364. In some embodiments, the enzymes used herein are stabilized by water-soluble sources of zinc(II), calcium(II) and / or magnesium(II) ions present in the finished composition providing such ions for the enzymes, along with other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II) and vanadium(IV)).

[0459] The protease variants of the present invention can also be incorporated into detergent formulations disclosed in WO 97 / 07202 (incorporated herein by reference).

[0460] The liquid cleaning composition of the present invention is characterized by having a high pH (pH 10 or above). The high-pH liquid cleaning composition may further comprise a high amount of water. In a preferred aspect of the invention, the liquid cleaning composition comprises:

[0461] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0462] (b) Water ranging from 40% to 95% wt, preferably from about 70% to about 90% wt;

[0463] (c) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or alkali metal hydroxide or a mixture thereof, to provide a pH from about 11 to 13.5; and

[0464] (d) Protease variants ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt.

[0465] Surfactants can be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In one specific embodiment, the cleaning composition comprises one or more nonionic surfactants and a compound with one or more anionic surfactants. Surfactants are typically present at levels of about 0.1% to 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10% by weight. When included in a cleaning composition according to the invention, the surfactant will generally be contained from about 1% to about 40% by weight, such as from about 1% to about 20%, preferably from about 0% to about 5% by weight. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkane sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkane-2,3-dimethylbis(sulfate), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) (e.g., sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0466] When included in a cleaning composition according to the invention, it will typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0467] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)), together with products and combinations thereof available under the trade names SPAN and TWEEN.

[0468] Non-limiting examples of semi-polar surfactants include amine oxides (AOs) (e.g., alkyl dimethyl amine oxides), N-(cocoylalkyl)-N,N-dimethyl amine oxides and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides and combinations thereof.

[0469] Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

[0470] Preferred surfactants include LAS, MES, alcohol ether sulfates, ethoxylated lauryl alcohol, or sodium dodecylbenzene sulfonate.

[0471] The liquid high-pH cleaning composition according to the invention typically comprises at least 20% and up to 95% water by weight, such as up to about 70% wt, up to about 65% wt, up to about 55% wt, up to about 45% wt, or up to about 35% wt. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in the liquid cleaning composition. The cleaning composition may contain from 0% to 30% organic solvents.

[0472] The cleaning composition may also contain a co-hydrophilic solvent, which is a compound that dissolves a hydrophobic compound in an aqueous solution (or conversely, a polar substance in a nonpolar environment). Typically, co-hydrophilic solvents possess both hydrophilic and hydrophobic characteristics (such as the so-called amphiphilic properties known from surfactants); however, the molecular structure of co-hydrophilic solvents generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12:121-128. Co-hydrophilic solvents do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and the formation of micelles, thin layers, or other well-defined intermediate phases by lipids occur. Instead, many co-hydrophilic solvents exhibit a continuous type of aggregation process, where the size of the aggregates increases with increasing concentration. However, many co-hydrophilic solvents alter the phase behavior, stability, and colloidal properties of systems containing substances with both polar and nonpolar characteristics, including mixtures of water, oils, surfactants, and polymers. Water-soluble solvents are classically used across industries, from pharmaceuticals, personal care, and food to technical applications. The use of water-soluble solvents in cleaning compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity. Cleaning compositions according to the invention may contain 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5% wt, of a water-soluble solvent. Any water-soluble solvent known in the art for use in detergents may be used. Non-limiting examples of water-soluble solvents include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthylformate, sodium hydroxynaphthylsulfonate, sodium ethylhexylsulfonate, and combinations thereof. Cleaning compositions according to the invention may contain about 0-65% by weight, such as about 5% to about 45%, of a detergent builder or co-builder, or a mixture thereof. In dishwashing detergents, the level of builder agents is typically 40%-65%, particularly 50%-65%. Builder agents and chelating agents soften wash water, for example, by removing metal ions from the liquid. Builder agents and / or co-builder agents can be, in particular, chelating agents that form water-soluble complexes with Ca and Mg. Any builder agent and / or co-builder agent known in the art for use in laundry detergents can be utilized.Non-limiting examples of detergent builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium silicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2'-meta-aminotriethanol), and carboxymethyl inulin (CMI) and combinations thereof.

[0473] The cleaning compositions according to the invention may further comprise 0-20% by weight, such as about 5% to about 10% wt, of a detergent co-agent, or a mixture thereof. The detergent composition may comprise only the co-agent, or in combination with a co-agent, such as a zeolite co-agent. Non-limiting examples of co-agents include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-dibutanoic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta-(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), and iminodisuccinic acid (IDS). N-(2-sulfomethyl)-aspartic acid (IDA), N-(2-sulfoethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), o-amino acid N,N-diacetic acid (ANDA), sulfanilamide-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), sulfonyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and their salts. Further exemplary builders and / or co-builders are described, for example, in WO 2009 / 102854 and US 5,977,053.

[0474] The cleaning compositions according to the invention may contain 0-50% by weight, such as about 0.1% to about 25%, of a bleaching system. Bleaching systems often remove color through oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Any bleaching system known in the art for use in laundry detergents can be utilized. Suitable bleaching system components include bleaching catalysts, hypochlorites, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, pre-formed peracids, and mixtures thereof.

[0475] Some non-limiting examples of bleaching catalysts that can be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), specifically Me3-TACN, such as the binuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2”-nitrotris(ethane-1,2-diylazalkylene-κN-methylene)triphenol-κ3O]manganese(III). The bleaching catalyst can also be other metal compounds, such as iron or cobalt complexes.

[0476] Suitable preformed peracids include, but are not limited to: peroxycarboxylic acids and their salts, percarbonates and their salts, perimidic acids and their salts, peroxymonosulfate and their salts (e.g., potassium persulfate (Oxone(R))), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems that combine with peracids to form bleaching activators, which may contain, for example, inorganic salts, including alkali metal salts such as sodium salts of perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, and persilicates. The term bleaching activator herein refers to a compound that reacts with a peroxide bleaching agent (like hydrogen peroxide) to form a peracid. The resulting peracid structure... Activated bleaching agents. Suitable bleaching activators to be used herein include those belonging to the ester amide, imide, or acid anhydride classes. Suitable examples are tetraacetyl ethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), dipperoxylauric acid, 4-(dodecyloxy)benzenesulfonate (LOBS), 4-(decyloxy)benzenesulfonate, 4-(decyloxy)benzoate (DOBS), 4-(nonanoyloxy)benzenesulfonate (NOBS) and / or those disclosed in WO Those listed in 98 / 17767. Specific families of bleaching activators are disclosed in EP 624154, and particularly acetylacetic acid triethyl ester (ATC). ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly because it eventually degrades into citric acid and alcohol. Furthermore, acetylacetic acid triethyl ester and triacetin exhibit good hydrolytic stability in the product during storage, and are effective bleaching activators. Finally, ATC provides good washing ability for garment additives. Alternatively, the bleaching system may include, for example, amides, imides, or sulfone-type peroxy acids. The bleaching system may also include peracids such as 6-(phthalimide)percapanoic acid (PAP). The bleaching system may also include a bleaching catalyst.

[0477] In some embodiments, the bleaching component may be an organic catalyst selected from organic catalysts having the following chemical formulas:

[0478]

[0479] (iii) and its mixtures; wherein each R 1 Independently, it is a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably, each R 1 Independently, it is a branched alkyl group containing 9 to 18 carbons or a straight-chain alkyl group containing 11 to 18 carbons; more preferably, each R 1Independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isonyl, isodecyl, iso-tridecyl, and iso-pentadecanyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, and WO2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc phthalocyanine.

[0480] One specific bleaching agent is hypochlorite, such as sodium hypochlorite (NaClO). Hypochlorite is stable due to its high pH, ​​and furthermore, it is an antibacterial agent. Typically, cleaning compositions according to the invention contain 3%-8% sodium hypochlorite and 0.01%-0.05% sodium hydroxide; sodium hydroxide is used to slow the decomposition of sodium hypochlorite into sodium chloride and sodium chlorate. Hypochlorite may not be part of the detergent formulation, but is added separately during the washing process.

[0481] In one aspect of the invention, the high pH cleaning composition of the invention does not contain any bleach, i.e., the cleaning composition may be bleach-free.

[0482] The cleaning compositions according to the invention may contain 0-10% by weight, such as 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1% of a polymer. Any polymer known in the art for use in detergents may be used. The polymer may function as a co-cleaning agent as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or anti-foaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicone, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered. The cleaning compositions of the present invention may also include fabric toners, such as dyes or pigments, which, when formulated in a detergent composition, can deposit on the fabric when the fabric comes into contact with a detergent liquid comprising the detergent composition, and thus alter the color of the fabric by absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric toners alter the color of a surface because they absorb at least a portion of the visible light spectrum. Suitable fabric toners include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of the following dyes that fall under the Colour Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red or mixtures thereof, for example as described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276 and EP 1876226 (incorporated by reference).The detergent composition preferably includes a fabric toner from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt%. The composition may include 0.0001 wt% to 0.2 wt% of fabric toner, which may be particularly preferred when the composition is in the form of a unit-dosage pouch. Suitable toners are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0483] The cleaning compositions according to the invention may include one or more (additional) enzymes, such as amylase, hydrolase, arabinase, glycosylase, cellulase (e.g., endoglucanase), keratinase, galactanase, halogenated peroxidase, lipase, mannanase, oxidase, such as laccase and / or peroxidase, pectinase, pectin lyase, protease, DNase, xylanase, xanthan gumase, and xyloglucanase.

[0484] Generally, the properties of one or more selected enzymes should be compatible with the detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0485] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Apocytozoa*, such as fungal cellulases produced by *Pyrophyllus*, *Thermophyllus*, and *Fusarium* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0486] Alkaline or neutral cellulases, which offer color-care benefits, are particularly suitable. Commercially available cellulases include Celluzyme. TM and Carezyme TM (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM (Kao Corporation).

[0487] The composition may include one or more additional proteases, including those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. This includes chemically modified or protein-engineered mutants. It may be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases may be, for example, from the S1 family (e.g., trypsin) or the S8 family (e.g., subtilisin). Metalloproteinases may be, for example, thermophilic bacterial proteases from family M4 or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0488] Examples of metalloproteinases are neutral metalloproteinases as described in WO 2007 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0489] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm Durazym Tm , Ultra Ultra Ultra Ultra as well as (Novozymes), those sold under the following product names: Purafect Purafect Purafect Purafect as well as (Danisco / DuPont), Axapem TM (Gist-Brocades NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0490] Suitable lipases and keratins include those of bacterial or fungal origin. This also includes chemically modified or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermophilic*, such as those from *Thermophilic Hypotherium* (formerly named *Pyrophyte*) as described in EP 258068 and EP305216; cutinases from the genus *Pyrophyte*, such as *Pyrophyte Specific* (WO 96 / 13580); lipases from strains of the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *Alcaligenes* or *Alcaligenes-like* (EP 218272), *Pseudomonas cepacia* (EP331376), *Pseudomonas* strain SD705 (WO 95 / 06720 & WO 96 / 27002), *Pseudomonas wisconsinensis* (WO 96 / 12012); and GDSL-type *Streptomyces* lipases (WO 96 / 12012). 2010 / 065455); cutinase from *Oryza sativa* (WO 2010 / 107560); cutinase from *Pseudomonas mendoza* (US 5,389,536); lipase from *Thermobifida fusca* (WO 2011 / 084412); lipase from *Bacillus stearothermophilus* (WO 2011 / 084417); lipase from *Bacillus subtilis* (WO 2011 / 084599); and lipase from *Streptomyces griseus* (WO 2011 / 150157) and *S. pristinaespiralis* (WO 2012 / 137147). Preferred commercially available lipase products include Lipolase. TM Lipex™; Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0491] Other examples are lipases sometimes called acyltransferases or perhydrolases, such as acyltransferases homologous to Candida antarctica lipase A (WO 2010 / 111143), acyltransferases from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of Mycobacterium smegmatis perhydrolases (especially the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 2010 / 100028).

[0492] Suitable amylases can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Commercially available amylases include Duramyl. TM Special amylase TM Fungayl TM Stainzyme TM Stainzyme Plus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont).

[0493] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *Coprinus spp.*, and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0494] Commercially available peroxidases include Guardzyme. TM (Novozymes)

[0495] The one or more detergent enzymes can be included in the cleaning composition by adding a separate additive comprising one or more enzymes, or by adding a combination additive comprising all of these enzymes. The detergent additives of the present invention, i.e., individual or combined additives, can be formulated as, for example, granules, liquids, slurries, etc., with preferred detergent additive formulations being granules, especially dust-free granules; liquids, especially stabilized liquids; or slurries.

[0496] Any detergent component known in the art for use in laundry cleaning compositions may also be used. Other optional detergent components include preservatives, shrinkage inhibitors, anti-fouling redeposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC and / or polyols such as propylene glycol), fabric finishing agents (including clays), fillers / processing aids, optical brighteners / brighteners, foaming agents, foam (foam) regulators, fragrances, soil suspending agents, softeners, defoamers, dulling inhibitors, and wicking agents, used alone or in combination. Any ingredient known in the art for use in laundry detergents may be used. The selection of such ingredients is entirely within the skill of a person of ordinary skill.

[0497] The cleaning compositions of the present invention may also contain dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powder Detergents, Surfactant Science Series, Volume 71, Marcel Dekker.

[0498] The cleaning compositions of the present invention may further include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole, or mixtures thereof. When present in the subject composition, the dye transfer inhibitor may be present at the following levels by weight of the composition: from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%.

[0499] The cleaning compositions of the present invention will also preferably contain additional components that can color the items being cleaned, such as optical brighteners or brighteners. The brightener is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of optical brighteners include sodium salts of the following: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-diphenylamino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine 4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 2-(stilbene-4"-naphthalene-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate. Also preferred is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-aminoalkylcoumarin. Suitable fluorescent whitening agent levels range from a lower level of about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from a lower level of about 0.2 wt% to a higher level of 0.5 wt% or even 0.75 wt%.

[0500] The cleaning compositions of the present invention may also include one or more dirt-releasing polymers that help remove dirt from fabrics, such as cotton and polyester-based fabrics, particularly hydrophobic dirt from polyester-based fabrics. Dirt-releasing polymers may be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Powdered Detergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Dekker, Inc. Another type of dirt-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may comprise a polyalkylimide structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (which is hereby incorporated by reference). Furthermore, any graft copolymer is a suitable dirt-releasing polymer. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (which are hereby incorporated by reference). Other dirt-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, such as those described in EP 1867808 or WO 03 / 040279 (both of which are hereby incorporated by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0501] The cleaning compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under the category of waste-releasing polymers may also function as anti-redeposition agents.

[0502] Other suitable excipients include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, water-soluble solvents, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastics for liquid detergents.

[0503] The cleaning compositions of the present invention can be formulated into bags, which can be configured as single-compartment or multi-compartment bags. They can have any form, shape, and material suitable for preserving the composition, for example, preventing its release from the bag before contact with water. The bags are made of a water-soluble membrane encapsulating an internal volume. This internal volume can be divided into compartments within the bag. Preferred membranes are polymeric materials that form membranes or sheets, preferably polymers. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference M8630, as sold by Chris Craft In. Prod., Gary, Indiana, US), plus plasticizers like glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry detergent compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. The chamber for the liquid component may differ in composition from the chamber containing the solid component. See, for example, US2009 / 0011970.

[0504] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the washing solution can cause delayed dissolution of selected components.

[0505] The present invention also relates to methods of using the cleaning compositions according to the invention in the washing of textiles and fabrics, such as household laundry and industrial laundry. Such methods include using the compositions or combinations thereof according to the invention in cleaning hard surfaces such as floors, tables, walls, roofs, etc., as well as surfaces of hard objects such as automobiles (car washing) and tableware (tableware washing).

[0506] The protease variants of the present invention can be added to and thus become a component of the high-pH cleaning composition according to the present invention. Therefore, one aspect of the present invention relates to the use of the protease variants in high-pH cleaning processes such as laundry washing and / or hard surface cleaning at pH 10 or higher.

[0507] The cleaning compositions of the present invention can be formulated as, for example, a detergent composition for hand or machine washing, including a laundry detergent additive composition suitable for pre-treating stained fabrics and a fabric softener composition for rinsing, or a detergent composition for general household hard surface cleaning operations, or a detergent composition for hand or machine washing of dishes.

[0508] The cleaning process or textile care process can be, for example, a laundry process, a dishwashing process, or the cleaning of hard surfaces (such as bathroom tiles, floors, countertops, drains, sinks, and basins). The laundry process can be, for example, household laundry, but it can also be industrial laundry. Furthermore, the present invention relates to a method for washing fabrics and / or garments, wherein the method comprises treating the fabric with a washing solution containing a high-pH cleaning composition according to the invention and at least one protease variant of the invention. The cleaning process or textile care process can be, for example, performed by machine washing, hand washing, or a pre-spotter process. The washing solution can be, for example, an aqueous washing solution containing the cleaning composition.

[0509] This invention further relates to the use of the cleaning composition of this invention in the removal of protein stains. Protein stains may be stains such as food stains, including baby food, sebum, cocoa, eggs, blood, milk, ink, grass, or combinations thereof.

[0510] Preferred embodiments relate to a cleaning method comprising the step of contacting an object with a high-pH cleaning composition, including a protease variant of the present invention, under conditions suitable for cleaning the object. In a preferred embodiment, the cleaning composition is used in the washing of clothing or tableware.

[0511] Another embodiment relates to a method for removing stains from fabrics or tableware, the method comprising contacting the fabric or tableware with a high-pH cleaning composition including the protease of the present invention under conditions suitable for cleaning the object.

[0512] Compositions and methods for treating fabrics (e.g., desizing textiles) using the cleaning compositions of the present invention are also contemplated. High-pH cleaning compositions can be used in any fabric treatment method, methods well known in the art (see, for example, US 6,077,316). For example, in one aspect, a method for improving the feel and appearance of a fabric includes contacting the fabric with a protease in a solution. In another aspect, the fabric is treated with the solution under pressure.

[0513] The high-pH cleaning composition of the present invention is suitable for use in liquid laundry and liquid hard surface applications (including dishwashing and car washing). Therefore, the present invention includes a method for washing fabrics or hard surfaces. This method includes the step of contacting the fabric / dishware to be cleaned with a solution comprising the high-pH cleaning composition according to the present invention. Fabrics may include any fabric capable of being washed under normal consumer use conditions. Hard surfaces may include any tableware, such as earthenware, knives, ceramics, plastics (e.g., melamine), metals, porcelain, glass, acrylic resins, or other hard surfaces (e.g., automotive, floors, etc.). The solution preferably has a pH from about 9 to about 13.5. The composition can be used in the solution at concentrations from about 100 ppm, preferably 500 ppm, to about 15,000 ppm. The water temperature range is typically from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The water-to-fabric ratio is typically from about 1:1 to about 30:1.

[0514] In some preferred embodiments, these high-pH cleaning compositions provided herein are typically formulated such that, during aqueous cleaning operations, the wash water has a pH of from about 9 to about 13.5, or, in alternative embodiments, from about 10 to about 13.5, or even from about 11 to about 13.5. In some preferred embodiments, liquid laundry detergent products are formulated to have a pH of from about 12 to about 13.5. Techniques for controlling the pH at recommended usage levels include the use of buffers, acids, bases, etc., and are well known to those skilled in the art. In the context of this invention, the use of bases to adjust the pH to about 9 to 13.5, preferably about 10 to 13.5.

[0515] The invention is further summarized in the following paragraphs:

[0516] 1. A liquid cleaning composition having a pH of 10 or higher, comprising:

[0517] (a) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence with at least 60% sequence identity with the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C.

[0518] (b) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0519] 2. A liquid cleaning composition, comprising:

[0520] (a) Water from 20% to 95% wt, preferably from 40% to 95% wt, and more preferably from about 70% to about 90% wt;

[0521] (b) at least 0.01 wt% of a protease, wherein the protease is a variant of the parent protease, and wherein the protease variant has an amino acid sequence that is at least 60% sequence identical to the amino acid sequence of SEQ ID NO:2, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours at 40°C in a liquid detergent having a pH of 7.5 or higher, preferably 10.

[0522] (c) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0523] 3. The liquid cleaning composition as described in paragraph 1, wherein the pH is from about 12 to 13.5.

[0524] 4. The liquid cleaning composition as described in any one of paragraphs 1-3, wherein the protease is a variant of a protease having at least 60% identity with the amino acid sequence of SEQ ID NO:2, wherein the variant includes substitutions at one or more positions selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO:1).

[0525] 5. The liquid cleaning composition as described in any one of paragraphs 1-4, wherein the protease is a variant of a protease having at least 60% identity with the amino acid sequence of SEQ ID NO:2, wherein said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO:2). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D.

[0526] 6. The liquid cleaning composition as described in any one of paragraphs 1-5, wherein the variant comprises one or more substitutions corresponding to substitutions X209W, X262E, X76D, X194P, X204D, and X206L.

[0527] 7. The liquid cleaning composition according to any one of the preceding paragraphs, wherein the variant has an amino acid sequence that is at least 60% identical to SEQ ID NO:2, for example, having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0528] 8. The liquid cleaning composition according to any one of the preceding paragraphs, wherein the protease variant has greater storage stability at 40°C than at least one stable protease variant selected from the group consisting of:

[0529] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0530] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0531] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0532] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0533] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

[0534] S9E+N18S+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0535] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0536] S9E+N43R+N76D+A194P+Q206L+Y209W+S259D+N261W+L262ES9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0537] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0538] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0539] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0540] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

[0541] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0542] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0543] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0544] S9E+N76D+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0545] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0546] S9E+N43R+N76D+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0547] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0548] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0549] S9E+N43R+I72A+N76D+A194P+Q206L+L262E;

[0550] S9E+N43R+N76D+A194P+V205I+Q206L+L262E.

[0551] 9. The liquid cleaning composition according to any one of the preceding paragraphs, wherein the protease variant is selected from the group consisting of:

[0552] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0553] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0554] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0555] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0556] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0557] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0558] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0559] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0560] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0561] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0562] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0563] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0564] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0565] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0566] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0567] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0568] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0569] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0570] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0571] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0572] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0573] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0574] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0575] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0576] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0577] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0578] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0579] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0580] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0581] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0582] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0583] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0584] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0585] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0586] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0587] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0588] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0589] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0590] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0591] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0592] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0593] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0594] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0595] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0596] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0597] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0598] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0599] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0600] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0601] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0602] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0603] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0604] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0605] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0606] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0607] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0608] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0609] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0610] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0611] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0612] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0613] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0614] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0615] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[0616] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

[0617] 10. A liquid cleaning composition comprising:

[0618] (a) A detergent surfactant ranging from 0% to 20% wt, preferably from 0.1% to 2.5% wt;

[0619] (b) a protease variant ranging from 0.001% to 10% wt, preferably from 0.1% to 5% wt; and

[0620] (c1) Water from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt; and / or

[0621] (c2) An alkaline buffer system from 1% to 30% wt, the alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH of about 7.5 to 13.5, preferably a pH of about 12 to 13.5;

[0622] (d) Optionally, the protease inhibitor, preferably the protease inhibitor, is a peptidoprotease inhibitor.

[0623] 11. The liquid cleaning composition as described in paragraph 10, wherein the protease variant includes substitutions at one or more sites selected from positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261, and 262 corresponding to BPN' (SEQ ID NO: 1).

[0624] 12. The liquid cleaning composition according to paragraph 11, wherein the protease variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO). NO:1) replaces X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D.

[0625] 13. The liquid cleaning composition according to any one of paragraphs 10 to 12, wherein the protease variant has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[0626] 14. The liquid cleaning composition according to any one of paragraphs 10-13, wherein the protease variant is selected from the group consisting of:

[0627] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0628] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0629] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0630] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0631] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0632] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0633] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0634] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0635] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0636] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0637] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0638] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0639] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0640] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0641] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0642] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0643] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0644] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0645] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0646] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0647] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0648] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0649] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0650] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0651] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0652] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0653] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0654] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0655] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0656] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0657] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0658] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0659] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0660] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0661] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0662] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0663] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0664] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0665] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0666] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0667] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0668] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0669] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0670] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0671] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0672] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0673] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0674] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0675] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0676] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0677] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0678] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0679] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0680] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0681] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0682] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0683] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0684] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0685] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0686] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0687] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0688] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0689] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0690] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[0691] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

[0692] 15. The cleaning composition according to any one of the preceding paragraphs, wherein the cleaning composition comprises one or more additional enzymes selected from the group consisting of: amylase, catalase, cellulase (e.g., endoglucanase), keratinase, halogenated peroxidase, lipase, mannanase, pectinase, pectin lyase, peroxidase, protease, xanthan gumase, and xyloglucanase, or any mixture thereof.

[0693] 16. Use of the liquid cleaning composition as described in any of the preceding paragraphs in cleaning processes such as laundry or hard surface cleaning, including dishwashing and industrial cleaning.

[0694] 17. The use as described in paragraph 16, wherein the garment washing process is selected from the group consisting of: residential garment washing process, industrial garment washing process and public garment washing process.

[0695] The invention is further described through the following examples, but these should not be construed as limiting the scope of the invention.

[0696] Example

[0697] Materials and Methods

[0698] Suc-AAPF-pNA activity assay

[0699] Proteolytic activity can be determined using Suc-AAPF-PNA as a substrate. Suc-AAPF-PNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline and is a blocked peptide that can be cleaved by an endopeptide. Upon cleavage, a free PNA molecule is released, which is yellow in color and can therefore be measured spectrophotometrically at a wavelength of 405 nm. This Suc-AAPF-PNA substrate is manufactured by Bachem (catalog number L1400, dissolved in DMSO).

[0700] The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay was performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / mL in 100 mM Tris, pH 8.6). The solution was mixed at room temperature and the absorbance was measured at OD 405 nm over 5 minutes at 20-second intervals.

[0701] Under a given set of conditions, the slope (absorbance per minute) of the time-dependent absorption curve is directly proportional to the activity of the protease in question. The protease sample is diluted to a level where the slope is linear.

[0702] Example 1: Preparation and expression of protease variants

[0703] The introduction of mutations and expression cassettes into Bacillus subtilis is summarized below. All DNA manipulations were performed by PCR (e.g., Sambrook et al.; Molecular Cloning; Cold Spring Harbor Laboratory Press).

[0704] Recombinant Bacillus subtilis constructs encoding a protease variant were inoculated into shake flasks containing nutrient-rich medium (e.g., 100 g / L sucrose (Denisco catalog 109-0429), 40 g / L soybean hulls (soybean meal), 10 g / L Na₂HPO₄·12H₂O (Merck catalog 6579), and 0.1 ml / L Replacement-Dowfax 63N10 (Dow Chemicals)). Culture was typically carried out for 4 days at 30°C with shaking at 220 rpm.

[0705] Example 2: Purification of protease variants

[0706] The culture medium was centrifuged at 26000 x g for 20 min, and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter to remove any remaining Bacillus host cells. The pH of the 0.2 μm filtrate was adjusted to pH 8 with 3 M Tris base, and the pH-adjusted filtrate was applied to a MEP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris / HCl and 1 mM CaCl2 (pH 8.0). After washing the column with equilibration buffer, it was eluted stepwise with 20 mM CH3COOH / NaOH and 1 mM CaCl2 (pH 4.5). The fractions from the column were analyzed for protease activity using a Suc-AAPF-pNA assay at pH 9, and the peak fractions were combined. The pH of the pooled product from the MEP Hypercel column was adjusted to pH 6 using 20% ​​(v / v) CH3COOH or 3M Tris base, and the pH-adjusted pooled product was diluted with deionized water to the same conductivity as 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). The diluted pooled product was then applied to SP-agarose (Sepharose) equilibrated in 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). Fast flow column (GE Healthcare). After washing the column with equilibration buffer, the protease variant was eluted with a linear NaCl gradient (0-->0.5M) in the same buffer for five column volumes. The fractions from the column were analyzed for protease activity using a Suc-AAPF-pNA assay at pH 9, and the activity fractions were analyzed by SDS-PAGE. The fractions (only one band was observed on a Coomassie-stained SDS-PAGE gel) were combined into a purified formulation and used for further experiments.

[0707] Example 3: Accelerated Storage Stability Determination

[0708] The storage stability of the protease variant in liquid detergent was assessed using an accelerated assay after incubation at elevated temperatures for up to 24 hours.

[0709] Based on absorbance at 280 nm and theoretical extinction coefficient, all purified protease samples were diluted to concentrations of 0.2 and 0.1 mg / mL using 0.01% Triton X-100. For each variant, two wells were prepared with a high protease concentration and two wells with a low concentration. As a reference, Savinase (SEQ ID NO 2) or a stabilized protease variant was included in each microtiter plate. Using a magnetic wand (on a Zephyr pipette stage (Caliper LifeSciences) for 30 min), 30 μl of protease sample was mixed with 270 μl of detergent (Surf Sparkling Ocean, DC-2014-0009) in the wells of a microtiter plate (Nunc U96PP 0.5 mL). 20 μl of this mixture was then transferred to another microtiter plate (0.5 ml of Nunc U96PP with a magnetic rod) and mixed with 150 μl of 100 mM Tris (pH 8.6) (at least 5 min on a Zephyr). 30 μl of this dilution was transferred to Nunc F 96-MTP, and after adding 70 μl of substrate solution, the initial activity of the non-stressed sample was determined by measuring absorbance at 405 nm every 20 sec for 5 min (on a SpectraMax Plus). After sealing, the detergent plate was incubated (without shaking) at 50 °C and pH 11 in an Eppendorf Thermomixer. After incubation for 1, 3–4, and 23–24 hours, 20 μl samples were drawn, and the residual activity of the stressed sample was measured as with the initial non-stressed activation.

[0710] The decrease in activity during incubation with detergent was assumed to be exponential. Linear regression from Log(activity) to incubation time revealed the half-life (T1 / 2). Savinase (SEQ ID NO 2) has a very short half-life under the conditions of this experiment, and therefore the effects of these mutations were measured relative to already stable protease variants.

[0711] Accelerated storage stability of variant T1 / 2IF at 40°C: Improving half-life effect of single mutation

[0712] Mutant G61E

[0713] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T1 / 2: 0.9

[0714] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T1 / 2: 4.8

[0715] Mutant Y209W

[0716] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T1 / 2: 4.5

[0717] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T1 / 2: 13.0

[0718] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E, T1 / 2: 4.6

[0719] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T1 / 2: 12.4

[0720] Mutant M222S

[0721] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.0

[0722] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E, T1 / 2: 14.5

[0723] Mutant V205I

[0724] S9E+N43R+N76D+A194P+Q206L+T255E+S256D+S259D+T260E+N261W+L262E, T1 / 2: 5.0

[0725] S9E+N43R+N76D+A194P+V205I+Q206L+T255E+S256D+S259D+T260E+N261W+L262E, T1 / 2: 1.2

[0726] S9E+N43R+N76D+N117H+H120D+A194P+Q206L+S259D+L262E, T1 / 2: 5.0

[0727] S9E+N43R+N76D+N117H+H120D+A194P+V205I+Q206L+S259D+L262E, T1 / 2: 1.6

[0728] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E, T1 / 2: 3.0

[0729] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E, T1 / 2: 6.6

[0730] S9E+N18S+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T1 / 2: 1.0

[0731] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E, T1 / 2: 4.6

[0732] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T1 / 2: 0.9

[0733] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E, T1 / 2: 5.0

[0734] S9E+N43R+N76D+A194P+Q206L+Y209W+S259D+N261W+L262E, T1 / 2: 9.9

[0735] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T1 / 2: 13.0

[0736] Mutant S256D

[0737] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T1 / 2: 4.5

[0738] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E, T1 / 2: 6.6

[0739] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T1 / 2: 12.4

[0740] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E, T1 / 2: 13.3

[0741] Mutant S188E

[0742] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.0

[0743] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 13.7

[0744] Mutant T260E

[0745] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T1 / 2: 13.5

[0746] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E, T1 / 2: 15.1

[0747] Mutant H120T

[0748] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E, T1 / 2: 3.0

[0749] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E, T1 / 2: 5.4

[0750] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.0

[0751] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.2

[0752] Mutant G160P

[0753] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T1 / 2: 13.2

[0754] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T1 / 2: 14.3

[0755] Mutant Q182E

[0756] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T1 / 2: 4.5

[0757] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E, T1 / 2: 4.6

[0758] S9E+N76D+V205I+Q206L+Y209W+S256D+N261W+L262E, T1 / 2: 12.3

[0759] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T1 / 2: 13.2

[0760] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH, T1 / 2: 4.2

[0761] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH, T1 / 2: 4.6

[0762] S9E+N43R+N76D+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E, T1 / 2: 11.5

[0763] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E, T1 / 2: 14.0

[0764] Mutant N185E

[0765] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.0

[0766] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.4

[0767] Mutant N261M

[0768] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T1 / 2: 12.0

[0769] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E, T1 / 2: 12.7

[0770] Mutant S259D

[0771] S9E+N43R+I72A+N76D+A194P+Q206L+L262E, T1 / 2: 6.0

[0772] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E, T1 / 2: 7.2

[0773] S9E+N43R+N76D+A194P+V205I+Q206L+L262E, T1 / 2: 6.0

[0774] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E, T1 / 2: 6.5

[0775] Example 4: Full-scale washing results of the protease of the present invention

[0776] The washing performance of the protease of the present invention was tested in a full-scale wash. The washing performance was also tested in a liquid laundry detergent on 22 different stains using a dosage-response method.

[0777] After washing and rinsing, lay the small fabric sample flat and allow it to air dry overnight at room temperature. Evaluate all washes the following day. Evaluate the light reflectance of the small fabric sample using a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. Measurements are taken in the absence of UV in the incident light, and the reflectance at 460 nm is extracted. Measurements are performed on both unwashed and washed small fabric samples. Place the test fabric sample to be measured on top of another small fabric sample of the same type and color.

[0778] The enzyme effect, ΔRem, was calculated by subtracting measurements from enzyme-washed fabric samples from unwashed fabric samples. 酶 Washing performance is expressed as Δreflectance (ΔRem).

[0779] The experiments were conducted using a detergent composition and small pieces of fabric under the experimental conditions specified in Table 21 below.

[0780] Table 21: Experimental conditions for full-scale laundry washing experiments

[0781]

[0782] The water hardness was adjusted to 6.7°dH by adding CaCl2, MgCl2 and NaHCO3 (Ca2+:Mg2+:CO3-=3:1:6) to the test system.

[0783] Table 22: Δreflectance values ​​of detergents containing proteases compared to detergents without proteases at 15°C in all Free & Clear detergents (enzymes tested are listed below):

[0784] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0785] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0786] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0787] Δ (Contrast with blank space)

[0788]

[0789]

[0790] Table 23: Δ reflectance values ​​of detergents containing proteases compared to detergents without proteases at 30°C in all Free & Clear detergents (enzymes tested in the "Results (Enzyme-Blank)" section below are as follows):

[0791] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0792] 2: S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0793] 3: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0794] 4: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0795] All free & clear 30℃

[0796]

[0797]

[0798] Table 24: Δreflectance values ​​of detergents containing proteases compared to those without proteases at 30°C for all Arm & Hammer products (the tested enzymes are listed below).

[0799] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0800] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0801] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0802] Δ (Comparison with blank) Detergent Arm & Hammer

[0803]

[0804]

[0805] Table 25: Δ reflectance values ​​of detergents containing proteases compared to detergents without proteases in Purex at 30°C (the enzymes tested are listed below).

[0806] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0807] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0808] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0809] Purex

[0810]

[0811]

[0812]

[0813] Table 26: In Sun Triple Clean, at 30°C, the Δ reflectance values ​​of detergents containing proteases compared to detergents without proteases (the tested enzymes are listed below):

[0814] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0815] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0816] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0817] Δ (Comparison with blank) Detergent Sun Triple Clean

[0818]

[0819]

[0820]

[0821] Table 27: Δreflectance values ​​of detergents including proteases compared to detergents without proteases in Xtra at 30°C. The enzymes tested include the following:

[0822] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0823] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0824] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E:

[0825] Δ (Comparison blank) Detergent Xtra

[0826]

[0827]

[0828] Example 5: Testing of the subtilisin 309 variant in a microwash

[0829] Using a micro-washing system, the washing performance of the protease of the present invention was tested on a technical stain using a laundry detergent.

[0830] The microwash test is a test method in which contaminated textiles are continuously lifted and placed into a test solution and then rinsed.

[0831] Table 28: Washing experiments were conducted under the following specified experimental conditions:

[0832]

[0833] The test materials are from EMPA Testmaterials AG. 12, CH-9015 Street, Gallon, Switzerland) obtained.

[0834] The textiles were then air-dried and their washability was measured as the brightness of their colors. Brightness can also be expressed as reflectance (R), which is a measure of the amount of light reflected or emitted from a test material when illuminated with white light. The reflectance (R) of the textiles was measured at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. Measurements were performed according to the manufacturer's protocol.

[0835] The enzyme effect, ΔRem, was calculated by subtracting measurements from enzyme-washed fabric samples from unwashed fabric samples. 酶 .

[0836] As described in the micro-washing test for the washing method of clothing, the experiment was performed with the detergent composition and a small piece of fabric sample under the experimental conditions specified in Table 29 below.

[0837] Table 29: Experimental conditions for the micro-laundry washing experiment

[0838]

[0839]

[0840] By using CaCl2, MgCl2 and NaHCO3 (Ca 2+ :Mg 2+ CO3 - Add a 2:1:4.5 ratio to the test system to adjust the water hardness to 8.4°dH. After washing, rinse the textiles with tap water and dry them.

[0841] Table 30: Relative performance of the protease variant of Bacillus subtilisin 309 compared with detergents containing Bacillus subtilisin 309 (SEQ ID NO 2) at 25°C.

[0842]

[0843]

[0844]

[0845] The results in Table 30 show that, at 25°C, on blood / milk / ink, the subtilisin 309 variant exhibited improved or equivalent washing performance compared to subtilisin 309 (SEQ ID NO:2). sequence list <110> Novozymes A / S Nielsen, Jens Erik <120> Liquid cleaning compositions including protease variants <130> 13106-WO-PCT <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 275 <212> PRT <213> Bacillus amyloliquefaciens <400> 1 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 <210> 2 <211> 269 <212> PRT <213> Bacillus lentus <400> 2 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265

Claims

1. A liquid cleaning composition having a pH of 10 or higher, comprising: (a) at least 0.01 wt% of a protease, wherein the protease is a variant of a parent protease comprising the amino acid sequence of SEQ ID NO: 2, wherein the protease variant is selected from the group consisting of: S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+ L262E; S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E; S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+ 275aH; S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E; S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+ N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+ L262E+ 275aH+ 275bH; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+ N261W+L262E; S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E; S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E; S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+P131 +A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E, wherein the position corresponds to the position of BPN' in SEQ ID NO: 1, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours in a liquid detergent having a pH of 10 at 40°C; and (b) Optionally, a protease inhibitor.

2. The liquid cleaning composition of claim 1, wherein the protease inhibitor is a peptidase inhibitor.

3. A liquid cleaning composition comprising: (a) Water from 20% to 95% wt; (b) at least 0.01 wt% of a protease, wherein the protease is a variant of a parent protease comprising the amino acid sequence of SEQ ID NO: 2, wherein the protease variant is selected from the group consisting of: S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+ L262E; S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E; S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+ 275aH; S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E; S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+ N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+ L262E+ 275aH+ 275bH; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+ N261W+L262E; S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E; S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E; S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+P131 +A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E, wherein the position corresponds to the position of BPN' in SEQ ID NO: 1, and wherein the protease variant is characterized by having at least 10% higher residual activity than the parent protease when measured after 4 hours at 40°C in a liquid detergent having a pH of 7.5 or higher; and (c) Optionally, a protease inhibitor.

4. The liquid cleaning composition according to claim 3, comprising from 40% to 95% wt of water.

5. The liquid cleaning composition according to claim 3, comprising from 70% to 90% wt of water.

6. The liquid cleaning composition according to claim 3, wherein the pH is 10.

7. The liquid cleaning composition according to claim 3, wherein the protease inhibitor is a peptidaldehyde protease inhibitor.

8. The liquid cleaning composition according to claim 1 or 3, wherein the pH is from 10 to 13.

5.

9. The liquid cleaning composition of claim 8, wherein the pH is from 10 to 13, from 10 to 12.5, from 10 to 12, from 10 to 11.5, from 10 to 11, or from 10 to 10.5; or from 12 to 13.

5.

10. The liquid cleaning composition according to claim 1 or 3, wherein the pH is from 11 to 13.

11. A liquid cleaning composition comprising: (a) Detergent surfactants from 0% to 20% wt; (b) From 0.001% to 10% wt of a protease variant, wherein the protease variant is a variant of the parent protease comprising the amino acid sequence of SEQ ID NO: 2, wherein the protease variant is selected from the group consisting of: S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+ L262E; S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E; S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E; S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH; S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E; S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+ N261W+L262E; S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+ L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+ L262E+ 275aH+ 275bH; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+ S256D+N261W+L262E; S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E; S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E; S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+ 275aH+ 275bH; S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E; S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E; S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E; S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E; S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+P131 +A194P+Q206L+Y209W+S259D+L262E; S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+ 275aH+ 275bH; S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+ 275aH+ 275bH; S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E; S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E; where the position corresponds to the position of BPN' in SEQ ID NO: 1; (c1) Water from 20% to 95% wt; and / or (c2) An alkaline buffer system from 1% to 30% wt, comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof, to provide a pH from 7.5 to 13.5; and (d) Optionally, a protease inhibitor.

12. The liquid cleaning composition of claim 11, comprising 0.1% to 2.5% wt of detergent surfactant.

13. The liquid cleaning composition of claim 11, comprising 0.1% to 5% wt of a protease variant.

14. The liquid cleaning composition of claim 11, comprising from 40% to 95% wt water.

15. The liquid cleaning composition of claim 11, comprising from 70% to 90% wt water.

16. The liquid cleaning composition of claim 11, wherein the pH is from 10 to 13.

5.

17. The liquid cleaning composition of claim 11, wherein the pH is from 11 to 13.

5.

18. The liquid cleaning composition of claim 11, wherein the protease inhibitor is a peptidaldehyde protease inhibitor.

19. The cleaning composition according to claim 1, 3, or 11, wherein the cleaning composition comprises one or more additional enzymes selected from the group consisting of: Hydrolytic enzymes, amylases, catalases, cellulases, cutinases, halogenated peroxidases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, DNases, xanthan gumases, and xyloglucanases, or any mixture thereof.

20. The cleaning composition of claim 19, wherein the cellulase is an endoglucanase.

21. The cleaning composition according to claim 1, 3 or 11, comprising a peptidase inhibitor.

22. The cleaning composition according to claim 1, 3 or 11, wherein the protease variant has improved storage stability and / or improved washing performance compared to the protease of SEQ ID NO:

2.

23. Use of the liquid cleaning composition according to any one of the preceding claims in a cleaning process.

24. The use according to claim 23, wherein the cleaning process is laundry or hard surface cleaning.

25. The use according to claim 23 or 24, wherein the cleaning process is dishwashing and industrial cleaning.

26. The use according to claim 24, wherein the garment washing process is selected from residential garment washing processes, industrial garment washing processes, and public institution garment washing processes.

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