Liquid cleansing compositions comprising protease variants

Abstract

The present invention provides liquid cleansing compositions comprising protease variants. 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] This application is a divisional application of the Chinese invention patent application with the application date of December 3, 2015, the invention name of which is “Liquid cleaning composition comprising protease variants”, PCT application number: PCT / EP2015 / 078588, and patent application number: 201580063750.6.

[0002] References to sequence listings

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0004] The present 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. Background Art

[0005] In the detergent industry, enzymes have been used for decades in washing formulations. The enzymes used in such formulations include amylases, cellulases, lipases, mannosidases and proteases together with other enzymes, or mixtures thereof. The most important enzyme commercially is protease. Many proteases traditionally used in cleaning processes are alkaline proteases that usually have a pH optimum of about 9, and are therefore suitable for quite alkaline detergents. However, some specific liquid detergents have a pH higher than 10, and due to very harsh conditions and the resulting low storage stability of enzymes in such detergents, it has not been previously thought that these detergents are suitable for enzymes. In addition, in liquid detergents, particularly when the water content is high, enzymes tend to be more unstable.

[0006] Many useful protease variants have been described, many of which have improved activity, stability and solubility in different detergents.

[0007] However, these variants are not suitable for use in very high pH detergents, as the alkaline environment of these detergents will cause the enzymes to decompose after a very short time and thus reduce their activity. Summary of the invention

[0008] The present invention relates to protease variants that are stable under alkaline conditions and suitable for high pH liquid cleaning and detergent compositions comprising such variants.

[0009] The present invention relates to a liquid cleaning composition having a pH of 10 or more, 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 in 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 10.

[0010] In another embodiment, the present invention relates to a liquid cleaning composition having a pH of 10 or more, 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in 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 10; and (b) optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0011] In another embodiment, the present invention relates to a liquid cleaning composition comprising: (a) from 20% to 95%wt, preferably from 40% to 95%wt, further preferably from about 70% to about 90%wt of water; (b) at least 0.01wt% 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 in that it has 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 10; and (c) optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0012] The present invention also relates to a liquid cleaning composition comprising:

[0013] (a) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0014] (b) from 40% to 95%wt, preferably from about 70% to about 90%wt, water;

[0015] (c) from 1% to 30% by weight of an alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof to provide a pH of from about 11 to 13.5; and

[0016] (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of the protease variant.

[0017] In another embodiment, the present invention further relates to a liquid cleaning composition comprising:

[0018] (a) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0019] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of the protease variant; and

[0020] (c1) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or

[0021] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0022] (e) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0023] In the embodiment, (c1) and (c2) may be used as substitutes for each other, or they may be used in combination with each other.

[0024] definition

[0025] The term "allelic variant" means any one of two or more alternative forms of a gene occupying the same chromosomal site. Allelic variation is naturally produced by mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0026] The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. The cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The early initial RNA transcript is a precursor to the mRNA that is processed through a series of steps, including splicing, before appearing as a mature spliced ​​mRNA.

[0027] The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA or a combination thereof.

[0028] The term "control sequence" means a nucleic acid sequence required for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence can be native (i.e., from the same gene) or exogenous (i.e., from different genes) to the polynucleotide encoding the variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At least, the control sequence includes a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction enzyme sites that are conducive to connecting these control sequences to the coding region of the polynucleotide encoding the variant, these control sequences can be provided with multiple joints.

[0029] The term "detergent component" is defined herein to mean the type of chemicals that can be used in a cleaning composition, such as a detergent composition. Examples of detergent components are surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfumes, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizing agents. The detergent composition may include one or more detergent components of any type.

[0030] The term "cleaning composition" includes "detergent compositions" and, unless otherwise specified, includes all forms of detergent compositions, such as bars, uniform tablets, tablets having two or more layers, bags 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 (HDL), delicate fabric liquid detergents, liquid and / or solid laundry detergents and delicate fabric detergents; hard surface cleaning formulations, for example, glass, wood, ceramic and metal countertops and windows; carpet cleaners; stove cleaners; fabric fresheners ; fabric softeners; textile and clothing pre-spotters, together with dishwashing detergents, such as hand dishwashing detergents, light-duty dishwashing detergents, machine dishwashing detergents; general or heavy-duty cleaners, general cleaners in liquid, gel or paste form, liquid cleaning and disinfectants, including antibacterial hand-washing types, cleaning rods, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair rinses; shower gels, foam baths; metal cleaners; together with cleaning aids, such as bleach additives and "stain-sticks" or pre-treatment types. The bag can be configured as a single or multiple chambers. It can have any form, shape and material suitable for storing the composition, such as not allowing the composition to be released from the bag before contact with water. The bag is made of a water-soluble film that encapsulates an inner volume. The inner volume can be divided into chambers with bags. Preferred films are polymeric materials that form films 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, polymethacrylate, most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the level of polymer in film such as PVA is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. Film can also be a blended composition, which includes a hydrolytically degradable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under trade reference M8630, such as sold by MonoSol LLC, Indiana, USA) plus a plasticizer, such as glycerol, ethylene glycol, propylene glycol, sorbitol and mixtures thereof. These bags can include solid laundry cleaning compositions or partial components and / or liquid cleaning compositions or partial components separated by water-soluble films. The chamber for the liquid component may differ in composition from the chamber comprising the solid: US 2009 / 0011970 A1.

[0031] The detergent ingredients (or components) can be physically separated from each other by compartments in water-soluble bags or different layers of tablets. Therefore, bad storage interactions between components can be avoided. In the washing solution, the different dissolution curves of each compartment can also cause delayed dissolution of the selected components.

[0032] The cleaning compositions (e.g., liquid cleaning compositions) as described herein are suitable for use in cleaning processes such as laundry or hard surface cleaning, including dishwashing and industrial cleaning. Exemplary laundry processes can be selected from the group consisting of a residential laundry process, an industrial laundry process, and an institutional laundry process.

[0033] In addition to comprising the protease variant of the present invention, the detergent formulation may also comprise one or more additional enzymes (such as perhydrolases, amylases, catalases, cellulases (e.g., endoglucanases), cutinases, haloperoxygenases, lipases, mannanases, pectinases, xyloglucanases, pectinases, pectinase lyases, peroxidases, proteases, DNases, xanthanases, and xyloglucanases, or any mixture thereof), and / or components such as surfactants, builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfumes, tarnish 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 solubilizing agents.

[0034] 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 crockery, such as plates, cups, glasses, bowls, all forms of eating utensils, such as spoons, knives, forks, and serving utensils as well as ceramics, plastics (such as melamine), metals, porcelain, glass and acrylic.

[0035] The term "dishwashing composition" refers to all forms of compositions used for cleaning hard surfaces. The present invention is not limited to any particular type of dishwashing composition or to any particular detergent.

[0036] 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.

[0037] The term "expression vector" means a linear or circular DNA molecule that includes a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

[0038] The term "hard surface cleaning" is defined herein as cleaning hard surfaces, where hard surfaces may include floors, tables, walls, roofs, etc., as well as 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 dining utensils (e.g., spoons, knives, forks), serving utensils, ceramics, plastics (e.g., melamine), metals, porcelain, glass, and acrylics.

[0039] The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0040] The term "improved properties" means features associated with a protease variant that are improved relative to a parent protease. Such improved properties include, but are not limited to, wash 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.

[0041] The term "stability" includes storage stability and stability during use, such as stability during cleaning, and reflects the stability of the protease variants according to the present invention as a function of time, such as how much activity can be retained when the protease variants are placed in solution, especially in a detergent solution. This stability is affected by many factors, such as pH, temperature, detergent composition, such as the amount of builder, surfactant, etc. The stability of the protease can be measured using an assay as described in Example 3. The term "improved stability" or "increased stability" is defined herein as a variant protease showing increased stability in solution relative to the stability of the parent protease (e.g., relative to SEQ ID NO: 2).

[0042] In a particular aspect of the invention, the improved stability is an improved stability in a liquid detergent having a pH of 10 or higher. The term "detergent stability" or "improved detergent stability" refers in particular to the improved stability of the 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 a temperature between 15°C and 50°C, such as 20°C, 30°C or 40°C.

[0043] The term "improved wash performance" is defined herein as the wash performance of a protease variant according to the invention relative to a parent protease, e.g. showing improved wash performance by increased stain removal. The term "wash performance" includes laundry washing, but also includes, for example, wash performance in dishwashing. Wash performance can be quantified as described under the definition of "wash performance" herein.

[0044] The term "isolated" means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (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 more or all of the naturally occurring components with which it is essentially associated; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). The isolated substance may be present in a fermentation broth sample.

[0045] The term "laundry" refers to both domestic and industrial laundry and means the process of treating textiles and / or fabrics with a solution comprising a detergent composition of the present invention. The laundry process can be carried out, for example, using, for example, a domestic or industrial washing machine or can be carried out manually.

[0046] The term "mature polypeptide" means 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, the 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 a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0047] The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having protease activity.

[0048] The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not originally found in nature to contain a segment of nucleic acid, or is synthetic, and includes one or more control sequences.

[0049] The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0050] The term "parent" means a protease to which changes are made to produce enzyme variants of the present invention. It should be understood that the expression of "having identical amino acid sequences" in the context relates to 100% sequence identity. In a specific embodiment, the parent is a protease with 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.

[0051] The term "protease" is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses). EC numbers refer to the 1992 enzyme nomenclature of the NC-IUBMB Academic Press in San Diego, California, including publications in Eur. J. Biochem. 1223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem. 237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem. 264: Supplements 1-5 of 610-650 (1999). The most widely used protease in detergent industry such as clothing and dishwashing is serine protease or serine peptidase, which is a subgroup of the following protease, which is characterized by having serine in the active site, and the serine forms a covalent adduct with the substrate. In addition, subtilase (and serine protease) is characterized by having two active site amino acid residues, i.e. histidine residue and aspartic acid residue, in addition to serine. Subtilase refers to the serine protease subgroup according to Siezen et al., 1991, protein engineering (ProteinEngng.) 4:719-737 and Siezen et al., 1997, protein science (ProteinScience) 6:501-523. Subtilase can be divided into 6 subgroups, i.e., subtilisin family, thermophilic protease (Thermitase) family, proteinase K family, lantibiotic peptidase family, Kexin family and Pyrolysin family. The term "protease activity" means proteolytic activity (EC 3.4). Proteases that can be used in detergents are mainly endopeptidases (EC 3.4.21). There are several protease activity types: the three main activity types are: trypsin-like, in which there is cleavage of the amide substrate at P1 after Arg or Lys; chymotrypsin-like, in which cleavage occurs at P1 after one of the hydrophobic amino acids; and elastase-like, in which cleavage occurs after Ala at P1. For the purposes of the present 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 the present invention have at least 20%, such as 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 mature polypeptide of the parent enzyme.In a specific aspect, the protease variant of the invention has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the enzymatic activity of the polypeptide of SEQ ID NO: 2.

[0052] The term "protease activity" means proteolytic activity (EC 3.4). The proteases of the present invention are endopeptidases (EC 3.4.21). There are several types of protease activity: the three main types of activity are: trypsin-like, in which there is cleavage of an amide substrate at P1 after Arg or Lys; chymotrypsin-like, in which cleavage occurs at P1 after one of the hydrophobic amino acids; and elastase-like, in which 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%, such as 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% of the protease activity of the polypeptide of SEQ ID NO: 2.

[0053] The term "residual activity" in this context means the protease activity left or remaining after storage, especially in a liquid detergent. After the protease is added to the detergent, when the residual activity is measured, the activity of the protease is measured at t1. Then after a certain amount of storage time, typically minutes or weeks, the residual activity is measured at t2. The protease variants of the present invention preferably have a 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 in a high pH liquid detergent at a pH of 10, before storage at a temperature of 30°C (at t1), and when measured at t=4 hours or more. 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 present invention are preferably more stable, i.e. have a higher residual activity after storage in a high pH liquid detergent (pH=10 or higher) compared to the parent protease (e.g., a protease with SEQ ID NO 2), and / or the protease variants according to the present invention are more stable, i.e. have a higher residual activity after storage in a liquid detergent (i.e., a liquid detergent comprising 40% water or more water) compared to the parent protease (e.g., a protease with SEQ ID NO 2). Preferably, the residual activity of the protease variants of the present invention 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%, higher when measured after storage for 4 hours at 30°C in a liquid detergent with a pH of 10 and / or in a liquid detergent with at least 40% water compared to the residual activity of the parent protease, e.g., compared to SEQ ID NO 2. As described in Example 3.

[0054] In particular, the relative stability of the protease variants according to the present invention and their corresponding parent proteases at high pH, ​​above or at pH 10, is different from their relative stability at neutral pH, above or at pH 7.

[0055] The correlation between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) Needle program, preferably 5.0.0 version or later. The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) was used as the percent identity and was calculated as follows:

[0056] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0057] For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wengsch algorithm (Needleman and Wengsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output (obtained using the -non-simplified (nobrief) option) labeled as "longest consistency" is used as a percentage consistency and is calculated as follows:

[0058] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in alignment)

[0059] Different stringency conditions are defined below.

[0060] The term "substantially pure variant" means a preparation containing 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% by weight of other polypeptide materials that are naturally or recombinantly associated therewith. Preferably, the variant is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, and 100% pure, by weight of the total polypeptide material present in the preparation. These variants of the present invention are preferably present in substantially pure form. For example, this can be accomplished by preparing the variant via well-known recombinant methods or via classical purification methods.

[0061] The term "substantially pure polynucleotide" means a polynucleotide preparation that does not contain other external or unwanted nucleotides and is in a form suitable for use in a genetically engineered polypeptide production system. Thus, a substantially pure polynucleotide contains 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% other polynucleotide materials naturally or recombinantly associated with the polynucleotide. However, a substantially pure polynucleotide may include naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Preferably, a substantially pure polynucleotide is at least 90% pure by weight, such as at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure. These polynucleotides of the present invention are preferably present in substantially pure form.

[0062] The term "textile" means any textile material including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, as well as fabrics made from these materials, such as garments, clothing and other articles. When the term fabric or clothing is used, it is intended to also include the broad term textile.

[0063] The term "variant" means a polypeptide having protease activity comprising changes (i.e., substitutions, insertions and / or deletions) at three or more (e.g., several) positions. Substitution means that the amino acid occupying the position is replaced by a different amino acid; deletion means the removal of the amino acid occupying the position; and insertion means the addition of one or more (e.g., several) amino acids (e.g., 1, 2, 3, 4 or 5 amino acids) adjacent to and immediately following the amino acid occupying a position. The term protease variant may be a variant of a subtilase parent, i.e., a protease variant is a subtilase comprising changes (i.e., substitutions, insertions and / or deletions) at three or more (e.g., several) positions compared to a parent protease.

[0064] The term "wash performance" is used as the ability of an enzyme to remove stains present on the object to be cleaned during, for example, washing, such as laundry washing or hard surface cleaning. The improvement in wash performance can be quantified by calculating the so-called intensity value (Int) defined in the AMSA assay.

[0065] The term "wild-type protease" means a protease expressed by a naturally occurring organism, such as a bacterium, archaea, yeast, fungus, plant or animal found in nature. An example of a wild-type subtilase is subtilisin Savinase, amino acids 1 to 269 of SEQ ID NO:2.

[0066] Variant naming conventions

[0067] For the purposes of the present invention, the subtilisin BPN' (sequence of amino acids 1-275 of SEQ ID NO: 1 (Siezen et al., 1991, Protein Eng. 4: 719-737)) is used to determine the corresponding amino acid residues in another protease. The amino acid sequence of another protease is aligned with the mature polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Package, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 5.0.0 or later. The parameters used were gap opening penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0068] The identification of the corresponding amino acid residue in another protease can be determined by aligning multiple polypeptide sequences using several computer programs using their corresponding default parameters, including but not limited to MUSCLE (Multiple Sequence Comparison 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; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology). Biology 537:39-64; Kato and Asato, 2010, Bioinformatics 26:1899-1900); and EMBOSS EMMA (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Res. 22:4673-4680) using ClustalW.

[0069] When other enzymes deviate from the mature polypeptide of SEQ ID NO:1 so that traditional sequence-based comparison methods cannot detect their mutual relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other paired sequence comparison algorithms can be applied. Greater sensitivity in sequence-based searches can be obtained using search programs that use probabilistic representations (profiles) of polypeptide families to search databases. 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 a family or superfamily of polypeptides has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from different sources (PSI-BLAST, secondary structure predictions, structural alignment spectra, and solvation potential) as input to a neural network that predicts the structural fold of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments, in turn, can be used to generate homology models for polypeptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0070] For proteins of known structure, several tools and resources can be obtained for retrieving and generating structural comparisons. For example, the SCOP superfamily of proteins has been structurally compared, and those comparisons are accessible and downloadable. Multiple 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 compare two or more protein structures, and the implementation of these algorithms can be used in addition to query structural databases with structures of interest to find possible structural homologues (e.g., Holm and Parker, 2000, Bioinformatics 16:566-567).

[0071] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. Accepted IUPAC single-letter and three-letter amino acid abbreviations are employed.

[0072] replace :For amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the threonine at position 226 is replaced by alanine as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), such as "Gly205Arg+Ser411Phe" or "G205R+S411F" represents that glycine (G) is replaced by arginine (R) at position 205 and position 411, and serine (S) is replaced by phenylalanine (F). The "X" before the position means that any original amino acid at the position can be replaced.

[0073] Missing : For amino acid deletions, the following nomenclature is used: initial amino acid, position, * Thus, a deletion of glycine at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by plus signs ("+"), e.g., "Gly195 * +Ser411 * " or "G195 * +S411 * ”.

[0074] insert: The insertion of additional amino acid residues, such as, for example, 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, for example, Lys and Ala can be inserted after G195 as: Gly195GlyLysAla or G195GKA. In such cases, the inserted one or more amino acid residues can also be numbered by adding lowercase letters to the amino acid residue position number before the inserted one or more amino acid residues, in this example: * 195aK * 195bA. In the above example, the sequence 194 to 196 is thus:

[0075] Subtilisin 309

[0076] Variants

[0077] Where the substitution and insertion occur at the same position, this may be represented as S99SD+S99A, or abbreviated as S99AD. The same modification may also be represented as S99A+ * 99aD.

[0078] In cases where an amino acid residue identical to an existing amino acid residue is inserted, degeneracy in the nomenclature is evident. If, for example in the above example, a glycine is inserted after the glycine, it would be represented as G195GG or *195GaG. For the following changes, the same actual change could also be represented simply as A194AG or * 194aG, from:

[0079] Subtilisin 309

[0080] arrive:

[0081] Variants

[0082] Such cases will be apparent to the skilled person and therefore the expression G195GG and corresponding expressions of this type of insertion are intended to include such equivalent degenerate expressions.

[0083] Multiple changes: Variants comprising multiple changes are separated by plus signs ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine at positions 170 and 195, respectively, by tyrosine and glutamic acid. Alternatively, spaces or commas may separate multiple changes, e.g., A170Y G195E or A170Y, G195E, respectively.

[0084] Different changes : When different changes can be introduced at one position, these different changes are separated by a comma, for example "Arg170Tyr,Glu" represents that the arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0085] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0086] Alternatively, the various alterations or optional substitutions may be indicated by brackets, for example Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or abbreviated as R170[Y, G] or R170{Y, G}. DETAILED DESCRIPTION

[0087] Alkaline liquid detergents with high pH are widely used for cleaning, such as laundry and dishwashing. Liquid detergents with elevated pH are particularly commonly used by consumers, especially in North America. High pH cleaning compositions are also used in industrial cleaning processes.

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

[0089] Examples of commercial 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 Products), Purex Free and Clear (Henkel), and Xtra Tropical Passion (Church & Dwight).

[0090] The addition of enzymes to detergents is highly advantageous because the specific activity of these enzymes effectively removes specific stains from surfaces such as fabrics and dishes. 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] The present invention relates to high pH liquid cleaning compositions comprising alkaline stable protease variants suitable for use in such compositions.

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

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

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

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

[0096] (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of the protease variant.

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

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

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

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

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

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

[0103] The compositions of the present invention preferably contain an alkaline buffer system to provide a pH of at least about 7.5, at least about 8, at least about 9, preferably a pH of 10 or higher. Preferably, the pH is from about 9 to about 13. In order to achieve a high pH, ​​it is necessary to have an alkali metal hydroxide, especially sodium hydroxide or potassium hydroxide, usually in an amount of 0.1% to about 30% by weight of the composition (weight percentage, abbreviated wt%), and preferably 1.0% to 2.5%, or higher amounts of a suitable alkali metal silicate, such as a metal silicate, depending on the desired pH of the product.

[0104] The composition of the present invention preferably comprises 20% to 95%wt, preferably 40% to 95%wt, further preferably from about 70% to about 90%wt of water. Preferably from about 75% to 80%wt of water, preferably from about 80% to 85%wt of water, preferably from about 70%-75%wt of water, or preferably from about 75%-80%wt of water. Preferably, the composition of the present invention includes 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 weight percentage (wt) of water.

[0105] These protease variants may be added to the high pH cleaning composition according to the invention in an amount corresponding to 0.01-200 mg enzyme protein per liter of washing liquor, preferably 0.05-50 mg enzyme protein per liter of washing liquor, in particular 0.1-10 mg enzyme protein per liter of washing liquor.

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

[0107] A composition for use in an automatic dishwasher (ADW) may for example comprise 0.0001%-50%, such as 0.001%-30%, such as 0.01%-20%, such as 0.5%-15% enzyme protein by weight of the composition.

[0108] The present invention relates to a liquid cleaning composition having a pH of 10 or more, 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 in 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 10.

[0109] The protease variants added to the cleaning compositions according to the present invention show an improved half-life to a certain extent, making it possible to store and use the protease in high pH (pH>10.0) liquid cleaning compositions such as detergents. In addition, the protease variants show significant washing performance in high pH liquids, and thus open the possibility of improving the washing performance of high pH liquid detergents and / or reducing the concentration of detergents present in these detergent formulations used to achieve the same washing performance. In addition, the protease variants show significant washing performance in high pH liquids, and thus open the possibility of improving the washing performance of high pH liquid detergents and / or reducing the concentration of proteases present in these detergent formulations used to achieve the same washing performance.

[0110] One embodiment of the present invention relates to a liquid cleaning composition having a pH of 10 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention provides a liquid cleaning composition having a pH of 10 or more and comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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 having a pH from about 7 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 7 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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 having a pH from about 8 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 8 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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).

[0113] One embodiment of the present invention relates to a liquid cleaning composition having a pH from about 9 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 9 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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 having a pH from about 10 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 10 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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).

[0115] One embodiment of the present invention relates to a liquid cleaning composition having a pH from about 11 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution at one or more of the following positions selected from the positions corresponding to 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). In another embodiment, the present invention relates to a liquid cleaning composition having a pH from about 11 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to 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 corresponding to 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).

[0116] One embodiment of the present invention relates to a liquid cleaning composition having a pH of from about 7 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacement of 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 of from about 7 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W, preferably, the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W and a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W. ID NO: 1) is substituted with 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 having a pH of from about 8 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacement of 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 of from about 8 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W, preferably, the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W and a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W. ID NO: 1) is substituted with 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 having a pH of from about 9 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: NO: 1) replacement of 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 of from about 9 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W, preferably, the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W and a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W. ID NO: 1) is substituted with 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 having a pH of from about 10 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: NO: 1) replacement of 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 of from about 10 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W, preferably, the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W and a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W. NO: 1) is substituted with 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 having a pH of from about 11 to about 13 or higher comprising a protease, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: NO: 1) replacement of 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 of from about 11 to about 13 or higher, wherein the protease is a variant protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W, preferably, the variant comprises a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W and a substitution corresponding to BPN' (SEQ ID NO: 1) of substitutions X205I and X209W. NO: 1) is substituted with 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:

[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 so that they have a pH of from about 7.5 to about 13.5, or from about 8.0 to about 13.5, from about 8.5 to about 13.5, from 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, from about 10 to about 10.5. In some preferred embodiments, liquid cleaning compositions are formulated so that the pH is from about 10 to about 13.5. In other preferred embodiments, liquid cleaning compositions are formulated so that the pH is from about 12 to 13.5.

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

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

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

[0191] (c) from 1% to 30% by weight of an alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof to provide a pH of 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 a substitution at one or more positions selected from the positions corresponding to 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0195] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt, of a protease variant, wherein the variant comprises a substitution at one or more positions selected from the group consisting of 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) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or

[0197] (c2) from 1% to 30% wt of an alkaline buffer system 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 from about 10 to 13.5;

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

[0199] In a preferred embodiment, the protease variant comprises the substitutions 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

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

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

[0203] (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO 1), preferably, the variant comprises a substitution 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 corresponding to BPN' (SEQ ID NO: 1), further preferably, the variant comprises the substitutions X205I and X209W corresponding to BPN' (SEQ ID NO 1) and the substitutions X205I and X209W corresponding to BPN' (SEQ ID NO 1) is substituted with 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0206] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO:1), 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 variant comprises a BPN′ corresponding to (SEQ ID NO: 1), further preferably, the variant comprises the substitutions X205I and X209W corresponding to BPN' (SEQ ID NO: 1) and the substitutions X205I and X209W corresponding to BPN' (SEQ ID NO: 1). NO: 1) is substituted with 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) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or

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

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

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

[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] The protease variants of the present invention have improved stability in liquid cleaning compositions having a pH of 10 or higher when compared to a parent protease, such as the protease having SEQ ID NO: 2. This makes these variants suitable for use in high pH liquid detergents, for example, for laundry or dishwashing.

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

[0278] In one embodiment the protease variant has improved stability in high pH liquid cleaning compositions and equivalent or improved wash performance compared to the parent protease, wherein storage stability can be measured using the 'Accelerated Storage Stability Assay' and wash performance can be measured using the 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 one 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, at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity to 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, at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0282] In one aspect, 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, most preferably between 3 and 8 changes. In another aspect, 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 parent or precursor protease may be any protease, or even more preferably any subtilase as defined below. The parent protease is preferably a protease having at least 60% identity to SEQ ID NO: 2, but the parent may be any serine protease. Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds and have an essential serine residue at the active site (White, Handler and Smith, 1973, "Principles of Biochemistry", Fifth Edition, McGraw-Hill Book Company, New York, pp. 271-272). The molecular weight range of bacterial serine proteases is 20,000 to 45,000 daltons. They are inhibited by diisopropyl fluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, which is also a serine protease. An even more preferred parent is an alkaline protease, which encompasses a subgroup reflecting the high pH optimum of some serine proteases from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41:711-753).

[0284] The parent protease may be a subtilase, which is a subgroup of serine proteases proposed by Siezen et al. (1991), Protein Eng. 4: 719-737 and Siezen et al. (1997), Protein Science 6: 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously known as subtilisin-like proteases. Subtilisin was previously generally defined as a serine protease produced by gram-positive bacteria or fungi, but is now a subgroup of subtilases according to Siezen et al. A wide variety of subtilases have been identified, and the amino acid sequences of many subtilases have been determined. For a more detailed description of such subtilases and their amino acid sequences, see Siezen et al. (1997). Subtilisin is a subgroup of subtilases as described above, which are serine proteases from the S8 family, in particular from the S8A subfamily, as defined in the MEROPS database (http: / / merops.sanger.ac.uk / cgi-bin / famsum?family=S8). Examples of subtilisin are subtilisin BPN' (SEQ ID NO: 1) and subtilisin 309 (SEQ ID NO: 2), which have MEROPS numbers S08.034 and S08.003, respectively. The parent protease may also be a subtilase isolated from a natural source, wherein, while retaining the characteristics of the subtilase, it has been subsequently modified (e.g., one or more replacements, one or more substitutions, one or more deletions and / or one or more insertions of one or more amino acid side chains). Furthermore, the parent protease may be a subtilase prepared by DNA shuffling techniques as described by Ness et al., 1999, Nature Biotechnology, 17: 893-896.

[0285] Alternatively, the term "parent protease" may be referred to as a "precursor protease" and is used to describe the starting protease in which mutations are made to obtain the variants of the invention. The parent protease preferably belongs to the subtilisin subgroup.

[0286] A subgroup of subtilases, I-S1 or "true" subtilisins, includes "standard" subtilisins such as subtilisin 168 (BSS168), subtilisin BPN', subtilisin Carlsberg ( Novozymes), and subtilisin DY (BSSDY). BPN' is subtilisin BPN' from Bacillus amyloliquefaciens, subtilisin BPN' having the amino acid sequence of SEQ ID NO:1. Siezen et al. (supra) identified another subgroup of subtilases, I-S2 or high alkaline subtilisins. Subgroup I-S2 proteases are described as strongly alkaline subtilisins and include enzymes such as: subtilisin PB92 (BAALKP) ( Genencor International, Inc.), subtilisin 147 (BLS147) ( Novozymes), alkaline elastase YaB (BSEYAB) and subtilisin 309 having the amino acid sequence SEQ ID NO 2 ( Novozymes).

[0287] The homology between two amino acid sequences is in the context of being described by the parameter "identity" for the purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as described above. The results from the program calculate the "percent identity" between the two sequences in addition to the amino acid alignment.

[0288] Based on this description it will be fairly straightforward for a person skilled in the art to identify suitable homologous subtilases that may be modified according to the present invention.

[0289] The parent protease can be a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO: 1, which has protease activity. In one aspect, the amino acid sequence of the parent differs from 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 can be a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO: 2, and it has protease activity. In one aspect, the amino acid sequence of the parent differs from 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 may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.

[0292] The parent protease may be obtained from microorganisms of any genus. For the purposes of the present 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 the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

[0293] The parent may be a bacterial protease. For example, the parent may 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] In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis protease.

[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 Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Netherlands Type Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Agricultural Research Service, Northern Regional Research Center (NRRL).

[0296] Can use above-mentioned probe from other sources, comprise from nature (for example, soil, compost, water etc.) separated microorganism or directly from natural material (for example, soil, compost, water etc.) DNA sample identification and obtain this parent.Be used for directly separating microorganism and DNA from natural living environment technology is well known in the art.Then can be by similarly screening in the genomic DNA or cDNA library of another microorganism or mixed DNA sample to obtain the polynucleotide of encoding parent.Once detected the polynucleotide of encoding parent with one or more probes, then can be by utilizing technology known to those of ordinary skill in the art to separate or clone this polynucleotide (referring to for example, Sarah Brooke (Sambrook) et al., 1989, see 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 the substitutions 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 NO:1 position, and

[0299] (b) recovering the variant.

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

[0301] Site-directed mutagenesis is a technique for introducing one or more (eg, several) mutations at one or more defined sites in the polynucleotide encoding the parent.

[0302] Site-directed mutagenesis can be achieved in vitro by using PCR involving oligonucleotide primers containing the desired mutation. Site-directed mutagenesis in vitro can also be performed by cassette mutagenesis, which involves cutting by a restriction enzyme at a site in a plasmid including a polynucleotide encoding a parent and then connecting the oligonucleotide containing the mutation in the polynucleotide. Generally, the restriction enzyme that digests the plasmid is the same as the restriction enzyme of the oligonucleotide to allow the sticky ends of the plasmid and the inserted fragment to be connected to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

[0303] Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, e.g., 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 may be used in the present invention.There are many commercially available kits that can be used to prepare variants.

[0305] Synthetic gene construction requires in vitro synthesis of designed polynucleotide molecules to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as multiplexed microchip-based techniques described by Tian et al. (2004, Nature 432: 1050-1054), and similar techniques in which oligonucleotides are synthesized and assembled 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 shuffling, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 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 / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of single amino acid residues in polypeptides to be determined rapidly.

[0308] A plurality of aspects that realize semisynthetic gene construction by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or reorganization.Semisynthetic construction typically utilizes the process of synthetic polynucleotide fragments in conjunction with PCR technology.Therefore, the region of gene restriction can be synthesized from scratch, and other regions can use site-specific mutagenic primers to increase, and other regions can be subjected to fallibility PCR or non-fallibility PCR amplification.Then polynucleotide subsequence can be reorganized.

[0309] In addition to the protease according to the present invention, the liquid cleaning composition according to the present invention may also include additional 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 stains, the temperature at which cleaning is performed, and the formulation of the detergent product.

[0310] In a particular embodiment, the high pH liquid cleaning composition comprises a protease variant of the present invention and one or more detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric hueing agents, fabric conditioners, suds boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfumes, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0311] Conventional stabilizers and protease inhibitors can be used to stabilize the protease variants of the cleaning composition of the present invention, for example, polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, various salts (such as NaCl, KCl), lactic acid, formic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters; or phenylboronic acid derivatives such as 4-formylphenylboronic acid (e.g., as described in WO 96 / 41859)), or peptide aldehydes (such as 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 with one 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 described in WO 2009 / 118375, WO 2013 / 004635), or as described in WO 2005 / 105826, WO 2009 / 118375, WO 98 / 13459) or protein type protease inhibitors such as RASI, BASI, WASI (rice, barley and wheat bifunctional α-amylase / subtilisin inhibitor) (e.g., as in WO 2009 / 095425) or CI2 or SSI.

[0312] In addition, the use of peptide aldehydes 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 peptide aldehydes Phe-Gly-Ala-PheH and Phe-Gly-Ala-LeuH for stabilizing subtilisin type proteases. In order to stabilize chymotrypsin type proteases, WO 94 / 04651 discloses Leu-Leu-TyrH as a suitable peptide aldehyde. In addition, WO 94 / 04651 suggests the use of methyl carbamate or methyl urea as the N-terminal protecting group of the peptide aldehyde. WO 98 / 13460 discloses peptide protease inhibitors, the use of peptide aldehydes or trifluoromethyl ketones, wherein the peptide chain comprises 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 sulfonamide or amidophosphate. As an example, CH3SO2Phe-Gly-Ala-LeuH can be used.

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

[0314]

[0315] 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 includes an N-terminal protecting group, with the proviso that if B1 is leucine, then B2 cannot be leucine; 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 formula B2-B1-B0-R, wherein:

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

[0318] B0 is a phenylalanine residue with 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] Said protease inhibitors of formula B2-B1-B0-R as described above may be used as optional protease inhibitors in the sense of the present invention (eg in combination with or as part of any embodiment of the invention as disclosed herein).

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

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

[0324] (b) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes 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) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water;

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

[0328] (c) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

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

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

[0333] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0334] (d) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0335] In the embodiment, (c1) and (c2) may be used as substitutes for each other or they may be used in combination with each other.

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

[0337] (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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% greater residual activity than the parent protease when measured after 4 hours at 40°C in a liquid detergent at pH 10, preferably, the variant comprises a substitution at one or more positions selected from the positions corresponding to 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes 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) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water;

[0341] (b) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% higher residual activity than the parent protease when measured after 4 hours at 40°C in a liquid detergent at pH 10, preferably, the variant comprises a substitution at one or more positions selected from the positions corresponding to 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0345] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, preferably, the variant comprises a substitution at one or more positions selected from the positions corresponding to 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); and

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

[0347] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0348] (d) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0349] In the embodiment, (c1) and (c2) may be used as substitutes for each other or they may be used in combination with each other.

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

[0351] (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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% higher residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent at pH 10, preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1), further 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 the group consisting of positions corresponding to BPN' (SEQ ID NO: 2 NO: 1) 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;

[0352] (b) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes 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) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water;

[0355] (b) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% higher residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent at pH 10, preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1), further 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 the group consisting of: 1) 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;

[0356] (c) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0359] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, preferably, the variant comprises substitutions at positions corresponding to positions 205 and 209 of BPN' (SEQ ID NO: 1), further 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 corresponding to 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); and

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

[0361] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0362] (d) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0363] In the embodiment, (c1) and (c2) may be used as substitutes for each other or they may be used in combination with each other.

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

[0365] (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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% higher residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent at pH 10, preferably, the variant comprises one or more substitutions corresponding to BPN′ (SEQ ID NO: 1) replaced by 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes 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) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water;

[0369] (e) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% higher residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent at pH 10, preferably, the variant comprises one or more substitutions corresponding to BPN′ (SEQ ID NO: 1) replaced by 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0373] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, preferably, said variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacements 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) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or

[0375] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0376] (d) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0377] In the embodiment, (c1) and (c2) may be used as substitutes for each other or they may be used in combination with each other.

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

[0379] (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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that when measured after 4 hours at 40° C. in a liquid detergent at pH 10, it has at least 10% higher residual activity than the parent protease, preferably, the variant comprises a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO 1), further preferably, the variant comprises a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO 1) and a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO 1). 1) is substituted with 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes 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) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water;

[0383] (b) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that when measured after 4 hours at 40° C. in a liquid detergent at pH 10, it has at least 10% higher residual activity than the parent protease, preferably, the variant comprises a substitution corresponding to substitutions X205I and X209W of BPN' (SEQ ID NO 1), further preferably, the variant comprises a substitution corresponding to substitutions X205I and X209W of BPN' (SEQ ID NO 1) and a substitution corresponding to BPN' (SEQ ID NO 1) is substituted with 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, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0387] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, preferably, the variant comprises a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO: 1), further preferably, the variant comprises a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO: 1) and a substitution corresponding to the substitutions X205I and X209W of BPN' (SEQ ID NO: 1). 1) is substituted with 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

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

[0389] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

[0390] (d) Optionally, a protease inhibitor, preferably, the protease inhibitor is a peptide aldehyde protease inhibitor, further preferably, the protease inhibitor is 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, B2 optionally includes an N-terminal protecting group, provided that if B1 is leucine, then B2 cannot be leucine.

[0391] In the embodiment, (c1) and (c2) may be used as substitutes for each other or they may be used in combination with each other.

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

[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 compositions can be formulated as described in, for example, WO 92 / 19709, WO 92 / 19708, and US 6,472,364. In some embodiments, the enzymes utilized herein are stabilized by water-soluble sources of zinc (II), calcium (II), and / or magnesium (II) ions, 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) present in the finished composition to provide such ions to the enzymes.

[0459] The protease variants of the present invention may also be incorporated into detergent formulations as 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 (at or above pH 10). The liquid high pH cleaning composition may further include a high amount of water. In a preferred aspect of the present invention, the liquid cleaning composition comprises:

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

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

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

[0464] (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of the protease variant.

[0465] Surfactant can be anionic and / or cationic and / or nonionic and / or semipolar and / or zwitterionic, or its mixture.In a specific embodiment, this cleaning composition comprises one or more nonionic surfactants and the compound with one or more anionic surfactants.Surfactant typically exists with the level 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%.When being included in the cleaning composition according to the present invention, surfactant will comprise by weight from about 1% to about 40%, such as from about 1% to about 20%, preferably by weight from about 0% to about 5%. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) (e.g., sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS) , alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soap, and combinations thereof.

[0466] When included in the cleaning composition according to the present invention, it will generally contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethylethanol quaternary ammonium (ADMEAQ), cetyl 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 polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)), as well as products available under the SPAN and TWEEN trade names, and combinations thereof.

[0468] Non-limiting examples of semi-polar surfactants include amine oxides (AO) such as alkyl dimethyl amine oxides, N-(cocoyl alkyl)-N,N-dimethyl amine oxides and N-(tallow-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 betaines, alkyl dimethyl betaines, sulfobetaines, and combinations thereof.

[0470] Particularly 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 present invention typically contains at least 20% by weight and up to 95% by weight of water, such as up to about 70%wt of water, up to about 65%wt of water, up to about 55%wt of water, up to about 45%wt of water, up to about 35%wt of water. 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%-30% of an organic solvent.

[0472] The cleaning composition may also include a hydrotrope, which is a compound that dissolves hydrophobic compounds in aqueous solution (or conversely, dissolves polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties known from surfactants); however, the molecular structure of hydrotropes is generally not conducive to spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12: 121-128. Hydrotropes do not show a critical concentration above which self-aggregation as found for surfactants and lipids forming micelles, lamellae or other well-defined mesophases will occur. On the contrary, many hydrotropes show a continuous type of aggregation process, in which the size of the aggregates grows with increasing concentration. However, many hydrotropes change the phase behavior, stability, and colloidal properties of systems containing substances with polar and non-polar characteristics (including mixtures of water, oil, surfactants, and polymers). Classically, hydrotropes are used across industries from pharmaceuticals, personal care, and food to technical applications. The use of hydrotropes in cleaning compositions allows, 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. The cleaning composition according to the present invention may include 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5%wt of a hydrotrope. Any hydrotrope known in the art for use in detergents can be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfonate, and combinations thereof. The cleaning composition according to the present invention may include 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 is typically 40%-65%, especially 50%-65%. Builders and chelating agents soften wash water, for example by removing metal ions from the liquid. Builders and / or co-builders may especially be chelating agents that form water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium silicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2"-nitrotriethanol), and carboxymethylinulin (CMI), and combinations thereof.

[0473] Cleaning compositions according to the present invention can also include 0-20% by weight, such as about 5% to about 10%wt of detergent co-builders, or mixtures thereof. The detergent composition can include only co-builders, or in combination with builders, such as zeolite builders. Non-limiting examples of co-builders 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 acid. Further specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic 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 acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP ... acid)(IDA), N-(2-sulfomethyl)-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 In some embodiments, the present invention relates to benzoic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta (methylenephosphonic acid) (DTPMP), aminotri (methylenephosphonic acid) (ATMP) and combinations and salts thereof. Additional exemplary builders and / or co-builders are described, for example, in WO 2009 / 102854 and US 5,977,053.

[0474] Cleaning compositions according to the present invention may contain 0-50% by weight, such as from about 0.1% to about 25% of a bleaching system. Bleach systems often remove color by 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 may be utilized. Suitable bleaching system components include bleach catalysts, hypochlorites, photobleaches, bleach activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, preformed peracids, and mixtures thereof.

[0475] Some non-limiting examples of bleach 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 binuclear manganese complexes [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2"-nitrilotri(ethane-1,2-diylazanylidene-κN-methylidene)triphenol-κ3O]manganese (III). The bleach 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 salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts (e.g., potassium hydrogen persulfate (Oxone(R)), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems in combination with a peracid-forming bleach activator, which may comprise, for example, inorganic salts, including alkali metal salts such as sodium perborate (usually mono- or tetrahydrate), percarbonates, persulfates, perphosphates, persilicates. The term bleach activator is used herein to mean a compound that reacts with a peroxide bleach (such as hydrogen peroxide) to form a peracid. The peracid-forming activator thus formed is a bleaching agent that reacts with a peroxide bleach (such as hydrogen peroxide) to form a peracid. Activated bleaching agents. Suitable bleach activators to be used herein include those belonging to the class of ester amides, imides or anhydrides. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), diperoxylauric acid, 4-(dodecyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS) and / or the bleach activators disclosed in WO 98 / 17767. The specific family of bleach activators is disclosed in EP 624154 and particularly acetyl triethyl citrate (ATC). ATC or short chain triglycerides (like triacetin) have the following advantages, it is environmentally friendly, because it eventually degrades to citric acid and alcohol. In addition, acetyl triethyl citrate and triacetin have good hydrolytic stability in product when stored, and it is an effective bleach activator. Finally, ATC provides good washing ability for clothing additives. Alternatively, the bleaching system can include the peroxyacid of for example amide, imide or sulfone type. The bleaching system can also include peracid, such as 6-(phthalimido) perhexanoic acid (PAP). The bleaching system can 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 formula:

[0478]

[0479] (iii) and mixtures thereof; wherein each R 1 is independently a branched alkyl group containing from 9 to 24 carbons or a straight chain alkyl group containing from 11 to 24 carbons. Preferably, each R 1 is independently a branched alkyl group containing from 9 to 18 carbons or a straight chain alkyl group containing from 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, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described in, for example, WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242. Suitable photobleaching agents can for example be sulfonated zinc phthalocyanine.

[0480] One specific bleaching agent is a hypochlorite, such as sodium hypochlorite (NaClO). Hypochlorites are stabilized by high pH, ​​and in addition, hypochlorites are antibacterial agents. Typically, the cleaning composition according to the invention comprises 3%-8% sodium hypochlorite and 0.01%-0.05% sodium hydroxide; the sodium hydroxide is used to slow down the decomposition of the sodium hypochlorite into sodium chloride and sodium chlorate. Hypochlorites may not be part of the detergent formula, but a separate product is added during the washing process.

[0481] In one aspect of the present invention, the high pH cleaning compositions of the present invention do not include any bleaching agents, ie, the cleaning compositions may be bleach-free.

[0482] Cleaning compositions according to the present invention may contain 0-10% by weight, such as 0.5%-5%, 2%-5%, 0.5%-2% or 0.2%-1% polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, greasy cleaning and / or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethylene imine), 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 silicones, copolymers of terephthalic acid and oligoethylene glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Additional exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and ethoxysulfate diquaternary ammonium salts. Other exemplary polymers are disclosed in, for example, WO 2006 / 130575. Salts of the above-mentioned polymers are also contemplated. The cleaning compositions of the present invention may also include fabric hueing agents, such as dyes or pigments, which, when formulated in a detergent composition, may be deposited on the fabric when the fabric is contacted with a washing solution comprising the detergent composition, and thus the color of the fabric is changed by absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, because they absorb at least a portion of the visible light spectrum, fabric hueing agents change the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of the following dyes falling into 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 (hereby incorporated by reference).The detergent composition preferably includes from about 0.00003wt% to about 0.2wt%, from about 0.00008wt% to about 0.05wt%, or even from about 0.0001wt% to about 0.04wt% of a fabric hueing agent. The composition may include from 0.0001wt% to 0.2wt% of a fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit dose bag. Suitable hueing agents are also disclosed in, for example, WO 2007 / 087257 and WO 2007 / 087243.

[0483] The cleaning composition according to the invention may comprise one or more (additional) enzymes, such as amylases, perhydrolases, arabinanases, carbohydrases, cellulases (e.g. endoglucanases), cutinases, galactanases, haloperoxygenases, lipases, mannanases, oxidases, such as laccases and / or peroxidases, pectinases, pectin lyases, proteases, DNases, xylanases, xanthanases, and xyloglucanases.

[0484] In general, the properties of the enzyme or enzymes selected should be compatible with the detergent (ie, pH optimum, compatibility with other enzymes and non-enzymatic ingredients, etc.), and the enzyme or enzymes should be present in an effective amount.

[0485] Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, such as the fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum described in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89 / 09259.

[0486] Particularly suitable cellulases are the alkaline or neutral cellulases having color care benefits. 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 plant or microbial origin. Preferably microbial origin. Including chemically modified or protein engineered mutants. It can be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease can be, for example, an S1 family (such as trypsin) or an S8 family (such as subtilisin). Metalloprotease can be, for example, a thermolysin or other metalloprotease from, for example, family M4, such as those from M5, M7 or M8 families.

[0488] Examples of metalloproteases are neutral metalloproteases 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 trade 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 (alkaliphilic Bacillus subtilisin) from Kao Corporation (Kao).

[0490] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipases from thermophilic fungi, e.g., from Thermomyces lanuginosus (formerly named Humicola lanuginosus) as described in EP 258068 and EP 305216; cutinases from Humicola, e.g., Humicola insolens (WO 96 / 13580); lipases from strains of Pseudomonas (some of these now renamed Burkholderia), e.g., Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (EP 331376), Pseudomonas strain SD705 (WO 95 / 06720 & WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (WO 2010 / 065455); cutinase from Magnaporthe oryzae (WO 2010 / 107560); cutinase from Pseudomonas mendocinae (US 5,389,536); lipase from Thermobifida fusca (WO 2011 / 084412); Geobacillus stearothermophilus lipase (WO 2011 / 084417); lipase from Bacillus subtilis (WO 2011 / 084599); and lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces pristinaespiralis (WO 2012 / 137147). Preferred commercial lipase products include Lipolase TM 、Lipex TM ;Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor) and Lipomax (from Gist-Brocades).

[0491] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, such as acyltransferases with homology 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 the Mycobacterium smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0492] Suitable amylases may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Commercially available amylases are Duramyl TM , Amylase TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM 、Natalase TM , Liquozyme X and BAN TM (from Novozymes), and Rapidase TM 、Purastar TM / Effect 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. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, for example from Coprinus cinereus, and variants thereof, 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 combined additive comprising all of these enzymes. The detergent additive of the present invention, i.e., a separate or combined additive, can be formulated into, for example, granules, liquids, slurries, etc., preferably the detergent additive formulation is granules, especially dust-free granules; liquids, especially stabilized liquids; or slurries.

[0496] Any detergent component known in the art for use in laundry detergent compositions can also be utilized. Other optional detergent components include preservatives, shrink-proofing agents, anti-soil redeposition agents, wrinkle-resistant agents, bactericides, adhesives, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC and / or polyols such as propylene glycol), fabric finishing agents (including clay), fillers / processing aids, fluorescent whitening agents / optical brighteners, foaming agents, foam (foam) regulators, spices, dirt suspending agents, softeners, foam suppressants, tarnish inhibitors and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents can be utilized. The selection of such ingredients is fully within the technical scope of ordinary technicians.

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

[0498] Cleaning compositions of the present invention can also include one or more dye transfer inhibitors. Suitable polymer dye transfer inhibitors include but are not limited to copolymers, polyvinyl oxazolidone and polyvinyl imidazole or their mixtures of polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, N-vinyl pyrrolidone and N-vinylimidazole. When present in the subject composition, the dye transfer inhibitor can exist at the following level 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 composition of the present invention will also preferably include additional components that can color the items being cleaned, such as fluorescent whitening agents or optical brighteners. Wherein the brightener is preferably present at a level of about 0.01% to about 05%. Any fluorescent whitening agent suitable for use in laundry detergent compositions can be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and diphenyl-distyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents 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 -6-ylamino)stilbene-2,2'-disulfonate, 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-(distyryl-4"-naphthalene-1.,2':4,5)-1,2,3-triazole-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-styryl) disulfonate. Also preferred fluorescent whitening agents are commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazoline and 7-aminoalkyl coumarin. Suitable fluorescent whitening agent levels include from about 0.01wt%, from 0.05wt%, from about 0.1wt% or even from about 0.2wt% lower levels to 0.5wt% or even 0.75wt% higher levels.

[0500] Cleaning compositions of the present invention can also include one or more soil release polymers, which help remove dirt from fabrics (such as cotton and polyester-based fabrics), particularly remove hydrophobic dirt from polyester-based fabrics. Soil release polymers can be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, referring to, for example, powdered detergents (PowderedDetergents), surfactant science series (Surfactant science series) volume 71, chapter 7, Marcel Dekker, Inc.). Another type of soil release polymer is an amphiphilic alkoxylated oily cleaning polymer comprising a core structure and a plurality of alkoxylated groups connected to the core structure. The core structure can include a polyalkyl imine structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (combined hereby by reference). In addition, any graft copolymer is a suitable soil release 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 soil release 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 cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionic modified cellulose and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, ester carboxymethylcellulose and mixtures thereof.

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

[0502] Other suitable adjuvants include, but are not limited to, shrink-proofing agents, wrinkle-proofing agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, foam suppressants, solvents, and structurants and / or structural elastic agents for liquid detergents.

[0503] The cleaning composition of the present invention can be formulated into bags, which can be configured as single compartments or multiple compartments. It can have any form, shape and material suitable for preserving the composition, such as before contacting with water, the composition is not allowed to be released from the bag. The bag is made of a water-soluble film that encapsulates the inner volume. The inner volume can be divided into the compartments of the bag. The preferred film is a polymeric material that forms a film 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, polymethacrylate, most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the level of polymer in film such as PVA is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. The film can also be a blend composition comprising a hydrolytically degradable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under trade reference M8630, such as sold by Chris Craft In. Prod., Gary, Indiana, US) plus a plasticizer, such as glycerol, ethylene glycol, propylene glycol, sorbitol and mixtures thereof. These bags can include solid laundry detergent compositions or partial components and / or liquid cleaning compositions or partial components separated by water-soluble films. The chamber for the liquid component can be different in composition from the chamber comprising the solid. See, for example, US2009 / 0011970.

[0504] The detergent ingredients can be physically separated from each other by compartments in water-soluble bags or different layers of tablets. Thus, bad storage interactions between components can be avoided. In the washing solution, the different dissolution curves of each compartment can also cause delayed dissolution of the selected components.

[0505] The present invention is also directed to methods of using the cleaning compositions according to the invention in textile and fabric washing, such as domestic laundry washing as well as industrial laundry washing. Such methods include methods of using the compositions according to the invention or compositions thereof in cleaning hard surfaces such as floors, tables, walls, roofs, etc., as well as surfaces of hard objects such as cars (car washing) and dishes (dishwashing).

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

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

[0508] The cleaning process or textile care process can be, for example, a laundry process, a dishwashing process or a hard surface (such as bathroom tiles, floors, desktops, drains, sinks and washbasins) cleaning. The laundry process can be, for example, a household laundry process, but it can also be an industrial laundry process. In addition, the present invention relates to a method for washing fabrics and / or clothing, wherein the method comprises treating fabrics with a washing solution comprising a high pH cleaning composition according to the present invention and at least one protease variant of the present invention. The cleaning process or textile care process can be, for example, carried out in a machine washing process or in a hand washing process or in a pre-spotter process. The washing solution can, for example, be a washing solution comprising a cleaning composition.

[0509] The present invention further relates to the use of the cleaning composition of the present invention in the process of removing protein stains. The protein stains may be stains such as food stains, such as baby food, sebum, cocoa, eggs, blood, milk, ink, grass, or a combination thereof.

[0510] Preferred embodiments relate to methods of cleaning comprising the step of contacting the object with a high pH cleaning composition comprising a protease variant of the present invention under conditions suitable for cleaning the object. In preferred embodiments, the cleaning composition is used in a laundry or dishwashing process.

[0511] Still another embodiment is directed to a method for removing stains from fabric or dishware comprising contacting the fabric or dishware with a high pH cleaning composition comprising a 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, which methods are well known in the art (see, e.g., US 6,077,316). For example, in one aspect, the feel and appearance of a fabric are improved by a method comprising contacting the fabric with a protease in a solution. In one aspect, the fabric is treated with the solution under pressure.

[0513] The high pH cleaning compositions of the present invention are 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 washing hard surfaces. The method includes a step of contacting the fabric / dishware to be cleaned with a solution comprising a high pH cleaning composition of the present invention. The fabric may include any fabric that can be washed under conventional consumer use conditions. The hard surface may include any tableware, such as pottery, cutlery, ceramics, plastics (such as melamine), metals, porcelain, glass, acrylic resins or other hard surfaces (such as cars, floors, etc.). The solution preferably has a pH from about 9 to about 13.5. The composition may be used in a solution at a concentration of from about 100 ppm, preferably 500 ppm to about 15,000 ppm. The water temperature typically ranges 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 so that in the aqueous cleaning process, the wash water has the following pH: from about 9 to about 13.5, or in alternative embodiments, or from about 10 to about 13.5, even from about 11 to about 13.5. In some preferred embodiments, the liquid laundry detergent product is formulated to have a pH from about 12 to about 13.5. The technology used to control pH at the recommended use level includes using buffers, acids, alkalis, etc., and is well known to those of ordinary skill in the art. In the context of the present invention, alkali is used to adjust the pH to about 9 to 13.5, preferably from about 10 to 13.5.

[0515] The present 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 a parent protease, and wherein the protease variant has an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% greater residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent having a pH of 10

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

[0519] 2. A liquid cleaning composition comprising:

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

[0521] (b) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in 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, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

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

[0524] 4. A liquid cleaning composition as described in any of paragraphs 1-3, wherein the protease is a variant of a protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, wherein the variant comprises a substitution at one or more positions selected from the positions corresponding to 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).

[0525] 5. A liquid cleaning composition as described in any of paragraphs 1-4, wherein the protease is a variant of a protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacement of 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 of any of paragraphs 1-5, wherein the variant comprises one or more substitutions corresponding to substitutions X209W, X262E, X76D, X194P, X204D, X206L.

[0527] 7. A liquid cleaning composition according to any 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, at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0528] 8. The liquid cleaning composition according to any of the preceding paragraphs, wherein the protease variant has a storage stability at 40°C greater 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 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) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

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

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

[0621] (c2) from 1% to 30% wt of an 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 from about 12 to 13.5;

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

[0623] 11. A liquid cleaning composition as described in paragraph 10, wherein the protease variant comprises a substitution at one or more positions selected from the positions corresponding to 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).

[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: 1) replacement of 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. A liquid cleaning composition according to any of paragraphs 10 to 12, wherein the protease variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 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 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. A cleaning composition according to any of the preceding paragraphs, comprising one or more additional enzymes selected from the group consisting of amylases, catalases, cellulases (e.g., endoglucanases), cutinases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, and xyloglucanases, or any mixture thereof.

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

[0694] 17. The use of paragraph 16, wherein the laundry process is selected from the group consisting of a residential laundry process, an industrial laundry process, and an institutional laundry process.

[0695] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0696] Example

[0697] Materials and Methods

[0698] Suc-AAPF-pNA activity assay

[0699] Proteolytic activity can be determined by a method using Suc-AAPF-PNA as a substrate. Suc-AAPF-PNA is an abbreviation for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and is a blocked peptide that can be cleaved by an endoprotease. After cleavage, a free PNA molecule is released, which has a yellow color and can therefore be measured by visible light spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is produced by Bachem (Cat. No. L1400, dissolved in DMSO).

[0700] Protease samples to be analyzed are diluted in residual activity buffer (100mM Tris pH 8.6). The assay is 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 100mM Tris pH 8.6). The solution is mixed at room temperature and absorbance is measured at OD 405nm every 20 seconds for 5 minutes.

[0701] Under a given set of conditions, the slope of the time-dependent absorbance curve (absorbance per minute) 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 mutations and introduction of the expression cassette into B. subtilis are summarized below.All DNA manipulations were performed by PCR (eg, Sambrook et al.; Molecular Cloning; Cold Spring Harbor Laboratory Press).

[0704] The recombinant Bacillus subtilis construct encoding the protease variant is used to inoculate a shake flask containing a nutrient-rich medium (e.g., 100 g / L sucrose (Danisco catalog number 109-0429), 40 g / L soybean hulls (soy flour), 10 g / L Na2HPO4.12H2O (Merck catalog number 6579), 0.1 ml / L replacement-Dowfax 63N10 (Dow Chemical). Cultivation is typically performed at 30°C with shaking at 220 rpm for 4 days.

[0705] Example 2: Purification of protease variants

[0706] The culture fluid was centrifuged at 26000 x g for 20 minutes, and the supernatant was carefully separated from the precipitate decantation. The supernatant was filtered by a 0.2 μm filter to remove the remaining bacillus host cells. The pH in the 0.2 μm filtrate was adjusted to pH 8 with 3M Tris alkali, and the pH-adjusted filtrate was applied to 20mM Tris / HCl, 1mM CaCl2 (pH 8.0) in the MEP Hypercel post (Pall Corporation) balanced. After the post was washed with equilibrium buffer, 20mM CH3COOH / NaOH, 1mM CaCl2 (pH 4.5) were progressively eluted. The fraction from the post was analyzed for protease activity using the Suc-AAPF-pNA assay under pH 9, and the peak fractions were merged. The pH of the pool from the MEP Hypercel column was adjusted to pH 6 with 20% (v / v) CH3COOH or 3M Tris base, and the pH adjusted pool was diluted with deionized water to the same conductivity as 20mM MES / NaOH, 2mM CaCl2 (pH 6.0). The diluted pool was applied to SP-Sepharose equilibrated in 20mM MES / NaOH, 2mM CaCl2 (pH 6.0). The proteinase variants were eluted using a linear NaCl gradient (0-->0.5 M) in the same buffer using a fast flow column (GE Healthcare). After the column was washed with an equilibrium buffer, the proteinase variants were eluted using a linear NaCl gradient (0-->0.5 M) in the same buffer over five column volumes. The fractions from the column were analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9, and the active fractions were analyzed by SDS-PAGE. The fractions (only one band was observed on the SDS-PAGE gel of Coomassie stain) were combined into purified preparations and used for additional experiments.

[0707] Example 3: Accelerated Storage Stability Determination

[0708] The storage stability of the protease variants in liquid detergents was evaluated using an accelerated assay with incubation at elevated temperature for up to 24 hours.

[0709] Based on the absorbance at 280nm and theoretical extinction coefficient, all purified protease samples were diluted to a concentration of 0.2 and 0.1mg / ml using 0.01% Triton X-100. For each variant, 2 holes with high protease concentration and 2 holes with low concentration were included. As a reference, Savinase (SEQ ID NO 2) or stabilized protease variants were included on each microtiter plate. Using a magnetic bar (on Zephyr pipetting station (Caliper LifeSciences company) for 30min), 30 μl protease samples were mixed with 270 μl detergents (Surf (Surf) Sparkling Ocean (SparklingOcean), DC-2014-0009) in the hole of a microtiter plate (Nunc U96 PP 0.5ml). 20 μl of this mixture was then transferred to another microtiter plate (Nunc U96 PP 0.5 ml with a magnetic bar) and mixed with 150 μl 100 mM Tris (pH 8.6) (at least 5 min on 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 the absorbance at 405 nm every 20 sec for 5 min (on SpectraMax Plus). After sealing, the detergent plate was incubated (without shaking) at 50 ° C and pH 11 in an Eppendorf Thermomixer. After 1, 3-4 and 23-24 hours of incubation, 20 μl samples were taken and the residual activity of the stressed sample was measured like the initial non-stressed activity.

[0710] The decrease in activity during incubation with detergent was assumed to be exponential. The half-life (T1 / 2) was found from the linear regression of Log(activity) versus incubation time. 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 the already stable protease variants.

[0711] Accelerated storage stability of variant T1 / 2IF at 40°C: half-life improvement impact of single mutations

[0712] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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] Mutation 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 Example 4: Full-scale washing results of the protease of the present invention

[0775] The wash performance of the proteases of the invention was tested in a full scale wash. The wash performance was tested in a laundry liquid detergent on 22 different stains in a dose response.

[0776] After washing and rinsing, the small pieces of cloth are spread out and allowed to air dry overnight at room temperature. All washings are evaluated on the next day of washing. Use a Macbeth Color Eye 7000 reflection spectrophotometer with a very small aperture to evaluate the light reflection of the small pieces of cloth. Measure and extract the reflection at 460nm without UV in the incident light. Measure on unwashed and washed small pieces of cloth. The test small piece of cloth to be measured is placed on the top of another small piece of cloth of the same type and color.

[0777] The enzyme effect, ΔRem, for each stain was calculated by taking the measurement from the swatch washed with enzyme and subtracting it from the measurement from the swatch not washed with enzyme. 酶 The washing performance is expressed as the Δ reflectance value (ΔRem).

[0778] The experiments were performed using the detergent compositions and swatches under the experimental conditions specified in Table 21 below.

[0779] Table 21: Experimental conditions for full-scale laundry washing experiments

[0780]

[0781] 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.

[0782] Table 22: Delta reflectance values ​​for detergents including protease compared to detergents without protease at 15°C in all free & clear (enzymes tested were as follows:

[0783] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0784] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0785] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0786] Δ(Compared to blank)

[0787]

[0788]

[0789] Table 23: Delta reflectance values ​​for detergents including protease compared to detergents without protease at 30°C in All Free & Clear (The enzymes tested in "Results (Enzyme - Blank)" below are as follows:

[0790] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0791] 2: S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0792] 3: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0793] 4: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0794] All free&clear 30℃

[0795]

[0796]

[0797] Table 24: Delta reflectance values ​​for detergents including protease compared to detergents without protease at 30°C in all Arm & Hammer (enzymes tested were as follows:

[0798] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0799] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0800] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0801] Δ(Compared to blank) detergent Arm&Hammer

[0802]

[0803]

[0804] Table 25: Delta reflectance values ​​for detergents including protease compared to detergent without protease in Purex at 30°C (enzymes tested were as follows:

[0805] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0806] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0807] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0808] Purex

[0809]

[0810]

[0811] Table 26: Delta Reflectance values ​​for detergents including protease compared to detergents without protease in Sun Triple Clean at 30°C (Enzymes tested were as follows:

[0812] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0813] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0814] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E);

[0815] Δ(Compared to blank) detergent Sun Triple Clean

[0816]

[0817]

[0818] Table 27: Delta reflectance values ​​for detergents including proteases compared to detergents without proteases at 30°C in Xtra. The enzymes tested included the following:

[0819] 1: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0820] 2: S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0821] 3: S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E:

[0822] Δ(Compared to blank) Detergent Xtra

[0823]

[0824]

[0825] Example 5: Testing of Subtilisin 309 Variants in the Microwash

[0826] The cleaning performance of the protease of the present invention was tested on a technical stain using a laundry liquid detergent using a mini-wash system.

[0827] The mini-wash test is a test method in which soiled textiles are successively lifted into a test solution and subsequently rinsed.

[0828] Table 28: Washing experiments were performed under the following specified experimental conditions:

[0829]

[0830] Test materials were obtained from EMPA Testmaterials AG. 12, CH-9015 Rue des Galles, Switzerland).

[0831] The textiles were then air-dried and the wash performance was measured as the brightness of the color of these textiles. Brightness can also be expressed as reflectance (R), which is a measure of the 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 521VIS spectrophotometer. Measurements were performed according to the manufacturer's protocol.

[0832] The enzyme effect, ΔRem, for each stain was calculated by taking the measurement from the swatch washed with enzyme and subtracting it from the measurement from the swatch not washed with enzyme. 酶 .

[0833] The experiments were performed with the detergent compositions and swatches as described for the laundry method in the mini-wash assay under the experimental conditions specified in Table 29 below.

[0834] Table 29: Experimental conditions for the micro-washing clothing washing experiment

[0835] Detergent dosage YPE, 2g / L Test solution volume 50mL pH According to the original situation Washing time 20min soaking + 18min main wash temperature 25℃ Water hardness 8.4°dH Protease concentration 0-20-40-80nM Small fabric swatch EMPA 117EH

[0836] By mixing CaCl2, MgCl2 and NaHCO3 (Ca 2+ Mg 2+ :CO3 - =2:1:4.5) was added to the test system to adjust the water hardness to 8.4° dH. After washing, the textiles were rinsed with tap water and dried.

[0837] Table 30: Relative performance of protease subtilisin 309 variants compared to detergent with subtilisin 309 (SEQ ID NO 2) at 25°C

[0838]

[0839]

[0840]

[0841] Results Table 30 shows that the subtilisin 309 variants show improved or equivalent wash performance compared to subtilisin 309 (SEQ ID NO: 2) on blood / milk / ink at 25°C.

[0842] 1. A liquid cleaning composition having a pH of 10 or higher, comprising:

[0843] (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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in that it has at least 10% greater residual activity than the parent protease when measured after 4 hours at 40° C. in a liquid detergent having a pH of 10

[0844] (b) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0845] 2. A liquid cleaning composition comprising:

[0846] (a) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water;

[0847] (b) 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 to the amino acid sequence of SEQ ID NO: 2, and wherein the protease variant is characterized in 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;

[0848] (c) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0849] 3. The liquid cleaning composition of item 1, wherein the pH is from about 12 to 13.5.

[0850] 4. A liquid cleaning composition as described in any of items 1-3, wherein the protease is a variant of a protease having at least 60% identity to the amino acid sequence of SEQ ID NO:2, wherein the variant comprises a substitution at one or more positions selected from positions corresponding to 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).

[0851] 5. A liquid cleaning composition as described in any one of items 1 to 4, wherein the protease is a variant of a protease having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, wherein the variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacement of 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.

[0852] 6. The liquid cleaning composition of any one of items 1-5, wherein the variant comprises one or more substitutions corresponding to the substitutions X209W, X262E, X76D, X194P, X204D, X206L.

[0853] 7. A liquid cleaning composition according to any of the preceding items, wherein the variant has an amino acid sequence that is at least 60% identical to SEQ ID NO: 2, for example, at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0854] 8. The liquid cleaning composition according to any of the preceding items, wherein the protease variant has a storage stability at 40°C greater than at least one stable protease variant selected from the group consisting of:

[0855] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0856] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0857] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0858] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0859] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

[0860] S9E+N18S+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0861] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0862] S9E+N43R+N76D+A194P+Q206L+Y209W+S259D+N261W+L262ES9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0863] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0864] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0865] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0866] S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

[0867] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0868] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0869] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

[0870] S9E+N76D+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0871] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0872] S9E+N43R+N76D+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0873] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0874] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0875] S9E+N43R+I72A+N76D+A194P+Q206L+L262E;

[0876] S9E+N43R+N76D+A194P+V205I+Q206L+L262E.

[0877] 9. The liquid cleaning composition according to any of the preceding items, wherein the protease variant is selected from the group consisting of:

[0878] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0879] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E; S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0880] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0881] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0882] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0883] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0884] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0885] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0886] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0887] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0888] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0889] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0890] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0891] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0892] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0893] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0894] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0895] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0896] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0897] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0898] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0899] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0900] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0901] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0902] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0903] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0904] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0905] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0906] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0907] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0908] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0909] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0910] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0911] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0912] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0913] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0914] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0915] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0916] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0917] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0918] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0919] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0920] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0921] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0922] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0923] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0924] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0925] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0926] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0927] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[0928] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0929] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[0930] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0931] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0932] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E; S9E+N4 3R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

[0933] 10. A liquid cleaning composition comprising:

[0934] (a) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant;

[0935] (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of the protease variant; and

[0936] (c1) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or

[0937] (c2) from 1% to 30% wt of an 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 from about 11 to 13.5;

[0938] (d) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

[0939] 11. A liquid cleaning composition as described in claim 10, wherein the protease variant comprises a substitution at one or more positions selected from the positions corresponding to 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).

[0940] 12. A liquid cleaning composition according to item 11, wherein the protease variant comprises one or more substitutions corresponding to BPN' (SEQ ID NO: 1) replacement of 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.

[0941] 13. A liquid cleaning composition according to any one of items 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.

[0942] 14. The liquid cleaning composition according to any one of items 10-13, wherein the protease variant is selected from the group consisting of:

[0943] S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

[0944] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0945] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0946] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

[0947] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0948] S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0949] S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

[0950] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0951] S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

[0952] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0953] S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0954] S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

[0955] S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

[0956] S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0957] S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0958] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

[0959] S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

[0960] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

[0961] S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

[0962] S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

[0963] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

[0964] S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

[0965] S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

[0966] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0967] S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0968] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0969] S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0970] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[0971] S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

[0972] S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0973] S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0974] S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0975] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;

[0976] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;

[0977] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0978] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;

[0979] S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0980] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;

[0981] S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;

[0982] S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;

[0983] S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0984] S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0985] S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0986] S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0987] S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0988] S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;

[0989] S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;

[0990] S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;

[0991] S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;

[0992] S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0993] S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;

[0994] S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;

[0995] S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0996] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;

[0997] S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[0998] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;

[0999] S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[1000] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[1001] S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;

[1002] S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

[1003] S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

[1004] S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

[1005] S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

[1006] S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;

[1007] S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

[1008] 15. A cleaning composition according to any preceding claim, comprising one or more additional enzymes selected from the group consisting of perhydrolases, amylases, catalases, cellulases (e.g., endoglucanases), cutinases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, DNases, xanthanases, and xyloglucanases, or any mixture thereof.

[1009] 16. Use of a liquid cleaning composition as described in any preceding claim in a cleaning process such as laundry or hard surface cleaning, including dishwashing and industrial cleaning.

[1010] 17. The use of item 16, wherein the laundry process is selected from the group consisting of a residential laundry process, an industrial laundry process, and an institutional laundry process.

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 as set forth in SEQ ID NO: 2, and wherein the protease variant is substituted relative to the parent protease as follows: S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E; or S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, Wherein the position numbering is based on SEQ ID NO:1 (BPN'), and wherein the protease variant has 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 10; and (b) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

2. A liquid cleaning composition comprising: (a) from 20% to 95% wt, preferably from 40% to 95% wt, more preferably from about 70% to about 90% wt of water; (b) at least 0.01 wt % of a protease, wherein the protease is a variant of a parent protease as set forth in SEQ ID NO: 2, and wherein the protease variant is substituted relative to the parent protease as follows: S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E; or S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, Wherein the position numbering is based on SEQ ID NO:1 (BPN'), and wherein the protease variant has at least 10% greater residual activity than the parent protease when measured after 4 hours at 40°C in a liquid detergent having a pH of 7 or greater; and (c) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

3. The liquid cleaning composition of claim 1, wherein the pH is from about 12 to 13.

5.

4. The liquid cleaning composition of claim 2, wherein the pH is from about 7 to 13.5, such as 7-9, 7.5-13.5, 8-13.5, 10-13.

5.

5. A liquid cleaning composition comprising: (a) from 0% to 20%wt, preferably 0.1% to 2.5%wt, of a detergent surfactant; (b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a protease variant, wherein the protease is a variant of a parent protease as shown in SEQ ID NO: 2, and wherein the protease variant is substituted relative to the parent protease as follows: S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E; S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E; or S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the positions are numbered according to SEQ ID NO: 1 (BPN'); and (c1) from 20% to 95% wt, preferably from 40% to 95% wt, further preferably from about 70% to about 90% wt of water; and / or (c2) from 1% to 30% wt of an alkaline buffer system comprising an alkali metal silicate or an alkali metal hydroxide or a mixture thereof to provide a pH of about 7 to 13.5, preferably a pH of from about 7 to 9, 7.5 to 13.5, 8 to 13.5 or 11 to 13.5; and (d) Optionally, a protease inhibitor, preferably the protease inhibitor is a peptide aldehyde protease inhibitor.

6. A cleaning composition according to any one of the preceding claims, comprising one or more additional enzymes selected from the group consisting of: Perhydrolases, amylases, catalases, cellulases (e.g., endoglucanases), cutinases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, DNases, xanthanases, and xyloglucanases, or any mixture thereof.

7. Use of a liquid cleaning composition as claimed in any preceding claim in a cleaning process such as laundry or hard surface cleaning, including dishwashing and industrial cleaning.

8. The use as claimed in claim 7, wherein the laundry washing process is selected from the group consisting of: Residential laundry processes, industrial laundry processes and institutional laundry processes.

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