Cleaning compositions comprising a protease

Abstract

In the present invention new cleaning compositions comprising a protease are provided. In addition, methods of making the cleaning compositions and using the cleaning compositions for having an improved storage stability are provided.

Description

Field of the invention

[0001] In the present invention liquid cleaning compositions comprising a protease are provided. In addition, methods of making the cleaning compositions and using the cleaning compositions for improving the storage stability are provided.Background of the invention

[0002] Enzymes are increasingly used in cleaning compositions. Enzymes are biodegradable and can be catalytically active already at lower temperatures, which results in reduction of energy consumption during cleaning. Proteases are enzymes capable of hydrolyzing proteins. Thus, proteases have been employed in the removal of protein stains and have been added to cleaning compositions for this purpose. However, in cleaning compositions, proteases are often unstable and tend to denature upon storage of the protease-containing cleaning composition leading to a reduction of wash performance over time. Protease stability in cleaning compositions highly depends on the interaction of the protease with the detergent components. Thus, protease stability in cleaning compositions depends on the protease itself as well as the specific composition of the cleaning composition. The inventors of the present application identified that the use of a particular type of protease in combination with specific detergent components results in a cleaning composition that has improved protease stability during storage of the cleaning composition.Brief summary of the invention

[0003] The present invention is directed to a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising one or more weak builder and one or more strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

[0004] The present invention is further directed to a method of making such cleaning composition and to the use of such cleaning composition for improving the storage stability of the protease variant in the cleaning composition and for improving the wash performance of the cleaning composition after storage.Detailed description of the invention

[0005] The present invention may be understood more readily by reference to the following detailed description of the embodiments described herein and the examples included herein. Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.Definitions

[0006] Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0007] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all compounds, methods and uses described herein.

[0008] As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

[0009] In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±1 0 %, and even more preferably ±5 %.

[0010] Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below. Throughout this application, various publications are referenced. The disclosure of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0011] It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of members, this is meant to also encompass a group which consists of these members only.

[0012] "Amino acid substitutions" are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated as "His120Ala" or "H120A". Substitutions can also be described by merely naming the resulting amino acid in the variant without specifying the amino acid of the parent at this position, e.g., by using "X120A" or "120A" or "Xaa120Ala" or "120Ala".

[0013] Variants comprising multiple substitutions are separated by "+", e.g., "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple substitutions may be separated by space or a comma, e.g., "R170Y G195E" or "R170Y, G195E" respectively. Where different alternative substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., "Arg170Tyr,Glu" and "R170T,E", respectively, represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternative substitutions at a particular position can also be indicated as "X120A,G,H", "120A,G,H", "X120A / G / H", or "120A / G / H". Alternatively, different substitutions may be indicated in brackets, e.g., "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or in short "R170 [Y,G]" or"R170 {Y,G}".

[0014] The numbering of the amino acid residues of the proteases described herein is as commonly used for proteases in the field (cf. P.N. Bryan, Biochimica et Biophysica Acta 1543 (2000), 203-222, cf. p. 204, left col., 3 rd< para.) according to the numbering of the BPN' subtilisin protease from Bacillus amyloliquefaciens the sequence of which is shown in SEQ ID NO: 2 (i.e., according to the numbering of SEQ ID NO: 2 or according to "BPN' numbering"). In an alternative way, one can describe the amino acid positions with reference to the numbering of SEQ ID NO: 1 or SEQ ID NO: 3, i.e., according to the numbering of SEQ ID NO: 1 or according to the numbering of SEQ ID NO: 3. As an example, position 101 of the protease shown in SEQ ID NO: 1 according to the numbering of SEQ ID NO: 2 corresponds to position 99 according to the numbering of SEQ ID NO: 1 or 3.

[0015] Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity when compared to a parent sequence. Sequence identity usually is provided as "% sequence identity" or "% identity". For calculation of sequence identities, in a first step a sequence alignment is produced. According to this invention, a pairwise global alignment is produced, meaning that two sequences are aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.

[0016] According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48, p. 443-453). Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). After aligning two sequences, in a second step, an identity value is determined from the alignment produced. For this purpose, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of the present invention over its complete length multiplied with 100: %-identity = (identical residues / length of the alignment region which is showing the respective sequence of the present invention over its complete length) *100.

[0017] A special aspect concerning amino acid substitutions are conservative mutations which often appear to have a minimal effect on protein folding resulting in substantially maintained enzyme properties of the respective enzyme variant compared to the enzyme properties of the parent enzyme. Conservative mutations are those where one amino acid is exchanged with a similar amino acid. Such an exchange most probably does not change enzyme properties. Herein the following conservative exchanges are considered: Amino acid A is similar to amino acids S Amino acid D is similar to amino acids E; N Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acids W; Y Amino acid H is similar to amino acids N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acids K; Q Amino acid S is similar to amino acids A; N; T Amino acid T is similar to amino acids S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acids F; Y Amino acid Y is similar to amino acids F; H; W

[0018] Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. Preferably, such mutations are not pertaining the functional domains of an enzyme, more preferably conservative mutations are not pertaining the catalytic centers of an enzyme. "Protein formulation" (or "enzyme preparation" or "polypeptide formulation"), e.g., "protease formulation", means any non-complex formulation comprising a small number of ingredients, preferably, 2-6 components, wherein the ingredients serve the purpose of stabilizing the proteins comprised in the protein formulation and / or the stabilization of the protein formulation itself. Preferably, the non-complex protein formulation comprises the protein in higher concentrations than the complex formulation, e.g., than a detergent formulation. Thus, preferably the non-complex protein formulation is a concentrated protein formulation. Preferably, non-complex protein formulations comprise 20 to 120 mg / g active enzyme, whereas complex formulations, like cleaning compositions, comprise 0.002 to 10 mg / g active enzyme. In contrast to a non-complex formulation, a complex formulation means herein a formulation comprising a higher number of ingredients, preferably, 7-30 components, wherein the ingredients serve the purpose of stabilizing the proteins comprised in the protein formulation and / or the stabilization of the protein formulation itself, but additionally the complex formulation comprises components that serve the purpose of the complex formulation, e.g., a cleaning composition. An example for a non-complex protein formulation is a concentrated enzyme composition that is used as a stock-solution to prepare a complex formulation, e.g., a cleaning composition, wherein in the cleaning compositions other compounds are present that serve the cleaning purpose of the cleaning composition, e.g., surfactants and / or chelating agents.

[0019] A "liquid" composition has a physical form that is not solid or gas. A "liquid" composition has a definite volume but no a definite shape. This means that a liquid will take on the shape of the container it is placed in, but the amount of space it takes up will not change. Unlike gases, liquids are not easily compressible and have a higher density. Thus, liquid compositions include gel-type compositions. An "aqueous liquid" composition is a composition in which more than 50 weight% of the sum of all solvents is water.

[0020] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability in the course of time, thermostability, pH stability, and chemical stability. "Enzymatic activity" or "catalytic activity" means the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzyme's actual function, e.g., proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, amylases activity involves hydrolysis of glycosidic linkages in polysaccharides, etc.

[0021] The term "enzyme stability" according to the current invention relates to the retention of enzymatic cleaning performance as a function of time during storage or operation. Retention of enzymatic cleaning performance as a function of time during storage is called "storage stability".

[0022] To determine and quantify changes in enzymatic cleaning performance of enzymes stored under certain conditions over time, the enzymatic cleaning performance is measured as described herein in the examples.

[0023] "Enzyme inhibitors" as used herein are compounds that slow down or halt enzymatic activity. Enzyme inhibitors frequently also stabilize the enzyme in its three-dimensional structure. Hence, enzyme inhibitors usually also act as "enzyme stabilizers".

[0024] The term "detergent stability" (also called herein "residual activity in a detergent", or "residual enzymatic wash performance", or "storage stability in a cleaning composition") refers to the ability of an enzyme to exert cleaning performance after storage in a cleaning composition, preferably, at a temperature of 15 °C, 30 °C, 37 °C, 45 °C, or 50 °C for up to 86 days in a cleaning composition (preferably, in any of the detergent compositions F1, F3, F4, F6, F7, F8 and F9 shown in Table 1 comprising a builder composition as described herein). Most preferably, detergent stability is determined by measuring enzyme stability of the protease after storage at a temperature of at 37°C for 56 days in a cleaning composition as described herein.

[0025] As used herein, "wash performance" (also called herein "cleaning performance") of an enzyme refers to the contribution of the enzyme to the cleaning performance of a cleaning composition, i.e. the cleaning performance added to the cleaning composition by the performance of the enzyme. The term "wash performance" is used herein similarly for laundry and hard surface cleaning. Wash performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a detergent market segment. The term "improved wash performance" is used to indicate that a better end result is obtained in stain removal under relevant washing conditions, or that less enzyme, on weight basis, is needed to obtain the same end result relative to the corresponding control conditions.

[0026] As used herein, the term "specific performance" refers to the cleaning and removal of specific stains or soils per unit of active enzyme. In some embodiments, the specific performance is determined using stains or soils such as egg, egg yolk, milk, grass, minced meat blood, chocolate sauce, baby food, sebum, etc.

[0027] The terms "cleaning composition" (also called herein "cleaning formulation", "detergent composition", "detergent formulation" or "detergent") as used herein includes compositions and formulations designed for cleaning soiled material. Such compositions and formulations include those designed for cleaning soiled material or surfaces of any kind. Cleaning compositions can be for industrial and institutional cleaning or for fabric and home care. Compositions for "industrial and institutional cleaning" includes such cleaning compositions being designed for use in industrial and institutional cleaning, such as those for use of cleaning soiled material or surfaces of any kind, such as hard surface cleaners for surfaces of any kind, including tiles, carpets, PVC-surfaces, wooden surfaces, metal surfaces, lacquered surfaces.

[0028] "Compositions for fabric and home care" include cleaning compositions and formulations including but not limited to laundry cleaning compositions and detergents, fabric softening compositions, fabric enhancing compositions, fabric freshening compositions, laundry prewash, laundry pretreat, laundry additives, spray products, dry cleaning agent or composition, laundry rinse additive, wash additive, post-rinse fabric treatment, ironing aid, dish washing compositions, hard surface cleaning compositions, unit dose formulation, delayed delivery formulation, detergent contained on or in a porous substrate or nonwoven sheet, and other suitable forms that may be apparent to one skilled in the art in view of the teachings herein and detailed herein below when describing the compositions. Such compositions may be used as a pre-laundering treatment, a post-laundering treatment, or may be added during the rinse or wash cycle of the laundering operation, preferably during the wash cycle of the laundering or dish washing operation, and as further detailed herein below when describing the use and application of the inventive compound(s) and compositions comprising such polymers.

[0029] Cleaning compositions are complex formulations as further defined herein. Cleaning compositions according to the invention include cleaning compositions for different applications such as laundry and hard surface cleaning. Cleaning compositions according to the invention also include biodegradable cleaning compositions. Cleaning compositions according to the invention are composed of several adjunct cleaning additives.

[0030] Cleaning compositions are further described in Reference RF1 and Reference RF2, in particular in paragraphs

[0004] and

[0007] of Reference RF1.

[0031] The publication IPCOM000274907D concerning cleaning compositions published on www.IP.com is regarded as Reference RF1, which is incorporated herein by reference in its entirety. The publication Prior Art Disclosure; Issue 684; paragraphs

[3000] to

[3061] ; ISSN: 2198-4786; published: February 12, 2024 also concerning cleaning compositions will be regarded as Reference RF2, which is incorporated herein by reference in its entirety.

[0032] The term "adjunct cleaning additive" is defined herein to mean a type of chemical, which can be used in cleaning compositions. A typical adjunct cleaning additive is a surfactant. "Surfactant" means an organic chemical that is not a soap and when added to a liquid, changes the properties of that liquid at an interface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, zwitterionic, or amphoteric. A "surfactant composition" refers to the totality of all surfactants in the cleaning composition not including soaps. "Soaps" are understood herein as salts of saturated and unsaturated C12-C18 fatty acids.

[0033] The term "effective amount of an adjunct cleaning additive" includes amounts of certain components to provide effective stain removal and / or effective cleaning conditions (e.g. pH, temperature, water hardness, quantity of foaming), amounts of certain components to effectively provide optical benefits (e.g. optical brightening, dye transfer inhibition, color care), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants).

[0034] The term "laundry" or "laundering" relates to both household laundering and industrial laundering and means the process of treating textiles and / or fabrics with a solution containing a cleaning composition. The laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.

[0035] The term "textile" means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics made of these materials such as garments, cloths and other articles. The terms "fabric" refers to a textile made by weaving, knitting or felting fibers. The term "garment" refers to any article of clothing made of textile. The term "fibers" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheep's wool, angora, mohair, cashmere). Examples for fibers of synthetic origin are polyurethane fibers such as Spandex ®< or Lycra ®< , polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, woven or non-woven fabrics.

[0036] The term "hard surface cleaning" relates to both household hard surface cleaning and industrial hard surface cleaning and means the process of treating hard surfaces with a solution containing a cleaning composition. Hard surfaces may include any hard surfaces in the household or industry, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes and medical devices such as diagnostic instruments, trays, pans, holders, racks, forceps, scissors, shears, saws (e.g. bone saws and their blades), hemostats, knives, chisels, rongeurs, files, nippers, drills, drill bits, rasps, burrs, spreaders, breakers, elevators, clamps, needle holders, carriers, clips, hooks, gouges, curettes, retractors, straightener, punches, extractors, scoops, keratomes, spatulas, expressors, trocars, dilators, cages, glassware, tubing, catheters, cannulas, plugs, stents, endoscopes, arthoscopes and related equipment. A particular form of hard surface cleaning is dishwashing, particularly automatic dishwashing (ADW).

[0037] The term "dish wash" refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.

[0038] The term "medical device cleaning" refers to the cleaning step in reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories, manual and mechanical / automated cleaning methods. Manual cleaning is used when mechanical units are not available or medical devices to be cleaned are too fragile or difficult to clean with a mechanical unit. Mechanical / automated cleaning methods remove soiling and microorganisms through an automated cleaning and rinsing process, this includes ultrasonic cleaning and washing. In the field of detergency, usually the term "stains" is used with reference to laundry, e.g., cleaning of textiles, fabric, or fibers, whereas the term "soils" is usually used with reference to hard surface cleaning, e.g., cleaning of dishes and cutlery. However, herein the terms "stain" and "soil" shall be used interchangeably.

[0039] "Builders" (also called "complexing agent", "chelating agent", or "chelator") are chemical compounds capable of binding cations, in particular magnesium and calcium cations. A "builder composition" refers to the totality of all builders in the cleaning composition and is a mixture of at least two different builders.

[0040] A "sequestering builder" as used herein is different from a precipitating builder in that no significant amount of precipitate is formed when the builder is used in an amount sufficient to combine with all of the calcium ions in an aqueous solution with 7 °dH hardness (German hardness) initially at neutral pH. A "strong builder" is classified as high efficiency chelator that can bind the divalent cations such as Ca2+ strongly with a logarithmic stability constant (Log K Ca ) of the cation / chelator complex of above 4, particular above 5, above 6 or above 7. The stability constants are determined at an ionic strength of 0.1 M and at a temperature of 25°C. A "strong sequestering builder" combines both of the above-mentioned properties. Further details on strong / weak and sequestering / precipitating builders are provided in paragraphs

[0060] to

[0070] of EP2989117B1, which are incorporated herein by reference.

[0041] A "biodegradable" compound (such as a surfactant or complexing agent) is a compound that is readily biodegradable in an aerobic aqueous medium according to the OECD guideline 301. "Readily biodegradable" is defined according to the Detergents Regulation as the ability of a product to biodegrade more than 60% within 28 days according to OECD 301A-F / ASTM.

[0042] The term "effective amount of a detergent component" includes amounts of certain components to provide effective stain removal and / or effective cleaning conditions (e. g. pH, temperature, water hardness, quantity of foaming), amounts of certain components to effectively provide optical benefits (e. g. optical brightening, dye transfer inhibition, color care), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants).

[0043] A composition "essentially devoid" of a compound shall mean herein that the respective compound is not added to the composition on purpose, meaning that at most non-effective amounts are present, most preferably 0% of the compound are contained in the composition.Detailed description

[0044] Herein, new cleaning compositions are provided, which have beneficial properties.Cleaning compositions

[0045] Specifically, the present invention is directed to a cleaning composition comprising an effective amount of a protease variant, a surfactant composition as described herein, and a builder composition as described herein. As understood herein, the cleaning composition is a complex formulation as described herein.Protease variant

[0046] The cleaning composition described herein comprises a protease variant as described herein. Protease variants have "proteolytic activity" (also referred to as "protease activity"). This property is related to hydrolytic activity of a protease (i.e., proteolysis, which means hydrolysis of peptide bonds linking amino acids together in a polypeptide chain) on protein containing substrates, e.g., casein, haemoglobin, and BSA. Quantitatively, proteolytic activity is related to the rate of degradation of protein by a protease or proteolytic enzyme in a defined course of time. The methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). For instance, proteolytic activity can be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD405.

[0047] To determine changes in proteolytic activity over time, the "initial enzymatic activity" of a protease is measured under defined conditions at time zero and at a certain point in time later. By di-viding the latter activity with the activity at time point zero the residual activity can be calculated (x%). By comparison of the 100%-value with the x%-value, a potential loss of proteolytic activity can be determined in its extent.

[0048] The protease variant is preferably a protease variant of the parent protease shown in SEQ ID NO: 1. Preferably, the protease variant comprises an amino acid sequence which is at least 60%, preferably at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 (BPN' numbering, i.e., wherein the positions are numbered by their correspondence to the amino acid sequence of subtilisin BPN' of B. amyloliquefaciens, established as SEQ ID NO: 2). Preferably, the protease variant comprises as a catalytic triad the amino acids aspartate, histidine, and serine, preferably the protease variant is a subitilisin protease.

[0049] Preferably, the protease variant has at least 60%, preferably at least 80%, but less than 100% sequence identity to SEQ ID NO: 1 as described herein and comprises compared to SEQ ID NO: 1 at least two, three, or four additional negative charges, more preferably three additional negative charges, most preferably two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 compared to the region of SEQ ID NO: 1 corresponding to residues 98 to 104 of SEQ ID NO: 2.

[0050] Preferably, the at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of substitutions, deletions and insertions, preferably by substitutions.

[0051] Preferably, in the protease variant the at least one, preferably at least two, additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are caused by one or more amino acid substitutions at amino acid position selected from the group consisting of 98, 99, 100, 101, 102, 103, and 104, preferably at position 101, according to the numbering of SEQ ID NO: 2.

[0052] Preferably, the at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of D99E, R101D and R101E.

[0053] In a preferred embodiment, the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D, preferably R101E, according to the numbering of SEQ ID NO: 2. In an alternative embodiment, the at least one, preferably at least two, additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are not caused by the amino acid substitution R101E or R101D.

[0054] In a preferred embodiment, the loop sequence 98-104 that has compared to SEQ ID NO: 1 two additional negative charges comprises a sequence selected from the group consisting of ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGEGSV, and ASEGEGSV with longer sequences having an insertion in the loop sequence.

[0055] In one embodiment, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

[0056] Preferably, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101E, according to the numbering of SEQ ID NO: 2. Preferably, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101E, according to the numbering of SEQ ID NO: 2.

[0057] In one embodiment, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2.

[0058] The protease variant may comprise one or more, preferably 1-10, more preferably 1-5 conservative amino acid substitutions. Thus, in one embodiment, the protease variant has at least 80% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the protease variant comprises one or more conservative amino acid exchanges as described herein.

[0059] Preferably, compared to SEQ ID NO: 1 the protease variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 20, at least 30 or at least 40 conservative amino acid exchanges. Thus, compared to SEQ ID NO: 1 a protease variant described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid exchanges in addition to the modifications resulting in at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104, preferably in addition to the substitution R101D or R101E, preferably R101E, and optionally in addition to one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2. Preferably, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the remaining difference in amino acid sequence is due to conservative amino acid exchanges as described herein.

[0060] In another embodiment, the protease variant comprises an amino acid sequence which is at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 ac-cording to the numbering of SEQ ID NO: 2 and the protease variant comprises compared to SEQ ID NO: 1 one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 3, 4, 9, 15, 24, 27, 33, 36, 45, 55, 57, 58, 59, 61, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 117, 118, 120, 123, 124, 128, 129, 130, 131, 136, 137, 143, 154, 156, 160, 161, 163, 167, 170, 171, 172, 185, 194, 195, 199, 205, 206, 209, 217, 218, 222, 224, 232, 235, 236, 238, 244, 245, 248, 252, 261, 262, and 274.

[0061] In one embodiment, the protease variant is not mutated at positions Asp32, His64 and Ser221 (according to the numbering of SEQ ID NO: 2).

[0062] In one embodiment, said protease variant comprises one or more substitutions selected from: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T, or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) combinations of two or more amino acid substitutions according to (a) to (h), according to the numbering of SEQ ID NO: 2. In one embodiment, said protease variant has at least 80%, but less than 100% identical to SEQ ID NO: 1 and is characterized by comprising one amino acid (according to (a)-(h)) or combinations according to (i) together with the amino acid substitution 101E, 101D, 101N, 101Q, 101A, 101G, or 101S (according to the numbering of SEQ ID NO: 2). In one embodiment, the protease variant is at least 80%, but less than 100% identical to SEQ ID NO: 1 and is characterized by comprising the mutation R101E, or S3T + V4I + V205I, or S3T + V4I + R101E + V205I or S3T + V4I + V199M + V205I + L217D (according to the numbering of SEQ ID NO: 2). In another embodiment, the protease variant comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and being further characterized by comprising S3T + V4I + S9R + A15T + V68A + D99S + R101S + A103S + I104V + N218D (according to the numbering of SEQ ID NO: 2).

[0063] In another embodiment, the protease variant comprises an amino acid sequence which is at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and the protease variant comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D, preferably R101E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D according to the numbering of SEQ ID NO: 2.

[0064] In this embodiment, preferably, the protease variant comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and the protease variant comprises compared to SEQ ID NO: 1 the amino acid substitutions R101E and S156D and / or L262E, and optionally at least one further mutation selected from I104T, H120D, Q137H, S141H, R145H and S163G according to the numbering of SEQ ID NO: 2.

[0065] In another embodiment, the protease variant has at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2, and one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 76, 138, 145, 156, 166, 167, 169, 177, 187, 189, 191, 206, 209, 215, 218, and 262, preferably selected from the group consisting of N76D / E / Q, A138Q, R145L / W / Y, S156D / Q, S166G, Y167T, A169G, V177 L, A187 D, F189R, Q191R, Q206A / L / S / T, Y209K / V / W, A215K / W, N218S / T, and L262D / E / Q

[0066] In this embodiment, preferably, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2, and one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 156, 166, 187, 189, 191, 206, 209, 215 and 262, preferably selected from the group consisting of S156D, S166G, A187 D, F189R, Q191R, Q206L, Y209W, A215K und L262E.

[0067] In this embodiment, particularly preferred, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 according to the numbering of SEQ ID NO: 2 the amino acid substitutions (i) S3T-V4I-R101E-V199I-Q206L-Y209W; (ii) S3T-V4I-R101E-V199I-N218S; (iii) S3T-V4I-R101E-V199I-N76D; (iv) S3T-V4I-R101E-V199I-S156D-L262E; (v) S3T-V4I-R101E-V199I-Q206L-Y209W-S156D-L262E; (vi) S3T-V4I-R101E-V199I-N76D-Q206L-Y209W; (vii) S3T-V4I-R101E-V199I-N76D-S156D-Q206L-Y209W-L262E; (viii) S3T-V4I-R101E-V199I-N76D-N218S; (ix) S3T-V4I-R101E-V199I-N76D-S156D-Y209W-L262E; (x) S3T-V4I-R101E-V199I-N76D-Y209W; (xi) S3T-V4I-R101E-V199I-N76D-S156D-Q206L-L262E; (xii) S3T-V4I-R101E-V199I-N76D-Q206L; (xiii) S3T-V4I-R101E-V199I-S156D-Q206L-Y209W; (xiv) S3T-V4I-R101E-V199I-Q206L-Y209W-L262E; (xv) S3T-V4I-R101E-V199I-A138Q-R145W-Y167 T-Q206L; (xvi) S3T-V4I-R101E-V199I-N76D-R145Y-A215W-N218S-L262E; (xvii)S3T-V4I-R101E-V199I-A138Q-S156D-V177 L-Q206L; (xviii) S3T-V4I-R101E-V199I-Q206L-Y209W-A215K-S156D-L262E; (xix) S3T-V4I-R101E-V199I-S156D-S166G-Q191R-Q206L-Y209W-L262E; or (xx) S3T-V4I-R101E-V199I-S156D-A187 D-F189R-Q206L-Y209W-L262E.

[0068] More preferably, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises the amino acid substitution R101E according to the numbering of SEQ ID NO: 2.

[0069] Particularly preferred, the protease variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3 and comprises the amino acid substitution R101E according to the numbering of SEQ ID NO: 2.

[0070] Particularly preferred, the protease variant comprises a glutamic acid (E) at position 101 according to the numbering of SEQ ID NO: 2 and differs from SEQ ID NO: 3 only by 1-20, preferably 1-10, more preferably 1-5, amino acid substitutions, preferably conservative amino acid substitutions.

[0071] Most preferably, the protease variant has 100% sequence identity to SEQ ID NO: 3.

[0072] Preferably, the total amount of protease in the cleaning composition is 0.005-0.075 weight%, preferably 0.01-0.06 weight%.Surfactant composition

[0073] The cleaning composition described herein comprises a surfactant composition as described herein. The surfactant composition of the cleaning composition comprises one or more surfactants, which may be anionic, cationic, non-ionic, semi-polar, zwitterionic, or a mixture thereof. Suitable surfactants are described in more detail in

[0008] to

[0013] of Reference RF1, excluding soaps, and in

[3008] to

[3024] and

[3026] to

[3034] of Reference RF2.

[0074] The surfactant composition of the cleaning composition is characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) in the cleaning composition to the total amount of all surfactants in the cleaning composition is 0 to 0.33.Non-ionic surfactants (NIO)

[0075] In one embodiment, the cleaning composition comprises one or more non-ionic surfactants (NIO). Suitable non-ionic surfactants are described in more detail in

[00010] of Reference RF1 and in

[3009] to

[3020] of Reference RF2. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE orAEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkyl phenol ethoxylates (APE), nonyl phenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanola-mides (FAM), fatty acid diethanol amides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid am-ides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FA-GA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0076] In one embodiment, the surfactant composition comprises one or more compound of the formula (NIS1a), (NIS1b), or (NIS1c):

[0077] The variables of the formula (NIS1a), (NIS1b) and (NIS1c) are defined as follows: R1 is selected from H, C1-C23 alkyl and C2-C23 alkenyl, wherein alkyl and / or alkenyl are linear (straight-chain; n-) or branched; examples are n-C7H15, n-C8H17, n-C9H19, n-C11H23, n-C13H27, n-C15H31, n-C17H35, i-C9H19, i-C12H25. R2 is selected from H, C1-C20 alkyl and C2-C20 alkenyl, wherein alkyl and / or alkenyl are linear (straight-chain; n-) or branched. R3 and R4, each independently selected from H, C1-C16 alkyl, wherein alkyl is linear (straightchain; n-) or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl. R5 is selected from H and C1-C18 alkyl, wherein alkyl is linear (straight-chain; n-) or branched. R6 is selected from (wherein the dotted line indicates binding to NIS1c):

[0078] The integers of the formula (NIS1a), (NIS1b) and (NIS1c) are defined as follows: m is in the range of zero to 200, preferably 1-80, more preferably 3-20; n and o, each independently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; o preferably is in the range of 1 to 50, more preferably 4 to 25. The sum of m, n and o is at least one, preferably the sum of m, n and o is in the range of 5 to 100, more preferably in the range of from 9 to 50.

[0079] Surfactants according to formula NIS1a, NIS1b, and NIS1c are also called alcohol ethoxylates (AEO).

[0080] Preferably, the surfactant composition comprises one or more alcohol ethoxylates. Preferably, the surfactant composition comprises one or more ethoxylated C10-C18 alcohol, preferably oxoalcohol, preferably C13-C15-oxoalcohol, preferably with 6-8 EO. Preferably, the surfactant composition comprises one or more non-ionic surfactant according to the formula (NIS1a). Preferably, the surfactant composition comprises one or more alcohol ethoxylate, preferably, one or more ethoxylated C10-C18 alcohol, preferably oxoalcohol, according to the formula (NIS1a), preferably one or more ethoxylated C13-C15-oxoalcohol, preferably with 6-8 EO, according to the formula (NIS1a). Preferably, the surfactant composition comprises one or more AEO of formula (NIS1a), wherein R1 is C9-C17 alkyl, preferably saturated alkyl, preferably C11-C13 alkyl, R2 is H, R5 is H, m = 6-8, n and o being 0.Anionic surfactants

[0081] In one embodiment, the surfactant composition of the cleaning composition comprises one or more anionic surfactants as described herein, wherein soaps, as defined herein, are not understood as anionic surfactants. Suitable anionic surfactants are described in more detail in

[0009] of Reference RF1, excluding soaps, and in

[3021] to

[3024] and

[3026] to

[3029] of Reference RF2. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkyl benzene sulfonates (LAS), isomers of LAS, branched alkyl benzene sulfonates (BABS), phenyl alkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxy alkane sulfonates and disulfonates, alkyl sulfates such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, or diesters and monoesters of sulfo succinic acid, and combinations thereof.Alkyl benzene sulfonates (AS)

[0082] In one embodiment, the surfactant composition comprises one or more alkyl benzene sulfonates (AS) as described herein. Preferably, the alkyl benzene sulfonate is a C9-C20 alkyl benzene sulfonate, more preferably a C10-C15 alkyl benzene sulfonate, most preferably a C10-C13 alkyl benzene sulfonate. Most preferably, the alkyl benzene sulfonate is selected from compounds of the formula (AS2a) wherein R1 in formula (AS2a) is linear, branched, or a mixture of linear and branched C10-C20, preferably C10-C13 alkyl; M+ being preferably Na+ or Ca2+.

[0083] In one embodiment, the one or more anionic surfactant is a linear alkyl benzene sulfonate (LAS), i.e., with R1 in formula (AS2a) being linear alkyl. In another embodiment, the one or more anionic surfactant is a branched alkyl benzene sulfonate (BAS), i.e., with R1 in formula (AS2a) being branched alkyl. In one embodiment, the alkyl benzene sulfonate is a mixture of LAS and BAS. Preferably, the alkyl benzene sulfonate is a C9-C20 alkyl benzene sulfonate, more preferably a C10-C15 alkyl benzene sulfonate, most preferably a C10-C13 alkyl benzene sulfonate. Preferably, the alkyl benzene sulfonate is a linear alkyl benzene sulfonate with C9-C20 alkyl, more preferably C10-C15 alkyl, most preferably C10-C13 alkyl. Preferably, the surfactant composition comprises one or more alkyl benzene sulfonate according to the formula (AS2a). Preferably, the surfactant composition comprises one or more alkyl benzene sulfonate according to the formula (AS2a) with R1 being linear and / or branched, preferably linear, C9-C20 alkyl, more preferably C10-C15 alkyl, most preferably C10-C13 alkyl and M+ being preferably Na+ or Ca2+.

[0084] In another embodiment, the surfactant composition is essentially devoid of alkyl benzene sulfonates. In one embodiment, the surfactant composition does not comprise alkyl benzene sulfonates.Sulfates and carboxylates

[0085] In one embodiment, the surfactant composition comprises one or more compounds of the formula (AS1a) or (AS1b):

[0086] The variables in formula (AS1a and AS1b) are defined as follows: R1 is selected from C1-C23-alkyl (such as 1-, 2-, 3-, 4- C1-C23-alkyl) and C2-C23-alkenyl, wherein alkyl and / or alkenyl are linear (straight-chain; n-) or branched, and wherein 2-, 3-, or 4-alkyl; examples are n-C7H15, n-C9H19, n-C11H23, n-C13H27, n-C15H31, n-C17H35, i-C9H19, i-C12H25. R2 is selected from H, C1-C20-alkyl and C2-C20-alkenyl, wherein alkyl and / or alkenyl are linear (straight-chain; n-) or branched. R3 and R4, each independently selected from C1-C16-alkyl, wherein alkyl is linear (straightchain; n-) or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, nhexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl. A- is selected from -RCOO-, -SO3- and -RSO3-, wherein R is selected from linear (straightchain; n-) or branched C1-C8-alkyl and C1-C4 hydroxyalkyl. Compounds are called (fatty) alcohol / alkyl (ethoxy / ether) sulfates [(F)A(E)S] when A- is -SO3- or -RSO3- and (fatty) alcohol / alkyl (ethoxy / ether) carboxylate [(F)A(E)C] when A- is -RCOO-. Compounds of formula AS1a or AS1b, which are alkyl ether sulfates with A- being -SO3- are also called AES. M+ is selected from H and salt forming cations. Salt forming cations may be monovalent or multivalent; hence M+ equals 1 / v Mv+. Examples include but are not limited to sodium, potassium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanolamine.

[0087] The integers of the formula (AS1a) and (AS1b) are defined as follows: m is in the range of zero to 200, preferably 1-80, more preferably 3-20; n and o, each independently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; o preferably is in the range of 1 to 50, more preferably 4 to 25. The sum of m, n and o is in the range of 0 to 100. In one embodiment, the sum of m, n and o is 0. In another embodiment the sum of m, n and o is at least one, preferably the sum of m, n and o is in the range of 9 to 50.

[0088] Anionic surfactants of the formula (AS1a) and (AS1b) may be of any structure, block copolymers or random copolymers.

[0089] Preferably, the surfactant composition comprises one or more alkyl ether sulfates (AES). Preferably, the surfactant composition comprises one or more C12-C15 alcohol ether sulfate, linear or branched, preferably with 1-3 EO.

[0090] Preferably, the surfactant composition comprises one or more compounds of formula (AS1a). Preferably, the surfactant composition comprises one or more alkyl ether sulfates of formula (AS1a). Preferably, the surfactant composition comprises one or more C12-C15 alcohol ether sulfate, linear or branched, preferably with 1-3 EO, according to the formula (AS1a). Preferably, the surfactant composition comprises one or more C12-C14 fatty alcohol ether sulfate, preferably with 1-3 EO, according to the formula (AS1a).

[0091] Preferably, the surfactant composition comprises one or more AES of formula (AS1a), wherein R1 is C9-C15 alkyl, preferably saturated alkyl, preferably C11-C13 alkyl, R2 is H, A- is -SO3-, m = 1-3, n and o being 0 and M+ is preferably Na+.Other surfactants

[0092] The surfactant composition may comprise one or more cationic, semipolar, zwitterionic, or amphoteric surfactant. Preferably, the surfactant composition may comprise one or more amphoteric surfactant. Suitable amphoteric surfactants are described in more detail in

[00011] of Reference RF1 and in

[3030] to

[3034] of Reference RF2. Suitable zwitterionic surfactants are described in more detail in

[00012] of Reference RF1. Suitable cationic surfactants are described in more detail in

[00013] of Reference RF1. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyl dimethyl amine oxide, N-(coco alkyl)-N,N-dimethyl amine oxide and N-(tallow-alkyl)-N,N-bis-(2-hydroxy ethyl) amine oxide, fatty acid alkanol amides and ethoxylated fatty acid alkanol amides, and combinations thereof. Non-limiting examples of zwitterionic surfactants include betaine, alkyl dimethyl betaine, sulfo betaine, and combinations thereof.Builder composition

[0093] The cleaning composition described herein comprises a builder composition described herein. The builder composition of the cleaning composition comprises one or more weak builder and one or more strong builder. Preferably, the ratio between the total amount of all weak builders and the total amount of all strong builders in the cleaning composition is in the range 1.0 : 0.001 to 1.0 : 0.5, preferably 1.0 : 0.005 to 1.0 : 0.2.

[0094] Preferably, the total amount of all builders in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%.

[0095] Preferably, the total amount of weak builder in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%.

[0096] Preferably, the total amount of strong builder in the cleaning composition is 0.0001-4.5 weight%, preferably 0.001-0.3 weight%.

[0097] Suitable builders are described in more detail in paragraphs

[0014] to

[0018] of Reference RF1 and in paragraphs

[3001] to

[3005] of Reference RF2.Weak builder

[0098] Preferably, the weak builder is selected from the group consisting of citric acid, amino-tris-methylene-phosphonic acid (AMP), succinate, acetate, and salts thereof. Preferably, the weak builder is a weak sequestering builder. Preferably, the weak builder is citrate. Citrates include the mono- and the dialkali metal salts and in particular the mono- and preferably the trisodium salt of citric acid, ammonium or substituted ammonium salts of citric acid as well as citric acid as such. Citrate can be used as the anhydrous compound or as the hydrate, for example as sodium citrate dihydrate. Most preferred is sodium citrate.Strong builder

[0099] Preferably, the strong builder is selected from the group consisting of phosphates, silicates, carbonates, phosphonates, amino carboxylates and polycarboxylates. Preferably, the strong builder is a strong sequestering builder. Preferably the strong builder is selected from the group consisting of methylglycinediaceticacid (MGDA), ethylenediaminedisuccinic acid (EDDS), diaminetetramethylenephosphonate (EDTMP) diethylenetriaminepentamethylenephosphonate (DTPMP), glutamic acid diacetate (GLDA), 1-hydroxyethane-1,1-diphosphonate (HEDP), ethylenediaminetetraacetic acid (EDTA), nitrilotrimethylenephosphonic acid (NTMP), nitrilotriacetic acid (NTA), pyrophosphate, sodium tripolyphosphate (STPP), iminodisuccinic acid (IDS), and salts thereof. Preferably, the one or more strong builder is selected from the group consisting of MGDA, EDDS, DTPMP and salts thereof. Preferably, the one or more strong builder is a phosphonate, preferably DTPMP.

[0100] Preferably, the builder composition of the cleaning composition comprises citrate and one or more compound selected from the group consisting of MGDA, EDDS, DTPMP and salts thereof. Preferably, the builder composition of the cleaning composition comprises citrate and a phosphonate, preferably DTPMP.Further components

[0101] The cleaning compositions described herein including the compounds described herein may contain further adjunct cleaning additives (also abbreviated herein as "adjuncts"). Suitable additional adjunct cleaning additives are further described in Reference RF1 and Reference RF2.

[0102] Preferably, the cleaning composition comprises one or more additional adjunct cleaning additive selected from the group consisting of polymers, further enzymes, enzyme stabilizing systems, soaps, structurants or thickeners, clay soil removal / anti-redeposition agents, solubilizing agents, bleaching compounds, bleaching agents, bleach activators, bleach catalysts, brighteners, malodor control agents, pigments, dyes, opacifiers, hueing agents, dye transfer inhibiting agents, chelating agents, suds boosters, suds suppressors (antifoams), color speckles, silver care, anti-tarnish and / or anti-corrosion agents, alkalinity sources, pH adjusters, pH-buffer agents, hydrotropes, scrubbing particles, antibacterial agents, anti-oxidants, softeners, carriers, processing aids, pro-perfumes, dye fixation agent, and perfumes.Soaps

[0103] In one embodiment, the surfactant composition comprises one or more soaps (AS4). In one embodiment, AS4 are selected from salts (M+) of saturated and unsaturated C12-C18 fatty acids (AS4a), such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, (hydrated) erucic acid. M+ is selected from salt forming cations. Salt forming cations may be monovalent or multivalent; hence M+ equals 1 / v Mv+.

[0104] Examples include but are not limited to sodium, potassium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanolamine.

[0105] In one embodiment, AS4 are salts (M+) derived from natural fatty acids (AS4b) such as tallow (mainly C14-C18), coconut oil (mainly C12-C18), palm kernel oil (mainly C8-C18), olive oil (mainly C16-C18) or canola oil (mainly C18). Preferably, the surfactant composition comprises one or more salt of a coco-fatty acid, preferably one or more salt of a coco-fatty acid with C12-C18.Additional enzymes

[0106] In preferred embodiments, the cleaning compositions comprise one or more additional enzyme different to the protease variant described herein. The composition described herein can comprise more than one enzyme of different types, e.g., an amylase and a protease, or more than one enzyme of the same type, e.g., two or more different proteases, or mixtures thereof, e.g., an amylase and two different proteases of which one is the protease variant described herein. "Additional enzymes" are defined in more detail in paragraphs

[0020] to

[0027] of Reference RF1.

[0107] These additional enzymes may include hydrolases, such as additional proteases, amylases, lipases, DNases, cellulases, hemicellulases, phospholipases, esterases, mannanases, xylanases, dispersins, oxidoreductases, cutinases, pectate lyases, pectinases, lactases and peroxidases. Preferably, the additional enzyme is selected from the group consisting of amylases, lipases, proteases other than the protease variant described herein, cellulases, mannanases, and combinations of at least two of the foregoing types. Most preferably, the one or more additional enzyme is an amylase, preferably, an alpha-amylase, preferably an amylase as described in more detail in paragraphs

[0022] Reference RF1.Enzyme stabilizing system

[0108] In a preferred embodiment, the cleaning composition comprises an enzyme stabilizing system as described herein or in the chapter "Enzyme stabilizing system" of Reference RF1.

[0109] Preferably, the enzyme stabilizing system comprises at least one compound selected from the group consisting of polyols (preferably, glycerol, 1,2-propanediol, 1,3-propanediol, ethylene glycol, or sorbitol, preferably 1,2-propanediol), inorganic salts (preferably, CaCl2, MgCl2, or NaCl), short chain (preferably, C1-C3) carboxylic acids or salts thereof (preferably, formic acid, formate (preferably, sodium formate), acetic acid, acetate, or lactate), borate, boric acid, boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes, peptide acetals, and peptide aldehyde hydrosulfite adducts, preferably peptide aldehydes, preferably the enzyme stabilizing system comprises a protease inhibitor, preferably a peptide aldehyde , more preferably a tripeptide aldehyde, most preferably a tripeptide aldehyde (preferably, Z-VAL-H or Z-GAY-H) and 1,2-propanediol.

[0110] Preferably, the protease inhibitor is a peptide aldehyde, preferably a tripeptide aldehyde, preferably selected from a compound according to formula (I) wherein R1 and R2 are groups such that NH-CHR1-CO and / or NH-CHR2-CO are non-polar amino acids, R3 is a group such that NH-CHR3-CO is a non-polar amino acid; and Z is an N-terminal protection group, preferably selected from benzyloxycarbonyl (Cbz), p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), formyl, acetyl (Ac), methyloxy, alkoxycarbonyl, methoxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc), and tert-butyloxycarbonyl (Boc). Preferably, R1 and R2 of formula (I) is a group such that NH-CHR1-CO and NH-CHR2-CO is an L- or D-amino acid residue of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gin, Tyr, Cys, Lys, Arg, His, Asn, Glu, m-tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle, preferably independently from each other selected from an L- or D-amino acid residue of Ala, Val, Gly and Leu. Preferably, R3 of formula (I) is a group such that NH-CHR3-CO is an L- or D-amino acid residue of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gin, Tyr, Cys, Lys, Arg, His, Asn, Glu, m-tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle, or wherein R3 is (CH3)3SiCH2, preferably independently from each other selected from an L- or D-amino acid residue of Tyr, Phe, Val, Ala and Leu. Most preferably, the protease inhibitor is Z-VAL-H or Z-GAY-H.

[0111] In one embodiment, the cleaning composition comprises a protease inhibitor, preferably a peptide aldehyde, , in a molar ratio to the protease of 3:1 to 0.5:1, preferably 2:1 to 1:1.

[0112] In preferred embodiments, the cleaning compositions comprise a polymer, preferably cleaning polymers and / or soil release polymers. "Cleaning polymers and soil release polymers" are defined in more detail in paragraphs

[0032] to

[0034] of Reference RF1. These polymers include polycarboxylates, alkoxylated polyalkylenamines, alkoxylated polyalkylenimines, polyether-based polymers, rheology-modifying polymers, dye inhibition polymers and soil release polymers as defined in more detail in paragraphs

[3035] to

[3044] of Reference RF2.

[0113] Polymers may include, without limitation, "multifunctional alkoxylated polyethylene imines", "multifunctional alkoxylated diamines" and also terephthalic acid-based polyesters like Clariant's TexCare ®< , such as TexCare ®< SRN 170, TexCare ®< SRN 172, TexCare ®< SRN 260, TexCare ®< SRN 260 SG Terra and TexCare ®< SRA 300 as well as distinct combinations of all of the before mentioned polymers. Also included are graft polymers comprising a polyalkylene oxide based backbone with grafted side chains of vinyl ester monomer and optionally N-vinylpyrrolidone monomers.

[0114] In preferred embodiments, the cleaning compositions comprise the inventive enzyme(s) and a biocide. "Biocides" are defined in more detail in paragraphs

[0035] and

[0036] of Reference RF1. These biocides also include compounds as defined in more detail in paragraphs

[3006] and

[3007] of Reference RF2. Biocides may include, without limitation, 2-phenoxyethanol and 4,4'-dichoro 2-hydroxydiphenylether.

[0115] Further adjunct cleaning additives are included and described in more detail in paragraphs

[0005] ,

[0006] ,

[0019] ,

[0028] to

[0031] and

[0037] to

[0039] of Reference RF1.Preferred cleaning compositions

[0116] Preferably, the cleaning composition is a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

[0117] Preferably the liquid cleaning composition is a laundry or hard surface cleaning composition, preferably a liquid laundry cleaning composition, preferably an aqueous liquid laundry composition.

[0118] Preferably, the total amount of all non-aqueous solvents in the cleaning composition is 0-10 weight%, preferably 2-8 weight%.

[0119] Preferably, the cleaning composition comprises a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.3, preferably 0 to 0.2.

[0120] Preferably, the cleaning composition comprises a surfactant composition characterized in that the weight ratio of the total amount of all linear alkyl benzene sulfonates (LAS) in the cleaning composition to the total amount of all surfactants in the cleaning composition is 0 to 0.33.

[0121] Preferably, the cleaning composition comprises a surfactant composition characterized in that the weight ratio of the total amount of all linear alkyl benzene sulfonates (LAS) to the total amount of all surfactants in the cleaning composition is 0 to 0.3, preferably 0 to 0.2.

[0122] in one embodiment, the surfactant composition comprises at least one NIO as described herein, preferably at least one alcohol ethoxylate (AEO). In one embodiment, the surfactant composition comprises at least one anionic surfactant as described herein, preferably at least one alkylbenzene sulfonates (AS) and / or at least one AES. In one embodiment, the surfactant composition comprises at least one anionic surfactant as described herein and at least one NIO as described herein.

[0123] In one embodiment, the cleaning composition comprises one or more AS, preferably a mixture of LAS and BAS. In another embodiment, the cleaning composition is essentially devoid of alkylbenzene sulfonates. In one embodiment, the cleaning composition does not comprise any alkylbenzene sulfonates.

[0124] In one embodiment, the cleaning composition comprises one or more AES. In one embodiment, the surfactant composition comprises one or more AS as described herein and one or more AES as described herein.

[0125] In one embodiment, the surfactant composition comprises at least one NIO as described herein and at least one AS as described herein. In one embodiment, the surfactant composition comprises at least one NIO as described herein and at least AES as described herein. In one embodiment, the surfactant composition comprises or consists of at least one NIO as described herein, at least one AS, and at least AES as described herein.

[0126] In one embodiment, the cleaning composition comprises at least one soap, preferably a salt of a coco-fatty acid as described herein. In one embodiment, the cleaning composition comprises at least one NIO as described herein and at least one soap, preferably a salt of a coco-fatty acid as described herein. In one embodiment, the cleaning composition comprises at least one NIO as described herein, at least one AS, and at least one soap, preferably a salt of a coco-fatty acid as described herein. In one embodiment, the surfactant composition comprises at least one NIO as described herein, at least one AES, and at least one soap, preferably a salt of a coco-fatty acid as described herein. In one embodiment, the cleaning composition comprises or consists of at least one NIO as described herein, at least one AS, at least one AES, and preferably at least one soap, preferably a salt of a coco-fatty acid, preferably with C12-C18, as described herein.

[0127] Preferably, the total amount of all surfactants in the cleaning composition is 5-35 weight%, preferably 5-30 weight%, more preferably 10-25 weight%, most preferably 15-20 weight%.

[0128] Preferably, the total amount of all AS in the cleaning composition is 0-11.5 weight% or 1-11.5 weight%, preferably 0-20 weight%, more preferably 0-10 weight% or preferably 1-10 weight%.

[0129] Preferably, the total amount of all AES in the cleaning composition is 0-35 weight% or 1-35 weight%, preferably 0-20 weight%, more preferably 0-15 weight% or preferably 1-15 weight%.

[0130] Preferably, the total amount of all NIO in the cleaning composition is 0-35 weight% or 1-35 weight%, preferably 0-20 weight%, more preferably 0-15 weight% or preferably 1-15 weight%.

[0131] Preferably, the total amount of protease in the cleaning composition is 0.005-0.075 weight%, preferably 0.01-0.06 weight%.

[0132] Preferably, the total amount of all builders in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%.

[0133] Preferably, the total amount of weak builder in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%.

[0134] Preferably, the total amount of strong builder in the cleaning composition is 0.0001-4.5 weight%, preferably 0.001-0.3 weight%.

[0135] Preferably, the ratio between the total amount of all weak builders and the total amount of strong builders is 1.0 : 0.001 to 1.0 : 0.5, preferably 1.0 : 0.005 to 1.0 : 0.2.

[0136] Preferably, the cleaning composition is a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) in the cleaning composition to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2, wherein the cleaning composition preferably comprises one or more alcohol ethoxylate (AEO) and preferably one or more anionic surfactant, preferably selected from the group consisting of alkyl benzene sulfonate (AS) and alkyl ether sulfates (AES).

[0137] Preferably, the cleaning composition is a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all linear alkyl benzene sulfonates (LAS) in the cleaning composition to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2, wherein the cleaning composition preferably comprises one or more alcohol ethoxylate (AEO) and preferably one or more anionic surfactant, preferably selected from the group consisting of alkyl benzene sulfonate (AS) and alkyl ether sulfates (AES).

[0138] Preferably, the cleaning composition is a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 3 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2, wherein the cleaning composition preferably comprises one or more NIO, preferably one or more AEO, and preferably one or more anionic surfactant, preferably selected from the group consisting of alkyl benzene sulfonate (AS) (preferably, the AS being a mixture of LAS and BAS) and alkyl ether sulfates (AES), preferably wherein the cleaning composition comprises an enzyme stabilizing system, preferably comprising a tripeptide aldehyde and 1,2-propanediol.

[0139] Preferably, the cleaning composition comprises one or more surfactant selected from the group consisting of one or more AEO, one or more AS, and one or more AES, wherein preferably the AEO is an ethoxylated C10-C18 alcohol, preferably oxoalcohol, preferably C13-C15-oxoalcohol, preferably with 6-8 EO, wherein preferably the AS is a C9-C20 linear alkyl benzene sulfonate, preferably C10-C13 linear alkyl benzene sulfonate, and wherein preferably the AES is a C12-C15 alcohol ether sulfate, linear or branched, preferably with 1-3 EO.

[0140] Preferably, the weak builder comprises or consists of citrate and the strong builder is selected from the group consisting of MGDA, DTPMP, and EDDS, preferably the strong builder comprises or consists of DTPMP.

[0141] Preferably, the cleaning composition comprises one or more soap, preferably one or more coco-fatty acid, preferably one or more C12-C18 coco-fatty acid. Preferably, the total amount of all soap in the cleaning composition is 0-10 weight%, preferably 0.5-8 weight%, more preferably 1-5 weight%.

[0142] Preferably, the cleaning composition comprises an enzyme stabilizing system, preferably comprising or consisting of a tripeptide aldehyde and 1,2-propanediol.

[0143] Preferably, the liquid cleaning composition comprises a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, I. wherein the total amount of all surfactants in the cleaning composition is 5-35 weight%, preferably 5-30 weight%, more preferably 10-25 weight%, most preferably 15-20 weight%, II. wherein the total amount of all AS in the cleaning composition is 0-11.5 weight%, preferably 0-20 weight%, more preferably 0-10 weight% or preferably 1-10 weight%, III. wherein the total amount of all AES in the cleaning composition is 0-35 weight%, preferably 0-20 weight%, more preferably 0-15 weight% or preferably 1-15 weight%, IV. wherein the total amount of all NIO in the cleaning composition is 0-35 weight% or 1-35 weight%, preferably 0-20 weight%, more preferably 0-15 weight% or preferably 1-15 weight%, b) a builder composition comprising one or more weak builder and one or more strong builder, I. wherein the total amount of all builders in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, II. wherein the total amount of weak builder in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, III. wherein the total amount of strong builder in the cleaning composition is 0.0001-4.5 weight%, preferably 0.001-0.3 weight%, and c) a protease variant, preferably in an amount of 0.005-0.075 weight%, comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2.

[0144] Preferably, the liquid cleaning composition comprises a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, I. wherein the total amount of all surfactants in the cleaning composition is 5-35 weight%, preferably 5-30 weight%, more preferably 10-25 weight%, most preferably 15-20 weight%, II. wherein the total amount of all AS in the cleaning composition is 0-11.5 weight%, preferably 0-20 weight%, more preferably 0-10 weight% or preferably 1-10 weight%, III. wherein the total amount of all AES in the cleaning composition is 0-35 weight%, preferably 0-20 weight%, more preferably 0-15 weight% or preferably 1-15 weight%, IV. wherein the total amount of all NIO, preferably AEO, in the cleaning composition is 1-35 weight%, preferably 1-15 weight%, b) a builder composition comprising citrate and one or more builder selected from the group consisting of MGDA, DTPMP, and EDDS, I. wherein the total amount of all builders in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, II. wherein the total amount of citrate in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, III. wherein the total amount of all builders selected from the group consisting of MGDA, DTPMP, and EDDS in the cleaning composition is 0.0001-4.5 weight%, preferably 0.001-0.3 weight%, and c) a protease variant, preferably in an amount of 0.005-0.075 weight%, comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2.

[0145] Preferably, the liquid cleaning composition comprises a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, I. wherein the total amount of surfactants all in the cleaning composition is 5-35 weight%, preferably 5-30 weight%, more preferably 10-25 weight%, most preferably 15-20 weight%, II. wherein the total amount of all AS, preferably C9-C20 linear and / or branched alkyl benzene sulfonate, preferably a mixture of C10-C13 linear and branched alkyl benzene sulfonate, in the cleaning composition is 1-11.5 weight%, preferably 1-10 weight%, III. wherein the total amount of all AES, preferably C12-C15 alcohol ether sulfate, linear or branched, preferably with 1-3 EO, in the cleaning composition is 1-35 weight%, preferably 1-15 weight%, IV. wherein the total amount of all NIO, preferably AEO, preferably ethoxylated C10-C18 alcohol, preferably oxoalcohol, preferably C13-C15-oxoalcohol, preferably with 6-8 EO, in the cleaning composition is 1-35 weight%, preferably 1-15 weight%, b) a builder composition comprising citrate and one or more builder selected from the group consisting of MGDA, DTPMP, and EDDS, I. wherein the total amount of all builders in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, II. wherein the total amount of citrate in the cleaning composition is 0.1-9 weight%, preferably 0.1-7 weight%, 1-6 weight%, 2-6 weight%, 2-4 weight%, or preferably 2.5-3.5 weight%, III. wherein the total amount of all builders selected from the group consisting of MGDA, DTPMP, and EDDS in the cleaning composition is 0.0001-4.5 weight%, preferably 0.001-0.3 weight%, and c) a protease variant, preferably in an amount of 0.005-0.075 weight%, comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2, d) 0-10 weight%, preferably 0.5-8 weight%, more preferably 1-5 weight% of soap, preferably one or more coco-fatty acid, preferably one or more C12-C18 coco-fatty acid, preferably, wherein the total amount of all non-aqueous solvents in the cleaning composition is 0-10 weight%, preferably 2-8 weight%.Method of making

[0146] The present invention also relates to a method for making a cleaning composition comprising the steps of mixing a) a protease as described herein, b) a surfactant composition as described herein, and c) a builder composition as described herein.

[0147] The present invention also refers to a method for making a cleaning composition with improved protease stability and / or for providing a cleaning composition with improved wash performance comprising the steps of mixing a) a protease as described herein, b) a surfactant composition as described herein, and c) a builder composition as described herein.

[0148] In one embodiment, the present invention is directed to a method for providing a cleaning composition with improved storage stability and / or wash performance of a protease in the cleaning composition, comprising the steps of mixing a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

[0149] The cleaning compositions described herein may be in any form of a liquid cleaning composition. Preferably, the cleaning composition is a liquid laundry cleaning composition. Preferably, the cleaning composition is an aqueous liquid laundry cleaning composition.Method of use

[0150] The cleaning composition described herein can be used in various cleaning applications.

[0151] Preferably, the cleaning composition described herein is used in laundry or hard surface cleaning, preferably for home care or |&| cleaning. The cleaning composition described herein may be used in various industrial and institutional cleaning applications, including commercial laundry such as on premise laundry or tunnelwashing, mechanical ware wash such as in a hood machine or in a tunnelwasher, manual dish wash cleaning, carpet cleaning, open plant cleaning (outside pipe cleaning), cleaning in place (inside pipe cleaning), membrane cleaning such as in dairy, food, beverage or water treatment, vehicle care such as pad cleaning, microorganism removal, virus removal, insect removal or malodor removal or veterinary cleaning.

[0152] Preferably, the cleaning composition described herein is used for laundry cleaning.

[0153] In one embodiment, the cleaning composition described herein is used for providing an improved wash performance of the cleaning composition, preferably after storage of the cleaning composition, preferably after storage of the cleaning composition at a temperature of 15 °C, 30 °C, 37 °C, 45 °C, or 50 °C for 14, 28, 56, or 86 days, preferably at 37 °C for 56 days.

[0154] In a further embodiment, the cleaning composition described herein is used for providing an improved storage stability of the protease variant (as described herein) in the cleaning composition, preferably after storage of the cleaning composition, preferably after storage of the cleaning composition at a temperature of 15 °C, 30 °C, 37 °C, 45 °C, or 50 °C for 14, 28, 56, or 86 days, preferably at 37 °C for 56 days.

[0155] Thus, one embodiment is directed to the use of a liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising one or more weak builder and one or more strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, for improving the storage stability of the protease variant in the cleaning composition and / or for improving the wash performance of the cleaning composition after storage.

[0156] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims. The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention.Examples Material and Methods

[0157] Protease wash performance was determined in various detergent formulations.

[0158] The tested detergent formulations (Table 1) were prepared with the listed ingredients (1)-(8) according to the following procedure. Ingredient (3), (5) and (8) were filled into a beaker and heated to 50-55°C. While stirring with a blade stirrer at around 120 rpm, ingredient (4), which was preheated and homogenized, was slowly added. Sodium hydroxide was added for adjusting the pH value to pH 7.5 until the solution was clear and homogenous. Ingredient (2) was added and stirred until the formulation was homogenous. Heating was turned off and ingredients (1) and (6) were added when temperature was below 30°C. Table 1: Tested detergent formulationsFormulation number Ingredient concentration in weight% active F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 (1) AEO1.858.314.89.714.88.282.8812.65.4(2) AES136.49.73.2006.489.7206.48(3) AS3.26.4008.33.23.25.45.46.12(4) Coco-fatty acid2222222222(5) 1,2-Propanediol6666666666(6) Ethanol1111111111(7) Protease0.040.040.040.040.040.040.040.040.040.04(8) Deionized WaterUp to 100%Up to 100%Up to 100%Up to 100%Up to 100%Up to 100%Up to 100%Up to 100%Up to 100%Up to 100%(1): AEO: C13-C15-oxoalcohol (7 EO) (Lutensol AO 7, BASF) (2): AES: sodium laureth-2-sulfate = C12-C14 fatty alcohol ether sulfate (2 EO), sodium salt (Texapon N 70, BASF) (3): AS: alkylbenzylsulfonic acid, alkyl = C10-C13, linear (Maranil DBS / LC, BASF) (4): Coco fatty acid, C12-C18, C18 unsaturated (Edenor K 12-18, Edenor Technology Sdn Bhd)

[0159] Formulations F1, F3, F4, F6, F7, F8 and F9 are formulations according to the present invention.

[0160] Formulations F2, F5, and F10 are reference formulations.

[0161] For evaluation of the effect of the builder composition, all formulations shown in Table 1 were tested without a builder composition with 3% sodium citrate and 0.12% DTPMP with 6% sodium citrate and 0.24% DTPMP

[0162] Therefore, the builder composition was added to the formulations and the pH value was adjusted using sodium hydroxide to pH 8.0.

[0163] Subsequently, the protease (ingredient (7)) with tripeptide aldehyde protease inhibitor was added to the individual formulations.

[0164] All formulations shown in Table 1 were tested with Protease A (invention) and Protease B (reference).

[0165] Protease A (invention): SEQ ID NO: 3.

[0166] Protease B (reference): SEQ ID NO: 4.

[0167] The formulations were stored at 37°C for 56 days and subsequently measured for wash performance.

[0168] Eight soiled fabric swatches (Table 2) were washed with steel balls in wash liquor containing the above formulations after storage in the launder-o-meter (ratio height to diameter 1:1.5, size <2000 mL, filling rate <80%) under the washing conditions shown in Table 3. Table 2: Soiled swatches obtained from CFT, Vlaardingen, NetherlandsPC-05Blood / Milk / InkSWI-E-116Blood / Milk / InkC-S-39Full egg with pigment, agedC-S-38Egg yolk with pigment, agedC-S-01Blood, agedC-11Milk with carbon blackC-S-07Grass, pureSWI-E-112Cocoa Table 3: Wash conditions Water250 mLSteel balls20 pieces with 6 mm diameter and 0,9 gWash temperature30°CWash duration20 minDetergent dosing3 g / LWashing cycles1Ballast fabric15 g cotton fabric 283Sum ballast fabric + soiled swatches20 gWater hardness2,5 mmol / L; Ca 2+< : Mg 2+< : HCO 3 = 4 : 1 : 8

[0169] After washing, the fabric swatches were rinsed, spin-dried and dried in the air. The wash performance for the single stains were determined as follows: The achieved stain removal effect (ΔE) was determined by measuring reflectance values of the stains using a sphere reflectance spectrometer (SF500 type from Datacolor, USA, wavelength range 360-700nm). With the aid of the CIE-Lab color space classification, the brightness L*, the value a* on the red-green color axis and the b* value on the yellow-blue color axis, was measured before and after washing. △E is calculated according to the following formula: Δ E ab ∗ = Δ L ∗ 2 + Δ a ∗ 2 + Δ b ∗ 2

[0170] The results of the protease wash performance (measured as area under the curve, AUC) is shown in Table 4. AUC was calculated by ∑ i = 2 n time points ΔE i − ΔE i − 1 2 × days n − days n − 1 , with n = number of time points measured. Table 4: Total protease wash performance in launder-o-meter after storage at 37°C for 56 days expressed as area under curve (AUC) in [△E*days].Formulation number Superior wash performance of Protease A Wash performance [in AUC] without builder composition Wash performance [in AUC] with 3% citrate and 0.12% DTPMP Wash performance [in AUC] with 6% citrate and 0.24% DTPMP Protease A Protease B Protease A Protease B Protease A Protease B F1 (invention)Yes519161206763629073356499F2 (reference)No614964587053693972127789F3 (invention)Yes464654955913552575866761F4 (invention)Yes536548105802503958635297F5 (reference)No468443865234530657446448F6 (invention)Yes614555276912611275256753F7 (invention)Yes659462597802681780436657F8 (invention)Yes736365657832774485918031F9 (invention)Yes607658786699667977096996F10 (reference)No566957885862647361277170

[0171] It can be derived from Table 4 that the Protease A (invention) compared to Protease B (reference) shows a better total wash performance in formulations comprising a weak and a strong sequestering building with a surfactant composition described herein (cf. F1, F3, F4, F6, F7, F8 and F9) compared to formulations with a different surfactant composition (cf. F2, F5, and F10).

Claims

1. A liquid cleaning composition comprising a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising one or more weak builder and one or more strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

2. The cleaning composition of claim 1, wherein the protease variant comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D according to the numbering of SEQ ID NO: 2.

3. The cleaning composition of any of the preceding claims, wherein the surfactant composition comprises one or more non-ionic surfactant (NIO), preferably wherein the NIO is an alcohol ethoxylate (AEO).

4. The cleaning composition of any of the preceding claims, wherein the surfactant composition comprises one or more anionic surfactant, preferably selected from the group consisting of AS, alkyl ether sulfates (AES), and mixtures thereof.

5. The cleaning composition of any of the preceding claims, wherein a) the total amount of all surfactants in the cleaning composition is 5-35 weight%, b) the total amount of all AS is 0-11.5 weight%, c) the total amount of all AES is 0-35 weight%, and d) the total amount of all NIO is 0-35 weight%.

6. The cleaning composition of any of the preceding claims, wherein the surfactant composition comprises or consists of one or more AS, one or more NIO, and one or more AES.

7. The cleaning composition of any of the preceding claims, wherein the NIO is an ethoxylated C10-C18 alcohol, preferably oxoalcohol, preferably C13-C15-oxoalcohol, preferably with 6-8 EO, wherein the AS is a C9-C20 linear alkyl benzene sulfonate, preferably a C10-C13 linear alkyl benzene sulfonate, and wherein the AES is a C12-C15 alcohol ether sulfate, linear or branched, preferably with 1-3 EO.

8. The cleaning composition of any of the preceding claims, wherein the weak builder is a weak sequestering builder, preferably citrate.

9. The cleaning composition of any of the preceding claims, wherein the strong builder is a strong sequestering builder, preferably one or more selected from the group consisting of MGDA, DTPMP, and EDDS.

10. The cleaning composition of any of the preceding claims, wherein the cleaning composition comprises an enzyme stabilizing system, preferably wherein the enzyme stabilizing system comprises at least one compound selected from the group consisting of polyols (preferably, glycerol, 1,2-propanediol, 1,3-propanediol, ethylene glycol, or sorbitol, preferably 1,2-propanediol), inorganic salts (preferably, CaCl2, MgCl2, or NaCl), short chain (preferably, C1-C3) carboxylic acids or salts thereof (preferably, formic acid, formate (preferably, sodium formate), acetic acid, acetate, or lactate), borate, boric acid, boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde hydrosulfite adducts, preferably peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H), preferably the enzyme stabilizing system comprises a protease inhibitor, preferably a peptide aldehyde, more preferably a tripeptide aldehyde, most preferably a tripeptide aldehyde and 1,2-propanediol.

11. The cleaning composition of any of the preceding claims, wherein the cleaning composition comprises an additional enzyme, preferably selected from the group consisting of amylases, lipases, proteases other than the protease variant of any of the preceding claims, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxy-genases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, nucleases, ribonucleases (RNAses), deoxyribonucleases (DNAses), phosphodiesterases, phytases, carbohydrases, galactanases, xanthanases, xyloglucanases, oxidoreductases, perhydrolases, aminopeptidases, asparaginases, carbohydrases, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, alpha-galactosidases, beta-galactosidases, glucoamylases, alpha-glucosidases, beta-glucosidases, invertases, transglutaminases, dispersins, and combinations of at least two of the foregoing types, preferably, selected from amylases, lipases, cellulases, mannanases, and combinations of at least two of the foregoing types, most preferably amylases.

12. The cleaning composition of any one of preceding claims, wherein the cleaning composition is a laundry cleaning composition or a hard surface cleaning composition, preferably a biodegradable cleaning composition, preferably a laundry cleaning composition, more preferably a liquid laundry cleaning composition.

13. Method for providing a cleaning composition with improved storage stability and / or wash performance comprising the steps of mixing a) a surfactant composition characterized in that the weight ratio of the total amount of all alkyl benzene sulfonates (AS) to the total amount of all surfactants in the cleaning composition is 0 to 0.33, b) a builder composition comprising a weak builder and a strong builder, and c) a protease variant comprising an amino acid sequence which is at least 80%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease variant comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.

14. Use of a cleaning composition according to any of claims 1 to 12 for improving the storage stability of the protease variant in the cleaning composition.

15. Use of a cleaning composition according to any of claims 1 to 12 for improving the wash performance of the cleaning composition after storage.

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