WO2015049370A1 - Detergent composition and use of detergent composition - Google Patents

Detergent composition and use of detergent composition

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Publication number
WO2015049370A1
WO2015049370A1 PCT/EP2014/071231 EP2014071231W WO2015049370A1 WO 2015049370 A1 WO2015049370 A1 WO 2015049370A1 EP 2014071231 W EP2014071231 W EP 2014071231W WO 2015049370 A1 WO2015049370 A1 WO 2015049370A1
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Patent type
Prior art keywords
composition
acid
lipase
seq
id
Prior art date
Application number
PCT/EP2014/071231
Other languages
French (fr)
Inventor
Ana Maria Pardos BLASCO
Original Assignee
Novozymes A/S
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Classifications

    • CCHEMISTRY; METALLURGY
    • C11ANIMAL AND VEGETABLE OILS, FATS, FATTY SUBSTANCES AND WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16Organic compounds
    • C11D3/38Products with no well-defined composition, e.g. natural products
    • C11D3/386Preparations containing enzymes, e.g. protease, amylase
    • C11D3/38627Preparations containing enzymes, e.g. protease, amylase containing lipase
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL AND VEGETABLE OILS, FATS, FATTY SUBSTANCES AND WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16Organic compounds
    • C11D3/38Products with no well-defined composition, e.g. natural products
    • C11D3/386Preparations containing enzymes, e.g. protease, amylase
    • C11D3/38645Preparations containing enzymes, e.g. protease, amylase containing cellulase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2434Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)

Abstract

The present invention concerns a detergent composition for removing or releasing greasy stains, the use of a lipase and a cellulase for removing or releasing greasy stains, a method for removing or releasing greasy stains and an item treated according to the method.

Description

DETERGENT COMPOSITION AND USE OF DETERGENT COMPOSITION

Reference to a Sequence Listing

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

Field of the invention

The present invention concerns a detergent composition for removing or releasing greasy stains, the use of a lipase and a cellulase for removing or releasing greasy stains, a method for removing or releasing greasy stains and an item treated according to the method.

Background of the Invention

Cellulases are enzymes involved in the hydrolysis of cellulose. Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria, but they are also synthesized by plants. A very important industrial use of cellulases is for laundry, where cellulases are used for stain removal and anti- fuzz treatment of cotton textile.

Lipases are useful, e.g., as detergent enzymes to remove lipid or fatty stains from clothes and other textiles. Lipase enzymes have been used in detergents since the late 1980s for removal of greasy and oily soils by breakdown of soil into tri-glycerides. Current detergents e.g. laundry detergents compositions include a complex combination of active ingredients such as surfactants, enzymes bleaching agents, a builder system, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes. Lipolytic enzymes, including lipases and cutinases, have been employed in detergent cleaning compositions for the removal of greasy or oily stains.

One challenge for developing detergent compositions products for the low temperature area is that the detergent compositions need to perform optimally at both warm and cold wash conditions due to marked expectations. Therefore only chemicals and enzymes which in their functionality are robust towards a change in temperature will find their way into such products.

Currently available detergent composition products have a higher wash performance at

40°C as compared to 20°C and the detergency becomes even worse when the temperature is lowered from 20°C to 10°C. Especially dirt and stains comprising grease, oil or fat are difficult to remove at low temperatures, as the temperature helps melting the grease and thereby it is easier to remove the greasy dirt and stains.

It would be advantageous to optimize detergent compositions both powder and liquid and wash processes whereby stain removal may be improved without simultaneously also reducing the cleaning efficiency and particularly in light of the growing desire to reduce the overall energy consumption.

Summary of the Invention

The present invention concerns a detergent composition for removing or releasing greasy stains, which composition comprises a surfactant, a lipase and a cellulase. In addition the invention concerns the use of a lipase and a cellulase for removing or releasing greasy stains from a textile, a dish or a hard surface. The invention also concerns a method for removing or releasing greasy stains from an item having a greasy stain, which method comprises exposing the item to an aqueous solution of a lipase and a cellulase. Further, the invention concerns an item treated according to the method.

Brief Description of the Figures

Figure 1 shows the effect obtained in Example 1 after washing with a powder English base. Figure 2 shows the effect obtained in Example 1 after washing with a powder Italian base.

Figure 3 shows the effect obtained in Example 2 with the powder German base 2.

Figure 4 shows the effect obtained in Example 2 with the powder English base.

Figure 5 shows the effect obtained in Example 2 with the liquid Spanish base 2.

Figure 6 shows the effect obtained in Example 2 with the liquid Italian base 2.

Figure 7 shows the synergy obtained in Example 3 with the powder English base.

Figure 8 shows the synergy obtained in Example 3 with the liquid Spanish base 2.

Definitions

Enzyme Detergency benefit: The term "enzyme detergency benefit" is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and/or cleaning, prevention or reduction of redeposition of soils released in the washing process (an effect that also is termed anti- redeposition), restoring fully or partly the whiteness of textiles which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance (an effect that also is termed whitening). Textile care benefits, which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric (an effect that is also termed dye transfer inhibition or anti-backstaining), removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling), improvement- of the fabric-softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.

Textile care benefit: "Textile care benefits, which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one textile to another textile or another part of the same textile (an effect that is also termed dye transfer inhibition or anti-backstaining), removal of protruding or broken fibers from a textile surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling), improvement of the textile-softness, colour clarification of the textile and removal of particulate soils which are trapped in the fibers of the textile. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching component such as hydrogen peroxide or other peroxides or other bleaching species."

The term "greasy stain" means a stain comprising a lipid compound such as a fats, waxes, sterols, monoglycerides, diglycerides, triglycerides, phospholipids and/or fat-soluble vitamins,. The lipid compound can origin from a vegetable oil, oil from animal source, mineral oil or artificial oil. The lipid compound is generally soluble in organic solvents and generally insoluble in water. The greasy stain comprises one or more lipids and optionally other stain components such as particulate soil, dye, colorant, dairy components, or the like. The terms lipid and grease are used interchangeably.

When a greasy stain is said to have a "high content of lipids" or "the lipid content of the stain is high", it is meant that the stain consists almost entirely of lipids, such as at least 80% of the stain consists of lipids or at least 85% of the stain consists of lipids or at least 90% of the stain consists of lipids. Examples of such stains are stains having a high content of vegetable oil, mineral oil and/or animal fat, such as vegetable fat, butter or cooked butter.

When a greasy stain is said to have a "medium content of lipids", it is meant that the stain comprises a lipid such that at least 40% and up to 80% of the stain consists of lipids or at least 45% of the stain consists of lipids or at least 50% of the stain consists of lipids, or at least 55% of the stain consists of lipids, or at least 60% of the stain consists of lipids, or at least 65% of the stain consists of lipids, or at least 70% of the stain consists of lipids, or at least 75% of the stain consists of lipids. Examples of such stains are stains having a medium content of vegetable oil, mineral oil and/or, animal fat, such as compositions comprising vegetable oil, mineral oil and/or animal fat, e.g. lipstick or compositions comprising vegetable oil/animal fat and milk.

When a greasy stain is said to have a "low-medium content of lipids", it is meant that the stain comprises a lipid such that at least 15% and up to 40% of the stain consists of lipids, or at least 25% of the stain consists of lipids, or at least 25% of the stain consists of lipids, or at least 30% of the stain consists of lipids, or at least 35% of the stain consists of lipids. Examples of such stains are stains having a low-medium content of vegetable oil, mineral oil and/or animal fat, such as compositions comprising vegetable oil, mineral oil and/or animal fat, e.g. ice cream such as chocolate ice cream or compositions of comprising vegetable oil/animal fat and milk.

By the term "particulate soil" is meant particles with a heterogeneous nature with varying surface energies. The particles can be trapped in pores on the surfaces e.g. a textile.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having lipase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 807 of SEQ ID NO: 1 .

Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity. In one aspect, a subsequence contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of nucleotides 1 to 807 of SEQ ID NO: 1.

Substitutions. For an amino acid substitution, the following nomenclature is used:

Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g., "Gly205Arg + Ser41 1 Phe" or "G205R + S41 1 F", representing substitutions at positions 205 and 41 1 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by addition marks ("+"), e.g., "Gly195* + Ser41 1 *" or "G195* + S41 1 *".

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated "Gly195Glyl_ys" or "G195GK". An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195Glyl_ysAla" or "G195GKA". In such cases, the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

Figure imgf000006_0001

Multiple alterations. Variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., "Arg170Tyr,Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala + Arg170Gly,Ala" designates the following variants:

"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

Textile: The term "textile" means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling. The textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g. originating from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell), lyocell or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g. polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, aramid fiber), and/or cellulose-containing fiber (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fiber, lyocell). Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well.

Improved wash performance: The term "improved wash performance" is defined herein as the combination of cellulase and lipase displaying an increased wash performance relative to the wash performance of the base detergent composition or the base detergent composition comprising either lipase or cellulase, e.g. by increased stain removal. The term "wash performance" includes wash performance in laundry but also e.g. in dish wash.

Whiteness: The term "Whiteness" is defined herein as a broad term with different meanings in different regions and for different consumers. Loss of whiteness can e.g. be due to greying, yellowing, or removal of optical brighteners/hueing agents. Greying and yellowing can be due to soil redeposition, body soils, colouring from e.g. iron and copper ions or dye transfer. Whiteness might include one or several issues from the list below: colourant or dye effects; incomplete stain removal (e.g. body soils, sebum etc.); redeposition (greying, yellowing or other discolourations of the object) (removed soils reassociate with other parts of textile, soiled or unsoiled); chemical changes in textile during application; and clarification or brightening of colours.

Detergent Composition: The term "detergent composition" refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles, dishes, and hard surfaces. The detergent composition may be used to e.g. clean textiles, dishes and hard surfaces for both household cleaning and industrial cleaning. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish wash detergents). In addition to containing a bifunctional compound of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

Dish washing composition: The term "dish washing composition" refers to compositions including ADW compositions (Automated Dish Wash) intended for cleaning dishes, table ware, pots, pans, cutlery, plates, cups, glasses, bowls, cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass, acrylics and all forms of compositions for cleaning hard surfaces areas in kitchens. The present invention is not restricted to any particular type of dish wash composition. Dish wash: The term "dish wash" refers to all forms of washing dishes, e.g. by hand or automatic dish wash (ADW). 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, metals, china, glass and acrylics.

Hard surface cleaning: The term "Hard surface cleaning" is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash). Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass and acrylics.

Hard surface cleaning: The term "Hard surface cleaning" is defined herein as cleaning of hard surfaces, wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash).

Laundering: The term "laundering" relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a cleaning or detergent composition of the present invention. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand. Detailed Description of the Invention

The present invention concerns a detergent composition for removing or releasing greasy stains, which composition comprises a surfactant, a lipase and a cellulase. The present invention further comprises the use of a lipase in combination with cellulase for releasing removing greasy stains and a method for removing or releasing greasy stains from an item having a greasy stain. The method comprises exposing the item to an aqueous solution of a lipase and a cellulase.

Lipases are traditionally known as enzymes that remove greasy stains and are widely used in detergent composition. The lipases catalyse the breakdown of lipids in the greasy stain, which are then released from the item and removed with the waste water/rinse water. When the temperature at the same time is above the melting temperature of the lipids, the removal of the greasy stain is further eased.

The inventor has found that by using a lipase and a cellulase in combination and optionally with other enzymes, the removal or release of greasy stains are increased. When the combination of lipase and cellulase is used at low temperatures, e.g. below the melting point of the lipids contained in the greasy stain, the stain release and stain removal is increased.

The lipase may be of bacterial or fungal origin and it may be chemically modified or protein engineered. The stain treated according to the invention may comprise vegetable oil, mineral oil, animal fat or a combination thereof. For example the stain can comprise a vegetable oil such as a mayonnaise, a dressing or the like having a high content of lipids. In one embodiment the stain comprises an animal fat such as for example fat from pig, chicken, turkey, cow or fish. In one embodiment of the invention the stain may be a combination of vegetable oil and animal fat.

In one embodiment of the invention, the greasy stain has a high content of lipid. For example the stain can comprise lipstick, which comprises a combination of waxes and pigments, or a stain comprising lard.

In one embodiment, lipase is of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (W010/065455), cutinase from Magnaporthe grisea (W010/107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W01 1/084412), Geobacillus stearothermophilus lipase (W01 1/084417), lipase from Bacillus subtilis (W01 1/084599), and lipase from Streptomyces griseus (W01 1/150157) and S. pristinaespiralis (W012/137147).

Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541 , W094/25578, W095/14783, WO95/30744, W095/35381 , W095/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include include Lipolase™, Lipex™, Lipolex™ and

Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/1 1 1 143), acyltransferase from Mycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (W010/100028).

In one embodiment of the invention, the lipase is a polypeptide having the amino acid sequence which:

a) has at least 90% identity with the wild-type lipase derived from Humicola lanuginosa strain DSM 4109; b) compared to said wild-type lipase, comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three-dimensional structure within 15 A of E1 or Q249 with a positively charged amino acid; and c) comprises a peptide addition at the C-terminal; and/or

d) meets the following limitations:

i. comprises a negative amino acid in position E210 of said wild-type lipase; ii. comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said wild-type lipase; and

iii. comprises a neutral or negative amino acid at a position corresponding to N94 of said wild-type lipase and/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said wild-type lipase.

In one embodiment, the substitution mentioned under b) is within 10 A of E1 or Q249. In one embodiment said substitution is within 15 A (preferably 10 A) of E1 . In one embodiment the lipase comprises 2-4 of said substitutions, where 2 substitutions are preferred. In one embodiment the substitution is of an electrically neutral or negatively charged amino acid within 10 A of E1 with R. In one embodiment a substitution of amino acid S3, S224, P229, T231 , N233, D234 or T244 with a positively charged amino acid, preferably R.

In one embodiment, 1 -5 amino acids have been added to the C-terminal of the peptide. In one embodiment, the amino acids consist of electrically neutral (preferably hydrophobic) amino acids and are most preferably PGL or PG.

In one embodiment of the invention, the amino acids added at the C-terminal consist of neutral (preferably hydrophobic) amino acids and C, and the lipase further comprises substitution of an amino acid with C so as to form a disulfide bridge with the C of the peptide addition.

In one embodiment, the lipase comprises amino acids with negative or unchanged electric charge in at least two of positions N94, D96 and E99 of said wild-type lipase.

In one embodiment, the lipase comprises a substitution G91A, N94D/E/K/R, D96E/I/L/N/S/W, E99N/Q/K/R/H, N 101 S, R209P/S or Q249R/K/H.

In one embodiment of the invention, the lipase comprises one of the following sets of substitutions, optionally combined with Q249R/K/H and/or K98X:

G91 G/A/S/T + N94 (neutral or negative) + D96D/E/C/Y + E99E/D/C/Y,

G91 G/A/S/T + N94 (neutral or negative) + D96D/E/C/Y + E99N + N101 S,

G91 G/A/S/T + N94R/K/H + D96D/E/C/Y + E99E/D/C/Y,

G91 G/A/S/T + N94 (neutral or negative) + D96 (neutral or positive) + E99E/D/C Y, or

G91 G/A/S/T + N94 (neutral or negative) + D96D/E/C Y + E99 (neutral or positive). In one embodiment, the lipase comprises one of the following sets of substitutions, optionally combined with R209 (neutral or negative):

E99R/K/H + Q249R/K/H,

N94R/K/H + Q249R/K/H, or

D96 (neutral or positive) + Q249R/K/H.

In one embodiment of the invention, the lipase is a variant of a parent lipase which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T, G38A, D27R, and N33Q of SEQ ID NO: 2.

In one embodiment, the said variant in comparison with the parent lipase has increased stability. The stability is stability under storage conditions, protease stability, chemical stability, oxidation stability, pH stability, and/or thermostability.

In one embodiment, the variant is selected from the group consisting of:

a) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 2;

b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ; and

d) a fragment of the polypeptide of SEQ ID NO: 2.

In one embodiment, the lipase is a variant having activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2 selected from the group of:

a) D96E T231 R N233R;

b) N33Q D96E T231 R N233R;

c) N33Q T231 R N233R;

d) N33Q D1 1 1A T231 R N233R; e) N33Q T231 R N233R P256T;

f) N33Q G38A G91 T G163K T231 R N233R D254S;

g) N33Q G38A G91T D96E D1 1 1 A G163K T231 R N233R D254S P256T; h) D27R N33Q G38A D96E D1 1 1 A G163K T231 R N233R D254S P256T; i) D27R N33Q G38A G91T D96E D1 1 1A G163K T231 R N233R P256T; j) D27R N33Q G38A G91T D96E D1 1 1A G163K T231 R N233R D254S; k) D27R G38A G91T D96E D1 1 1A G163K T231 R N233R D254S P256T; I) D96E T231 R N233R D254S;

m) T231 R N233R D254S P256T;

n) G163K T231 R N233R D254S;

o) D27R N33Q G38A G91T D96E G163K T231 R N233R D254S P256T; p) D27R G91 T D96E D1 1 1 A G163K T231 R N233R D254S P256T;

q) D96E G163K T231 R N233R D254S;

r) D27R G163K T231 R N233R D254S;

s) D27R G38A G91 T D96E D1 1 1A G163K T231 R N233R D254S;

t) D27R G38A G91 T D96E G163K T231 R N233R D254S P256T;

u) D27R G38A D96E D1 1 1A G163K T231 R N233R D254S P256T:

v) D27R D96E G163K T231 R N233R D254S;

w) D27R D96E D1 1 1 A G163K T231 R N233R D254S P256T;

x) D27R G38A D96E G163K T231 R N233R D254S P256T.

The parent lipase may be (a) a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 , (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lipase activity. In one aspect, the amino acid sequence of the parent differs by up to 40 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 from the polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 2 containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids of SEQ ID NO: 2.

In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID

NO: 2.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium- high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 , (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 ; (ii) the polypeptide coding sequence of SEQ ID NO: 1 ; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X ray film or any other detection means known in the art. In one aspect, the nucleic acid probe is the polypeptide coding sequence of SEQ ID NO: 1 . In another aspect, the nucleic acid probe is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of nucleotides of SEQ ID NO: 1 . In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide having a sequence identity to the polypeptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N terminus or the C terminus of a region of another polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N terminus or the C terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982- 987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly. The parent may be a bacterial lipase. For example, the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis lipase.

In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus lipase.

In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans lipase.

In another aspect, the parent is a Thermobifida alba or Thermobifida fusca (formerly known as Thermomonaspora fusca) lipase.

The parent may be a fungal lipase. For example, the parent may be a yeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria lipase.

In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis lipase.

In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusahum negundi, Fusarium oxysporum, Fusahum reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusahum sporotrichioides, Fusahum sulphureum, Fusahum torulosum, Fusahum trichothecioides, Fusahum venenatum, Humicola ghsea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

In another aspect, the parent is a Thermomyces lanuginosus lipase, e.g., the lipase of SEQ ID NO: 2.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining lipase variants comprising: (a) introducing substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2; (b) selecting the variant which has lipase activity and in comparison with the parent lipase has improved stability; and (c) recovering the variant. The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent lipase.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent lipase and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US2004/0171 154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; W095/17413; or W095/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 1988, DNA 7: 127).

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

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

In one embodiment of the invention, the lipase is a lipase variant, comprising a substitution at one or more positions corresponding to positions T37A,D,E,F,G,H,I,L,N,P,Q,R,S,V,W,Y, N39A,C,D,E,F,G,I,K,L,M,P,Q,R,T,V,W,Y, and G91 D,H,I,P,Q of the mature polypeptide of SEQ ID NO: 4 , wherein the variant has lipase activity.

The variant of a parent lipase can be selected from the group consisting of:

a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 4;

b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 60 % identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and

d) a fragment of the mature polypeptide of SEQ ID NO: 4, which has lipase activity. In one embodiment of the invention, the lipase 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 the amino acid sequence of the parent lipase. The number of substitutions in the lipase variant is 1 -20, e.g., 1 -10 and 1 -5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions. The lipase variant may comprises a substitution at one or more positions corresponding to positions D96, T143, A150, E210, G225, T231 , N233 and

P250. In one embodiment the substitution is selected from D96G, T143A, A150G, E210Q, G225R, T231 R, N233R and P250R.

In one embodiment of the invention, the lipase variant is:

a) G91 Q +T143A +E210Q +T231 R +N233R +P250R;

b) G91 Q +A150G +E210Q +T231 R +N233R +P250R;

c) T37R +N39R +G91A +D96G +T231 R +N233R;

d) G91 Q +E210Q +T231 R +N233R +P250R; or e) G91 1 +E210Q +T231 R +N233R +P250R;

In one embodiment of the invention, the lipase variant is:

a) G91A +D96G +T231 R +N233R;

b) G91A +D96G +G225R +T231 R +N233R;

c) G91 N +E21 OQ +T231 R +N233R +P250R;

d) G91 L +E210Q +T231 R +N233R; or

e) G91A +D96G +A150G +T231 R +N233R.

The parent lipase may be (a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3.

In an aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lipase activity. In one aspect, the amino acid sequence of the parent differs by no more than 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the mature polypeptide of SEQ ID NO: 4.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ I D NO: 4. In another aspect, the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO: 4.

In another aspect, the parent is a fragment of the mature polypeptide of SEQ ID NO: 4 containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% of the number of amino acids of the mature polypeptide.

In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 4.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full- length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,

2d edition, Cold Spring Harbor, New York). The polynucleotide of SEQ ID NO: 3 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 4 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard

Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for

DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 3 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 3; (ii) the mature polypeptide coding sequence of SEQ ID NO: 3; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another aspect, the nucleic acid probe is nucleotides 67 to 873 of SEQ ID NO: 3. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 4; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 3.

In another embodiment, the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N terminus or the C terminus of a region of another polypeptide.

The variants may be prepared as described in the paragraph "preparation of variants" beginning on page 25 of WO 2013/1 13622.

In one embodiment of the invention, the cellulase is comprised by the enzyme classification EC 3.2.1.4. The cellulase is of bacterial or fungal origin and can be chemically modified or protein engineered. The cellulase can be derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia or Acremonium. In one embodiment of the invention the cellulase is derived from Humicola insolens, Myceliophthora thermophila, Sporotrichum pulverulentum, Fusarium oxysporum, Trichoderma reesei, Thielavia terrestris,

, Acremonium sp., Acremonium sp. CBS 478.94, Macrophomina phaseolina CBS 281.96, Crinipellis scabella CBS 280.96, Volutella colletotrichoides or Sordaria fimicola ATCC 52644 or Bacillus SP-KSMS237.

In one embodiment of the invention, the cellulase is an enzyme exhibiting endo-beta- 1 ,4-glucanase activity (EC 3.2.1.4), which is selected from one of

a) a polypeptide encoded by the DNA sequence of positions 1 to 2322 of SEQ ID NO:5;

b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO:5 under conditions wherein the DNA sequence is expressed;

c) an endo-beta-1 ,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:6; or a fragment thereof that has endo-beta-1 ,4-glucanase activity, when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1 .

In one embodiment, the cellulase exhibiting endo-beta-1 ,4-glucanase activity (EC

3.2.1.4) has a polypeptide having endo-beta-1 ,4-glucanase activity that is encoded by a polynucleotide that hybridizes with the nucleotide sequence shown in positions 1 -2322 of SEQ ID NO:5 under hybridization conditions comprising 5 x SSC at 45°C and washing conditions comprising 2 x SC at 60°C. The cellulase may comprise a polypeptide endogeneous to Bacillus sp., DSM 12648. In one embodiment of the invention the cellulase is active at a pH at least in the range of 4-1 1 , preferably 5.5-10.5.

In one embodiment of the invention, an isolated polynucleotide will hybridize to similar sized regions of SEQ ID NO: 5 or a sequence complementary thereto, under at least medium stringency conditions.

In particular, polynucleotides will hybridize to a denatured double-stranded DNA probe comprising either the full sequence encoding the catalytic domain of the enzyme which sequence is shown in positions 1 -2322 of SEQ ID NO: 5 or any probe comprising a subsequence of SEQ ID NO: 5 having a length of at least about 100 base pairs under at least medium stringency conditions, but preferably at high stringency conditions as described in detail below. Suitable experimental conditions for determining hybridization at medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 μg ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity higher than 1 x 109 cpm/microgram) probe for 12 hours at about 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least 60°C (medium stringency), still more preferably at least 65°C (medium/high stringency), even more preferably at least 70°C (high stringency), and even more preferably at least 75°C (very high stringency).

Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. DNA and RNA encoding genes of interest can be cloned in Gene Banks or DNA libraries by means of methods known in the art.

Polynucleotides encoding polypeptides having endo-glucanase activity of the invention are then identified and isolated by, for example, hybridization or PCR.

The present invention further provides counterpart polypeptides and polynucleotides from different bacterial strains (orthologs or paralogs). Of particular interest are endo- glucanase polypeptides from gram-positive alkalophilic strains, including species of Bacillus.

Species homologues of a polypeptide with endo-glucanase activity of the invention can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a DNA sequence of the present invention can be cloned using chromosomal DNA obtained from a cell type that expresses the protein. Suitable sources of DNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from chromosomal DNA of a positive cell line. A DNA sequence of the invention encoding an polypeptide having endo-glucanase activity can then be isolated by a variety of methods, such as by probing with probes designed from the sequences disclosed in the present specification and claims or with one or more sets of degenerate probes based on the disclosed sequences. A DNA sequence of the invention can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the DNA library can be used to transform or transfect host cells, and expression of the DNA of interest can be detected with an antibody (monoclonal or polyclonal) raised against the endo-glucanase cloned from B. sp., DSM 12648, expressed and purified as described in Materials and Methods and Examples 1 and 2 of WO 2002/099091 , or by an activity test relating to a polypeptide having endo-glucanase activity.

The endo-glucanase encoding part of the DNA sequence shown in SEQ ID NO:5 and/or an analogue DNA sequence of the invention may be cloned from a strain of the bacterial species Bacillus sp., preferably the strain DSM12648, producing the enzyme with endo-glucanase activity, or another or related organism as described herein.

How to use a sequence of the invention to get other related sequences: The disclosed sequence information herein relating to a polynucleotide sequence encoding an endo-beta- 1 ,4-glucanase of the invention can be used as a tool to identify other homologous endo- glucanases. For instance, polymerase chain reaction (PCR) can be used to amplify sequences encoding other homologous endo-glucanases from a variety of microbial sources, in particular of different Bacillus species.

The sequence of amino acids in position 1 to position 773 of SEQ ID NO:6 is a mature endo-glucanase sequence with a calculated molecular weight of 86 kDa. It is believed that positions 1 to about 340 of SEQ ID NO: 6 are the catalytically active domain of the of the present endo-glucanase enzyme. It is also believed that positions from about 340 to about 540 are the cellulose binding domain of the present endo-glucanase enzyme. The function of the remainder of the sequence, i.e., from about position 540 to position 773, is at present unknown.

The present invention provides an endo-glucanase enzyme comprising (i) the amino acid sequence of position 1 to position 773 of SEQ ID NO: 6, or a fragment thereof that has endo-glucanase activity.

A fragment of position 1 to position 773 of SEQ ID NO: 6 is a polypeptide, which have one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. In an embodiment the present invention provides an endo-glucanase enzyme comprising (ii) the amino acid sequence of positions 1 to about 340 of SEQ ID NO: 6, since it is contemplated that such a mono-domain endo-glucanase is also useful in the industrial applications described herein. In another embodiment the present invention provides an endo-glucanase enzyme comprising (iii) the amino acid sequence of positions 1 to from between about 540 and 773 of SEQ ID NO: 6, since it is contemplated that such an endo- glucanase comprising the catalytically active domain and the cellulose binding domain is also useful in the industrial applications described herein. In a preferred embodiment such fragment is a polypeptide which consists of position 1 to position 663 ±50 amino acids, preferably 1 to 663 ± 25 amino acids.

The present invention also provides endo-glucanase polypeptides that are substantially homologous to the polypeptide of (i), (ii), or (iii) above and species homologs (paralogs or orthologs) thereof. The term "substantially homologous" is used herein to denote polypeptides being at least 97%, preferred 98%, more preferred 98.5% identical, and most preferably 99% or more identical to the sequence shown in amino acids nos. 1 -773 of SEQ ID NO: 6, or a fragment thereof that has endo-glucanase activity, or its orthologs or paralogs. Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) as disclosed in Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1 .

Sequence identity of polynucleotide molecules is determined by similar methods using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 1 ) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991. See, in general Ford et al., Protein Expression and Purification 2: 95-107, 1991 , which is incorporated herein by reference.

DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA).

However, even though the changes described above preferably are of a minor nature, such changes may also be of a larger nature such as fusion of larger polypeptides of up to 300 amino acids or more both as amino- or carboxyl-terminal extensions to a polypeptide of the invention having endo-glucanase activity. Table 1

Conservative amino acid substitutions

Basic: arginine

lysine

histidine

Acidic: glutamic acid

aspartic acid

Polar: glutamine

asparagine

Hydrophobic: leucine

isoleucine

valine

Aromatic: phenylalanine

tryptophan

tyrosine

Small: glycine

alanine

serine

threonine

methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a-methyl serine) may be substituted for amino acid residues of a polypeptide according to the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the endo-glucanase polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 -1085, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e endo- glucanase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271 :4699-4708, 1996. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with polypeptides which are related to a polypeptide according to the invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination and/or shuffling followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-57, 1988), Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989), W095/17413, or WO 95/22625. Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, or recombination/shuffling of different mutations (WO 95/17413, WO 95/22625), followed by selecting for functional a polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Mutagenesis/shuffling methods as disclosed above can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially homologous to the polypeptides of (I), (II), or (III) above and retain the endo-glucanase activity of the wild-type protein.

The endo-glucanase enzyme of the invention may, in addition to the enzyme core comprising the catalytically active domain, i.e. positions 1 -about 340 of SEQ ID NO: 6, also comprise a cellulose binding domain (CBD), the cellulose binding domain and the catalytically active domain being operably linked. The cellulose binding domain (CBD) may exist as an integral part of the encoded enzyme as described above and in the appended SEQ ID NO:6, or be a CBD from another origin, introduced into the endo-glucanase thus creating an enzyme hybrid. In this context, the term "cellulose-binding domain" is intended to be understood as defined by Peter Tomme et al. "Cellulose-Binding Domains: Classification and Properties" in "Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definition classifies more than 120 cellulose-binding domains into 10 families (l-X), and demonstrates that CBDs are found in various enzymes such as cellulases (endo-glucanases), xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases. CBDs have also been found in algae, e.g. the red alga Porphyra purpurea as a non-hydrolytic polysaccharide- binding protein, see Tomme et al., op.cit. However, most of the CBDs are from cellulases and xylanases, CBDs are found at the N and C termini of proteins or are internal. Enzyme hybrids are known in the art, see e.g. WO 90/00609 and WO 95/16782, and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose-binding domain ligated, with or without a linker, to a DNA sequence encoding the endo-glucanase and growing the host cell to express the fused gene. Enzyme hybrids may be described by the following formula:

CBD - MR - X

wherein CBD is the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the cellulose-binding domain; MR is the middle region (the linker), and may be a bond, or a short linking group preferably of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40 carbon atoms; or is preferably from about 2 to about 100 amino acids, more preferably of from 2 to 40 amino acids; and X is an N-terminal or C- terminal region of a polypeptide corresponding at least to the catalytically active domain encoded by the DNA sequence of the invention.

In a similar way, the cellulose binding domain corresponding to from about position 340 to about position 540 of SEQ ID NO:6 can be used to form hybrids with endo-glucanases from sources other than Bacillus sp. AA349 and with other proteins. Examples of endo- glucanases from other sources replacing the endo-glucanase of positions 1 to about 340 of SEQ ID NO:6 include endo-glucanases from: (a) Bacillus lautus for instance Bacillus lautus NCIMB 40250 disclosed in W091 10732, (b) Humicola insolens DSM1800 disclosed in W091 17243 (c) Fusarium oxysporium DSM2672 disclosed in W091 17243 and (d) Bacillus sp. AC13 NCIMB 40482 disclosed in EP0651785. The enzymes can be prepared as described in WO 2002/099091 .

In one embodiment, the lipase and the cellulase is used together with further ingredients, such as hydrotropes, builders, co-builders, a bleaching systems, polymers, fabric hueing agents, mediators and/or enzymes. In one embodiment at least one additional enzyme is used. The further ingredients including the at least one additional enzyme may be comprised in a composition comprising the lipase and the cellulase. The additional enzyme may be selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectate lyase, pectinase, mannanase, arabinase, galactanase, and/or xylanase.

The concentration of the lipase is in the range of 0.01 -1 ,0 % w/w enzyme per detergent composition, in the range of 0.02-1 .0% w/w enzyme per detergent composition, in the range of 0.03-0.9% w/w enzyme per detergent composition, in the range of 0.04-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.8% w/w enzyme per detergent composition, in the range of 0.05-0.7% w/w enzyme per detergent composition, in the range of 0.05-0.6% w/w enzyme per detergent composition, in the range of 0.05-0.5 % w/w enzyme per detergent composition or in the range of 0.05-0.2 % w/w enzyme per detergent composition. The lipase and the cellulase may be used for removing or releasing greasy stains at a temperature in the range of 0°C to 90°C, in the range of 0°C to 80°C, in the range of 0°C to 70°C, in the range of 0°C to 60°C, in the range of 0°C to 50°C, in the range of 0°C to 40°C, in the range of 0°C to 30°C, in the range of 5°C to 35°C, in the range of 10°C to 30°C, in the range of 15°C to 25°C or in the range of 15°C to 20°C.

The use of the lipase and a cellulase for removing or releasing greasy stains may result in a cellulase effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points. Or the use of the lipase and the cellulase for removing or releasing greasy stains may result in a synergy effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points.

In one embodiment of the invention, the lipase and the cellulase is used as a composition in the form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

The composition comprises granules, which granules comprise a core and a protective coating, wherein:

a) the core comprises an enzyme, which is lipase and/or cellulase, and

b) the core or the coating comprises a reducing agent or a peroxide decomposing catalyst or an antioxidant, and

c) the core comprises a salt of a multivalent cation, and

d) the core or the coating comprises an acidic buffer component, and

e) the coating comprises a salt.

The core comprises the enzyme and the salt of a multivalent cation, and it may also comprise the reducing agent/antioxidant/peroxide decomposing catalyst and/or the acidic buffer component, typically as a homogenous blend. The blend may also include binders (such as synthetic polymer, wax, fat, or carbohydrate). The blend may further include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

The core can be prepared by granulating the blend, e.g. by use of granulation techniques including: crystallisation, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.

The core may consist of an inert particle with the blend absorbed into it, or with the blend applied on to the surface e.g. via fluid bed coating.

The core particle may have a diameter of 20-2000 μηη, particularly 50-1500 μηη, 100- 1500 m or 250-1200 μηι.

The composition comprises granules comprising lipase and granules comprising cellulase. In one embodiment of the invention the composition further comprises additional enzymes in the granules. The lipase, the cellulase and optionally additional enzymes are comprised in the same granule, or the composition may comprise granules, which comprises the lipase, the cellulase and optionally additional enzymes in separate granules.

The granules of the invention typically include between about 0.005 to about 500 mg/g on a dry weight basis of the enzyme component relative to the core (as active enzyme protein). For instance, the amount of enzyme in embodiments of the invention comprises about 0.05 to 300 mg/g, about 0.1 to 250 mg/g, about 0.5 to 200 mg/g, about 0.5 to 200 mg/g, about 1 .0 to 150 mg/g in the granule, or about 5.0 to 150 mg/g relative to the core.

In one embodiment of the invention, the acidic buffer component of the granule is present in an amount of 0.1 -10 % by weight relative to the core. The coating of the granule makes up 5-70 % by weight relative to the core and comprises at least 60% by weight w/w of a salt having a constant humidity at 20°C of at least 60%.

The granule contains a reducing agent, a peroxide decomposing catalyst and/or an antioxidant (a molecule capable of slowing or preventing the oxidation of other molecules) in the core and/or in the coating. Examples are sulfites, thiosulfates, erythorbates, ascorbates and nitrites, e.g. as salts of alkali metals and earth alkali metals. Other suitable materials are methionine, cysteine, propyl gallate, tert-butyl hydroquinone, tocopherols, thiodipropionic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) or tannic acid.

Further suitable examples are transition metals as reducing agents and/or peroxide decomposing catalysts like e.g. V, Co, Mn and Fe, typically as salts, e.g. sulfate-, acetate-, nitrate or chloride- salts or oxides, e.g. FeS04, FeCI3, CoS04, MnS04 or Mn02. Water soluble salts of the transition metals are preferred. As peroxide decomposing catalysts also an enzyme can be used, e.g. catalase.

The amount of the antioxidant, peroxide decomposing catalyst or reducing agent may be at least 0.1 % by weight relative to the core, particularly at least 0.2 %, at least 0.5 %, at least 1 %, or at least 1 % The amount may be at most 10% by weight relative to the core, particularly at most 5%, at most 4 %, at most 3 % or at most 2 %. Here, the amount of a salt is calculated in anhydrous form. Peroxide decomposing catalysts can be efficient in even lower concentrations, e.g. at least 0.001 %, or at least 0.01 %; the amount may be at most 5 % or at most 1 %.

The granule contains a salt of a multivalent cation in the core, particularly a divalent or trivalent cation, e.g. a salt of Mg, Zn, Cu, Mn, Ca or Al. The salt may include an organic or inorganic anion such as sulfate, chloride or acetate. Particular salts include magnesium sulfate and zinc sulfate, e.g. magnesium sulfate heptahydrate.

The salt may be used in an amount of at least 0.1 % by weight of the core, particularly at least 0.5% by weight, e.g. at least 1 % by weight. The amount may be at most 15 %, 10 % or 5 %. The percentage indicates the amount of the salt in anhydrous form.

The multivalent cation may be used in an amount of at least 0.02% by weight of the core, particularly at least 0.1 % by weight, e.g. at least 0.2 % by weight. The amount may be at most 6 %, at most 4 % or at most 2 %. The percentage indicates the amount of the multivalent cation. Acidic buffer component

The granule contains an acidic buffer component (acidic buffering agent) in the core or the coating. The amount may be at least 0.1 by weight of the core, particularly at least 1 % by weight. The amount is typically at most 10% by weight of the core, particularly at most 5% by weight. The percentage indicates the amount in anhydrous form.

The acidic buffer component has a pH below 7 when measured as a 1 % by weight aqueous solution (or alternatively a 10% solution). The acidic buffer component may have a pH of 1 to below 7, e.g. a pH of 3 to below 7, particularly a pH of 4 to 5. The acidic buffer component is typically a mixture comprising a weak acid and the corresponding base; it is at least partly in its acid form

Furthermore, the acidic buffer component has a pKa from 2 to 9, in particular a pKa from 4 to 9, in particular a pKa from 5 to 8, in particular a pKa from 2 to 6, in particular a pKa from 2 to 5, in particular a pKa from 2 to 4, in particular a pKa from 5 to 7. To utilize most of the potential buffer capacity the pH of an aqueous solution is in general below the pKa.

Particularly suitable acidic buffer components are salts of H3P04 e.g. NaH2P04,

KH2P04, and Ca(H2P04)2, polyphosphates e.g. sodium hexametaphosphate, polyacrylic acid and partly neutralized polyacrylic acid and co-polymers thereof, simple organic acids (less than 10 carbon atoms e.g. 6 or less carbon atoms) such as citric acid and salts thereof such as hydrogen citrate, e.g. disodium hydrogen citrate, malonic, succinic, glutaric, adipic acid.

In a particular embodiment, the acidic buffer components are selected from the group consisting of polyacrylic acid and partly neutralized polyacrylic acid and co-polymers thereof, citric acid and Na3-citrate. The granule comprises a core surrounded by at least one coating. The coating may comprise at least 60% by weight w/w of a salt, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight w/w.

The coating may be applied in an amount of at least 5 % by weight of the core, e.g. at least 10%, 10% or 15%. The amount may be at most 70 %, 50 %, 40 % or 30%.

To provide acceptable protection, the salt coating is preferably at least 1 μηη thick, particularly at least 2 μηη, at least 4 μηη or at least 8 μηη. The thicker the coating the more time consuming and expensive it gets to produce the granule. In a particular embodiment the thickness of the salt coating is below 100 μηη. In a more particular embodiment the thickness of the salt coating is below 60 μηη. In an even more particular embodiment the total thickness of the salt coating is below 40 μηη.

The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating has few or none uncoated areas. The layer or coating should in particular be homogenous in thickness. The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles is less than 50 μηη, such as less than 10 μηη or less than 5 μηη.

The salt coating is especially effective if it is applied in a fluid bed under relatively high humidity conditions.

The reducing agent/antioxidant/peroxide decomposing catalyst may be part of the salt coating, either as a homogeneous part of the entire salt coating, or as part of this coating, e.g. only as an inner layer of salt and /antioxidant/peroxide decomposing catalyst. Using e.g. FeS04 as a reducing agent/peroxide decomposing catalyst may induce a color change as the metal is oxidized, which can be hidden by having the component as an inner layer.

The salt coating can further contain other materials as known in the art, e.g. fillers, anti- sticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 grams in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g. at least 1 g per 100 g water, e.g. at least 5 g per 100 g water.

The salt may be an inorganic salt, e.g. salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms e.g. 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used. Specific examples include NaH2P04, Na2HP04, Na3P04, (NH4)H2P04, K2HP04, KH2P04, Na2S04, K2S04, KHS04,

ZnS04, MgS04, CuS04, Mg(N03)2, (NH4)2S04, sodium borate, magnesium acetate and sodium citrate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2S04), anhydrous magnesium sulfate (MgS04), magnesium sulfate heptahydrate (MgS04(7H20)), zinc sulfate heptahydrate (ZnS04(7H20)), sodium phosphate dibasic heptahydrate (Na2HP04(7H20)), magnesium nitrate hexahydrate (Mg(N03)2(6H20)), sodium borate decahydrate, sodium citrate dihydrate and magnesium acetate tetrahydrate.

The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85% by weight, or it may be another hydrate form of such a salt (e.g. anhydrate). The salt coating may be according to WO 00/01793, which is hereby incorporated by reference.

Specific examples of suitable salts are NaCI (CH20°c=76% by weight), Na2C03 (CH20°c=92% by weight), NaN03 (CH20°C=73% by weight), Na2HP04 (CH20°C=95% by weight), Na3P04 (CH25°C=92% by weight), NH4CI (CH20°C = 79.5% by weight), (NH4)2HP04 (CH20°c = 93,0% by weight), NH4H2P04 (CH20°C = 93.1 % by weight), (NH4)2S04 (CH20°c=81.1 % by weight), KCI (CH20°C=85% by weight), K2HP04 (CH20°C=92% by weight), KH2P04 (CH20°c=96.5% by weight), KN03 (CH20°C=93.5% by weight), Na2S04 (CH20°C=93% by weight), K2S04 (CH20°C=98% by weight), KHS04 (CH20°C=86% by weight), MgS04

(CH20°c=90% by weight), ZnS04 (CH20°C=90% by weight) and sodium citrate (CH25°C=86% by weight).

In a particular embodiment, the salt is selected from the group consisting of NaCI, Na2C03, NaN03, Na2HP04, Na3P04, NH4CI, (NH4)2HP04, NH4H2P04, (NH4)2S04, KCI, K2HP04, KH2P04, KN03, Na2S04, K2S04, KHS04, MgS04, ZnS04, NaCI and sodium citrate or mixtures thereof. In a more particular embodiment the salt is selected from the group consisting of NaCI, Na2C03, NaN03, Na2HP04, Na3P04, NH4CI, (NH4)2HP04, NH4H2P04, (NH4)2S04, KCI, K2HP04, KH2P04, KN03, Na2S04, K2S04, KHS04, NaCI and sodium citrate or mixtures thereof.

In a particular embodiment, the salt comprised in the coating of the granule is selected from the group consisting of NaCI, Na2C03, NaN03, Na2HP04, Na3P04, NH4CI, (NH4)2HP04, NH4H2P04, (NH4)2S04, KCI, K2HP04, KH2P04, KN03, Na2S04, K2S04, KHS04, MgS04, ZnS04, NaCI and sodium citrate or mixtures thereof.

Preferably the salt it applied as a solution of the salt e.g. using a fluid bed. In one embodiment of the invention, the granule comprises:

a) an enzyme, which is lipase and/or cellulase,

b) the core comprises Na-thiosulfate or methionine in an amount of 0.5-5% by weight relative to the core,

c) the core comprises magnesium sulfate or zinc sulfate in an amount of 2-8% by weight of the core,

d) the core comprises a mixture of citric acid and a citrate in an amount of 1 -5% by weight relative to the core, and

e) the salt coating makes up 10-30% by weight relative to the core, and comprises at least 75% by weight sodium sulfate.

Optionally, the granule may include an additional coating on the outside of the salt coating, e.g. in an amount of at least 0.5% by weight of the core, particularly at least 1 %, e.g. at most 20 % or 10 %. The additional coating may comprise polyethylene glycol (PEG), hydroxypropyl methyl cellulose (HPMC or MHPC), polyvinyl alcohol (PVA) or other film forming agents and can further contain fillers, antisticking agents, pigment, dye, plasticizers etc. Other additional coatings on the inside or outside of the salt coatings may be applied as known for people skilled in the art.

The lipase and the cellulase may be used for removing or releasing greasy stains in laundry, dishwashing, e.g. ADW, for hard surface cleaning or as a personal care cleaning product.

Surfactants

The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and may include any conventional surfactant(s) known in the art.

When included therein the detergent will usually contain from about 1 % to about 40% by weight of an anionic surfactant, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 15% to about 20%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonat.es (LAS), isomers of LAS, branched alkylbenzenesulfonat.es (BABS), phenylalkanesulfonat.es, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonat.es and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (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 alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or salt of fatty acids (soap), and combinations thereof.

When included therein the detergent will usually contain from about from about 1 % to about 40% by weigh of a cationic surfactant, for example from about 0.5% to about 30%, in particular from about 1 % to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12% or from about 10% to about 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.

When included therein the detergent will usually contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, in particular from about 1 % to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or /V-acyl /V-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121 -128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-10% by weight, for example 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof. Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1 -ol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethan-1 -ol), triethanolamine (TEA, also known as 2,2',2"-nitrilotriethan-1 -ol), and (carboxymethyl)inulin (CMI), and combinations thereof.

The detergent composition may also contain 0-50% by weight, such as about 5% to about 30%, of a detergent co-builder, or a mixture thereof. The detergent composition may include include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non- limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA PMA). Further non- limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2,2',2"- nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-/V,/V'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-/V,/V-diacetic acid (GLDA), 1 -hydroxyethane-1 ,1 - diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2- hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-/V-monoacetic acid (ASMA), aspartic acid- Λ/,/V-di acetic acid (ASDA), aspartic acid-/V-monopropionic acid (ASMP), iminodisuccinic acid (IDA), /V-(2-sulfomethyl)-aspartic acid (SMAS), /V-(2-sulfoethyl)-aspartic acid (SEAS), /V-(2-sulfomethyl)- glutamic acid (SMGL), /V-(2-sulfoethyl)-glutamic acid (SEGL), /V-methyliminodiacetic acid (Ml DA), a-alanine-/V,/V-diacetic acid (a-ALDA), serine-/V,/V-diacetic acid (SEDA), isoserine-/V,/V-diacetic acid (ISDA), phenylalanine-/V,/V-diacetic acid (PHDA), anthranilic acid-/V,/V-diacetic acid (ANDA), sulfanilic acid-/V,/V-diacetic acid (SLDA) , taurine-/V,/V-diacetic acid (TUDA) and sulfomethyl-/V,/V- diacetic acid (SMDA), /V-(2-hydroxyethyl)ethylenediamine-/V,/V',/V"-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854, US 5977053

Bleaching Systems

The detergent may contain 0-50% of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate, sodium perborates and hydrogen peroxide— urea (1 :1 ), preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, diperoxydicarboxylic acids, , perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non- limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator. The term bleach activator is meant herein as a compound which reacts with hydrogen peroxide to form a peracid via perhydrolysis. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters, amides, imides or anhydrides. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5- trimethylhexanoyl)oxy]benzene-1 -sulfonate (ISONOBS), 4-(dodecanoyloxy)benzene-1 -sulfonate (LOBS), 4-(decanoyloxy)benzene-1 -sulfonate, 4-(decanoyloxy)benzoate (DOBS or DOBA), 4- (nonanoyloxy)benzene-l -sulfonate (NOBS), and/or those disclosed in W098/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particulary preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmentally friendly Furthermore acetyl triethyl citrate and triacetin have good hydrolytical stability in the product upon storage and are efficient bleach activators. Finally ATC is multifunctional, as the citrate released in the perhydrolysis reaction may function as a builder. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6- (phthalimido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:

Figure imgf000037_0001

wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 1 1 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 1 1 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2- propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl. Other exemplary bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242. Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), polyvinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers , hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-/V-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated. Fabric hueing agents

The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.

Additional enzymes

The detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP1350843, EP0495257, EP0531372, W096/1 1262, W096/29397, WO98/08940, WO9634108 and WO9734005. Other examples are cellulase variants such as those described in WO94/07998, EP0531315, US 5457046, US 5686593, US 5763254, W095/24471 , WO98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.

The term "subtilases" refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501 -523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140). Other useful proteases may be those described in W092/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, W094/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in W095/23221 , and variants thereof which are described in WO92/21760, W095/23221 , EP1921 147 and EP1921 148.

Examples of metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int.) such as those derived from Bacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: W092/19729, WO96/034946, WO98/201 15, WO98/201 16, WO99/01 1768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, W01 1/036263, W01 1/036264, especially the variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 106, 1 18, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using the BPN' numbering. More preferred the subtilase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101 G,M,R S103A, V104I,Y,N, S106A, G1 18V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, DuralaseTm, DurazymTm, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, PreferenzTm, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, EffectenzTm, FN2®, FN3® , FN 4®, Excellase®, , Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown in Figure 29 of US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Amylases:

Suitable amylases which can be used together with the cellulase and lipase of the invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 or variants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 21 1 , 243, 264, 304, 305, 391 , 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprising residues 1 -33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181 , N190, M197, 1201 , A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1 -33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181 T+N190F+I201 F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ I D NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181 , 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering. More preferred variants are those having a deletion in two positions selected from 181 , 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO

08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201 , 207, 21 1 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90% sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131 , T165, K178, R180, S181 , T182, G183, M201 , F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E,R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N 128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N 128C+T131 I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181 .

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R1 18, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R1 18K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.

Other examples are amylase variants such as those described in WO201 1/098531 , WO2013/001078 and WO2013/001087.

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

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct materials

Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

Dispersants - The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents - The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine /V-oxide polymers, copolymers of /V-vinylpyrrolidone and /V-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01 % to about 5% or even from about 0.1 % to about 3% by weight of the composition.

Fluorescent whitening agent - The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulfonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6- ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'- disulfonate, 4,4'-bis-(2-anilino-4-(/V-methyl-/V-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1 ,2,3-triazol-2-yl)stilbene-2,2'-disulfonate and sodium 5-(2/-/-naphtho[1 ,2-c ][1 ,2,3]triazol-2-yl)-2-[(£)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s- triazin-6-ylamino) stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl- styryl)-disulfonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7- alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt%.

Soil release polymers - The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/1 13314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.

Anti-redeposition agents - The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents. Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents. Formulation of detergent products

The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

Detergent formulation forms: Layers (same or different phases), Pouches, versus forms for

Machine dosing unit.

Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009/001 1970 A1.

Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

Definition/characteristics of the forms :

A liquid or gel detergent , which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Laundry soap bars

The enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.

The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na+, K+ or NH4 + and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.

The laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, lipase and cellulase, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and and the mixture is then plodded. The combination of lipase and cellulase and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.

Granular detergent formulations

A granular detergent may be formulated as described in WO09/092699, EP1705241 ,

EP1382668, WO07/001262, US6472364, WO04/074419 or WO09/102854. Other useful detergent formulations are described in WO09/124162, WO09/124163, WO09/1 17340, WO09/1 17341 , WO09/1 17342, WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/1 12296, WO09/1 12298, WO09/103822, WO09/087033, WO09/050026, WO09/047125, WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375, WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545, WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/1 15391 , WO09/1 15392, WO09/074398, WO09/074403, WO09/068501 , WO09/065770, WO09/021813, WO09/030632, and WO09/015951.

WO201 1025615, WO201 1016958, WO201 1005803, WO201 1005623, WO201 1005730, WO201 1005844, WO201 1005904, WO201 1005630, WO201 1005830, WO201 1005912, WO201 1005905, WO201 1005910, WO201 1005813, WO2010135238, WO2010120863, WO2010108002, WO20101 1 1365, WO2010108000, WO2010107635, WO2010090915, WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979, WO2010030540, WO2010030541 , WO2010030539, WO2010024467, WO2010024469, WO2010024470, WO2010025161 , WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051 , WO2010102861 ,

WO2010099997, WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957, WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO201 1023716, WO2010142539, WO20101 18959, WO20101 15813,

WO2010105942, WO2010105961 , WO2010105962, WO2010094356, WO2010084203,

WO2010078979, WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486, WO2010066631 , WO2010066632, WO2010063689, WO2010060821 , WO2010049187, WO2010031607, WO2010000636,

Uses

The present invention includes a method for cleaning any surface including treating a textile or a hard surface or other surfaces in the field of fabric and/or home care. In one aspect of the invention, the method comprises the step of contacting the surface to be treated in a pre- treatment step or main wash step of a washing process, most preferably for use in a textile washing step or alternatively for use in dishwashing including both manual as well as automated/mechanical dishwashing. In one embodiment of the invention the lipase and cellulase and other components are added sequentially into the method for cleaning and/or treating the surface. Alternatively, the lipase and cellulase and other components are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, and mechanical agitation. Washing may be conducted with a foam composition as described in WO08/101958 and/or by applying alternating pressure (pressure/vaccum) as an addition or as an alternative to scrubbing and mechanical agitation. Drying of such surfaces or fabrics may be accomplished by any one of the common means employed either in domestic or industrial settings. The cleaning compositions of the present invention are ideally suited for use in laundry as well as dishwashing applications. Accordingly, the present invention includes a method for cleaning an object including but not limiting to fabric, tableware, cutlery and kitchenware. The method comprises the steps of contacting the object to be cleaned with a said cleaning composition comprising at least one embodiment of Applicants' cleaning composition, cleaning additive or mixture thereof. The fabric may comprise most any fabric capable of being laundered in normal consumer or institutional use conditions. The solution may have a pH from 8 to 10.5. The compositions may be employed at concentrations from 500 to 15.000ppm in solution. The water temperatures typically range from 5°C to 90°C. The water to fabric ratio is typically from 1 :1 to 30:1 . Use in detergents

The lipase and cellulase may be added to and thus become a component of a detergent composition. The detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the present invention provides a detergent additive comprising a polypeptide of the present invention as described herein.

The invention is further summarized in the following paragraphs:

1 . Detergent composition for removing or releasing greasy stains, which composition comprises a surfactant, a lipase and a cellulase.

2. Composition according to paragraph 1 , wherein the lipase is of bacterial or fungal origin. 3. Composition according to any of the preceding paragraphs, wherein the lipase is chemically modified or protein engineered.

4. Composition according to any of the preceding paragraphs, wherein the lipase is derived from Thermomyces, e.g. from T. lanuginosus, cutinase from Humicola, e.g. H. insolens, strains of Pseudomonas, e.g. P. alcaligenes or P. pseudoalcaligenes, P. cepacia, P. sp. strain SD705, P. wisconsinensis, GDSL-type Streptomyces lipases, cutinase from

Magnaporthe grisea, cutinase from Pseudomonas mendocina, Thermobifida fusca, Geobacillus stearothermophilus, Bacillus subtilis, Streptomyces griseus and S. pristinaespiralis.

5. Composition according to any of the preceding paragraphs, wherein the lipase is a polypeptide having an amino acid sequence which:

a) has at least 90 % identity with tSEQ ID NO: 2

b) compared to said SEQ ID NO: 2, comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three- dimensional structure within 15 A of E1 or Q249 with a positively charged amino acid; and

c) comprises a peptide addition at the C-terminal; and/or

d) meets the following limitations:

i. comprises a negative amino acid in position E210 of said SEQ ID NO:

2;comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said SEQ ID NO: 2; and

ii. comprises a neutral or negative amino acid at a position corresponding to N94 of said SEQ ID NO: 2 and/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said SEQ ID NO: 2.

6. Composition according to paragraph 5, wherein the said substitution is within 10 A of E1 or Q249.

7. Composition according to any of paragraphs 5-6, wherein said substitution is within 15 A

(preferably 10 A) of E1 .

8. Composition according to any of paragraphs 5-7, wherein the lipase comprises 2-4 (preferably two) of said substitutions.

9. Composition according to any of paragraphs 5-8, wherein the lipase comprises a substitution of an electrically neutral or negatively charged amino acid within 10 A of E1 with R.

10. Composition according to any of paragraphs 5-9, wherein the lipase comprises a substitution of amino acid S3, S224, P229, T231 , N233, D234 or T244 with a positively charged amino acid, preferably R.

1 1 . Composition according to any of paragraphs 5-10, wherein the peptide addition consists of 1 -5 amino acids.

12. Composition according to any of paragraphs 5-1 1 , wherein the peptide addition consists of electrically neutral (preferably hydrophobic) amino acids, and is most preferably PGL or PG.

13. Composition according to any of paragraphs 5-12, wherein the peptide addition consists of neutral (preferably hydrophobic) amino acids and C, and the lipase further comprises substitution of an amino acid with C so as to form a disulfide bridge with the C of the peptide addition.

14. Composition according to any of paragraphs 5-13, wherein the lipase comprises amino acids with negative or unchanged electric charge in at least two of positions N94, D96 and E99 of said SEQ ID NO: 2.

15. Composition according to any of paragraphs 5-14, wherein the lipase comprises a substitution G91A, N94D/E/K/R, D96E/I/L/N/S/W, E99N/Q/K/R/H, N101 S, R209P/S or Q249R/K/H.

16. Composition according to any of paragraphs 5-15, wherein the lipase comprises one of the following sets of substitutions, optionally combined with Q249R/K H and/or K98X: a) G91 G/A/S/T + N94(neutral or negative) + D96D/E/C/Y + E99E/D/C/Y, b) G91 G/A/S/T + N94(neutral or negative) + D96D/E/C/Y + E99N + N101 S, c) G91 G/A/S/T + N94R/K/H + D96D/E/C/Y + E99E/D/C/Y,

d) G91 G/A/S/T + N94(neutral or negative) + D96(neutral or positive) + E99E/D/C/Y, or

e) G91 G/A/S/T + N94 (neutral or negative) + D96D/E/C/Y + E99(neutral or positive).

Composition according to any of paragraphs 5-16, wherein the lipase comprises one of the following sets of substitutions, optionally combined with R209 (neutral or negative): a) E99R/K/H + Q249R/K/H,

b) N94R/K/H + Q249R/K/H, or

c) D96(neutral or positive) + Q249R/K/H.

Composition according to any of the preceding paragraphs, wherein the lipase is a variant of a parent lipase which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R of SEQ ID NO: 2.

Composition according to any of the preceding paragraphs, wherein the lipase is a variant of a parent lipase which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R or N33Q of SEQ ID NO: 2.

Composition according to any of the preceding paragraphs, wherein said variant in comparison with the parent lipase has increased stability.

Composition according to any of the preceding paragraphs, wherein the stability is stability under storage conditions, protease stability, chemical stability, oxidation stability, pH stability, and/or thermostability.

Composition according to any of the preceding paragraphs, wherein the variant is selected from the group consisting of:

a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to

SEQ ID NO: 2;

b. a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i);

c. a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ; and

d. a fragment of the polypeptide of SEQ ID NO: 2.

Composition according to any of the preceding paragraphs, wherein the lipase is a variant having activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231R+N233R and at least one or more (e.g., several) of D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2 selected from the group of:

a) D96E T231R N233R;

b) N33Q D96E T231R N233R;

c) N33QT231R N233R;

d) N33Q D111AT231R N233R;

e) N33Q T231R N233R P256T;

f) N33QG38AG91TG163KT231RN233RD254S;

g) N33Q G38A G91T D96E D111 A G163K T231 R N233R D254S P256T;

h) D27R N33Q G38A D96E D111 A G163K T231 R N233R D254S P256T;

i) D27R N33Q G38AG91T D96E D111A G163K T231 R N233R P256T;

j) D27R N33Q G38A G91T D96E D111A G163K T231R N233R D254S;

k) D27RG38AG91TD96E D111AG163KT231RN233RD254SP256T;

I) D96E T231R N233R D254S;

m) T231R N233R D254S P256T;

n) G163KT231R N233R D254S;

o) D27R N33Q G38AG91T D96E G163K T231R N233R D254S P256T;

p) D27R G91T D96E D111 A G163K T231 R N233R D254S P256T;

q) D96E G163KT231R N233R D254S;

r) D27R G163KT231R N233R D254S;

s) D27R G38AG91T D96E D111A G163K T231 R N233R D254S;

t) D27R G38AG91T D96E G163K T231R N233R D254S P256T;

u) D27RG38AD96ED111AG163KT231RN233RD254S P256T:

v) D27R D96E G163K T231R N233R D254S;

w) D27R D96E D111 A G163K T231R N233R D254S P256T;

x) D27R G38A D96E G163K T231 R N233R D254S P256T.

Composition according to any of the preceding paragraphs, wherein the lipase is a lipase variant, comprising a substitution at one or more positions corresponding to positions T37A,D,E,F,G,H,I,L,N,P,Q,R,S,V,W,Y, N39A,C,D,E,F,G,I,K,L,M,P,Q,R,T,V,W,Y, and G91 D,H,I,P,Q of the mature polypeptide of SEQ ID NO: 4 , wherein the variant has lipase activity.

Composition according to any of paragraphs 1-4 and 23, which is a variant of a parent lipase selected from the group consisting of:

e) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 4; f) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full-length complement of (i);

g) a polypeptide encoded by a polynucleotide having at least 60 % identity to the mature polypeptide coding sequence of SEQ ID NO: 3; and

h) a fragment of the mature polypeptide of SEQ ID NO: 4, which has lipase activity.

26. Composition according to any of the preceding paragraphs, wherein the lipase 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 the amino acid sequence of the parent lipase.

27. Composition according to any of the preceding paragraphs, wherein the number of substitutions in the lipase variant is 1 -20, e.g., 1 -10 and 1 -5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions.

28. Composition according to any of the preceding paragraphs, wherein the lipase variant further comprises a substitution at one or more positions corresponding to positions D96,

T143, A150, E210, G225, T231 , N233 and P250.

29. Composition according to any of the preceding paragraphs, wherein the substitution is selected from D96G, T143A, A150G, E210Q, G225R, T231 R, N233R and P250R.

30. Composition according to any of the preceding paragraphs, wherein said variant is: f) G91 Q +T143A +E210Q +T231 R +N233R +P250R;

g) G91 Q +A150G +E210Q +T231 R +N233R +P250R;

h) T37R +N39R +G91A +D96G +T231 R +N233R;

i) G91 Q +E210Q +T231 R +N233R +P250R; or

j) G91 1 +E210Q +T231 R +N233R +P250R;

31 . Composition according to any of the preceding paragraphs, wherein said variant is: f) G91A +D96G +T231 R +N233R;

g) G91A +D96G +G225R +T231 R +N233R;

h) G91 N +E210Q +T231 R +N233R +P250R;

i) G91 L +E210Q +T231 R +N233R; or

j) G91A +D96G +A150G +T231 R +N233R.

32. Composition according to any of the preceding paragraphs, wherein the cellulase is comprised by the enzyme classification EC 3.2.1 .4.

33. Composition according to any of the preceding paragraphs, wherein the cellulase is of bacterial or fungal origin.

34. Composition according to any of the preceding paragraphs, wherein the cellulase is chemically modified or protein engineered.

35. Composition according to any of the preceding paragraphs, wherein the cellulase is derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia or Acremonium.

Composition according to any of the preceding paragraphs, wherein the cellulase is derived from Humicola insolens, Myceliophthora thermophila, Sporotrichum pulverulentum, Fusarium oxysporum, Trichoderma reesei, Thielavia terrestris, ,

Acremonium sp., Acremonium sp. CBS 478.94, Macrophomina phaseolina CBS 281.96, Crinipellis scabella CBS 280.96, Volutella colletotrichoides or Sordaria fimicola ATCC 52644 or Bacillus SP-KSMS237.

Composition according to any of the preceding paragraphs, wherein the cellulase is an enzyme exhibiting endo-beta-1 ,4-glucanase activity (EC 3.2.1.4), which is selected from one of

a) a polypeptide encoded by the DNA sequence of positions 1 to 2322 of SEQ ID NO:5;

b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO:5 under conditions wherein the DNA sequence is expressed;

c) an endo-beta-1 ,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:6; or a fragment thereof that has endo-beta-1 ,4-glucanase activity, when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

Composition according to any of the preceding paragraphs, wherein the cellulase exhibiting endo-beta-1 ,4-glucanase activity (EC 3.2.1 .4) has a polypeptide having endo- beta-1 ,4-glucanase activity that is encoded by a polynucleotide that hybridizes with the nucleotide sequence shown in positions 1 -2322 of SEQ ID NO:5 under hybridization conditions comprising 5 x SSC at 45°C and washing conditions comprising 2 x SC at

60°C.

Composition according to any of the preceding paragraphs, wherein the cellulase comprises a polypeptide endogeneous to Bacillus sp., DSM 12648.

Composition according to any of the preceding paragraphs, wherein the cellulase is active at a pH at least in the range of 4-1 1 , preferably 5.5-10.5.

Composition according to any of the preceding paragraphs, wherein the composition further comprises: hydrotropes, builders, co-builders, a bleaching systems, polymers, fabric hueing agents, mediators and/or enzymes.

Composition according to any of the preceding paragraphs, wherein composition comprises at least one additional enzyme.

Composition according to any of the preceding paragraphs, wherein the additional enzyme is selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectate lyase, pectinase, mannanase, arabinase, galactanase, and/or xylanase.

44. Composition according to any of the preceding paragraphs, wherein the concentration of the lipase is in the range of 0.01 -1 ,0 % w/w enzyme per detergent composition, in the range of 0.02-1 .0% w/w enzyme per detergent composition, in the range of 0.03-0.9% w/w enzyme per detergent composition, in the range of 0.04-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.8% w/w enzyme per detergent composition, in the range of 0.05-0.7% w/w enzyme per detergent composition, in the range of 0.05-0.6% w/w enzyme per detergent composition, in the range of 0.05-0.5 % w/w enzyme per detergent composition or in the range of 0.05-0.2% w/w enzyme per detergent composition.

45. Composition according to any of the preceding composition paragraphs, wherein the concentration of the lipase is in the range of 0.05-0.15 % w/w enzyme per detergent composition.

46. Composition according to any of the preceding paragraphs, wherein the concentration of the cellulase is in the range of 0.01 -1 ,0 % w/w enzyme per detergent composition, in the range of 0.02-1 .0% w/w enzyme per detergent composition, in the range of 0.03-0.9% w/w enzyme per detergent composition, in the range of 0.04-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.9% w/w enzyme per detergent composition, in the range of 0.05-0.8% w/w enzyme per detergent composition, in the range of 0.05-0.7% w/w enzyme per detergent composition, in the range of 0.05-0.6% w/w enzyme per detergent composition, in the range of 0.05-0.5 % w/w enzyme per detergent composition or in the range of 0.05-0.2 % w/w enzyme per detergent composition.

47. Composition according to any of the preceding paragraphs, wherein the composition is a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

48. Composition according to any of the preceding paragraphs, wherein the composition comprises granules, which granules comprises a core and a protective coating, wherein: a. the core comprises an enzyme, which is lipase and/or cellulase, and

b. the core or the coating comprises a reducing agent or a peroxide decomposing catalyst or an antioxidant, and

c. the core comprises a salt of a multivalent cation, and

d. the core or the coating comprises an acidic buffer component, and

e. the coating comprises a salt. 49. Composition according to any of the preceding paragraphs, wherein the composition comprises granules comprising lipase and granules comprising cellulase.

50. Composition according to any of the preceding paragraphs, wherein the composition further comprises granules comprising additional enzymes.

51 . Composition according to any of paragraphs 1 to 26, wherein the composition comprises granules, wherein the lipase, the cellulase and optionally additional enzymes are comprised in the same granule.

52. Composition according to any of the preceding paragraphs, wherein the acidic buffer component of the granule is present in an amount of 0.1 -10 % by weight relative to the core.

53. Composition according to any of the preceding paragraphs, wherein the coating of the granule makes up 5-70 % by weight relative to the core and comprises at least 60% by weight w/w of a salt having a constant humidity at 20°C of at least 60%.

54. Composition according to any of the preceding paragraphs, wherein the reducing agent or peroxide decomposing catalyst of the granule is a thiosulfate, cysteine, methionine or a transition metal salt.

55. Composition according to any of the preceding paragraphs, wherein the reducing agent of the granule is present in an amount of 0.1 -10 % by weight relative to the core.

56. Composition according to any of the preceding paragraphs, wherein the salt of a multivalent cation of the granule is magnesium sulfate or zinc sulfate.

57. Composition according to any of the preceding paragraphs, wherein the salt of a multivalent cation of the granule is present in an amount of 0.1 -15% as anhydrous salt by weight of the core, or 0.02-6% as multivalent cation by weight of the core.

58. Composition according to any of the preceding paragraphs, wherein the acidic buffer component of the granule comprises a mixture of citric acid and a citrate.

59. Composition according to any of the preceding paragraphs, wherein the acidic buffer component of the granule is present in an amount of 1 -5% by weight relative to the core.

60. Composition according to any of the preceding paragraphs, wherein the salt in the coating of the granule has a constant humidity of at least 80% by weight.

61 . Composition according to any of the preceding paragraphs, wherein the coating of the granule comprises at least 75% by weight of the salt.

62. Composition according to any of the preceding paragraphs, wherein the salt coating of the granule comprises sodium sulfate.

63. Composition according to any of the preceding paragraphs, wherein the coating of the granule constitutes 10-30% by weight relative to the core.

64. Composition according to any of the preceding paragraphs, wherein the granule comprises: a. an enzyme, which is lipase and/or cellulase,

b. the core comprises Na-thiosulfate or methionine in an amount of 0.5-5% by weight relative to the core,

c. the core comprises magnesium sulfate or zinc sulfate in an amount of 2-8% by weight of the core,

d. the core comprises a mixture of citric acid and a citrate in an amount of 1 -5% by weight relative to the core, and

e. the salt coating makes up 10-30% by weight relative to the core, and comprises at least 75% by weight sodium sulfate.

65. Composition according to any of the preceding paragraphs, where the granule further comprises an additional coating on the outside of the salt coating, wherein the additional coating comprises a film-forming agent, particularly polyethylene glycol, hydroxypropyl methyl cellulose (HPMC or MHPC), or polyvinyl alcohol (PVA).

66. Composition according to any of the preceding paragraphs, wherein the composition is a laundry cleaning composition, a dishwashing cleaning composition, a hard-surface cleaning composition and/or a personal care cleaning composition.

67. Use of a lipase and a cellulase for removing or releasing a greasy stain.

68. Use of a detergent composition according to any of paragraphs 1 -66 for removing or releasing a greasy stain

69. Use according to paragraph 68, wherein the greasy stain is removed or released from a textile having a greasy stain, from a dish having a greasy stain or from a hard surface having a greasy stain.

70. Use according to any of the preceding use paragraphs, wherein the stain comprises vegetable oil, animal fat, mineral oil or a combination thereof.

71 . Use according to any of the preceding use paragraphs, wherein the stain has a low- medium content of lipid.

72. Use according to any of the preceding use paragraphs, wherein the stain has a medium content of lipid.

73. Use according to any of the preceding use paragraphs, wherein the stain has a high content of lipid.

74. Use according to any of the preceding use paragraphs, wherein the textile comprises cotton and/or polyester.

75. Use according to any of the preceding use paragraphs, wherein the greasy stain is released or removed at a temperature in the range of 0°C to 90°C, in the range of 0°C to 80°C, in the range of 0°C to 70°C, in the range of 0°C to 60°C, in the range of 0°C to

50°C, in the range of 0°C to 40°C, in the range of 0°C to 30°C, in the range of 5°C to 35°C, in the range of 10°C to 30°C, in the range of 15°C to 25°C or in the range of 15°C to 20°C.

76. Use according to any of the preceding paragraphs, wherein the wash performance is increased.

77. Use according to any of the preceding paragraphs, wherein the cellulase effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points.

78. Use according to any of the preceding paragraphs, wherein the synergy effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points.

79. A method for removing or releasing greasy stains from an item having a greasy stain, which method comprises exposing the item to an aqueous solution of a lipase and a cellulase.

80. A method for removing or releasing greasy stains from an item having a greasy stain, which method comprises exposing the item to an aqueous solution of a detergent composition of paragraphs 1 -66.

81 . Method according to paragraph 80, wherein the item is a textile, a dish or a hard surface.

82. Method according to any of the preceding method paragraphs, wherein the stain comprises vegetable oil, animal fat or a mineral oil combination thereof.

83. Method according to any of paragraphs 79-82, wherein the stain comprises a particulate soil.

84. Method according to any of the preceding method paragraphs, wherein the stain has a low-medium content of lipid.

85. Method according to any of the preceding method paragraphs, wherein the stain has a medium content of lipid.

86. Method according to any of the preceding method paragraphs, wherein the stain has a high content of lipid.

87. Method according to any of the preceding method paragraphs, wherein the textile comprises cotton and/or polyester.

88. Method according to any of the preceding method paragraphs, wherein the greasy stain is released or removed at a temperature in the range of 0°C to 90°C, in the range of 0°C to 80°C, in the range of 0°C to 70°C, in the range of 0°C to 60°C, in the range of 0°C to 50°C, in the range of 0°C to 40°C, in the range of 0°C to 30°C, in the range of 5°C to 35°C, in the range of 10°C to 30°C, in the range of 15°C to 25°C or in the range of 15°C to 20°C.

89. Method according to any of the preceding paragraphs, wherein the item is soaked in an aqueous solution. 90. Method according to any of the preceding paragraphs, wherein the soaking step comprises exposing the item to the lipase and the cellulase.

91 . Method according to any of the preceding method paragraphs, wherein the item is rinsed after being exposed to the lipase and cellulase.

92. Method according to any of the preceding method paragraphs, wherein the item is rinsed with water optionally comprising a softener.

93. Item treated according to any of the preceding method paragraphs.

Examples

Full scale wash

This is the test method used to test the wash performance of enzymes e.g. proteases, amylases and lipases in full scale wash under EU conditions.

The test swatches and ballast are added to each wash together with detergent and enzyme. After wash, the test swatches are dried and the remission is measured on a MacBeth Color Eye spectrophotometer.

The enzymes are added on basis of weight percent of the detergent dosage in each wash,

Equipment used:

Washing machine: Miele Softtronic W2445

Water meters and automatically data collection system

MacBeth Color Eye spectrophotometer

Scanner-Epson Expression 10000XL

Color Vector software; ADT-AN-028

For the preparation and adjustment of water hardness the following ingredients are needed:

Calcium chloride (CaCI2 « 2H20)

Magnesium chloride (MgCL2 « 6H20)

· Sodium Hydrogen Carbonate (NaHC03)

The test swatches are commercial available ones. The most used suppliers are: CFT, EMPA, wfk and Equest. The same batch of stains is used in all washes in a trial. Different swatch sizes are used:

Commercial swatches cut into 4 x 9cm or equal to 36cm2 (standard)

Swatches at 10x10 cm and some with a circular spot e.g. the Equest swatches The swatches are attached to t-towels by sewing or by a stapler; the same swatch types are placed on different towels or in different positions on the towel. Each swatch is marked individually for identification and to indicate the front side.

For lipase trials: The swatches are attached to T-towels in three corners. The bigger swatch should be floating around in the wash, unattached.

Ballast

The ballast consists of clean white cloth without optical whitener made of cotton, polyester or cotton/polyester. The composition of the ballast is a mix of different items at a cotton/polyester ratio of 65/35 based on weight. The ballast weight, dryness and item composition must be the same in each wash.

After each wash the ballast is inactivated in an industrial washer at 85°C/15 min or in a 95°C wash (EU machine) without detergent

Ballast Example: (Standard EU ballast composition, total 3kg)

· 3 T-shirts (100% cotton)

10 shirts, short sleeves (55% cotton 45% polyester)

4 pillow cases (35% cotton, 65% polyester), 1 10x75cm

1 small bed sheets, size 100x75cm (100% cotton)

3 Tea towels (100% cotton)

· Socks (80% cotton 20% polyester) as balance

Wash conditions

Information about stain/test fabric, number of swatches, detergent, detergent dosage, eventual inactivation of detergent, how to add detergent, enzyme, enzyme dosage, wash temperature, washing time, way of drying, treatment of swatches after termination of trial and information for data treatment if relevant are found in the Study Plan.

• Temperature: (20, 30, 40 or 60°C) is given in the Study Plan.

• Washing programme: Normal cotton wash without pre-wash: "Cottons".

• Water level 13-14L with "water plus"

· Water hardness: std. EU conditions: 15°dH, Ca2+/Mg2+/HC03 - ratio = 4:1 :7.5

• Enzyme dosage: given in study plan. Liquid enzymes: the enzyme amount is solubilised in 100 ml deionised water. Granulated enzymes: the enzyme is added directly or dissolved in deionised water before use; if dissolved, stir 10 min. on a magnetic stirrer. Detailed steps to carry out full scale wash trial

• Select wash program as in study plan.

• The detergent is placed in the wash drum in a "washing ball" (both liquid and powder detergents). Place them at the bottom. • Place the ballast and test swatches in the wash drum.

• Put enzyme solution in a "washing ball" and place it carefully in the drum on the top of the ballast-make sure the enzyme stays in the ball until the water intake.

Note: enzyme should be added in 100ml deionised water before wash

• Start digital water meter

• Start the washer by pressing the knob START

• Measure pH after 20 min from the start. Open the front door and take out manually a small sample. There is 1 -1.5°C temperature increasing after the door is opened but no influence on wash program.

• After wash, take out all ballast and swatches, put swatches into drying room.

• After the wash is completed, the test swatches are removed from the T-towel and placed on the tray for drying, and make sure the swatches are dried in completely darkness, as many stains are sensitive to light.

Drying procedure

Drying room Put stains on tray or hang in line and dry at room temperature. The room has a de-humidifier working for 24h per day to keep the room dry

Measurement

The swatches are measured according to swatch/stain type

If Color Eye is used:

• Measure same swatch type in stacks on grey background

• Some lipase swatches at measured after 24 hours, precisely e.g.: CS-10 and DN-18 If Scanner is used (for greasy stains on blue textile):

· Place and scan 2 swatches at the same time

• Place swatch no. 1 approx. 5cm from edge

• Use the Color Vector program to transfer colours into numbers

Evaluation of stains

Wash performance is expressed as a remission value. After washing and rinsing the swatches were spread out flat and allowed to air dry at room temperature overnight. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches were done using a Macbeth Color Eye 7000 reflectance spectrophotometer with very small aperture. The measurements were made without UV in the incident light and remission at 460 nm was extracted. Measurements were made on unwashed and washed swatches. The test swatch to be measured was placed on top of another swatch of same type and colour (twin swatch). Two swatches of each kind were used per wash, therefore they are used together for measuring one on top of each other. With only one swatch of each kind per beaker, a swatch from a replicate wash was used in this way. Remission values for individual swatches were calculated by subtracting the remission value of the unwashed swatch from the remission value of the washed swatch. The total wash performance for each stained swatch set was calculated as the sum of individual ARem.

Evaluation of the cellulase effect on greasy stains

A specific performance is seen with each formulation, which is obtained as described above. In order to evaluate the additional cellulase effect apart from the lipase effect the following calculation is made:

Cellulase effect = Rem Ceiiuiase+ii ase - Rem u ase

To calculate the combination and the synergy effect the following calculation is made: Combination effect = Rem yipase + Rem Ceiiuiase - Rem base

Synergy effectCeiiuiase + li ase = Rem ceiiuiase+npase - Combination effect

By "base" is meant the detergent composition without any enzymes.

The values that are below 2 points difference are classified as a medium effect while the cells with values above 2 are classified as strong effect. Example 1

The experimental conditions for the experiments are specified in Table 1 , Table 2 and Table 3 below.

Table 1 : Experimental conditions for full scale

Figure imgf000062_0001
Figure imgf000062_0002
Danish base 1 (powder) DK 70 0,2 0,1

Italian base 1 (powder) IT 75 0,2 0,1

German base 1 (powder) DE 65 0,2 0,1

Danish color base 2 (powder) DK 70 0,2 0,1

Spanish base 1 (powder) ES 60 0,2 0,1

English base is composed of: anionic surfactants (5-15%), oxygen-based bleaching agents (5-15%), zeolite (<5%), non-ionic surfactants (<5%), soap (<5%), polycarboxylates (<5%), phosphonates (<5%), optical brighteners (<5%), perfume (<5%).

Danish base 1 is composed of: sodium aluminum silicate, sodium carbonate, sodium sulphate, fatty alcohol ethoxylate-7, polycarboxylates, sodium tallow soap, sodium citrate, CMC, protease, methylene phosphonate, amylase and silicone.

Italian base 1 was analysed and found to be composed of: 3,46% LAS and 2,1 1 % soap plus other components such as builders and co-builders.

German base 1 (WFK ECE-2) base is composed of: 9,7% linear sodium alkyl benzene sulfonate, 5,2% ethoxylated fatty alcohol C12-18 (7 EO), 3,6% sodium soap, 4,5% foam inhibitor (silicone based), 1 1 ,8% sodium aluminium silicate (Zeolith 4A), 1 1 ,8% sodium carbonate, 5,2% sodium salt of a copolymer from acrylic and maleic acid, 3,4% sodium silicate, 1 ,3% carboxymethylcellulose (CMC), 0,8% diethylene triamine penta (methylene phosphonic acid, 9,8% sodium sulphate and 12,2% water.

The Danish color base 1 is composed of 10,5% anionic surfactants, 6% non-ionic surfactants, 52% builders, 12% filler and rest are additives such as perfume or dye trasfer inhibitors.

Spanish base 1 was analysed and found to be composed of: 10,82% LAS and 0.88% soap plus other components such as builders and co-builders.

The remaining compositions are standard commercial detergent compositions for laundering.

Table 3: Swatches with stains used

Producer Code Name Fat content

Equest 64BKC hamburger grease (blue textile)

Equest 125BKC Vegetable oil (blue textile)

Equest 71 BKC Lard (blue textile)

Equest 1 1 BKC Bacon grease (blue textile)

Equest 17BKC Beef dripping (blue textile) WFK W20PF Pigment /veg. Fat >50%

WFK W10D Sebum w. pigment >50%

Equest 68KC Ice Cream chocolate- premium >15%

WFK W10LS Lipstick >80%

CFT C-H1 13 Lard + Sudan Red >90%

WFK W10PPM Pigment/vegt. oil/milk >20%

WFK W10B Oliveoil w. pigment >50%

Equest 132KC Cooked butter >80%

CFT C-S-62 Lard with colorant

CFT C-H126 Vegetable Oil/Violet Dye >90%

WFK W20D Pigment /Sebum >50%

Water hardness was adjusted by addition of CaCI2, MgCI2 and NaHC03 to the test system. After washing, the textiles were rinsed in a Miele washing machine using a rinse program (STIVN). The stained swatches are commercially available from CFT (Center for Testmaterials BV, AC Vlaardingen, the Netherlands) and Equest (Warwick Equest Ltd Consett, County Durham, UK). The ballast is commercially available from WFK (wfk Testgewebe GmbH, Christenfeld 10, D-41379 Brijggen, Germany).

The performance of the enzyme is evaluated by measuring the remission of the textile swatches using the ColorEye at 460 nm. The cellulase effect is shown in Table 4 below.

The underlined cells correspond to the stains where the effect was evident, meaning that the difference in the remission value between 2 textiles is higher than 2 points. For these remission values the human eye will be able to detect the difference. The values that are below 2 points difference are classified as a medium effect while the cells with values above 2 are classified as strong effect.

Table 4: CELLULASE EFFECT POWDER

Figure imgf000064_0001
Equest 17BKC Beef 0,97 -0,84 1 ,04 2,24 2,78 -1 ,26

dripping

WFK W20PF Pigmen 1 ,95 2,15 2,57 0,03 0,97 1 ,24

t /veg.

Fat

WFK W10D Sebum -0,20 1 ,00 1 ,16 -0,89 0,25 -0,27

w.

pigment

Equest 68KC Ice 2,99 -0,34 -2,67 6,61 -0,30 8,80

Cream

(chocol

ate) - premiu

m

WFK W10LS Lipstick 2,31 0,65 0,82 -1 ,23 -0,31 0,90

CFT C-H1 13 Lard + -0,88 -1 ,44 3,50 -1 ,07 2,08 -1 ,40

Sudan

Red

WFK W10PP Pigmen 1 ,89 -0,03 -1 ,52 -0,31 0,33 -0,96

M t/vegt.oi

l/milk

WFK W10B Oliveoil 0,64 0,40 1 ,21 -0,79 0,20 -0,39

w.

pigment

Equest 132KC Cooked 1 ,39 -0,16 1 ,36 -1 ,49 -2,67 -2,31

butter

CFT C-S-62 Lard -0,57 -0,69 0,30 -2,18 -2,39 0,78

with

colorant

CFT C-H126 Vegeta -0,70 1 ,92 1 ,54 2,22 -0,92 0,80

ble

OilA iol

et Dye

WFK W20D Pigmen -0,92 0,36 2,71 -0,65 -0,69 -0,74

t

/Sebum

The data is further illustrated in figures 1 and 2:

Figure 1 shows the remission values obtained after washing with the English base (powder) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is given as the sum of the remission at 460nm for five stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) premium), WFK W10LS (Lipstick), WFK W10PPM (Pigment/veg. oil/milk), Equest 132KC (Cooked butter).

Figure 2 shows the remission values obtained after washing with the Italian base (powder) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is given as the sum of the remission at 460nm for eight stains: WFK W20PF (Pigment/veg. fat), WFK W10D (Sebum w. pigment), CFT C-H1 13 (Lard + Sudan Red), WFK W10B (Olive oil w. pigment), WFK W10LS (Lipstick), WFK W20D (pigment/sebum), CFT C-H126 (Veg. oil/violet dye), Equest 132KC (Cooked butter).

Example 2

The experimental conditions for the experiments are specified in Table 5, Table 6 and

Table 7 below.

Table 5: Experimental conditions for full scale

Figure imgf000066_0001
Table 6: Formulations for full scale

Figure imgf000066_0002

English base is the same as described in Example 1 .

German base 2 was analysed and found to be composed of: 12,00% LAS and 1.13% soap plus other components such as builders and co-builders.

Italian base 2 is composed of: 1 1 .7% anionic surfactants, 4,5% non-ioinic surfactants,

0,5% sodium formate and other additives such as citric acid and preservative.

German base 3 was analysed and found to be composed of: 4,5% AES plus other additives and stabilizers.

Spanish base 2 was analysed and found to be composed of: 13.22% AES plus other additives and stabilizers. Italian base 3 was analysed and found to be composed of: 1 1.05% AES plus other additives and stabilizers

Table 6: Swatches with stains used

Figure imgf000067_0001

Water hardness was adjusted by addition of CaCI2, MgCI2, and NaHC03 to the test system. After washing, the textiles were rinsed in a Miele washing machine using a rinse program (STIVN). The stained swatches are commercially available from CFT. The ballast is commercially available from WFK.

The performance of the enzyme is evaluated by measuring the remission of the textile swatches using the ColorEye at 460 nm. The cellulase effect is shown in Table 7.

The underlined cells correspond to the stains where the effect was evident, meaning that the difference in the remission value between 2 textiles is higher than 2 points. For these remission values the human eye will be able to detect the difference. The values that are below 2 points difference are classified as a medium effect while the cells with values above 2 are classified as strong effect.

Table 7: CELLULASE EFFECT POWDER AND LIQUID

Producer Code Name English German Italian German Spanish Italian base base 2 base 1 base 3 base 2 base 2

Equest 64BKC hamburger 0,3 -1 ,9 4,7 1 ,4 2,8 5,3

Figure imgf000068_0001

beef fat

The data is further illustrated in figures 3-6:

Figure 3 shows the remission value obtained after washing with the German base 2 (powder) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for four stains: WFK W20PF (Pigment/veg. fat), Equest 132KC (Cooked butter), WFK W20D (Pigment/Sebum), CFT C-H078 (lipstick handmade),

Figure 4 shows the remission value obtained after washing with the English base (powder) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for four stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) - premium), CFT C-H078 (lipsiick handmade), Equest 022KC (Black olives).

Figure 5 shows the remission value obtained after washing with the Spanish base 2 (liquid) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for six stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) - premium), WFK W10LS (lipstick), CFT C-G1 13 (Lard + Sudan Red), Equest 132KC (Cooked butter) and Equest 131 KC (Cooked beef fat).

Figure 6 shows the remission value obtained after washing with the Italian base 2 (liquid) without any enzymes and with either lipase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for four stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) - premium), Equest 132KC (Cooked butter) and Equest 131 KC (Cooked beef fat).

Example 3:

The experimental conditions for the experiments are specified in Table 8, Table 9 and Table 10 below.

Figure imgf000069_0001

Table 8: Experimental conditions for full scale

Table 9: Formulations for full scale

Figure imgf000069_0002

The compositions of all the detergents used in this experiment have previously been shown in Example 1 and 2.

The lipase variant of SEQ ID NO: 2 has the following modifications: D27R G38A D96E D1 1 1A G163K T231 R N233R D254S P256T.

Table 10: Swatches with stains used

Figure imgf000069_0003
WFK W20PF Pigment /veg. Fat >50%

Equest 68KC Ice Cream (chocolate) - premium >15%

WFK W10LS Lipstick >80%

CFT C-H1 13 Lard + Sudan Red >90%

Equest 132KC Cooked butter >80%

CFT C-H126 Vegetable Oil/Violet Dye >90%

WFK W20D Pigment /Sebum >50%

EM PA 101 Oliveoil carbon

Equest 022KC Black olives >15%

Equest 131 KC Cooked beef fat >10%

CFT CS-34 Curry

CFT CS-88 Indian curry

Water hardness was adjusted by addition of CaCI2, MgCI2, and NaHC03 to the test system. After washing, the textiles were rinsed in a Miele washing machine using a rinse program (STIVN). The stained swatches are commercially available from CFT. The ballast is commercially available from WFK.

The performance of the enzyme is evaluated by measuring the remission of the textile swatches using the ColorEye at 460 nm. The synergy effect is shown in Table 1 1

The highlighted cells correspond to the stains where the effect was evident. It is commonly known that if the remission value between 2 textiles is higher than 2 points the human eye will be able to detect the difference. That is why the highlighted cells contain values around 2 and higher than 2.

The values that are below 2 points difference are classified as a medium effect while the cells with values above 2 are classified as strong effect. Table 1 1 : SYNERGY EFFECT

Figure imgf000070_0001
WFK W20D Pigment /Sebum 0,94 -0,62 -1 ,27

Equest 132KC Cooked butter -0,92 -0,17 -1 ,50

WFK W10LS Lipstick -0,99 0,89 0,27

CFT C-H1 13 Lard + Sudan Red -0,61 1 ,42 -2,53

Equest 022KC Black olives -0,63 0,91 1 ,51

Equest 131 KC Cooked beef fat -3,00 2,93 0,03

EM PA EMPA 101 Oliveoil carbon 0,75 0,09 -1 ,45

CFT CS-34 Curry -0,04 -0,35 -0,33

CFT CS-88 Indian curry -0,27 0,29 0,00

Figure 7 shows the remission value obtained after washing with the English base (powder) without any enzymes and with lipase, cellulase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for four stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) - premium), WFK W10LS (Lipstick) and CFT C-H126 (Veg. oil/violet dye).

Figure 8 shows the remission value obtained after washing with the Spanish base 2 (liquid) without any enzymes and with either lipase, cellulase or a combination of lipase and cellulase. Data is shown as the sum of the remission at 460nm for four stains: WFK W20PF (Pigment/veg. fat), Equest 68KC (Ice Cream (chocolate) - premium), WFK W10LS (Lipstick) and CFT C-H126 (Veg. oil/violet dye).

Overall it is observed that the following stains show a strong cellulase effect (Example 1 and Example 2) and synergy effect (Example 3). These stains are considered as high fatty stains.

WFK W20PF Pigment/veg.fat

Equest 68KC Ice Cream chocolate

CFT C-H126 Vegetable oil/Violet dye

In two out of the three examples above, the following stains were easier to remove if a lipase and cellulase were present than the enzymes separately: WFK W10LS Lipstick, CFT C- H1 13 Lard + sudan red, Equest 022KC Black olives, Equest 131 KC Cooked beef fat.

Claims

Claims
Detergent composition for removing or releasing greasy stains, which composition comprises a surfactant, a lipase and a cellulase.
Composition according to claim 1 , wherein the lipase is a polypeptide having an amino acid sequence which:
a) has at least 90% identity with SEQ ID NO: 2
b) compared to said SEQ ID NO: 2 comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three- dimensional structure within 15 A of E1 or Q249 with a positively charged amino acid; and
c) comprises a peptide addition at the C-terminal; and/or
d) meets the following limitations:
i. comprises a negative amino acid in position E210 of said SEQ ID NO: 2; comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said SEQ ID NO: 2; and
ii. comprises a neutral or negative amino acid at a position corresponding to N94 of said SEQ ID NO: 2 and/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said SEQ ID NO: 2.
Composition according to any of the preceding claims, wherein the lipase is a variant of a parent lipase which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R of SEQ ID NO: 2.
Composition according to any of the preceding claims, wherein the cellulase is comprised by the enzyme classification EC 3.2.1 .4.
Composition according to any of the preceding claims, wherein the cellulase is an enzyme exhibiting endo-beta-1 ,4-glucanase activity (EC 3.2.1.4), which is selected from one of
d) a polypeptide encoded by the DNA sequence of positions 1 to 2322 of SEQ ID NO: 5;
e) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO: 5 under conditions wherein the DNA sequence is expressed; f) an endo-beta-1 ,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 6; or a fragment thereof that has endo-beta-1 ,4-glucanase activity, when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
6. Composition according to any of the preceding claims, wherein the composition comprises granules, which granules comprises a core and a protective coating, wherein: f. the core comprises an enzyme, which is lipase and/or cellulase, and
g. the core or the coating comprises a reducing agent or a peroxide decomposing catalyst or an antioxidant, and
h. the core comprises a salt of a multivalent cation, and
i. the core or the coating comprises an acidic buffer component, and
j. the coating comprises a salt.
7. Use of a detergent composition according to any of claims 1 -6 for removing or releasing a greasy stain
8. Use according to claim 7, wherein the greasy stain is removed or released from a textile having a greasy stain, from a dish having a greasy stain or from a hard surface having a greasy stain.
9. Use according to any of the preceding use claims, wherein the stain has a low-medium content of lipid, a medium content of lipid or a high content of lipid.
10. Use according to any of the preceding claims, wherein the cellulase effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points.
1 1 . Use according to any of the preceding claims, wherein the synergy effect is at least 2 points, at least 2.2 points, at least 2.4 points, at least 2.6 points, at least 2.8 points, at least 3.0 points, at least 3.5 points, at least 4.0 points or at least 5.0 points.
12. A method for removing or releasing greasy stains from an item having a greasy stain, which method comprises exposing the item to an aqueous solution of a lipase and a cellulase, wherein the item is a textile, a dish or a hard surface
13. A method according to claim 12, which method comprises exposing the item to an aqueous solution of a detergent composition of claims 1 -6.
14. Method according to any of the preceding claims, wherein the item is soaked in an aqueous solution, optionally comprising lipase and cellulase, the item is then exposed to the aqueous solution of a lipase and a cellulase and the item is rinsed with water optionally comprising a softener.
15. Item treated according to any of the preceding method claims.
PCT/EP2014/071231 2013-10-03 2014-10-03 Detergent composition and use of detergent composition WO2015049370A1 (en)

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