WO2007087242A2

WO2007087242A2 - A composition comprising a lipase and a bleach catalyst

Info

Publication number: WO2007087242A2
Application number: PCT/US2007/001593
Authority: WO
Inventors: Philip Frank Souter; Neil Joseph Lant; Alan Thomas Brooker; Gregory Scot Miracle; Nicola Jane Binney; David Lee Daugherty
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 2006-01-23
Filing date: 2007-01-22
Publication date: 2007-08-02
Anticipated expiration: 2008-07-23
Also published as: US20070173430A1; US7786067B2; AR059154A1; EP1979456A2; CA2635927A1; WO2007087242A3; CN101370921B; JP2009523900A; CN101370921A

Abstract

The present invention relates to a composition comprising: (i) a lipase; and (ii) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate.

Description

A COMPOSITION COMPRISING A LIPASE AND A BLEACH CATALYST

FIELD OF THE INVENTION The present invention relates to a composition comprising a lipase and a bleach catalyst.

More specifically, the present invention relates to composition comprising a lipase and a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate. The compositions of the present invention are typically suitable for use as laundry detergent compositions and exhibit a good cleaning performance and a reduced malodor profile, especially on problematic residual dairy soils.

BACKGROUND OF THE INVENTION

Dingy soils such as body soils and other hydrophobic soils, including dairy soils, are extremely difficult to remove from fabric during a laundering process. The appearance of lipase enzymes suitable for detergent applications in the 1980's (e.g. Lipolase and Lipolase Ultra, ex Novo Nordisk — now Novozymes) gave the formulator a new approach to improve grease removal. Lipase enzymes catalyse the hydrolysis of triglycerides which form a major component of many commonly encountered fatty soils such as sebum, animal fats (e.g. lard, ghee, butter) and vegetable oils (e.g. olive oil, sunflower oil, peanut oil). However, these enzymes show limited performance in the first wash cycle (being effective mainly during the drying stage of the laundering process) and give rise to a post-wash malodor. Without wishing to be bound by theory, the malodor arises from fatty acids released by the hydrolysis of fats and is particularly noticeable for dairy soils like milk, cream, butter and yogurt; dairy fats contain triglycerides functionalized with short chain (e.g. C₄) fatty acyl units which release malodorous volatile fatty acids after lipolysis. For a general review of the use of lipases in solid laundry detergents see the following reference: Enzymes in Detergency, ed. J.H. van Ee et al, VoI 69 Marcel Dekker Surfactant Series, Marcel Dekker. New York, 1997, pp93-132 (ISBN 0-8247-9995-X).

More recently so-called 'first wash' lipases have been commercialised such as Lipoprime™ and Lipex™ (ex. Novozymes) which show performance benefits in the initial wash cycle. The Lipex™ enzyme is described in more detail in WO 00/60063 and US 6,939,702 Bl (Novozymes). Laundry detergent formulations comprising the Lipex™ enzyme are described in more detail in IP.com publication IP 6443D (Novozymes). However in order to better exploit lipase technology, both the odour profile on residual dairy stains and the cleaning performance on complex soils still needs to be improved.

Detergent manufacturers have also attempted to incorporate bleach catalysts, especially oxaziridium or oxaziridinium-forming bleach catalysts, in their detergent products in an attempt to provide a good bleaching performance. EP 0 728 181, EP 0 728 182, EP 0 728 183, EP 0 775 192, US 4,678,792, US 5,045,223, US 5,047,163, US 5,360,568, US 5,360,569, US 5,370,826, US 5,442,066, US 5,478,357, US 5,482,515, US 5,550,256, US 5,653,910, US 5,710,116, US 5,760, 222, US 5,785,886, US 5,952,282, US 6,042,744, WO95/13351, WO95/13353, WO97/10323, WO98/16614, WO00/42151, WO00/42156, WO01/16110, WO01/16263, WO01/16273, WO01/16274, WO01/16275, WO01/16276, WO01/16277 relate to detergent compositions comprising an oxaziriduium and/or an oxaziridinium-forming bleach catalyst.

There is a continuing need for laundry detergent compositions that exhibit a good overall cleaning profile, a good cold water temperature bleaching performance, good greasy soil cleaning performance and a reduced malodor profile on residual fatty soils, especially dairy soils. The Inventors have found that by using lipase in combination with a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate improves the cleaning performance of the detergent composition whilst maintaining a reduced malodor profile on residual fatty soils, especially dairy soils.

In another embodiment of the present invention, the Inventors have found that the rubber sump hose compatibility profile is improved when a diacyl and/or a tetraacyl peroxide species is in combination with a lipase.

In an especially preferred embodiment of the present invention, the Inventors have found that using a lipase in combination with (i) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate and (ii) a diacyl and/or tetraacyl peroxide species, significantly improves the cleaning performance of the composition, reduces the malodor profile of the composition and improves the rubber sump hose compatibility profile of the composition.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the alignment of lipases. SEQUENCE LISTINGS

SEQ ID NO: 1 shows the DNA sequence encoding lipase from Thermomyces lanoginosus.

SEQ ID NO: 2 shows the amino acid sequence of a lipase from Thermomyces lanoginosus.

SEQ ID NO: 3 shows the amino acid sequence of a lipase from Absidia reflexa. SEQ ID NO: 4 shows the amino acid sequence of a lipase from Absidia corymbifera.

SEQ ID NO: 5 shows the amino acid sequence of a lipase from Rhizomucor miehei.

SEQ ID NO: 6 shows the amino acid sequence of a lipase from Rhizopus orγzae.

SEQ ID NO: 7 shows the amino acid sequence of a lipase from Aspergillus niger.

SEQ ID NO: 8 shows the amino acid sequence of a lipase from Aspergillus tubingensis. SEQ ID NO: 9 shows the amino acid sequence of a lipase from Fusarium oxysporrum.

SEQ ID NO: 10 shows the amino acid sequence of a lipase from Fusarium heterosporum.

SEQ ID NO: 11 shows the amino acid sequence of a lipase from Aspergillus oryzae.

SEQ ID NO: 12 shows the amino acid sequence of a lipase from Penicillium camemberti.

SEQ ID NO: 13 shows the amino acid sequence of a lipase from Aspergillus foetidus. SEQ ID NO: 14 shows the amino acid sequence of a lipase from Aspergillus niger.

SEQ ID NO: 15 shows the amino acid sequence of a lipase from Aspergillus oryzae.

SEQ ID NO: 16 shows the amino acid sequence of a lipase from Landerina penisapora.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a composition comprising: (i) a lipase; and (ii) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate.

In a second embodiment, the present invention provides a composition comprising: (i) a lipase; and (ii) a diacyl and/or tetraacyl peroxide species.

DETAILED DESCRIPTION OF THE INVENTION

Composition The composition comprises: (i) a lipase; and (ii) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate. The lipase and the bleach catalyst are described in more detail below. The composition may be suitable for use as a laundry detergent composition, laundry additive composition, dish-washing composition, or hard surface cleaning composition. The composition is typically a detergent composition. The composition may be a fabric treatment composition. Preferably the composition is a laundry detergent composition. The composition can be any form such as liquid or solid, although preferably the composition is in solid form. Typically, the composition is in particulate form such as an agglomerate, a spray-dried powder, an extrudate, a flake, a needle, a noodle, a bead, or any combination thereof. The composition may be in compacted particulate form, such as in the form of a tablet or bar. The composition may be in some other unit dose form, such as in the form of a pouch, wherein the composition is typically at least partically, preferably essentially completely, enclosed by a water-soluble film such as polyvinyl alcohol. Preferably, the composition is in free-flowing particulate form; by free-flowing particulate form, it is typically meant that the composition is in the form of separate discrete particles. The composition may be made by any suitable method including agglomeration, spray-drying, extrusion, mixing, dry-mixing, liquid spray-on, roller compaction, spheronisation, tabletting or any combination thereof.

The composition typically has a bulk density of from 450g/l to l,000g/l, preferred low bulk density detergent compositions have a bulk density of from 550g/l to 650g/l and preferred high bulk density detergent compositions have a bulk density of from 750g/l to 900g/l. The composition may also have a bulk density of from 650g/l to 750g/l. During the laundering process, the composition is typically contacted with water to give a wash liquor having a pH of from above 7 to less than 13, preferably from above 7 to less than 10.5. This is the optimal pH to provide good cleaning whilst also ensuring a good fabric care profile.

Preferably, the composition comprises: (i) from 0% to less than 10%, preferably to 7%, or to 4%, or from 1%, or from 1.5%, by weight of the composition, of tetraacetylethylenediamine and/or oxybenzene sulphonate bleach activators. Most preferably, the composition is essentially free of tetraacetylethylenediamine and/or oxybenzene sulphonate bleach activators. By "is essential free of it is typically meant "comprises no deliberately incorporated". Keeping the levels of these types of bleach activators to a minimum maintains the good dye safety profile of the composition. Preferably, upon contact with water the composition forms a wash liquor having a pH of from 7 to 10.5. Compositions having this reserve alkalinity profile and pH profile exhibit a good stability profile for lipase. Preferably, the composition comprises from 0% or from 1%, or from 2%, or from 3%, or from 4%, or from 5%, and to 30%, or to 20%, or to 10%, by weight of the composition, of a source of carbonate anion. The above described levels of a source of carbonate anion ensure that the composition has a good overall cleaning performance and a good bleaching performance. Preferably, the composition comprises a dye transfer inhibitor. Suitable dye transfer inhibitors are selected from the group consisting of: polyvinylpyrrolidone, preferably having a weight average molecular weight of from 40,000Da to 80,000 Da, preferably from 50,000Dl to 70,000Da; polyvinylimidazole, preferably having a weight average molecular weight of from 10,000Da to 40,000 Da, preferably from 15,000Da to 25,000Da; polyvinyl pyridine N-oxide polymer, preferably having a weight average molecular weight of from 30,000Da to 70,000Da, preferably from 40,000Da to 60,000Da; a co-polymer of polyvinylpyrrolidone and vinyl imidazole, preferably having a weight average molecular weight of from 30,000Da to 7O₃OOODa, preferably from 40,000Da to 60,000Da; and any combination thereof. Compositions comprising a dye transfer inhibitor show a further improved dye safety profile. The composition may comprise from 0% to less than 5%, preferably to 4%, or to 3%, or to

2%, or even to 1%, by weight of the composition, of zeolite-builder. Whilst the composition may comprise zeolite-builder at a level of 5wt% or greater, preferably the composition comprises less than 5wt% zeolite-builder. It may be preferred for the composition to be essentially free of zeolite-builder. By: "essentially free of zeolite -builder", it is typically meant that the composition comprises no deliberately incorporated zeolite-builder. This is especially preferred when the composition is a solid laundry detergent composition and it is desirable for the composition to be very highly soluble, to minimize the amount of water-insoluble residues (for example, which may deposit on fabric surfaces), and also when it is highly desirable to have transparent wash liquor. Suitable zeolite-builders include zeolite A, zeolite X, zeolite P and zeolite MAP.

The composition may comprise from 0% to less than 10%, or less than 5%, preferably to 4%, or to 3%, or to 2%, or even to 1%, by weight of the composition, of phosphate-builder. Whilst the composition may comprise phosphate-builder at a level of 10wt% or greater, preferably the composition comprises less than 10wt% phosphate-builder. It may even be preferred for the composition to be essentially free of phosphate-builder. By: "essentially free of phosphate-builder", it is typically meant that the composition comprises no deliberately added phosphate-builder. This is especially preferred if it is desirable for the composition to have a very good environmental profile. Suitable phosphate-builders include sodium tripolyphosphate.

The composition may comprise from 0% to less than 5%, or preferably to 4%, or to 3%, or even to 2%, or to 1%, by weight of the composition, of silicate salt. Whilst the composition may comprise silicate salt at a level of 5wt% or greater, preferably the composition comprises less than 5wt% silicate salt. It may even be preferred for the composition to be essentially free of silicate salt. By: "essentially free from silicate salt", it is typically meant that the composition comprises no deliberately added silicate salt. This is especially preferred when the composition is a solid laundry detergent composition and it is desirable to ensure that the composition has very good dispensing and dissolution profiles and to ensure that the composition provides a clear wash liquor upon dissolution in water. The silicate salts include water-insoluble silicate salts. The silicate salts also include amorphous silicate salts and crystalline layered silicate salts (e.g. SKS- 6). The silicate salts include sodium silicate.

The composition typically comprises adjunct ingredients. These adjunct ingredients include: detersive surfactants such as anionic detersive surfactants, non-ionic detersive surfactants, cationic detersive surfactants, zwitterionic detersive surfactants, amphoteric detersive surfactants; preferred anionic detersive surfactants are alkoxylated anionic detersive surfactants such as linear or branched, substituted or unsubstituted C ₁₂- is alkyl alkoxylated sulphates having an average degree of alkoxylation of from 1 to 30, preferably from 1 to 10, more preferably a linear or branched, substituted or unsubstituted C12-₁₈ alkyl ethoxylated sulphates having an average degree of ethoxylation of from 1 to 10, most preferably a linear unsubstituted C₁₂-₁₈ alkyl ethoxylated sulphates having an average degree of ethoxylation of from 3 to 7, other preferred anionic detersive surfactants are alkyl sulphates, alkyl sulphonates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates or any mixture thereof, preferred alkyl sulphates include linear or branched, substituted or unsubstituted Cio-is alkyl sulphates, another preferred anionic detersive surfactant is a C_IO-_B linear alkyl benzene sulphonate; preferred non-ionic detersive surfactants are C_8- 18 alkyl alkoxylated alcohols having an average degree of alkoxylation of from 1 to 20, preferably from 3 to 10, most preferred are C12-1₈ alkyl ethoxylated alcohols having an average degree of alkoxylation of from 3 to 10; preferred cationic detersive surfactants are mono- Cδ-is alkyl mono-hydroxyethyl di-methyl quaternary ammonium chlorides, more preferred are mono-Cg-io alkyl mono-hydroxyethyl di-methyl quaternary ammonium chloride, mono-Cio-12 alkyl mono-hydroxyethyl di-methyl quaternary ammonium chloride and mono-Cio alkyl mono- hydroxyethyl di-methyl quaternary ammonium chloride; source of peroxygen'such as percarbonate salts and/or perborate salts, preferred is sodium percarbonate, the source of peroxygen is preferably at least partially coated, preferably completely coated, by a coating ingredient such as a carbonate salt, a sulphate salt, a silicate salt, borosilicate, or mixtures, including mixed salts thereof; bleach activators such as tetraacetyl ethylene diamine, oxybenzene sulphonate bleach activators such as nonanoyl oxybenzene sulphonate, caprolactam bleach activators, imide bleach activators such as N-nonanoyl-N-methyl acetamide; enzymes such as amylases, arabinases, xylanases, galactanases, glucanases, carbohydrases, cellulases, laccases, oxidases, peroxidases, proteases, glucanases, pectate lyases and mannanases, especially preferred are proteases; suds suppressing systems such as silicone based suds suppressors; fluorescent whitening agents; photobleach; filler salts such as sulphate salts, preferably sodium sulphate; fabric-softening agents such as clay, silicone and/or quaternary ammonium compounds, especially preferred is montmorillonite clay optionally in combination with a silicone; fJocculants such as polyethylene oxide; dye transfer inhibitors such as polyvinylpyrrolidone, poly A- vinylpyridine N-oxide and/or co-polymer of vinylpyrrolidone and vinylimidazole; fabric integrity components such as hydrophobically modified cellulose and oligomers produced by the condensation of imidazole and epichlorhydrin; soil dispersants and soil anti-redeposition aids such as alkoxylated polyamines and ethoxylated ethyleneimine polymers; anti-redeposition . components such as carboxymethyl cellulose and polyesters; perfumes; sulphamic acid or salts thereof; citric acid or salts thereof; carbonate salts, especially preferred is sodium carbonate; and dyes such as orange dye, blue dye, green dye, purple dye, pink dye, or any mixture thereof.

A second embodiment of the present invention relates to a composition comprising: (i) a lipase; and (ii) a diacyl peroxide.

Lipase

The lipase of the composition of the present invention is a lipase variant with no C- terminal extension but with mutations introduced in certain regions of a parent lipase whereby the tendency to odor generation is reduced. Parent lipase

The parent lipase may be a fungal lipase with an amino acid sequence having at least 50 % homology as defined in the section "Homology and augment" to the sequence of the T. lanuginosus lipase shown in SEQ ID NO: 2. The parent lipase may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia,

Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide.

In a preferred aspect, the parent lipase is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having lipase activity. In another preferred aspect, the parent lipase is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus turbigensis, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Thermomyces lanoginosus (synonym: Humicola lanuginose), Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium puφurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another preferred aspect, the parent lipase is a Thermomyces lipase. In a more preferred aspect, the parent lipase is a Thermomyces lanuginosus lipase. In an even more preferred embodiment the parent lipase is the lipase of SEQ ID NO: 2.

Identification of regions and substitutions. The positions referred to in Region I through Region TV below are the positions of the amino acid residues in SEQ ID NO:2. To find the corresponding (or homologous) positions in a different lipase, the procedure described in "Homology and alignment" is used.

Substitutions in Region I

Region I consists of amino acid residues surrounding the N-terminal residue El. In this region it is preferred to substitute an amino acid of the parent lipase with a more positive amino acid. Amino acid residues corresponding to the following positions are comprised by Region I: 1 to 11 and 223-239. The following positions are of particular interest: 1, 2, 4, 8, 11, 223, 227, 229, 231 , 233, 234 and 236. In particular the following substitutions have been identified: XlN/*, X4V, X227G, X23 IR and X233R.

In a preferred embodiment the parent lipase has at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2 . In a most preferred embodiment the parent lipase is identical to SEQ ID NO: 2.

Substitutions in Region II

Region II consists of amino acid residues in contact with substrate on one side of the acyl chain and one side of the alcohol part. In this region it is preferred to substitute an amino acid of the parent lipase with a more positive amino acid or with a less hydrophobic amino acid. Amino acid residues corresponding to the following positions are comprised by Region II: 202 to 211 and 249 to 269. The following positions are of particular interest : 202, 210, 211, 253, 254, 255, 256, 259. In particular the following substitutions have been identified: X202G, X210K/VWA, X255Y/V/A, X256K/R and X259G/M/Q/V.

In a preferred embodiment the parent lipase has at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2. In a most preferred embodiment the parent lipase is identical to SEQ ID NO: 2.

Substitutions in Region III

Region III consists of amino acid residues that form a flexible structure and thus allowing the substrate to get into the active site. In this region it is preferred to substitute an amino acid of the parent lipase with a more positive amino acid or a less hydrophobic amino acid. Amino acid residues corresponding to the following positions are comprised by Region III: 82 to 102. The following positions are of particular interest: 83, 86, 87, 90, 91, 95, 96, 99. In particular the following substitutions have been .identified: X83T, X86V and X90A/R.

Substitutions in Region IV

Region IV consists of amino acid residues that bind electrostatically to, a surface. In this region it is preferred to substitute an amino acid of the parent lipase with a more positive amino acid. Amino acid residues corresponding to the following positions are comprised by Region IV:

27 and 54 to 62. The following positions are of particular interest: 27, 56, 57, 58, 60. In particular the following substitutions have been identified: X27R, X58N/AG/T/P and

X60V/S/G/N/R/K7A/L.

Amino acids at other positions

The parent lipase may optionally comprise substitutions of other amino acids, particularly less than 10 or less than 5 such substitutions. Examples are substitutions corresponding to one or more of the positions 24, 37, 38, 46, 74, 81, 83, 115, 127, 131, 137, 143, 147, 150, 199, 200, 203, 206, 211, 263, 264, 265, 267 and 269 of the parent lipase. In a particular embodiment there is a substitution in at least one of the positions corresponding to position 81, 143, 147, 150 and 249. In a preferred embodiment the at least one substitution is selected from the group consisting of X81Q/E, X143S/C/N/D/A, X147M/Y, X150G/K and X249R/I/L.

The variant may comprise substitutions outside the defined Regions I to IV, the number of substitutions outside of the defined Regions I to 3V is preferably less than six, or less than five, or less than four, or less than three, or less than two, such as five, or four, or three, or two or one. Alternatively, the variant does not comprise any substitution outside of the defined Regions I to IV. Further substitutions may, e.g., be made according to principles known in the art, e.g. substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202. Parent lipase variants

In one aspect, said variant, when compared to said parent, comprising a total of at least three substitutions, said substitutions being selected from one or more of the following groups of substitutions: a) at least two, or at least three, or at least four, or at least five, or at least six, such as two, three, four, five or six, substitutions in Region I, b) at least one, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six, substitution in Region II, c) at least one, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six, substitution in Region III, d) and/or at least one, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six, substitution in Region IV.

The variant may comprise substitutions, compared to the variant's parent, corresponding to those substitutions listed below in Table 1.

Table 1 : Some particular variants.

In a further particular embodiment the parent lipase is identical to SEQ ID NO:2, and the variants of Table 1 will thus be:

Table 2: Some particular variants of SEQ ID NO:2 Nomenclature for amino acid modifications

In describing lipase variants according to the invention, the following nomenclature is used for ease of reference: Original amino acid(s):position(s): substituted amino acid(s) According to this nomenclature, for instance the substitution of glutamic acid for glycine in position 195 is shown as G195E. A deletion of glycine in the same position is shown as

G195*, and insertion of an additional amino acid residue such as lysine is shown as Gl 95GK.

Where a specific lipase contains a "deletion" in comparison with other lipases and an insertion is made in such a position this is indicated as *36D for insertion of an aspartic acid in position 36. Multiple mutations are separated by pluses, i.e.: R170Y+G195E, representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.

X231 indicates the amino acid in a parent polypeptide corresponding to position 231, when applying the described alignment procedure. X231R indicates that the amino acid is replaced with R. For SEQ ID NO:2 X is T, and X231R thus indicates a substitution of T in position 231 with R. Where the amino acid in a position (e.g. 231) may be substituted by another amino acid selected from a group of amino acids, e.g. the group consisting of R and P and Y, this will be indicated by X231R/P/Y.

In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.

Amino acid grouping

In this specification, amino acids are classified as negatively charged, positively charged or electrically neutral according to their electric charge at pH 10. Thus, negative amino acids are E, D₅ C (cysteine) and Y, particularly E and D. Positive amino acids are R, K and H, particularly R and K. Neutral amino acids are G, A, V, L, I₅ P, F₅ W₅ S, T, M, N₅ Q and C when forming part of a disulfide bridge. A substitution with another amino acid in the same group (negative, positive or neutral) is termed a conservative substitution.

The neutral amino acids may be divided into hydrophobic or non-polar (G, A, V, L, I, P₅ F, W and C as part of a disulfide bridge) and hydrophilic or polar (S, T₃ M, N, Q). Amino acid identity

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For purposes of the present invention, the alignment of two amino acid sequences is determined by using the Needle program from the EMBOSS package (http://emboss.org) version

2.8.0. The Needle program implements the global alignment algorithm described in Needleman,

S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453. The substitution matrix used is

BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the present invention ("invention sequence"; e.g. amino acids 1 to 269 of SEQ ID NO:2) and a different amino acid sequence ("foreign sequence") is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence" or the length of the "foreign sequence", whichever is the shortest. The result is expressed in percent identity.

An exact match occurs when the "invention sequence" and the "foreign sequence" have identical amino acid residues in the same positions of the overlap. The length of a sequence is the number of amino acid residues in the sequence (e.g. the length of SEQ ID NO:2 is 269).

The parent lipase has an amino acid identity of at least 50 % with the T. lanuginosus lipase (SEQ ID NO: 2), particularly at least 55 %, at least 60 %, at least 75 %, at least 85 % , at least 90 %, more than 95 % or more than 98 %. In a particular embodiment the parent lipase is identical to the T. lanuginosus lipase (SEQ ID NO:2).

The above procedure may be used for calculation of identity as well as homology and for alignment. In the context of the present invention homology and alignment has been calculated as described below.

Homology and alignment For purposes of the present invention, the degree of homology may be suitably determined 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 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. In the present invention, corresponding (or homologous) positions in the lipase sequences of Absidia reflexa, Absidia corymbefera, Rhizmucor miehei, Rhizopus delemar, Aspergillus niger, Aspergillus tubigensis, Fusarium oxysporum, Fusarium heterosporum, Aspergillus oryzea, Penicilium camembertii, Aspergillus foetidus, Aspergillus niger, Thermomyces lanoginosus (synonym: Humicola lanuginose) and Landerina penisapora are defined by the alignment shown in Figure 1.

To find the homologous positions in lipase sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in Figure 1. The new sequence is aligned to the present alignment in Figure 1 by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is 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 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-45). The following settings are used for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1 The parent lipase has a homology of at least 50 % with the T. lanuginosus lipase (SEQ

ID NO: 2), particularly at least 55 %, at least 60 %, at least 75 %, at least 85 % , at least 90 %, more than 95 % or more than 98 %. In a particular embodiment the parent lipase is identical to the T. lanuginosus lipase (SEQ ID NO:2).

Hybridization

The present invention also relates to isolated polypeptides having lipase activity which are encoded by polynucleotides which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 178 to 660 of SEQ ID NO: 1, (ii) the cDNA sequence contained in nucleotides 178 to 660 of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). A subsequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has lipase activity. For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42⁰C in 5X SSPE, 0.3% SDS, 200 ug/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC₅ 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50⁰C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70⁰C (very high stringency).

DNA sequence, Expression vector, Host cell, Production of lipase

The invention provides a DNA sequence encoding the lipase of the invention, an expression vector harboring the DNA sequence, and a transformed host cell containing the DNA sequence or the expression vector. These may be obtained by methods known in the art.

The invention also provides a method of producing the lipase by culturing the transformed host cell under conditions conducive for the production of the lipase and recovering the lipase from the resulting broth. The method may be practiced according to principles known in the art. Lipase activity

- Lipase activity on tributyrin at neutral pH (LU)

A substrate for lipase is prepared by emulsifying tributyrin (glycerin tributyrate) using gum Arabic as emulsifier. The hydrolysis of tributyrin at 30 ⁰C at pH 7 or 9 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 micro mol butyric acid/min at pH 7.

- Benefit Risk

The Benefit Risk factor describing the performance compared to the reduced risk for odour smell is defined as: BR = RP_aVg / R. Lipase variants described herein may have BRs greater than 1, greater than 1.1, or even greater than 1 to about 1000. -Average Relative Performance The procedure for calculating average relative performance (RPavg) is found in Example 5 of the present specification. Lipase variants described herein may have (RPavg) of at least 0.8, at least

1.1 , at least 1.5, or even at least 2 to about 1000.

« Bleach catalyst

The bleach catalyst is capable of accepting an oxygen atom from a peroxyacid and/or salt thereof, and transferring the oxygen atom to an oxidizeable substrate. Suitable bleach catalysts include, but are not limited to: iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acyl imines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof.

Suitable iminium cations and polyions include, but are not limited to, N-methyl-3,4- dihydroisoquinolinium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (see, for example, compound 4, p. 433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared as described in U.S. Pat. 5,360,569 (see, for example, Column 11, Example 1); and N-octyl-3,4-dihydrσisoquinolinium p-toluene sulphonate, prepared as described in U.S. Pat. 5,360,568 (see, for example, Column 10, Example 3).

Suitable iminium zwitterions include, but are not limited to, N-(3-sulfoρropyl)-3,4- dihydroisoquinolinium, inner salt, prepared as described in U.S. Pat. 5,576,282 (see, for example, Column 31, Example II); N-[2-(sulρhooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in U.S. Pat. 5,817,614 (see, for example, Column 32, Example V); 2-[3- [(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in WO05/047264 (see, for example, page 18, Example 8), and 2-[3-[(2- butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinoliniurn, inner salt.

Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1 ,2,3,4- tetrahydro-2-methyl-l -isoquinolinol, which can be made according to the procedures described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfer catalysts include, but are not limited to, sodium l-hydroxy-N-oxy-N-[2- (sulphooxy)decyl]-l,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but are not limited to, 3- methyl- 1 ,2-benzisothiazole 1 , 1 -dioxide, prepared according to the procedure described in the Journal of Organic Chemistry (1990), 55(4), 1254-61. Suitable N-phosphonyl imine oxygen transfer catalysts include, but are not limited to, [R- (E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)- phosphinic amide, which can be made according to the procedures described in the Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70. Suitable N-acyl imine oxygen transfer catalysts include, but are not limited to, [N(E)]-N-

(phenylmethylene)acetamide. which can be made according to the procedures described in Polish Journal of Chemistry (2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but are not limited to, 3- methyl-4-phenyl-l ,2,5-thiadiazole 1 ,1 -dioxide, which can be made according to the procedures described in U.S. Pat. 5,753,599 (Column 9, Example 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)- 2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride, which can be made according to the procedures described in Tetrahedron Letters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but are not limited to, l,2:4,5-di-0-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose as prepared in U.S. Pat. 6,649,085 (Column 12, Example 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonyl functional group and is typically capable of forming an oxaziridinium and/or dioxirane functional group upon acceptance of an oxygen atom, especially upon acceptance of an oxygen atom from a peroxyacid and/or salt thereof. Preferably, the bleach catalyst comprises an oxaziridinium functional group and/or is capable of forming an oxaziridinium functional group upon acceptance of an oxygen atom, especially upon acceptance of an oxygen atom from a peroxyacid and/or salt thereof. Preferably, the bleach catalyst comprises a cyclic iminium functional group, preferably wherein the cyclic moiety has a ring size of from five to eight atoms (including the nitrogen atom), preferably six atoms. Preferably, the bleach catalyst comprises an aryliminium functional group, preferably a bi-cyclic aryliminium functional group, preferably a 3,4-dihydroisoquinoIinium functional group. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary oxaziridinium functional group upon acceptance of an oxygen atom, especially upon acceptance of an oxygen atom from a peroxyacid and/or salt thereof.

Preferably, the bleach catalyst has a chemical structure corresponding to the following chemical formula

wherein: n and m are independently from O to 4, preferably n and m are both 0; each R¹ is independently selected from a substituted or unsubstituted radical selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fused heterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto, carboxylic₃ and carboalkoxy radicals; and any two vicinal R¹ substituents may combine to form a fused aryl, fused carbocyclic or fused heterocyclic ring; each R² is independently selected from a substituted or unsubstituted radical independently selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups, carboxyalkyl groups and amide groups; any R² may be joined together with any other of R to form part of a common ring; any geminal R² may combine to form a carbonyl; and any two R² may combine to form a substituted or unsubstituted fused unsaturated moiety; R³ is a Ci to C20 substituted or unsubstituted alkyl; R⁴ is hydrogen or the moiety Q_t-A, wherein: Q is a branched or unbranched alkylene, t = 0 or 1 and A is an anionic group selected from the group consisting of OSOs^", SO₃ ^", CO₂ ^", OCO₂ ^", OPO₃ ^2", OPO₃H^" and OPO₂ ^"; R⁵ is hydrogen or the moiety -CR¹ 'R¹²-Y-G_b-Y_c- [(CR⁹R¹⁰)y-O]_k-R⁸, wherein: each Y is independently selected from the group consisting of O, S, N-H₃ or N-R⁸; and each R⁸ is independently selected from the group consisting of alkyl, aryl and heteroaryl, said moieties being substituted or unsubstituted, and whether substituted or unsubsituted said moieties having less than 21 carbons; each G is independently selected from the group consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ are independently selected from the group consisting of H and C₁-C4 alkyl; R¹¹ and R¹² are independently selected from the group consisting of H and alkyl, or when taken together may join to form a carbonyl; b = O or 1 ; c can = O or 1 , but c must = O if b = O; y is an integer from 1 to 6; k is an integer from O to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety; said moieties being substituted or unsubstituted; and X, if present, is a suitable charge balancing counterion, preferably X is present when R⁴ is hydrogen, suitable X, include but are not limited to: chloride, bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate, borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has a structure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbon atoms (including the branching carbon atoms) or a linear alkyl group containing from one to 24 carbon atoms; preferably R¹³ is a branched alkyl group containing from eight to 18 carbon atoms or linear alkyl group containing from eight to eighteen carbon atoms; preferably R¹³ is selected from the group consisting of 2-propylheptyI, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl; preferably R¹³ is selected from the group consisting of 2-butyloctyl, 2-pentylnonyl₃ 2-hexyldecyl, iso-tridecyl and iso-pentadecyl .

Oxybenzene sulphonate and/or oxybenzoic bleach activators

The composition preferably comprises (i) oxybenzene sulphonate bleach activators and/or oxybenzoic bleach activators and (ii) a source of peroxygen. Typically, the oxybenzoic acid bleach activator is in its salt form. Preferred oxybenzene sulphonate bleach activators include bleach activators having the general formula:

R-(C=O)-L wherein R is an alkyl group, optionally branched, having, when the bleach activator is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and L is leaving group. Examples of suitable leaving groups are benzoic acid and derivatives thereof, especially salts thereof. Another especially preferred leaving group is oxybenzene sulphonate. Suitable bleach activators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate, a salt of decanoyl oxybenzoic acid, 3,5,5-trimethyl hexanoyloxybenzene sulphonate, nonanoylamidocaproyloxybenzene sulphonate, and nonanoyloxybenzene sulphonate (NOBS). Suitable bleach activators are also disclosed in WO 98/17767. The incorporation of these bleach activators into the composition is especially preferred when the composition comprises low levels of zeolite builder and phosphate builder. The Inventors have found that combining these bleach activators with a source of peroxygen and a bleach catalyst as described in more detail above and a lipase, especially in an under-built detergent composition (such as a detergent composition comprising low levels of zeolite-builder and phosphate-builder), improves the overall cleaning performance, improves the rubber sump hose compatibility profile^and reduces the malodor profile of the composition.

Diacyl peroxide In another embodiment the composition comprises: (i) a lipase; and (ii) a diacyl and/or tetraacyl peroxide species. The Inventors have found that these composition exhibit excellent rubber hose compatibility. Diacyl peroxides and also tetraacyl peroxides are known to attack rubber, such as the rubber sump hoses of automatic washing machines, and over multiple washing cycles this can lead to failure of the rubber sump hose. The Inventors have found that combining the diacyl peroxides and/or tetraacyl peroxides with lipase overcomes this problem of rubber sump hose incompatibility.

The diacyl peroxide bleaching species is preferably selected from diacyl peroxides of the general formula:

R^C(O)-OO-(O)C-R²

in which R^ represents a Cg-Cj g alkyl, preferably Cβ-Cj2 alkyl group containing a linear chain of at least 5 carbon atoms and optionally containing one or more substituents (e.g. -N⁺ (CH3)3, -COOH or -CN) and/or one or more interrupting moieties (e.g. -CONH- or -CH=CH-) interpolated between adjacent carbon atoms of the alkyl radical, and R^ represents an aliphatic group compatible with a peroxide moiety, such that R^ and R^ together contain a total of 8 to 30 carbon atoms. In one preferred aspect R^ and R² are linear unsubstituted Cg-C^ alkyl chains. Most preferably R^ and R^ are identical. Diacyl peroxides, in which both R^ and R^ are C6-Cj2 alkyl groups, are particularly preferred. Preferably, at least one of, most preferably only one of, the R groups (R] or R₂), does not contain branching or pendant rings in the alpha position, or preferably neither in the alpha nor beta positions or most preferably in none of the alpha or beta or gamma positions. In one further preferred embodiment the DAP may be asymmetric, such that preferably the hydrolysis of Rl acyl group is rapid to generate peracid, but the hydrolysis of R2 acyl group is slow. The tetraacyl peroxide bleaching species is preferably selected from tetraacyl peroxides of the general formula:

R3-C(O)-OO-C(O)-(CH₂)n-C(O)-OO-C(O)-R3

in which R^ represents a C\ -Cg alkyl, preferably C3 - C7 group and n represents an integer from 2 to 12, preferably 4 to 10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species is present in an amount sufficient to provide at least 0.5 ppm, more preferably at least 10 ppm, and even more preferably at least 50 ppm by weight of the wash liquor. In a preferred embodiment, the bleaching species is present in an amount sufficient to provide from about 0.5 to about 300 ppm, more preferably from about 30 to about 150 ppm by weight of the wash liquor.

Pre-formed peroxyacid

The pre-formed peroxyacid or salt thereof is typically either a peroxycarboxylic acid or salt thereof, or a peroxysulphonic acid or salt thereof.

The pre-formed peroxyacid or salt thereof is preferably a peroxycarboxylic acid or salt thereof, typically having a chemical structure corresponding to the following chemical formula:

O

_R ₁1_Λ4 _{c Q Q} θ Y φ

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; the R¹⁴ group can be linear or branched, substituted or unsubstituted; and Y is any suitable counter-ion that achieves electric charge neutrality, preferably Y is selected from hydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched, substituted or unsubstituted Cβ-9 alkyl. Preferably, the peroxyacid or salt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any salt thereof, or any combination thereof. Preferably, the peroxyacid or salt thereof has a melting point in the range of

The pre-formed peroxyacid or salt thereof can also be a peroxysulphomc acid or salt thereof, typically having a chemical structure corresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; the R¹⁵ group can be linear or branched, substituted or unsubstituted; and Z is any suitable counter-ion that achieves electric charge neutrality, preferably Z is selected from hydrogen, sodium or potassium. Preferably R^1S is a linear or branched, substituted or unsubstituted Cβ-9 alkyl.

EXAMPLES

LIPASE VARIANTS EXAMPLES

Chemicals used as buffers and substrates are commercial products of at least reagent grade.

- Media and Solutions: LAS (Surfac PS™) and Zeolite A (Wessalith P™). Other ingredients used are standard laboratory reagents.

- Materials. EMPA221 from EMPA St Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland

Example 1: Production of enzyme A plasmid containing the gene encoding the lipase is constructed and transformed into a suitable host cell using standard methods of the art.

Fermentation is carried out as a fed-batch fermentation using a constant medium temperature of 34°C and a start volume of 1.2 liter. The initial pH of the medium is set to 6.5. Once the pH has increased to 7.0 this value is maintained through addition of 10% H3PO4. The level of dissolved oxygen in the medium is controlled by varying the agitation rate and using a fixed aeration rate of 1.0 liter air per liter medium per minute. The feed addition rate is maintained at a constant level during the entire fed-batch phase.

The batch medium contained maltose syrup as carbon source, urea and yeast extract as nitrogen source and a mixture of trace metals and salts. The feed added continuously during the fed-batch phase contains maltose syrup as carbon source whereas yeast extract and urea is added in order to assure a sufficient supply of nitrogen.

Purification of the lipase may be done by use of standard methods known in the art, e.g. by filtering the fermentation supernatant and subsequent hydrophobic chromatography and anion exchange, e.g. as described in EP 0 851 913, Example 3.

Example 2: AMSA - Automated Mechanical Stress Assay - for calculation of Relative Performance (RP).

The enzyme variants of the present application are tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page

23-24. The containers, which contain the detergent test solution, consist of cylindrical holes (6 mm diameter, 10 mm depth) in a metal plate. The stained fabric (test material) lies on the top of the metal plate and is used as a lid and seal on the containers. Another metal plate lies on the top of the stained fabric to avoid any spillage from each container. The two metal plates together with the stained fabric are vibrated up and down at a frequency of 30 Hz with an amplitude of 2 mm.

The assay is conducted under the experimental conditions specified below:

0.5 g/1 LAS

0.52 g/1 Na2CO3

Test solution 1.07 g/1 Zeolite A -

0.52 g/1 Tn sodium Citrate

Table 3

Cream-turmeric swatches are prepared by mixing 5 g of turmeric (Santa Maria, Denmark) with 100 g cream (38% fat, Aria, Denmark) at 50⁰C, the mixture is left at this temperature for about 20 minutes and filtered (50⁰C) to remove any undissolved particles. The mixture is cooled to 20^cC) woven cotton swatches, EMPA221, are immersed in the cream- turmeric mixture and afterwards allowed to dry at room temperature over night and frozen until use. The preparation of cream-turmeric swatches is disclosed in the patent application PA 2005 00775, filed 27 May 2005.

The performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.

Color measurements are made with a professional flatbed scanner (PFU DL2400pro), which is used to capture an image of the washed textile samples. The scans are made with a resolution of 200 dpi and with an output color depth of 24 bits. In order to get accurate results, the scanner is frequently calibrated with a Kodak reflective IT8 target.

To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Color Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:

The wash performance (P) of the variants is calculated in accordance with the formula:

P = Int(v) - Int(r) where

Int(v) is the light intensity value of textile surface washed with tested enzyme and Int(r) is the light intensity value of textile surface washed without the tested enzyme.

A relative performance score is given as the result of the AMSA wash in accordance with the definition: Relative Performance scores (RP) are summing up the performances (P) of the tested enzyme variants against the reference enzyme: RP = P(test enzyme) / Preference enzyme).

RPavg indicates the average relative performance compared to the reference enzyme at all four enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep/1)

RPavg = avg(RP(0.125), RP(0.25) RP(0.5), RP(LO))

A variant is considered to exhibit improved wash performance, if it performs better than the reference. In the context of the present invention the reference enzyme is the lipase of SEQ ID NO:2 with the substitutions T23 IR + N233R.

Example 3: GC — Gas Chromatograph - for calculation of risk factor. The butyric acid release from the lipase washed swatches are measured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC) using the following method. Four textile pieces (5 mm in diameter), washed in the specified solution in Table 3 containing 1 mg/1 lipase, are transferred to a Gas Chromatograph (GC) vial. The samples are analysed on a Varian 3800 GC equipped with a Stabilwax- DA w/Integra-Guard column (30m, 0.32 mm ID and 0.25 micro- m df) and a Carboxen PDMS SPME fibre (75 micro-m). Each sample is preincubated for 10 min at 40⁰C followed by 20 min sampling with the SPME fibre in the head-space over the textile pieces. The sample is subsequently injected onto the column (injector temperature=250^t>C). Column flow = 2 ml Helium/min. Column oven temperature gradient: 0 min = 4O⁰C, 2 min = 4O⁰C, 22 min = 240⁰C, 32 min = 240⁰C. The butyric acid is detected by FID detection and the amount of butyric acid is calculated based on a butyric acid standard curve.

The Risk Performance Odour, R, of a lipase variant is the ratio between the amount of released butyric acid from the lipase variant washed swatch and the amount of released butyric acid from a swatch washed with the lipase of SEQ ID NO: 2 with the substitutions T231R + N233R (reference enzyme), after both values have been corrected for the amount of released butyric acid from a non-lipase washed swatch. The risk (R) of the variants is calculated in accordance with the below formula:

Odour =measured in micro g butyric acid developed at 1 mg enzyme protein / 1 corrected for blank (Xtest enzyme = OdOUT _tes, ^^^ - Blank

^reference enzyme vJ&OUr reference enzyme " -DlanlC ■K- ^test enzyme ' ^reference enzyme

A variant is considered to exhibit reduced odor compared to the reference, if the R factor is lower than 1.

Example 4: Activity (JLU) relative to absorbance at 280nm

The activity of a lipase relative to the absorbance at 280 nm is determined by the following assay LU/A280:

The activity of the lipase is determined as described above in the section Lipase activity. The absorbance of the lipase at 280 nm is measured (A280) and the ratio LU/A280 is calculated.

The relative LU/A280 is calculated as the LU/A280 of the variant divided by the LU/A280 of a reference enzyme. In the context of the present invention the reference enzyme is the lipase of SEQ ID NO:2 with the substitutions T231R + N233R.

Example 5: BR - Benefit Risk

The Benefit Risk factor describing the performance compared to the reduced risk for odour smell is thus defined as: BR = RP_avg / R

A variant is considered to exhibit improved wash performance and reduced odor, if the BR factor is higher than 1.

Applying the above methods the following results are obtained:

Table 4

The reference lipase and variants 7 and 8 in Table 4 are described in WO 2000/060063.

Example 6

BR - Benefit Risk

The Benefit Risk was measured for the variants listed in Table 5. The Benefit Risk factor was measured in the same way as described in Example 5 and it was found to be above 1 for all the listed variants.

Table 5

The reference lipase is described in WO 2000/060063.

BLEACH CATALYST EXAMPLES

Example 6: Preparation of Sulphuric acid mono-r2-f3.4-dihydro-isoαuinolin-2-yl)-l-f2- ethylhexyloxymethvD-ethyl] ester, internal salt

Preparation of 2-ethylhexyl glycidyl ether: To a flame dried, 500 mL round bottomed flask equipped with an addition funnel charged with epichlorohydrin (15.62 g, 0.17 moles), is added 2- ethylhexaπol (16.5 g, 0.127 moles) and stannic chloride (0.20 g, 0.001 moles). The reaction is kept under an argon atmosphere and warmed to 9O⁰C using an oil bath. Epichlorohydrin is , dripped into the stirring solution over 60 minutes followed by stirring at 90⁰C for 18 hours. The reaction is fitted with a vacuum distillation head and l-chloro-3-(2-ethyl-hexyloxy)-propan-2-ol is distilled under 0.2mm Hg. The l-chloro-3-(2- ethyl-hexyloxy)-propan-2-ol (4.46 g, 0.020 moles) is dissolved in tetrahydrofuran (50 mL) and stirred at room temperature under an argon atmosphere. To the stirring solution is added potassium tert-butoxide (2.52 g, 0.022 moles) and the suspension is stirred at room temperature for 18 hours. The reaction is then evaporated to dryness, residue dissolved in hexanes and washed with water (100 mL). The hexanes phase is separated, dried with Na2SC>4, filtered and evaporated to dryness to yield the crude 2-ethylhexyl glycidyl ether, which can be further purified by vacuum distillation. Preparation of Sulphuric acid mono-[2-(3,4-dihydro-isoquiπolin-2-yl)-l-(2- ethylhexyloxymethyl)-ethyl] ester, internal salt: To a flame dried 250 mL three neck round bottomed flask, equipped with a condenser, dry argon inlet, magnetic stir bar, thermometer, and heating bath is added 3,4-dihydroisoquinoline (0.40 mol.; prepared as described in Example I of U.S. 5₃576,282), 2-ethylhexyl glycidyl ether (0.38 mol, prepared as described above), SO₃-DMF complex (0.38 mol), and acetonitrile (500 mL). The reaction is warmed to 80⁰C and stirred at temperature for 72 hours. The reaction is cooled to room temperature, evaporated to dryness and the residue recrystallized from ethyl acetate and/or ethanol to yield the desired product. The solvent acetonitrile may be replaced with other solvents, including but not limited to, 1 ,2- dichloroethane.

Example 7: Preparation of Sulphuric acid mono-f2-f3.4-dihvdro-isoquinolin-2-yl')-l-(2-butyl- octyloxymethylVethylT ester, internal salt

The desired product is prepared according to Example 1 but substituting 2-butyloctanol for 2-hexyloctanol.

COMPOSITION EXAMPLE The lipase incorporated in the compositions below is the lipase variant 1 to 5 described in example 5 Table 4, and combinations thereof.

Example 8: Laundry detergent compositions

The following laundry detergent compositions A, B, C and D are suitable for use in the present invention. Typically, these compositions are dosed into water at a concentration of from 80g/l to 120g/l during the laundering process.

The following laundry detergent compositions E, F₅ G and H are suitable for use in the present invention. Typically, these compositions are dosed into water at a concentration of from 80g/l to 120g/l during the laundering process.

The following laundry detergent compositions I, J, K and L are suitable for use in the present invention. Typically, these compositions are dosed into water at a concentration of from 20g/l to 60g/l during the laundering process.

Bleaching detergent compositions having the form of granular laundry detergents are exemplified by the following formulations. Any of the below compositions is used to launder fabrics at a concentration of 600 - 10000 ppm in water, with typical median conditions of 2500ppm, 25°C, and a 25:1 wateπcloth ratio. The typical pH is about 10 but can be can be adjusted by altering the proportion of acid to Na- salt form of alkylbenzenesulfonate.

* Lipase variant 1 to 5 described in example 5 Table 4, and combinations thereof. ** Organic catalyst prepared according to Examples 6 or 7 or mixtures thereof. *** Diacyl peroxide is preferably dinonanoylperoxide.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. All documents cited are, in relevant part, incorporated herein by reference, the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

Claims

CLAIMS What is claimed is:

1. A composition comprising: a) a variant of a parent lipase, said variant, when compared to said parent, comprising a total of at least three substitutions, said substitutions being selected from one or more of the following groups of substitutions:

(i) at least two substitutions in Region I,

(ii) at least one substitution in Region II,

(iii) at least one substitution in Region III, and/or

(iv) at least one substitution in Region IV; and b) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate.

2. A detergent composition according to Claim 1 , wherein said substitutions in Region I comprise substitutions in the positions corresponding to the positions 231 and 233.

3. A detergent composition according to Claim 2, wherein said substitutions at positions 231 and 233 are substituted with an R.

4. A detergent composition according to Claim 2, wherein said variant comprises a substitution in the position corresponding to position 4 of SEQ ID NO:2.

5. A detergent composition according to Claim 4, wherein said variant corresponding to position 4 of SEQ ID NO:2 is V.

6. A detergent composition according to Claim 2, wherein said variant comprises a substitution in the position corresponding to position 227 of SEQ ID NO:2.

7. A detergent composition according to Claim 6, wherein said variant corresponding to position 227 of SEQ ID NO:2 is G.

8. A detergent composition according to Claim 1, wherein said at least one substitution in Region II comprises a substitution selected from the group consisting of substitutions in positions corresponding to the positions 202, 211, 255 and 256.

9. A detergent composition according to Claim 8, wherein said at least one substitution in Region II comprises a substitution selected from the group consisting of X202G, X21 IL, X255Y/V and X256K.

10. A detergent composition according to Claim 1, wherein said at least one substitution in Region II comprises a substitution in the position corresponding to the position 210.

11. A detergent composition according to Claim 10, wherein said substitution corresponding to position 210 comprises X210K.

12. A detergent composition according to Claim 1, wherein said at least one substitution in Region III comprises a substitution selected from the group consisting of substitutions in positions corresponding to the positions 86 and 90.

13. A detergent composition according to Claim 12, wherein said at least one substitution in Region III comprises a substitution selected from the group consisting of X86V and X90A/R.

14. A detergent composition according to Claim 1, wherein said at least one substitution in Region III comprises a substitution in the position corresponding to the position 83.

15. A detergent composition according to Claim 14, wherein said substitution corresponding to position 83 comprises X83T.

16. A detergent composition according to Claim 1 , wherein said at least one substitution in Region IV comprises a substitution selected from the group consisting of substitutions in positions corresponding to the positions 27, 58 and 60.

17. A detergent composition according to Claim 16, wherein said at least one substitution in Region IV comprises a substitution selected from the group consisting of X27R, X58N/A/G/P/T and X60S/V/G/N/R/K/A/L.

18. A detergent composition according to Claim 1 , comprising at least two substitutions in Region IV corresponding to the positions 27, 58 and 60.

19. A detergent composition according to Claim 18, composing at least two substitutions in Region IV selected from the group consisting of X27R, X58N/A/G/P/T and X60S/V/G/N/R/K/A/L.

20. A detergent composition according to Claim 1, wherein said variant comprises at least one substitution outside the defined Regions I to IV.

21. A detergent composition according to Claim 20, wherein said at least one substitution outside the defined Regions I to IV is selected from the group consisting of substitutions in positions corresponding to position 81, 147, 150 and 249.

22. A detergent composition according to Claim 20, wherein said at least one substitution outside the defined Regions I to IV is selected from the group consisting of X81Q/E, X147M/Y, Xl 5OG and X249R/I/L.

23. A detergent composition according to Claim 2, wherein said parent lipase is at least 90% identical to SEQ ID NO:2.

24. A detergent composition according to Claim 1 , wherein the parent lipase is identical to SEQ ID NO: 2 and said variant comprises one of the following groups of substitutions: a) T231R + N233R + I255Y b) I202G + T231R + N233R c) I86V + L227G + T231R + N233R+ P256K d) Q4V + S58N + V60S + T231R + N233R e) S58N + V60S + I90R + T231R + N233R f) I90A + T23 IR + N233R + I255V g) S58N + V60S + I86V + A150G + L227G + T231R + N233R+ P256K h) S58N + V60S + L147M + F211L + T231R + N233R i) Q4V + S58A + V60S + S83T + I86V + AI 5OG -t- E210K + L227G +

T23 IR + N233R + P256K j) S58N + V60S + I86V + A150G + L227G + T231R + N233R + P256K.

25. A detergent composition according to Claim 1, wherein the parent lipase is identical to SEQ ID NO: 2 and said variant comprises one of the following groups of substitutions: a) Q4V + S58A + V60S + S83T + I86V + Al 50G + E21 OK + L227G + T231 R + N233R + P256K b) S58N + V60S + I86V + Al 50G + L227G + T231R + N233R + P256K.

26. A detergent composition according to Claim 1, wherein the lipase variant is characterized in that the Benefit Risk, when measured as given in the specification, is larger than 1.

27. A detergent composition comprising: a) polypeptide having lipase activity and which further has a Average Relative Performance of at least 0.8 and a Benefit Risk of at least 1.1 at the test conditions given in the specification; and b) a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate.

28. A composition according to Claim 27, wherein the bleach catalyst comprises a moiety selected from the group consisting of iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulphonyl imin.es; N-ρhosphonyl imines; N-acyl imines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof..

29. A composition according to Claim 27, wherein the bleach catalyst comprises an iminium and/or a carbonyl functional group.

30. A composition according to Claim 1 , wherein the bleach catalyst comprises an oxaziridinium and/or a dioxirane functional group, and/or is capable of forming an oxaziridinium and/or a dioxirane functional group upon acceptance of an oxygen atom.

31. A composition according to Claim 1, wherein the bleach catalyst has a chemical structure corresponding to the chemical formula:

wherein: n and m are independently from O to 4; each R¹ is independently selected from a substituted or unsubstituted radical selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fused heterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto, carboxylic, and carboalkoxy radicals, and any two vicinal R¹ substituents may combine to form a fused aryl, fused carbocyclic or fused heterocyclic ring; each R² is independently selected from a substituted or unsubstituted radical independently ^■ selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylenes, heterocyclic ring, alkoxy, arylcarbonyl groups, carboxyalkyl groups and amide groups; any R² may be joined together with any other of R² to form part of a common ring; any geminal R² may combine to form a carbonyl; and wherein any two R² may combine to form a substituted or unsubstituted fused unsaturated moiety; R³ is a Ci to C20 substituted or unsubstituted alkyl; R⁴ is hydrogen or the moiety Qt-A, wherein: Q is a branched or unbranched alkylene, t = 0 or 1, and A is an anionic group selected from the group consisting of OSO₃ ^", SO₃ ^', CO₂ ^", OCO₂ ^', OPO₃ ^2", OPO₃H^' and OPO₂ ^"; R⁵ is hydrogen or the moiety - CR^{1 I}R^l2-Y-G_b-Y_c-[(CR⁹R^I0)_y-O]_k-R⁸, wherein: each Y is independently selected from the group consisting of O, S, N-H, or N-R⁸; and each R⁸ is independently selected from the group consisting of alkyl, aryl and heteroaryl, said moieties being substituted or unsubstituted, and whether substituted or unsubsituted said moieties having less than 21 carbons; each G is independently selected from the group consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ are independently selected from the group consisting of hydrogen and C₁-C4 alkyl; R¹ ' and R¹² are independently selected from the group consisting of hydrogen and alkyl, or when taken together may join to form a carbonyl; b = O or 1 ; c can = O or 1 , but c must =

O if b = O; y is an integer of from 1 to 6; k is an integer of from O to 20; R⁶ is H₇ or an alkyl, aryl or heteroaryl moiety; said moieties being substituted or unsubstituted; and X, if present, is a suitable charge balancing counterion.

32. A composition according to Claim 1 , wherein the bleach catalyst has a chemical structure corresponding to the chemical formula:

wherein R^!3 is a branched alkyl group containing from 3 to 24 carbons, or a linear alkyl group containing from 1 to 24 carbons.

33. A composition according to Claim 1 , wherein the bleach catalyst has a chemical structure corresponding to the chemical formula:

wherein R¹³ is selected from the group consisting of 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

34. A composition according to Claim 1, wherein the composition comprises less than 5%, by weight of the composition, of a source of peroxygen.

35. A composition according to Claim 1, wherein the composition comprises from 5% to 10%, by weight of the composition, of a source of carbonate anion.

36. A composition according to Claim 1, wherein the composition comprises a dye transfer inhibitor.

37. A composition according to Claim I₃ wherein the composition comprises: a) less than 5%, by weight of the composition, of zeolite builder; b) optionally, less than 5%, by weight of the composition, of phosphate builder; and c) optionally, less than 5%, by weight of the composition, of silicate salt.

38. A composition according to Claim 1 , wherein the composition comprises a diacyl and/or a tetraacyl peroxide species.

39. A composition according to Claim 1 , wherein the composition comprises an oxybenzene sulphonate bleach activator and a source of peroxygen.

40. A composition according to Claim 1, wherein the composition comprises apre-formed peroxyacid.

41. A composition comprising: a) lipase; said lipase comprising a variant of a parent lipase, said variant, when compared to said parent, comprising a total of at least three substitutions, said substitutions being selected from one or more of the following groups of substitutions:

(i) at least two substitutions in Region I,

(ii) at least one substitution in Region II,

(iii) at least one substitution in Region III, and/or

(iv) at least one substitution in Region IV; and b) a diacyl and/or tetraacyl peroxide species.

42. A composition according to Claim 41, wherein the composition comprises a bleach catalyst that is capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizeable substrate.

43. A composition according to Claim 41 , wherein: said lipase comprises a variant of a parent lipase, said variant, when compared to said parent, comprising a total of at least three substitutions, said substitutions being selected from one or more of the following groups of substitutions: a) at least two substitutions in Region I, b) at least one substitution in Region II, c) at least one substitution in Region III, and/or d) at least one substitution in Region IV.

44. A composition according to Claim 41, wherein the parent lipase is identical to SEQ ID NO: 2 and said variant comprises one of the following groups of substitutions: a) T231R + N233R + I255Y b) I202G + T231 R + N233R c) I86V + L227G + T231R + N233R + P256K d) Q4V + S58N + V60S + T231R + N233R e) S58N + V60S + I90R + T231R + N233R f) I90A + T231R + N233R + I255V g) S58N + V60S + I86V + A150G + L227G + T23 IR + N233R + P256K h) S58N + V60S + L147M + F211L + T231R + N233R i) Q4V + S58A + V60S + S83T + I86V + A150G + E210K+ L227G +

T231 R + N233R + P256K j) S58N + V60S + I86V + Al 50G + L227G + T231R + N233R + P256K.

45. A composition according to Claim 41, wherein the lipase variant is a polypeptide having lipase activity and which further has a Average Relative Performance of at least 0.8 and a Benefit Risk of at least 1.1 at the test conditions given in the specification.

46. A composition according to Claim 1, wherein said lipase variant is a variant of SEQ ID NO: 2 comprising at least one of the mutations Q4V, S58N/A/G/P/T, I90R or Q249I/L.