composition

Abstract

The invention relates to a composition comprising from 0.0005 to 6 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1; to a method of treatment of a fabric substrate with a sebum stain, said method comprising treatment of a fabric substrate with a sebum stain, with a composition comprising from 0.0005 to 6 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1; and to the use of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1 to improve cleaning of sebum stains of fabric.

Description

[0001] COMPOSITION

[0002] Field of Invention

[0003] The invention concerns a composition, in particular a detergent composition, comprising a novel lysophospholipase enzyme.

[0004] Background of the Invention

[0005] Sebum is an oily soil which has remained a difficult stain to remove from worn garments. With a drive to encourage consumers to wash at lower temperatures, the challenge for effective removal of sebum remains demanding. Sebum consists of a number of fats and esters including wax esters, cholesterol esters, squalene and many free fatty acids / alcohols. Sebum is liquid at body temperature, but solid at ambient temperature.

[0006] These properties are particularly important for collar / cuff soil removal because it is easier to remove a liquid body oil than solids from clothes. Current laundry enzymes are not able to degrade all the components of the sebum which makes removal from fabric difficult.

[0007] There is a problem with sebum removal in that detergents including current commercial enzymes do not remove sebum adequately.

[0008] It is an object of the invention to improve the efficacy of sebum removal from fabrics.

[0009] Summary of the Invention

[0010] We have found that particular lysophospholipase enzymes solves this problem.

[0011] In one aspect the present invention provides a composition comprising from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.01 to 2 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1.

[0012] Preferably the invention provides a detergent composition comprising:

[0013] (a) from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.001 to

[0014] 2 wt.% wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1, and,

[0015] (b) from 1 to 60 wt.% preferably from 2 to 50 wt.%, more preferably from 4 to 35 wt.% of a detersive surfactant. Preferably the lysophospholipase enzyme has a sequence identity of at least 75% with SEQ. ID. 1. More preferably the lysophospholipase enzyme has a sequence identity of at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1.

[0016] Preferably the detergent composition comprises anionic and / or nonionic surfactant, preferably comprising both anionic and nonionic surfactant.

[0017] Preferably the anionic surfactant is present at a level of from 1 to 50 wt.%, preferably from 2 to 40 wt.%, more preferably from 3 to 30 wt.% and is preferably selected from linear alkyl benzenesulphonate, secondary alkane sulphonate, sodium laureth ether sulphate, sodium lauryl sulphate, sodium oleyl sulphate and sodium oleyl ether sulphate, methyl ester sulphonate, secondary alkyl sulphate (SALS), cardanol ether sulphate and a rhamnolipid.

[0018] Preferably the nonionic surfactant is present at a level of from 1 to 30 wt.%, preferably from 2 to 20 wt.%, more preferably from 3 to 15 wt.% and is preferably selected from an alcohol ethoxylate, an alcohol propoxylate, a methyl ester ethoxylate and an alkyl poly glycoside.

[0019] A preferred detergent composition is a laundry detergent composition. Preferably the laundry detergent composition is in the form of a liquid, solid, powder, pastille, bead or paste. More preferably the composition is a liquid or a powder, most preferably a liquid detergent.

[0020] The laundry detergent preferably comprises an alkoxylated polyamine, preferably at a level of from 0.1 to 8 wt.%, more preferably from 0.2 to 6 wt.%, most preferably from 0.5 to 5 wt.%.

[0021] The laundry detergent preferably comprises soil release polymer, the soil release polymer preferably selected from copolyesters of dicarboxylic acids and polydiols, more preferably a copolyester formed by condensation of terephthalic acid ester and 1 ,2-propanediol, the soil release polymer preferably present at a level of from 0.1 to 8 wt.%, more preferably from 0.2 to 6 wt.%, most preferably from 0.5 to 5 wt.%.

[0022] Preferred detergent compositions, particularly laundry detergent compositions additionally comprises one or more further enzymes selected from the group consisting of: proteases, lipases, cellulases, alpha-amylases, peroxidases / oxidases, pectate lyases, and / or mannanases. Preferably the one or more enzymes comprises protease, amylase, lipase and / or cellulase, more preferably protease.

[0023] In another aspect the invention provides a method of treatment of a fabric substrate with a sebum stain, said method comprising treatment of a fabric substrate with a sebum stain, with a composition comprising from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.01 to 2 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70%, preferably at least 75%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1.

[0024] Preferably the composition used in the method of the second aspect of the invention is a detergent composition according to any of the preferred embodiments disclosed and / or claimed herein.

[0025] In another aspect the present invention provides the use of a lysophospholipase enzyme having a sequence identity of at least 70%, preferably at least 75%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1 to improve cleaning of sebum stains of fabric.

[0026] Detailed Description of the Invention

[0027] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0028] All % levels of ingredients in compositions (formulations) listed herein are in wt.% based on total formulation unless other stated.

[0029] It is understood that any reference to a preferred ingredient of the composition / detergent composition is envisaged to be combinable subject matter with any other preferred ingredient of the composition / detergent composition disclosed herein.

[0030] The composition / detergent composition can be applied to any suitable substrate. Particularly preferred substrates are textiles. Particularly preferred composition / detergent compositions are laundry detergent compositions. Laundry detergent compositions may take any suitable form. Preferably the laundry detergent composition is in the form of a liquid, solid, powder, pastille, bead or paste, preferably the composition is a liquid or a powder, more preferably a liquid detergent.

[0031] The enzyme is a lysophospholipase enzyme, which can be described as being of enzyme class EC 3.1.1.5. This enzyme belongs to the family of hydrolases, specifically those acting on carboxylic ester bonds. This class of enzymes is useful as it can break down some of the lipids present in sebum.

[0032] The lysophospholipase enzyme has a sequence identity of at least 70% with SEQ. ID. 1. Preferably the lysophospholipase enzyme has a sequence identity of at least 75%, preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1.

[0033] The composition comprises from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.001 to 2 wt.% of the lysophospholipase enzyme. Other preferred amounts include from 0.001 to 1 wt.% of the lysophospholipase enzyme.

[0034] Preferred lysophospholipase enzyme is from Psychrobacter sp.

[0035] Percentage Sequence Identity

[0036] Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (referred to by the SEQ ID No.) after aligning the sequences and introducing gaps as necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.

[0037] Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence. For example, where a given sequence comprises 100 amino acids and the candidate sequence comprises 10 amino acids, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:

[0038] (A)

[0039] Given seq: XXXXXXXXXXXXXXX (15 amino acids)

[0040] Comparison seq: XXXXXYYYYYYY (12 amino acids)

[0041] % sequence identity = the number of identically matching amino acid residues after alignment divided by the total number of amino acid residues in the longer given sequence, i.e. (5 divided by 15) x 100 = 33.3%

[0042] Where the comparison sequence is longer than the given sequence, sequence identity may be determined over the entire length of the given sequence. For example:

[0043] (B)

[0044] Given seq: XXXXXXXXXX (10 amino acids)

[0045] Comparison seq: XXXXXYYYYYYZZYZZZZZZ (20 amino acids)

[0046] % sequence identity = number of identical amino acids after alignment divided by total number of amino acid residues in the given sequence, i.e. (5 divided by 10) x 100 = 50%.

[0047] Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein / DNA ENDGAP = -1 , Protein / DNA GAPDIST = 4.

[0048] Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity may be determined as described above and illustrated in examples (A) and (B). The most preferred lysophospholipase enzymes are given by Sequence ID No. 1 (SEQ. ID. 1). Letters refer to amino acids of the protein sequence.

[0049] SEQ. ID. 1

[0050] MSTNTDTGNDHILSSDNIHYLHHNFFEPSHENTAVKATLLIVHGMAEHSGRYADFAQFLADN GIAVATYDHLGHGQTVKTEADLGFFGEEHPVQSLLKDVIVMADSLKNRHPDVPHFIMGQSM GSFIVRNVLKHHAHNFTGAILMGTADANPLTKVLLPVNKVLAKVAPRKPNTVLASVMNKVLN SKLEDRISSSEFAWLAEDSAAIEAFEADPLTGFDFTNNGFMTLFCLMETGLNKGWSTTIPKD FPMLFISGEDDPIGDMGNGIRKIVTRLNKQNFSQVDIQLYPNMRHEPLHEKDHQTVYEDILE WIESHTQDN

[0051] Surfactant

[0052] The composition is preferably a detergent composition. The detergent composition comprises surfactant (which may include a mixture of two or more surfactants). The composition preferably comprises from 1 to 60 wt.%, more preferably from 2 to 50 wt.%, most preferably from 4 to 35 wt.% of a detersive surfactant. Even more preferred levels of surfactant are from 6 to 35 wt.%, more preferably from 8 to 35 wt.%.

[0053] The detergent composition (preferably a laundry detergent composition) comprises anionic and / or nonionic surfactant, preferably comprising both anionic and nonionic surfactant.

[0054] Anionic Surfactant are described in Anionic Surfactants Organic Chemistry (Surfactant Science Series Volume 56) edited By H.W.Stache (Marcel Dekker 1996).

[0055] Preferably, the composition comprises from 1 to 50 wt.%, preferably from 2 to 40 wt.%, more preferably from 3 to 30 wt.% anionic surfactant based on the total weight of composition.

[0056] Non-soap anionic surfactants for use in the invention are typically salts of organic sulphates and sulphonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term “alkyl” being used to include the alkyl portion of higher acyl radicals. Examples of such materials include alkyl sulphates, alkyl ether sulphates, alkaryl sulfonates, alphaolefin sulfonates and mixtures thereof. The alkyl radicals preferably contain from 10 to 18 carbon atoms and may be unsaturated. The alkyl ether sulphates may contain from one to ten ethylene oxide or propylene oxide units per molecule, and preferably contain one to three ethylene oxide units per molecule. The counterion for anionic surfactants is generally an alkali metal such as sodium or potassium; or an ammoniacal counterion such as monoethanolamine, (MEA) diethanolamine (DEA) or triethanolamine (TEA). Mixtures of such counterions may also be employed.

[0057] The compositions according to the invention may include alkylbenzene sulfonates, particularly linear alkylbenzene sulfonates (LAS) with an alkyl chain length of from 10 to 18 carbon atoms. Commercial LAS is a mixture of closely related isomers and homologues alkyl chain homologues, each containing an aromatic ring sulphonated at the “para" position and attached to a linear alkyl chain at any position except the terminal carbons. The linear alkyl chain typically has a chain length of from 11 to 15 carbon atoms, with the predominant materials having a chain length of about C12. Each alkyl chain homologue consists of a mixture of all the possible sulphophenyl isomers except for the 1 -phenyl isomer. LAS is normally formulated into compositions in acid (i.e. HLAS) form and then at least partially neutralized in-situ.

[0058] Some alkyl sulphate surfactant (PAS) may be used, such as non-ethoxylated primary and secondary alkyl sulphates with an alkyl chain length of from 10 to 18.

[0059] Also commonly used in laundry liquid compositions are alkyl ether sulfates having a straight or branched chain alkyl group having 10 to 18, more preferably 12 to 14 carbon 30 atoms and containing an average of 1 to 3EO units per molecule. A preferred example is sodium lauryl ether sulfate (SLES) in which the predominantly C12 lauryl alkyl group has been ethoxylated with an average of 3EO units per molecule.

[0060] The alkyl ether sulphate may be provided in a single raw material component or by way of a mixture of components.

[0061] Preferred anionic surfactants also include the C16 / 18 alkyl ether sulphates. Preferred anionic surfactants also include rhamnolipids.

[0062] The anionic surfactant is preferably selected from linear alkyl benzenesulphonate, secondary alkane sulphonate, sodium laureth ether sulphate, sodium lauryl sulphate, sodium oleyl sulphate and sodium oleyl ether sulphate, methyl ester sulphonate, secondary alkyl sulphate (SALS), cardanol ether sulphate and a rhamnolipid.

[0063] Mixtures of any of the above described materials may also be used. Preferably the detergent composition comprises non-ionic surfactant, preferably from 1 to 30 wt.%, preferably from 2 to 20 wt.%, more preferably from 3 to 15 wt.% of non-ionic surfactant.

[0064] Suitable nonionic detergent compounds which may be used include, in particular, the reaction products of compounds having an aliphatic hydrophobic group and a reactive hydrogen atom, for example, aliphatic alcohols, acids or amides, especially ethylene oxide either alone or with propylene oxide. The nonionic surfactant is preferably selected from an alcohol ethoxylate, an alcohol propoxylate, a methyl ester ethoxylate and an alkyl poly glycoside.

[0065] Preferred nonionic detergent compounds are the condensation products of aliphatic Cs to Cis primary or secondary linear or branched alcohols with ethylene oxide.

[0066] Most preferably the nonionic detergent compound is the alkyl ethoxylated non-ionic surfactant is a Cs to Cis primary alcohol with an average ethoxylation of 7EO to 9EO units.

[0067] Preferably the surfactants used are saturated.

[0068] A composition of the invention may contain one or more amphoteric (such as zwitterionic surfactants), preferably wherein if present, the amphoteric surfactant is present at a level of from 0.1 to 15 wt.%, preferably from 0.5 to 10 wt.%, more preferably from 1 to 5 wt.% and is preferably selected from alkyl betaines and the alkyl sulphobetaines (sultaines), more preferably carbobetaines and lauramine oxide.

[0069] Further specific amphoteric surfactants include alkyl amine oxides, alkyl amidopropyl betaines, alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates.

[0070] The amphoteric surfactant preferably comprises alkyl radicals containing from about 8 to about 22 carbon atoms preferably selected from C12, C14, C16, C18 and C18: 1 , the term “alkyl” being used to include the alkyl portion of higher acyl radicals.

[0071] Mixtures of any of the above described materials may also be used. Soil release polymer

[0072] When the detergent composition is in the form of a laundry composition, it is preferred that a soil release polymer is included.

[0073] The soil release polymer is preferably present at a level of from 0.1 to 10 wt.%.

[0074] The levels of soil release polymer are preferably from 0.1 to 8 wt.%, more preferably from 0.2 to 6 wt.%, most preferably from 0.5 to 5 wt.%.

[0075] Preferably the soil release polymer is a polyester based soil released polymer. More preferably the polyester soil release polymer is selected from copolyesters of dicarboxylic acids and polydiols. More preferably the soil release polymer is a polyethylene and / or polypropylene terephthalate based soil release polymer, most preferably a polypropylene terephthalate based soil release polymer, most preferably a copolyester formed by condensation of terephthalic acid ester and 1,2-propanediol.

[0076] Alkoxylated polyamine

[0077] When the detergent composition is in the form of a laundry composition, it is preferred that an alkoxylated polyamine is included.

[0078] Preferred levels of alkoxylated polyamine range from 0.1 to 8 wt.%, preferably from 0.2 to 6 wt.%, more preferably from 0.5 to 5 wt.%. Another preferred level is from 1 to 4 wt.%.

[0079] The alkoxylated polyamine may be linear or branched. It may be branched to the extent that it is a dendrimer. The alkoxylation may typically be ethoxylation or propoxylation, or a mixture of both. Where a nitrogen atom is alkoxylated, a preferred average degree of alkoxylation is from 10 to 30, preferably from 15 to 25.

[0080] A preferred material is alkoxylated polyethylenimine, most preferably ethoxylated polyethyleneimine, with an average degree of ethoxylation being from 10 to 30 preferably from 15 to 25, where a nitrogen atom is ethoxylated.

[0081] Additional Enzymes

[0082] Additional enzymes, other than the specified lysophospholipase may be present in the detergent composition. It is preferred that additional enzymes are present in the preferred laundry detergent composition. If present, then the level of each additional enzyme in the laundry composition of the invention is from 0.0001 wt.% to 0.1 wt.%.

[0083] Levels of enzyme present in the composition preferably relate to the level of enzyme as pure protein.

[0084] Preferred further enzymes include those in the group consisting of: proteases, cellulases, alpha-amylases, lipases, peroxidases / oxidases, pectate lyases, and / or mannanases. Said preferred additional enzymes include a mixture of two or more of these enzymes.

[0085] Preferably the further enzyme is selected from: proteases, cellulases, lipases, and / or alphaamylases. Most preferably the additional enzyme comprises protease.

[0086] Protease enzymes hydrolyse bonds within peptides and proteins, in the laundry context this leads to enhanced removal of protein or peptide containing stains. Examples of suitable proteases families include aspartic proteases; cysteine proteases; glutamic proteases; aspargine peptide lyase; serine proteases and threonine proteases. Such protease families are described in the MEROPS peptidase database (htp: / / merops.sanger.ac.uk / ). Serine proteases are preferred. Subtilase type serine proteases are more preferred. The term "subtilases" refers to a sub-group of serine protease. 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.

[0087] Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus, Bacillus gibsonii, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 and protease PD138. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease, and the chymotrypsin proteases derived from Cellumonas.

[0088] Most preferably the protease is a subtilisins (EC 3.4.21.62). Preferably the subtilisin is derived from Bacillus, preferably Bacillus lentus, B. al kalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii. Most preferably the subtilisin is derived from Bacillus gibsonii or Bacillus Lentus.

[0089] Suitable commercially available protease enzymes include those sold under the trade names names Alcalase®, Blaze®; Duralase®, Durazym®, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® all could be sold as Ultra® or Evity® (Novozymes A / S).

[0090] Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) or from H. insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes, P. cepacia, P. stutzeri, P. fluorescens, Pseudomonas sp. strain SD 705, P. wisconsinensis, a Bacillus lipase, e.g. from B. subtilis, B. stearothermophilus or B. pumilus.

[0091] Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™, Lipex™ and LipocleanTM(Novozymes A / S).

[0092] The composition may use cutinase, classified in EC 3.1.1.74. The cutinase used according to the invention may be of any origin. Preferably cutinases are of microbial origin, in particular of bacterial, of fungal or of yeast origin.

[0093] Suitable amylases (alpha and / or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alphaamylases obtained from Bacillus, e.g. a special strain of B. licheniformis, or the Bacillus sp. strains. Commercially available amylases are Duramyl™, Termamyl™, Termamyl Ultra™, Natalase™, Stainzyme™, Amplify™, Fungamyl™ and BAN™ (Novozymes A / S), Rapidase™ and Purastar™ (from Genencor International Inc.).

[0094] 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, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum. Commercially available cellulases include Celluzyme™, Carezyme™, Celluclean™, Endolase™, Renozyme™ (Novozymes A / S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation). Celluclean™ is preferred.

[0095] 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. Commercially available peroxidases include Guardzyme™ and Novozym™ 51004 (Novozymes A / S).

[0096] Enzyme Stabilizers

[0097] Any enzyme present in the composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid.

[0098] Chelating Agent

[0099] Chelating agents may be present or absent from the detergent compositions.

[0100] If present, then the chelating agent is present at a level of from 0.01 to 5 wt.%.

[0101] Preferred chelating agents are phosphonic acid (or salt thereof) chelating agents, preferably selected from: 1-Hydroxyethylidene-1,1-diphosphonic acid (HEDP);

[0102] Diethylenetriaminepenta(methylenephosphonic acid) (DTPMP);

[0103] Hexamethylenediaminetetra(methylenephosphonic acid) (HDTMP);

[0104] Aminotris(methylenephosphonic acid) (ATMP); Ethylenediaminetetra(methylenephosphonic acid) (EDTMP); Tetramethylenediaminetetra(methylenephosphonic acid) (TDTMP); and, Phosphonobutanetricarboxylic acid (PBTC).

[0105] Perfume / Fragrances

[0106] As used herein the terms fragrance and perfume are used interchangeably.

[0107] The composition preferably comprises a fragrance.

[0108] Preferably the laundry composition comprises a fragrance, preferably wherein the fragrance is included between 0.001 and 2.0wt%, more preferably 0.01 and 1.5wt% and most preferably 0.1 and 1.0wt%. Preferably the fragrance comprises greater than 50wt% biodegradable materials, more preferably greater than 60wt% biodegradable materials, more preferably greater than 70wt% biodegradable materials, more preferably greater than 80wt% biodegradable materials, more preferably greater than 90% biodegradable materials and most preferably the fragrance consists of 100wt% biodegradable materials.

[0109] Preferably the fragrance comprises at least one note (compound) from: alpha-isomethyl ionone, benzyl salicylate; citronellol; coumarin; hexyl cinnamal; linalool; pentanoic acid, 2- methyl-, ethyl ester; octanal; benzyl acetate; 1,6-octadien-3-ol, 3,7-dimethyl-, 3-acetate; cyclohexanol, 2-(1 ,1 -dimethylethyl)-, 1-acetate; delta-damascone; beta-ionone; verdyl acetate; dodecanal; hexyl cinnamic aldehyde; cyclopentadecanolide; benzeneacetic acid, 2- phenylethyl ester; amyl salicylate; beta-caryophyllene; ethyl undecylenate; geranyl anthranilate; alpha-irone; beta-phenyl ethyl benzoate; alpa-santalol; cedrol; cedryl acetate; cedry formate; cyclohexyl salicyate; gamma-dodecalactone; and, beta phenylethyl phenyl acetate.

[0110] Polymers

[0111] The composition may comprise one or more further polymers. Examples are carboxymethylcellulose, poly (ethylene glycol), poly(vinyl alcohol), polycarboxylates such as polyacrylates, maleic / acrylic acid copolymers and lauryl methacrylate / acrylic acid copolymers.

[0112] Optional Ingredients

[0113] A composition of the invention may contain optional ingredients to enhance performance and I or consumer acceptability. Examples of such ingredients include anti-foams, fluorescers, shading dyes, preservatives, anti-microbials (e.g. bactericides), foam boosting agents, polyelectrolytes, anti-shrinking agents, anti-wrinkle agents, anti-oxidants, sunscreens, anticorrosion agents, drape imparting agents, anti-static agents, ironing aids, dyes I colorants, shading dyes, pearlisers and / or opacifiers and microcapsules. Each of these ingredients will be present in an amount effective to accomplish its purpose. Generally, these optional ingredients are included individually at an amount of up to 5% (by weight based on the total weight of the composition).

[0114] If an anti-foam is included it is preferably a fatty acid soap. Suitable fatty acids in the context of this invention include aliphatic carboxylic acids of formula RCOOH, where R is a linear or branched alkyl or alkenyl chain containing from 6 to 24, more preferably 10 to 22, most preferably from 12 to 18 carbon atoms and 0 or 1 double bond. Preferred examples of such materials include saturated C12-18 fatty acids such as lauric acid, myristic acid, palmitic acid or stearic acid; and fatty acid mixtures in which 50 to 100% (by weight based on the total weight of the mixture) consists of saturated C12-18 fatty acids. Such mixtures may typically be derived from natural fats and / or optionally hydrogenated natural oils (such as coconut oil, palm kernel oil or tallow).

[0115] Examples

[0116] The invention will be demonstrated by the following non-limiting examples.

[0117] PblysoPI is a putative lysophospholipase from a Psychrobacter sp. Characterisation of this enzyme has shown that it is active against p-nitrophenyl esters and triglycerides.

[0118] Differential scanning fluorimetry indicated a melting temperature of around 55°C and assays under various conditions found the pH optimum to be between 7 and 9. The temperature optimum for activity against p-nitrophenyl esters, was found to be 25°C, though activity is retained to at least 50°C.

[0119] SEQ. ID. 1 relates to the sequence structure of PblysoPI as labelled herein.

[0120] The reference to Lipex is Lipex Evity® 200L an enzyme sourced from Novozymes in Denmark.

[0121] Production of lysophospholipase enzyme

[0122] Expression and Purification

[0123] The gene encoding the sequence above for a putative lysophospholipase (PblysoPI) was synthesised (GenScript Biotech, NJ) and cloned into the pET28a+ vector for recombinant expression in E. coli.

[0124] The gene was transformed into BL21 (DE3) cells by the heat shock method and spread onto plates containing 50 .g / ml kanamycin to select for positive transformants.

[0125] Individual colonies were selected and grown in 10ml of LB media supplemented with 50 .g / ml kanamycin overnight at 37°C with 200rpm shaking. 10ml of this culture was transferred to a 2.5I flask containing 11 of LB media supplemented with kanamycin, incubated at 37°C with 200rpm shaking until and ODeoo of 0.7 was reached. IPTG was added for induction to a final concentration of 0.5mM and then incubated at 25°C with shaking at 200rpm for 24 hours.

[0126] Cells were harvested by centrifugation at 4500rpm for 30 minutes, and then resuspended in 10ml lysis buffer (50mM Tris-HCI, 500mM NaCI, pH 8) per 1g of wet cell paste. Lysis was carried out by sonication on ice, then the resulting lysate was centrifuged at 14500rpm for 30 minutes. The supernatant was collected and purified by immobilised metal affinity chromatography, using a 1ml HisTrap FF column, attached to an AKTA Start. After the protein was bound to the column, elution was carried out via the use of a linear gradient up to 100% of elution buffer (50mM Tris-HCI, 500mM NaCI, 500mM imidazole, pH 8).

[0127] Absorbance at 280nm was used to identify the fractions containing the desired protein, which was confirmed by SDS page. The fractions were pooled and concentrated before being applied to a Superdex 200 16 / 60 gel filtration column, attached to an AKTA pure. The system was run at a flow rate of 1ml / min and the enzyme eluted between 58 and 93ml. The fractions were pooled and concentrated using a Vivaspin 10,000 MWCO concentrator.

[0128] Determination of protein concentration and purity

[0129] The concentration of the purified protein was determined using the Bradford assay method.

[0130] A serial dilution of Bovine Serum albumin (starting concentration: 2mg / ml) was used to create a range of 8 standards. 20 .l of each of the standards was added to the wells of a 96- well microtitre plate, in triplicate along with 3 dilutions of the enzyme solution. 180 J of Bradford reagent was added to each well, the plate incubated at room temperature for 5 minutes. The absorbance of the samples at 595nm was measured using a Tecan infinite plate reader.

[0131] The purity of the enzyme was determined using SDS-PAGE. The protein sample was heated with SDS-PAGE loading buffer to 90°C for 10 mins and then loaded onto a 4-20% SurePAGE gel along with Spectra multicolour broad range molecular weight marker. The gel was run for 45 minutes at 160V and then stained using Quick Coomassie. A single band in the lane loaded with the purified enzyme showed high purity.

[0132] Biochemical determination of esterase and lipase activity Esterase activity was determined by assaying the enzyme against 4-nitrophenyl hexanoate (C6). The 4-nitrophenyl hexanoate was dissolved in methanol to a concentration of 8mM, then this stock solution was further diluted to 1 mM in acidified water.

[0133] In a 96-well microtitre plate, 20 .l of enzyme was added to wells, in triplicate, as well as 20 .l of buffer for negative control samples. 160 J of assay buffer (50mM Tris-HCI, 500mM NaCI, pH 8) was added to each well with 20 .l of substrate added last using a multi-channel pipette. The production of the product (4-nitrophenyl) was monitored by measuring absorbance at 405nm for 15 minutes.

[0134] Lipase activity was measured using a pH indicator (phenol red) and triacetin (C2) or tributyrin (C4) as the substrate to be hydrolysed. 20 .l of enzyme or buffer control was added, in triplicate, to the wells of a 96-well microtitre plate. 160 J of phenol red (35 .g / ml in 5mM Tris HCI) was added to each well before the substrate was added using a multichannel pipette. The colour change of the indicator was then monitored at 570nm for 1 hr where a decrease in absorbance at this wavelength signifies the release of the fatty acid product from the triglyceride substrates.

[0135] Application testing

[0136] Example 1 - Cleaning of artificial sebum on knitted cotton substrate

[0137] Artificial sebum stains were obtained from CFT in the form of a mixture of sebaceous compounds applied to a knitted cotton substrate. The enzyme was applied as a pretreatment at a concentration of 0.1mg / ml in combination with 50% detergent formulation. 500 .l of the enzyme / formulation mixture was applied to the stains, and a formulation-only mixture was used as a control.

[0138] The stain coated in the pre-treatment mixture was then incubated either for 1 hr or overnight before a wash phase was carried out. This was done at either 25°C or 40°C in a linitest for 1 hr, in a 200ml volume of water, containing 1g / l of detergent formulation.

[0139] After washing, the stains were rinsed to remove residual enzyme and wash liquor and allowed to air dry for 24-48 hrs. The dry stains were scanned using an X-rite to measure stain intensity when compared to unstained, white fabric. Three primary measures were taken; L*, a* and b* which can be used in the following equation to calculate deltaE: Equation 1.

[0140] Where AL, Aa, and Ab are the difference in darkness, redness, and yellowness, respectively, between a white fabric and the stained fabric.

[0141] Eq. 1 tells us that lower values of AL, Aa, and Ab will result in lower values of AE, wherein a value of 0 would correspond to a completely white fabric.

[0142] The efficacy of cleaning is then expressed as:

[0143] Equation 2. SRI = 100 - AE

[0144] Wherein higher SRI values correspond to cleaner fabric, and 100 corresponds to completely white fabric.

[0145] Performance in wash testing against artificial sebum

[0146] Wash studies with enzyme used as a pre-treatment identify that the lysophospholipase enzyme PblysoPI shows a clear improvement in the removal of artificial sebum when compared to both the no-enzyme control, and the current benchmark lipase; Lipex Evity under multiple conditions. The 5-9 SRI unit increase for the experimental enzyme compared to the commercial Lipex enzyme indicate a clearly visible improved cleaning effect over the control enzyme. These results are shown in table 1.

[0147] Table 1 - Wash studies comparing the lysophospholipase enzyme versus Lipex

[0148] The results shown in table 1 relating to example 1 show a statistically significant improvement in stain removal from sebum by the lysophospholipase enzyme in comparison to commercially available Lipex Evity® 200L enzyme. Example 2 - Cleaning of human sebum on knitted cotton substrate

[0149] Human sebum-stained t-shirt collars were cut into 1 ,5cm2swatches for pre-treatment with either an enzyme / formulation mix, or formulation only in a 12 well plate.

[0150] The swatches were scanned using an X-rite to give a measure of the colour before washing. Each swatch was treated with 500 .l either 0.1mg / ml enzyme and 50% formulation, or 50% formulation and buffer control. The stains were incubated with the pre-treatment overnight before being washed in 2ml of water containing 1g / l detergent formulation for 1 hr with 200rpm shaking at 25°C. The wash liquor was then removed, and the stains rinsed with 3x 2ml of dH2O.

[0151] After washing, the stains were allowed to dry for 24-48 hours before being scanned again using an X-rite to compare to the pre-wash data.

[0152] Performance in wash testing against human sebum

[0153] SRI values were calculated using the above equations and the difference in SRI value between the pre- and post-wash measurements were used as a measure of cleaning. The experiments were performed in duplicate and the ± indicates the standard deviation across the samples.

[0154] The data, displayed in table 2 shows a statistically significant improvement in the removal of human sebum when using the experimental enzyme compared to the formulation only control.

[0155] Table 2 - showing the performance of the lysophospholipase against human sebum

Claims

P0000875WQ CPL19CLAIMS1. A composition comprising from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.01 to 2 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1.

2. A composition according to claim 1, which is a detergent composition comprising:(a) from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.001 to 2 wt.% wt.% of a lysophospholipase enzyme having a sequence identity of at least 70% with SEQ. ID. 1 , and,(b) from 1 to 60 wt.% preferably from 2 to 50 wt.%, more preferably from 4 to 35 wt.% of a detersive surfactant.

3. A composition according to claim 1 or a detergent composition according to claim 2, wherein the lysophospholipase enzyme has a sequence identity of at least 75%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1.

4. A detergent composition according to claim 2 or claim 3, wherein the detergent composition comprises anionic and / or nonionic surfactant, preferably comprising both anionic and nonionic surfactant.

5. A detergent composition according to claim 4, wherein the anionic surfactant is present at a level of from 1 to 50 wt.%, preferably from 2 to 40 wt.%, more preferably from 3 to 30 wt.% and is preferably selected from linear alkyl benzenesulphonate, secondary alkane sulphonate, sodium laureth ether sulphate, sodium lauryl sulphate, sodium oleyl sulphate and sodium oleyl ether sulphate, methyl ester sulphonate, secondary alkyl sulphate (SALS), cardanol ether sulphate and a rhamnolipid.

6. A detergent composition according to claim 4 or claim 5, wherein the nonionic surfactant is present at a level of from 1 to 30 wt.%, preferably from 2 to 20 wt.%, moreP0000875WQ CPL20 preferably from 3 to 15 wt.% and is preferably selected from an alcohol ethoxylate, an alcohol propoxylate, a methyl ester ethoxylate and an alkyl poly glycoside.

7. A detergent composition according to any preceding claim, wherein the detergent composition is a laundry detergent composition.

8. A laundry detergent composition according to claim 7, wherein the laundry detergent composition is in the form of a liquid, solid, powder, pastille, bead or paste, preferably the composition is a liquid or a powder, more preferably a liquid detergent.

9. A laundry detergent composition according to claim 7 or claim 8, wherein the laundry detergent composition comprises an alkoxylated polyamine, preferably at a level of from 0.1 to 8 wt.%, more preferably from 0.2 to 6 wt.%, most preferably from 0.5 to 5 wt.%.

10. A laundry detergent composition according to any one of claims 7 to 9, wherein the laundry detergent composition comprises soil release polymer, the soil release polymer preferably selected from copolyesters of dicarboxylic acids and polydiols, more preferably a copolyester formed by condensation of terephthalic acid ester and 1 ,2-propanediol, the soil release polymer preferably present at a level of from 0.1 to 8 wt.%, more preferably from 0.2 to 6 wt.%, most preferably from 0.5 to 5 wt.%.

11. A composition according to any preceding claim, additionally comprising one or more further enzymes selected from the group consisting of: proteases, lipases, cellulases, alpha-amylases, peroxidases / oxidases, pectate lyases, and / or mannanases, preferably the one or more enzymes comprises protease, amylase, lipase and / or lipase, more preferably protease.

12. A method of treatment of a fabric substrate with a sebum stain, said method comprising treatment of a fabric substrate with a sebum stain, with a composition comprising from 0.0005 to 6 wt.%, preferably from 0.005 to 4 wt.%, more preferably from 0.01 to 2 wt.% of a lysophospholipase enzyme having a sequence identity of at least 70%, preferably at least 75%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1.P0000875WQ CPL2113. A method of treatment of a fabric substrate according to claim 12, wherein the composition is a detergent composition according to any one of claims 2 to 11.

14. Use of a lysophospholipase enzyme having a sequence identity of at least 70%, preferably at least 75%, more preferably 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, most preferably 100% with SEQ. ID. 1 to improve cleaning of sebum stains of fabric.

Images