CN111479912B

CN111479912B - Detergent composition comprising protease

Info

Publication number: CN111479912B
Application number: CN201880077676.7A
Authority: CN
Inventors: 朱虎; 张虹; 李慧; D·A·兰; M·L·汤普森
Original assignee: Unilever IP Holdings BV
Current assignee: Unilever IP Holdings BV
Priority date: 2017-11-30
Filing date: 2018-10-31
Publication date: 2021-08-10
Anticipated expiration: 2038-10-31
Also published as: EP3717616B1; CN111479912A; BR112020010648A2; EP3717616A1; WO2019105675A1

Abstract

The present invention provides a detergent composition comprising: (i)1 to 60 wt%, preferably 2 to 50 wt%, more preferably 4 to 50 wt% of a surfactant; (ii)0.0005 to 1 wt.%, preferably 0.005 to 0.6 wt.% of a protease having at least 90% sequence identity to SEQ ID NO 1; and methods and uses for improving enzymatic cleaning in water using the protease.

Description

Detergent composition comprising protease

Technical Field

The present invention relates to surfactant containing detergent compositions incorporating novel proteases.

Background

Water can be a scarce resource. Users who wish to use detergent compositions on substrates, especially laundry detergents on fabrics, may only be able to use water which is not optimal for cleaning. An example of this is the use of sometimes saline water (water with significant sodium chloride content), such as sea water.

Proteases are common ingredients in cleaning compositions. One problem with commercial proteases is that they function poorly under saline conditions.

Disclosure of Invention

We have found that the incorporation of a novel protease according to claim 1 in detergent compositions shows enhanced cleaning. In one aspect, the present invention provides a detergent composition comprising:

(i)1 to 60 wt%, preferably 2 to 50, more preferably 4 to 50 wt% of a surfactant;

(ii)0.0005 to 1 wt.%, preferably 0.005 to 0.6 wt.% of a protease having at least 90% sequence identity with SEQ ID NO 1.

Preferably, the protease has at least 95%, more preferably 97% sequence identity to SEQ ID NO 1. Most preferably, the protease has 1100% sequence identity to SEQ ID NO. Preferred detergent compositions are laundry detergent compositions. Preferably, the laundry detergent composition is a liquid or powder, more preferably, the detergent is a liquid detergent.

Preferably, the laundry detergent composition comprises anionic and/or nonionic surfactants, more preferably, the laundry detergent composition comprises anionic and nonionic surfactants.

The laundry detergent preferably comprises an alkoxylated polyamine. The laundry detergent preferably comprises a soil release polymer, more preferably a polyester based soil release polymer.

Preferably, the laundry detergent comprises a phosphonic acid (or salt thereof) chelant in an amount of less than 0.1 wt%, more preferably less than 0.01 wt%, most preferably the composition is free of phosphonic acid (or salt thereof) chelants.

Preferred detergent compositions, especially laundry detergent compositions, further comprise a further enzyme selected from the group consisting of: lipases, cellulases, alpha-amylases, peroxidases/oxidases, pectate lyases, mannanases, and/or additional proteases. In another aspect, the present invention provides a method of improving enzymatic cleaning in water having a sodium chloride content of 0.1 to 4%, preferably 0.25 to 3% by weight at 20 ℃, said method comprising introducing a protease having at least 90% sequence identity to SEQ ID No.1 into a detergent composition comprising 1 to 60% by weight of a surfactant; and subsequently treating a substrate, preferably a fabric, with said composition.

In another aspect, the invention provides the use of a protease having at least 90%, preferably 95%, more preferably 97%, most preferably 100% sequence identity to SEQ ID No.1 for improving enzymatic cleaning in water having a sodium chloride content of 0.1 to 4% at a temperature of 15 ℃ to 45 ℃.

Detailed Description

As used herein, the indefinite article "a" and "an" and its corresponding definite article "the" mean at least one, or one or more, unless otherwise specified.

Unless otherwise specified, all% levels of ingredients in the compositions (formulations) listed herein are by weight based on the total formulation.

The detergent composition may take any suitable form, for example a liquid, solid (including powder) or gel.

The detergent composition may be applied to any suitable substrate. A particularly preferred substrate is a fabric. Particularly preferred detergent compositions are laundry detergent compositions.

The laundry detergent composition may take any suitable form. The preferred form is a liquid or a powder, with a liquid being most preferred.

Surface active agent

The detergent composition comprises a surfactant (which comprises a mixture of two or more surfactants). The composition comprises from 1 to 60 wt%, preferably from 2 to 50 wt%, more preferably from 4 to 50 wt% of a surfactant. Even more preferred levels of surfactant are 6 to 30 wt%, more preferably 8 to 20 wt%.

The detergent composition (preferably, a laundry detergent composition) comprises anionic and/or nonionic surfactants, preferably both anionic and nonionic surfactants.

Suitable anionic detergent compounds which may be used are typically water-soluble alkali metal salts of organic sulphuric and sulphonic acids having an alkyl group containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the alkyl portion of higher alkyl groups.

Examples of suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulphates, in particular higher C by reaction, for example from tallow or coconut oil₈To C₁₈Those obtained by sulfating alcohols, alkyl C₉To C₂₀Sodium and potassium benzene-sulphonates, especially linear secondary alkyl C₁₀To C₁₅Sodium benzenesulfonate; and sodium alkyl glyceryl ether sulfates, particularly those ethers of higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum.

The anionic surfactant is preferably selected from: linear alkyl benzene sulfonate; an alkyl sulfate; alkyl ether sulfates; soap; alkyl (preferably methyl) ester sulfonates and mixtures thereof.

Most preferred anionic surfactants are selected from: linear alkyl benzene sulfonate; an alkyl sulfate; alkyl ether sulfates and mixtures thereof. Preferably, the alkyl ether sulphate is C with an average of 1 to 3 EO (ethoxylate) units₁₂-C₁₄N-alkyl ether sulfates.

Sodium Lauryl Ether Sulfate (SLES) is particularly preferred. Preferably, the linear alkylbenzene sulfonate is C₁₁To C₁₅Sodium alkyl benzene sulfonate. Preferably, the alkyl sulfates are linear or branched C₁₂To C₁₈Sodium alkyl sulfate. Sodium dodecyl sulfate is particularly preferred (SDS, also known as primary alkyl sulfate).

In liquid formulations, preferably two or more anionic surfactants are present, such as linear alkyl benzene sulphonate together with alkyl ether sulphate.

In liquid formulations, preferably, the laundry composition comprises, in addition to the anionic surfactant, an alkyl ethoxylated nonionic surfactant, preferably from 2 to 8 wt% of alkyl ethoxylated nonionic surfactant. 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 fatty alcohols, acids or amides, with especially ethylene oxide, alone or together with propylene oxide. Preferred nonionic detergent compounds are aliphatic C₈To C₁₈Condensation products of linear or branched primary or secondary alcohols with ethylene oxide.

Most preferably, the nonionic detergent compound is an alkyl ethoxylated nonionic surfactant which is a C having an average ethoxylation of from 7EO to 9EO units₈To C₁₈A primary alcohol.

Preferably, the surfactant used is saturated.

Protease enzyme

We have found that this protease works well under saline conditions. Thus, the protease may be considered salt tolerant (halotolerant). This means that it can function in a high salt environment.

We have also found that the protease works well at low temperatures of 20 ℃.

The protease outperforms commercial proteases in both high temperature at 40 ℃ and low temperature saline environments at 20 ℃.

The protease is present in an amount of 0.0005 to 1 wt.%, preferably 0.005 to 0.6 wt.%.

The protease has at least 90%, preferably 95%, or even 97% sequence identity to SEQ ID NO 1. Most preferably, the protease may have 1100% sequence identity to SEQ ID NO.

Alkoxylated polyamines

When the detergent composition is in the form of a laundry composition, it preferably comprises an alkoxylated polyamine. The preferred content of alkoxylated polyamine is in the range of 0.1 to 8 wt.%, preferably 0.2 to 6 wt.%, more preferably 0.5 to 5 wt.%. Another preferred content is 1 to 4 wt%.

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

One preferred material is an alkoxylated polyethyleneimine, most preferably an ethoxylated polyethyleneimine, having an average degree of ethoxylation of from 10 to 30, preferably from 15 to 25, wherein the nitrogen atoms are ethoxylated.

Soil release polymers

When the detergent composition is in the form of a laundry composition, it preferably comprises a soil release polymer.

Preferred levels of soil release polymer are in the range of 0.1 to 10 wt%, preferably 0.2 to 8 wt%, more preferably 0.25 to 7 wt%, most preferably 0.5 to 6 wt%.

Suitable polyester-based soil release polymers are described in WO 2014/029479 and WO 2016/005338.

Additional enzymes

In addition to the specified enzymes, additional enzymes may be present in the detergent composition. It is preferred that the additional enzyme is present in the preferred laundry detergent composition.

Each enzyme, if present, is present in the laundry composition of the present invention at a level of from 0.0001 wt% to 0.1 wt%.

The amount of enzyme present in the composition is preferably related to the amount of enzyme as pure protein.

Preferred further enzymes include those selected from the group consisting of: lipases, cellulases, alpha-amylases, peroxidases/oxidases, pectate lyases, mannanases, and/or additional proteases. The preferred additional enzyme comprises a mixture of two or more of these enzymes.

Preferably, the further enzyme is selected from: a lipase, a cellulase, an alpha-amylase, and/or another protease.

Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include those from: humicola (Humicola) (synonym thermophilic fungi (Thermomyces)), for example from h.lanuginosa (t.lanuginosus) as described in EP 258068 and EP 305216 or from h.insolens as described in WO 96/13580; pseudomonas lipases, for example from pseudomonas alcaligenes (p. alcaligenes) or pseudomonas pseudoalcaligenes (p. pseudoalcaligenes) (EP 218272), pseudomonas cepacia (p.cepacia) (EP 331376), pseudomonas stutzeri (GB 1,372,034), pseudomonas fluorescens (p. fluoroscens), pseudomonas strains SD 705(WO 95/06720 and WO 96/27002), p.wisconsinensis (WO 96/12012); bacillus lipases, for example from Bacillus subtilis (B.subtilis) (Dartois et al (1993), Biochemica et Biophysica Acta,1131,253-360), Bacillus stearothermophilus (B.stearothermophilus) (JP 64/744992) or Bacillus pumilus (B.pumilus) (WO 91/16422).

Further examples are lipase variants, such as those described in WO 92/05249, WO 94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202, WO 00/60063.

Preferred commercially available lipases include Lipolase^TMAnd Lipolase Ultra^TM、Lipex^TMAnd Lipoclean^TM(Novozymes A/S)。

The process of the invention may be carried out in the presence of a phospholipase classified under EC 3.1.1.4 and/or EC 3.1.1.32. As used herein, the term phospholipase is an enzyme that is active on phospholipids.

Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified at the outer (sn-1) and middle (sn-2) positions with two fatty acids and phosphorylated at the third position; phosphorus (P)The acid in turn can be esterified to an amino alcohol. Phospholipases are enzymes involved in phospholipid hydrolysis. Can distinguish between various types of phospholipase activity, including phospholipase A₁And A₂Which hydrolyses one fatty acyl group (at the sn-1 and sn-2 positions, respectively) to form lysophospholipids; and lysophospholipase (or phospholipase B), which can hydrolyze the remaining fatty acyl groups in lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol or phosphatidic acid, respectively.

Proteases hydrolyze the peptides and bonds within the protein, which results in enhanced removal of protein or peptide containing stains in a laundry environment. Examples of suitable protease families include aspartic proteases, cysteine proteases, glutamic proteases, asparagine (aspargegine) peptide lyases, serine proteases and threonine proteases. Such protease families are described in the MEROPS peptidase database (http:// polymers. sanger. ac. uk /). Serine proteases are preferred. The subtilisin type serine proteases are more preferred. The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al, Protein Engng.4(1991)719-737 and Siezen et al, Protein Science 6(1997) 501-523. Serine proteases are a subset of proteases characterized by a serine at the active site that forms a covalent adduct with a substrate. Subtilases can be divided into 6 sub-classes, namely the Subtilisin (Subtilisin) family, the thermolysin (thermolase) family, the proteinase K family, the lanthionine (Lantibiotic) peptidase family, the Kexin family and the Pyrrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US7262042 and WO09/021867, as well as subtilisin (subtilisin lentitus), subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279, and the protease PD138 described in WO 93/18140. Other useful proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO 02/016547. Examples of trypsin-like proteases are trypsin (e.g.of porcine or bovine origin) and fusarium protease as described in WO89/06270, WO94/25583 and WO05/040372, and chymotrypsin derived from Cellulomonas (Cellumonas) as described in WO05/052161 and WO 05/052146.

Most preferably, the protease is subtilisin (EC 3.4.21.62).

Examples of subtilases are those derived from Bacillus such as Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US7262042 and WO09/021867, as well as subtilisin tarda, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279, and protease PD138 described in WO 93/18140. Preferably, the subtilisin is derived from Bacillus, preferably Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii, as described in U.S. Pat. No. 6,312,936B 1, U.S. Pat. No. 5,679,630, U.S. Pat. No. 4,760,025, U.S. Pat. No. 7,262,042 and WO 09/021867. Most preferably, the subtilisin is derived from Bacillus gibsonii or Bacillus lentus.

Suitable commercially available proteases include those under the trade name

Duralase^TM，Durazym^TM，

Ultra，

Ultra，

Ultra，

Ultra，

And

those sold, all as

Or

(Novozymes A/S).

The compositions may use cutinases classified as EC 3.1.1.74. The cutinase to be used according to the invention may be of any origin. Preferably, the cutinase is of microbial origin, in particular of bacterial, fungal or yeast origin.

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, alpha-amylases obtained from Bacillus, such as particular strains of Bacillus licheniformis described in more detail in GB 1,296,839, or strains of Bacillus disclosed in WO 95/026397 or WO 00/060060. A commercially available amylase is Duramyl^TM、Termamyl^TM、Termamyl Ultra^TM、Natalase^TM、Stainzyme^TM、Fungamyl^TMAnd BAN^TM(Novozymes A/S)、Rapidase^TMAnd Purastar^TM(from Genencor International Inc.).

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from: bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. as disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757, WO89/09259, WO 96/029397 and WO 98/012307, the fungus cellulases produced by Humicola insolens, Thielavia terrestris, myceliophthora thermophila and Fusarium oxysporum. Commercially available cellulases include Celluzyme^TM、Carezyme^TM、Celluclean^TM、Endolase^TM、Renozyme^TM(Novozymes A/S)、Clazinase^TMAnd Puradax HA^TM(Genencor International Inc.) and KAC-500(B)^TM(Kao Corporation)。Celluzyme^TMIs preferred.

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., Coprinus cinereus, and variants thereof, such as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme^TMAnd Novozym^TM51004(Novozymes A/S)。

Further suitable enzymes are discussed in WO2009/087524, WO2009/090576, WO2009/107091, WO2009/111258 and WO 2009/148983.

The aqueous solution used in the process preferably has the enzyme present. The enzyme is preferably present in the aqueous solution used in the process at a concentration in the range of 0.01 to 10ppm, preferably 0.05 to 1 ppm.

Enzyme stabilizer

Any enzyme present in the composition may be stabilized using conventional stabilizers, for example polyols such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or a boric acid derivative, for example an aromatic borate ester, or a phenyl boronic acid derivative, for example 4-formylphenyl boronic acid, and the compositions may be formulated as described, for example, in WO 92/19709 and WO 92/19708.

Chelating agents

The chelant may or may not be present in the detergent composition.

Example phosphonic acid (or salts thereof) chelating agents are: 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP); diethylenetriamine penta (methylene phosphonic acid) (DTPMP); hexamethylenediamine tetra (methylene phosphonic acid) (HDTMP); aminotris (methylenephosphonic Acid) (ATMP); ethylenediaminetetra (methylenephosphonic acid) (EDTMP); tetramethylenediaminetetra (methylenephosphonic acid) (TDTMP); and phosphonobutane-tricarboxylic acid (PBTC).

Further materials

Further optional but preferred materials which the detergent composition (preferably a laundry detergent composition) may comprise include fluorescers, perfumes, shading dyes and polymers.

Fluorescent agent

The composition preferably comprises a fluorescent agent (optical brightener). Fluorescent agents are well known, and many such fluorescent agents are commercially available. Typically, these fluorescent agents are supplied and used in the form of their alkali metal salts, e.g., sodium salts.

The total amount of fluorescent agent or agents used in the composition is typically from 0.0001 to 0.5 wt%, more preferably from 0.005 to 2 wt%, more preferably from 0.01 to 0.1 wt%.

Preferred classes of fluorescers are: distyrylbiphenyl compounds such as Tinopal (trademark) CBS-X; diamine stilbene disulfonic acid compounds such as Tinopal DMS pure Xtra and Blankophor (trade Mark) HRH; and pyrazoline compounds such as Blankophor SN.

Preferred fluorescers are those having CAS-No 3426-43-5; CAS-No 35632-99-6; CAS-No 245765-13-7; CAS-No 12224-16-7; CAS-No 13863-31-5; CAS-No 4193-55-9; CAS-No 16090-02-1; CAS-No 133-66-4; CAS-No 68444-86-0; fluorescent agent of CAS-No 27344-41-8.

The most preferred fluorescent agents are: sodium 2 (4-styryl-3-sulfophenyl) -2H-naphthol [1,2-d ] triazole, disodium 4,4' -bis { [ (4-anilino-6- (N-methyl-N-2 hydroxyethyl) amino 1,3, 5-triazin-2-yl) ] amino } stilbene-2, 2' disulfonate, disodium 4,4' -bis { [ (4-anilino-6-morpholinyl-1, 3, 5-triazin-2-yl) ] amino } stilbene-2-2 ' disulfonate, and disodium 4,4' -bis (2-sulfostyryl) biphenyl.

The aqueous solution used in the method preferably has a fluorescent agent present. The fluorescent agent is preferably present in the aqueous solution used in the method in the range of 0.0001 to 0.1g/L, more preferably 0.001 to 0.02 g/L.

Perfume

The composition preferably comprises a perfume. Many suitable examples of fragrances are provided in CTFA (Cosmetic, Toiletry and Fragrance Association)1992 International layers Guide, published by CFTA Publications, and OPD 1993 Chemicals layers Directory 80th annular Edition, published by Schnell Publishing Co.

Preferably, the fragrance comprises at least one of the following notes (compounds): alpha-isomethyl ionone, benzyl salicylate; citronellol; coumarin; hexyl cinnamic aldehyde; linalool; 2-methyl pentanoic acid ethyl ester; octanal; benzyl acetate; 3, 7-dimethyl-1, 6-octadien-3-ol 3-acetate; 2- (1, 1-dimethylethyl) -cyclohexanol 1-acetate; delta-damascone (damascone); beta-ionone; tricyclodecenyl acetate (verdyl acetate); dodecanal; hexyl cinnamaldehyde (hexyl cinnnamic aldehyde); cyclopentadecanolide; 2-phenylethyl phenylacetate; amyl salicylate; beta-caryophyllene; ethyl undecylenate; geranyl anthranilate; α -irone; beta-phenylethyl benzoate; α -santalol; cedrol; cedryl acetate; cedryl formate (cedry format); cyclohexyl salicylate; gamma-dodecalactone, and beta-phenylethylphenyl acetate.

Useful components of perfumes include both materials of natural and synthetic origin. They include single compounds and mixtures. Specific examples of such components can be found in the literature, for example, in the Feraroli's Handbook of flavour Ingredients,1975, CRC Press; jacobs, Synthetic Food adjuns, 1947, edited by Van nonstrand; or s.arctander, Perfume and flavour Chemicals,1969, Montclair, n.j. (USA).

It is common for multiple perfume components to be present in a formulation. In the compositions of the present invention, it is envisaged that four or more, preferably five or more, more preferably six or more, or even seven or more different perfume components will be present.

In the perfume mixture, preferably 15 to 25% by weight is top notes. Top notes are defined by Poucher (Journal of the Society of Cosmetic Chemists 6(2):80[1955 ]). Preferred top notes are selected from citrus oil, linalool, linalyl acetate, lavender, dihydromyrcenol, rose oxide and cis-3-hexanol.

The international daily-use perfumery association has issued a list of fragrance ingredients (fragrances) in 2011. (http:// www.ifraorg.org/en-us/ingredients #. U7Z4 hPldWzk).

The international daily fragrance institute provides a database of fragrances (fragrances) with safety information.

Perfume top notes can be used to suggest the whiteness and brightness benefits of the present invention.

Some or all of the perfume may be encapsulated, typical perfume components which facilitate encapsulation include those having a relatively low boiling point, preferably a boiling point of less than 300 ℃, preferably 100 ℃ and 250 ℃. It is also advantageous to encapsulate perfume components having a low Clog P (i.e. those that will have a higher tendency to partition into water), preferably having a Clog P of less than 3.0. These materials having relatively low boiling points and relatively low CLog P have been referred to as "delayed blooming" perfume ingredients and comprise one or more of the following materials: allyl hexanoate, amyl acetate, amyl propionate, anisaldehyde, anisole, benzaldehyde, benzyl acetate, benzyl acetone, benzyl alcohol, benzyl formate, benzyl isovalerate, benzyl propionate, β - γ hexenol, camphor gum, l-carvone, d-carvone, cinnamyl alcohol, cinnamyl formate (cinamyl form), cis-jasmone, cis-3-hexenyl acetate, cuminol, cyclal c, dimethyl benzyl methanol acetate, ethyl acetoacetate, ethyl ethylacetoacetate, ethylamyl ketone, ethyl benzoate, ethyl butyrate, ethylhexyl ketone, ethylphenyl acetate, eucalyptol, eugenol, fenchyl acetate (fenchyl acetate), flor acetate (tricyclodecenyl acetate), tricyclodecene propionate, geraniol, hexenol, hexenyl acetate, hexyl acetate, Hexyl formate, solanol (hydroacetylalcohol), hydroxycitrocitronellal, indanone, isoamyl alcohol, isomenthone, isopulegyl acetate, isoquinolinone, ligustral, linalool oxide, linalyl formate, menthone, menthylacetone, methyl amyl ketone, methyl anthranilate, methyl benzoate, methyl benzyl acetate, methyl eugenol, methyl heptenone, methyl heptyne carbonate, methyl heptyne ketone, methyl hexyl ketone, methyl phenyl methyl acetate, methyl salicylate, methyl-n-methyl anthranilate, nerol, octolactone, octanol, p-cresol methyl ether, p-methoxyacetophenone, p-methylacetone, phenyl acetaldehyde, phenyl ethyl acetate, phenyl ethyl alcohol, Phenylethyldimethylcarbinol, prenyl acetate, propyl borate, pulegone, rose oxide, safrole, 4-terpinenol (4-terpinenol), alpha-terpinenol, and/or phenylacetaldehyde dimethanol acetal (viridine). It is common for multiple perfume components to be present in a formulation. In the compositions of the present invention, it is envisaged that there will be four or more, preferably five or more, more preferably six or more, or even seven or more different perfume components present in the perfume from the given list of delayed release perfumes given above.

Another group of fragrances that may be employed with the present invention are the so-called "aromatherapy" materials. These include many components that are also used in perfumes, including components of essential oils such as sage, eucalyptus, geranium, lavender, dried nutmeg skin (Mace) extract, neroli, nutmeg, spearmint, sweet violet leaves and valerian.

It is preferred that the laundry treatment composition is devoid of peroxygen bleach, such as sodium percarbonate, sodium perborate and peracids.

Shading dye

Preferably, when the composition is a laundry detergent composition then it comprises a hueing dye. Preferably, the hueing dye is present at 0.0001 to 0.1 wt% of the composition.

Dyes are described in Color Chemistry Synthesis, Properties and Applications of Organic Dyes and Pigments (H Zollinger, Wiley VCH, Surich, 2003) and Industrial Dyes Chemistry, Properties Applications (K Hunger (ed), Wiley-VCH Weinheim 2003).

Hueing dyes for laundry compositions preferably have a maximum absorption in the visible range (400-700nm) of greater than 5000L mol^-1cm^-1Preferably greater than 10000L mol^-1cm^-1The extinction coefficient of (a). The color of the dye is blue or violet.

Preferred shading dye chromophores are azo, azine, anthraquinone and triphenylmethane.

Azo, anthraquinone, phthalocyanine and triphenylmethane dyes preferably carry a net anionic charge or no charge. Azines preferably carry a net anionic or cationic charge. During the washing or rinsing step of the washing process, a blue or violet shading dye is deposited onto the fabric, providing a visible shade to the fabric. In this regard, the dye imparts a blue or violet color to the white cloth with a hue angle of 240 to 345, more preferably 250 to 320, most preferably 250 to 280. The white cloth used in this test was a bleached, non-mercerized woven cotton sheet.

Hueing dyes are discussed in WO2005/003274, WO2006/032327(Unilever), WO2006/032397(Unilever), WO2006/045275(Unilever), WO06/027086(Unilever), WO2008/017570(Unilever), WO2008/141880(Unilever), WO2009/132870(Unilever), WO2009/141173(Unilever), WO2010/099997(Unilever), WO2010/102861(Unilever), WO2010/148624(Unilever), WO2008/087497(P & G), WO2011/011799(P & G), WO2012/054820(P & G), WO2013/142495(P & 151g) and WO2013/151970(P & G).

The monoazo dyes preferably contain a heterocyclic ring, and are most preferably thiophene dyes. The monoazo dyes are preferably alkoxylated and are preferably uncharged or anionically charged at pH 7. Alkoxylated thiophene dyes are discussed in WO/2013/142495 and WO/2008/087497. Preferred examples of thiophene dyes are shown below:

the disazo dye is preferably a sulfonated disazo dye. Preferred examples of sulfonated bisazo compounds are direct violet 7, direct violet 9, direct violet 11, direct violet 26, direct violet 31, direct violet 35, direct violet 40, direct violet 41, direct violet 51, direct violet 66, direct violet 99 and alkoxylated forms thereof. Alkoxylated disazo dyes are discussed in WO2012/054058 and WO 2010/151906.

Examples of alkoxylated disazo dyes are:

thiophene dyes are available from Milliken under the trade names Liquitint Violet DD and Liquitint Violet ION.

The azine dye is preferably selected from sulphonated phenazine dyes and cationic phenazine dyes. Preferred examples are acid blue 98, acid violet 50, dyes having CAS number 72749-80-5, acid blue 59, and phenazine dyes selected from the group consisting of:

wherein:

X₃selected from: -H, -F, -CH₃，-C₂H₅，-OCH₃and-OC₂H₅；

X₄Selected from: -H, -CH₃，-C₂H₅，-OCH₃and-OC₂H₅；

Y₂Selected from: -OH, -OCH₂CH₂OH，-CH(OH)CH₂OH，-OC(O)CH₃And C (O) OCH₃。

The hueing dye is present in the composition in the range of 0.0001 to 0.5 wt%, preferably 0.001 to 0.1 wt%. Depending on the nature of the hueing dye, there is a preferred range depending on the potency of the hueing dye, which depends on the class and the specific potency within any particular class. As mentioned above, the hueing dye is a blue or violet hueing dye.

Mixtures of hueing dyes may be used.

Most preferably, the hueing dye is a reactive blue anthraquinone dye covalently linked to an alkoxylated polyethyleneimine. The alkoxylation is preferably selected from ethoxylation and propoxylation, most preferably propoxylation. Preferably, 80 to 95 mole% of the N-H groups in the polyethyleneimine are replaced by isopropanol groups by propoxylation. Preferably, the molecular weight of the polyethyleneimine is 600 to 1800 prior to reaction with the dye and propoxylation.

An example structure of a preferred reactive anthraquinone covalently linked to a propoxylated polyethyleneimine is:

(Structure I).

Polymer and method of making same

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

A sequence table: SEQ ID NO 1

Examples

The invention will be illustrated by the following non-limiting examples.

Isolation and culture of protease-producing microorganisms

By mixing the components in a mixture containing (g/L seawater): selective screening on skim milk agar plates of tryptone 5, yeast extract 2, skim milk powder 40, agar powder 16, protease-producing microbial strains were isolated from marine sediment samples collected in gulf of china (yellow sea, 36 ° 09'N, 120 ° 32' E). The plates were incubated at 20 ℃ for 48-72 hours to obtain bacterial colonies. Colonies with a transparent hydrolysis loop of casein in milk were evaluated as protease producers. Proteolytic LA-05 strain showing a larger hydrolytic loop was selected for further experiments. The seeds and fermentation medium for the culture of LA-05 strain consisted of 5g tryptone, 2g yeast extract and 1L seawater, pH 7.0. The medium was autoclaved at 121 ℃ for 20 minutes. First, the isolated LA-05 strain was inoculated with 2% (v/v) seed medium and cultured on a rotary shaker incubator at 20 ℃ and 150rpm for 12 hours. The culture was then transferred to a 2-L Erlenmeyer flask containing 400ml of fermentation medium and incubated at 15-30 ℃ and 150rpm for 78 hours.

To obtain maximum enzyme yield, the kinetics of growth and enzyme production were measured every 6 hours during the incubation period (78 hours). Cell density was monitored by measuring absorbance at 600 nm. Cell-free supernatants were recovered by centrifugation at 12000rpm for 20min at 4 ℃ and then used as crude enzyme preparations to determine protease activity.

Strain identification and phylogenetic analysis

For genus identification of LA-05 strain, Analytical characterization index (API) dipstick tests and 16S rRNA gene sequencing were performed. The physiological and biochemical properties of strain LA-05 were studied using an API test strip according to the manufacturer's instructions.

The 16S rRNA sequence was amplified by PCR using a forward primer (27F, 5'-AGAGTTTGATCMTGGCTCAG-3') and a reverse primer (1492R, 5 '-TACGGYTACCTTGTTACGACTT-3'). Genomic DNA of strain LA-05 was purified by TIANAmp Bacteria DNA Kit (TIANGEN, Beijing, China) and then used as a template for PCR amplification involving 30 cycles (cycle parameters: denaturation at 94 ℃ for 50s, primer annealing at 58 ℃ for 50s, and extension at 72 ℃ for 100 s).

The amplified product was cloned in pMD18-T vector (Takara, Dalian, China), and a recombinant plasmid pMD-16S was constructed. The recombinant plasmid was then transferred into competent cells of E.coli DH5 a. Recombinant clones of E.coli DH5a were cultured in LB broth medium containing ampicillin (60. mu.g/ml). The DNA fragment ligated to 16S rRNA in the recombinant plasmid was confirmed by commercial DNA sequencing (Sangon Biotech co., ltd., shanghai, china). Multiple sequence alignments were performed through the EzTaxon database using the recognition program. Model culture strains with a pairwise similarity higher than 97% were selected and phylogenetic and Molecular evolution Analysis was performed by the neighbor-joining method using Molecular Evolution Genetic Analysis (MEGA) software (version 7.0.9). Statistical evaluation of the tree topology was calculated by boot value analysis (bootstrap analysis) using 1000 replications.

Protease purification

All purification steps were performed at 4 ℃ unless otherwise specified.

Concentrating by ultrafiltration

400ml of 30-hour-old culture were centrifuged at 12,000rpm for 20 minutes at 4 ℃. The supernatant was filtered through a 0.22 μm filter to completely remove bacterial cells and media debris and recovered as a crude protease preparation. The prepared supernatant was concentrated by a Millipore's Amicon Ultra-4 centrifugal filter unit with a molecular weight cut-off of 3 kDa. After washing three times with ultrapure water, the buffer of the crude protease was replaced with 50mM Tris-HCl buffer (pH8.0) containing 0.1M NaCl.

Purification of

2ml of the concentrated solution was loaded on a gel filtration column (HiLoad 16/600 Superdex 200pg, GE Healthcare) equilibrated and eluted with 50mM Tris-HCl buffer (pH8.0) containing 0.1M NaCl. The column was eluted with 180ml of buffer at a flow rate of 1ml/min until the optical density of the eluate at 280nm was zero. Elution fractions of 3ml each were collected and analyzed for protease activity. Fractions showing protease activity were pooled and then used on a HiTrap Q FF (1ml) ion exchange column equilibrated with 50mM Tris-HCl buffer (pH8.0) containing 0.1M NaCl.

The column was then washed with the same buffer and the bound proteins were eluted with a linear gradient of NaCl in the range of 0.1-1M at a flow rate of 1 ml/min. Fractions eluted at 1ml each were collected and analyzed for protease activity. Fractions containing protease activity were pooled and stored at-80 ℃ for further study.

Determination of protease Activity

Protein concentrations were determined using the BCA protein assay kit (Sangon Biotech, shanghai, china) using Bovine Serum Albumin (BSA) as a reference. Protease activity was determined according to a modified method (Lagzian and Asoodeh, 2012). Briefly, 0.25ml aliquots of purified protease were incubated with 0.75ml of 50mM Tris-HCl buffer (pH8.0) containing 1% (w/v) casein at 45 ℃ for 10 minutes. The reaction was stopped by adding 0.5mL of 20% (w/v) trichloroacetic acid (TCA). The mixture was mixed with lab-dancer and left at room temperature for 20 minutes. Then, the precipitate was removed by centrifugation at 12,000rpm for 20 minutes, and the absorbance of the supernatant was measured at 280 nm. One unit (U) of protease activity is defined as the amount of enzyme that hydrolyses casein under the experimental conditions to release 1. mu.g tyrosine per minute. Protease activity units were calculated using tyrosine (0-100. mu.g/ml) as standard.

Electrophoresis, mass spectrometry, zymography (zymography) and isoelectric focusing

Molecular weight

The molecular weight of the purified protease was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad Mini-PROTEIN apparatus (Farhadian et al, 2015) according to standard protocols using (5%, w/v) stacking gel and (12%, w/v) separation gel. The gel was stained with Coomassie Brilliant blue R-250 and destained with methanol-acetic acid-water (5/1/4, v/v/v). The relative molecular weight of the purified enzyme was estimated by comparing its mobility to standard protein markers.

At the same time, a FlashDetector is used^TMInstrument (Bruker microflex)^TMLRF, Bruker daltons, USA), the precise molecular mass of the purified protease was analysed in linear forward mode by matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF/MS). Data were collected by FlexControl 3.4 and used with FlexAnalysis 3.4 software analysis.

Zymogram

The enzyme activity was visualized by zymography using a modified method (Machado et al, 2016). Briefly, samples were mixed with a β -mercaptoethanol-free gel loading buffer and loaded onto an electrophoresis gel without heating. Electrophoresis was performed in ice water. Next, the gel was immersed in 50mM Tris-HCl buffer (pH8.0) containing 2.5% Triton X-100 at 4 ℃ and shaken gently for 1 hour to remove SDS. To extract the residual Triton X-100, the gel was washed twice with 50mM Tris-HCl buffer (pH8.0) at 4 ℃ for 20 minutes each, and then incubated with 1% (w/v) casein in 50mM Tris-HCl buffer (pH8.0) at 25 ℃ for 1 hour. The gel was soaked in 20% (w/v) trichloroacetic acid (TCA) to stop the protease reaction, stained with Coomassie Brilliant blue R-250 for 2 hours, and destained overnight with methanol-acetic acid-water (5/1/4, v/v/v) to reveal the protease hydrolysis band.

Isoelectric focusing

Isoelectric focusing of purified proteases was performed on gel strips (Bio-Rad, USA) with a fixed pH gradient (IPG) of 3 to 10 using the Multiphor II electrophoresis system (GE Healthcare). Briefly, the purified protease was desalted and washed with 1% glycine buffer by ultrafiltration. After immobilization of the IPG strip, Pre-electrophoresis (Pre-electrophoresis) was started at 700V for 20min at 15 ℃. The samples and IEF standards were then subjected to electrophoresis at 2000V for 90 minutes at 15 ℃. Finally, the strips were fixed by TCA buffer treatment for 30 min, stained with coomassie blue R-250, and destained with methanol-acetic acid-water mixture. The pI values of the purified proteases were evaluated by ImageQuant TL Version 7.0 software.

Determination of N-terminal amino acid sequence

The protease purified by SDS-PAGE was transferred to polyvinylidene fluoride (PVDF) membrane in CAPS buffer according to the protocol of Matsudaira (Matsudaira, 1987). PVDF membrane was lightly stained with Coomassie Brilliant blue R-250 and the enzyme-containing band was excised. Next, the N-terminal amino acid sequence was analyzed by an automated Edman degradation method using a PPSQ-21A protein Sequencer (SHIMADZU). The first 20 amino acid residues were aligned with those in the UniProtKB/Swiss-Prot database and the Protein Data Bank Protein database using a BLAST homology search (NCBI, USA).

Identification and three-dimensional structural modeling of potential protease encoding genes

The protease-containing band obtained by denaturing SDS-PAGE was carefully excised and the protein was analyzed by tandem mass spectrometry (MALDI MS/MS) as described in the published protocol (Marchand et al, 2009). TOF/TOF Explorer Using default mode^TMThe software (AB SCIEX) processes all the obtained sample spectra. The identification of Peptide Mass Fingerprints (PMF) was searched against NCBI database (non-redundant protein sequences) using GPS Explorer (V3.6) with search engine MASCOT (2.3). Proteins with a protein score confidence interval (c.i.) above 95% are considered confidence recognitions.

Candidate proteins were selected based on MASCOT search results, proteolytic activity and secretion mechanism. To amplify the coding sequence of the protease by PCR, multiple sequence alignments of the coding sequences of the candidates were performed by DNAMAN software. Designing a pair of primers according to the conserved regions of the upstream and downstream coding sequences: 5'-ATGAACCAACAACGTCAACTAAGCTG-3', and 5'-CGGGTCAATCTAAACGCAACG-3'. The coding sequence was amplified and cloned into the pMD18-T vector using E.coli DH5a as the host strain for Sanger sequencing. Finally, the resulting nucleotide sequence is translated into an amino acid sequence, which is a mature protease having 321 amino acid residues. Trace metals in the purified protease were determined by flame atomic absorption spectroscopy.

Washing study in Mini-bottles

Cotton swatches stained with blood/milk/ink on cotton cloth E116(Centre for Testmaterials-Netherlands) were used for mini-bottle washing together with cotton ballast (cotton ballast). Stains were applied in triplicate in wash bottles. FH26 containing 1g/L laundry formulation (two types, labeled F1 and F2) was prepared using water, and protease (control or salt tolerant) was added to a concentration of 5mg/L in a total volume of 100 mL. The enzyme content is equal to the content of 0.5 wt% protease in the formulation. The control benchmark used was the leading commercial protease material (Carnival evaluation, from Novozymes). To replicate saline conditions, 2% final salt concentration (NaCl) was also used.

The washing was carried out at 20 ℃ and 40 ℃ with shaking at 250rpm for 1 hour. Washing at 20 ℃ shows the benefits of the invention even at low temperature conditions.

After washing, the stain was separated from the wash liquor, rinsed 2x in a beaker containing 1L FH26 water, then left to dry overnight. After drying, the stained plates were digitally scanned and their Δ Ε was measured. This value is used to indicate the cleaning effect and is defined as the color difference between the white cloth and the dyed cloth after washing.

Mathematically, Δ E is defined as:

wherein Δ L is a measure of the difference in darkness between the washed cloth and the white cloth; Δ a and Δ b are measures of the difference in red and yellow, respectively, between the two cloths. It is clear from this equation that the lower the Δ Ε value, the whiter the cloth. For such color measurement techniques, see the International Commission on illumination (Commission International de l' Eclairage) (CIE); recommendation on Uniform color Spaces, color difference equations, psychometric color terms, supplement No.2 to CIE Publication, No.15, Colormetric, Bureau Central de Ia CIE, Paris 1978.

Herein, the cleaning effect is expressed in the form of the Stain Release Index (SRI):

SRI＝100-ΔE

the higher the SRI, the cleaner the cloth, the SRI is 100 (white).

Formulations for use

Formulation 1(F1)	(wt%)
		Demineralized water	To 100
Nonionic surfactant (25-7)	4.365
		Tinopal 5BMGX	0.200
Acusol WR	0.700
		TEA	8.820
EU LAS acid	5.820
		Glycerol	2.000
Prifac 5908	0.860
		Dequest 2010	1.500
EU SLES	4.365
		BIT-Proxel	0.040
Citric acid	1.000

Preparation 2(F2)	(wt%)
		Demineralized water	To 100 percent
Tinopal CBS-X	0.090
		NaOH	0.457
TEA	1.500
		Citric acid	0.400
LAS acid (Indianuba)	4.500
		EPEI	2.325
SLES 3EO(Texapon N70LST)	13.500
		EPEI	0.775
BIT	0.0200
		MIT	0.0095
Soladona LA059-2012 uses	0.9800
		NaCl	1.5000
CAPB	1.5000

The two enzymes tested were added to formulations 1 and 2 to give an effective enzyme content of 0.5 wt% in the formulation. The protease is:

protease 1 (comparative) Carnival event, a commercially available enzyme from Novozymes.

Protease 2 (according to the invention) ═ protease with 1100% identity to SEQ ID NO

The results are shown in table 1.

The higher the SRI (stain release index), the better the cleaning.

The results of the wash study show that, as expected, both proteases gave cleaning benefits over the control-only formulation in all wash environments (both at 20 ℃ and 40 ℃, and in different formulations). Wash studies at 20 ℃ show the added benefit of improved cleaning even at low temperature wash conditions (20 ℃).

The performance benefit of this salt tolerant protease was clearly evident in FH26 water containing 2% salt (NaCl). The salt tolerant protease outperformed the control commercial protease in both formulations 1 and 2 at both 20 ℃ and 40 ℃. The cleaning benefit (above baseline protease 1) caused by this salt-tolerant protease is even more noticeable in laundry formulation 2 which does not contain a chelating agent (which is present in formulation 1). Interestingly, at 20 ℃ wash temperatures, the performance benefits are greatly improved, which further serves to highlight the potential applicability of this technology to improve stain cleaning in seawater conditions.

Claims

1. A detergent composition comprising:

(i)1 to 60 wt% of a surfactant;

(ii)0.0005 to 1% by weight of a protease having 1100% sequence identity to SEQ ID NO.

2. The detergent composition of claim 1, comprising from 2 to 50 wt% of a surfactant.

3. The detergent composition of claim 1, comprising from 4 to 50 wt% of a surfactant.

4. The detergent composition of claim 1, comprising 0.005 to 0.6 wt% of the protease.

5. The detergent composition according to any one of claims 1 to 4, wherein the detergent composition is a laundry detergent composition.

6. A laundry detergent composition according to claim 5, wherein the laundry detergent composition is a liquid or powder detergent.

7. A laundry detergent composition according to claim 5, wherein the laundry detergent composition is a liquid detergent.

8. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises an anionic and/or nonionic surfactant.

9. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises both anionic and nonionic surfactants.

10. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises an alkoxylated polyamine.

11. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises the alkoxylated polyamine at a level of from 0.1 to 8 wt%.

12. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises the alkoxylated polyamine at a level of from 0.2 to 6 wt%.

13. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises the alkoxylated polyamine at a level of from 0.5 to 5 wt%.

14. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises a soil release polymer.

15. A laundry detergent composition according to claim 5, wherein the laundry detergent composition comprises a polyester-based soil release polymer.

16. A laundry detergent composition according to claim 5, wherein the chelating agent for phosphonic acid or a salt thereof is present at a level of less than 0.1 wt%.

17. A laundry detergent composition according to claim 5, wherein the chelating agent for phosphonic acid or a salt thereof is present at a level of less than 0.01 wt%.

18. A laundry detergent composition according to claim 5, wherein the composition is free of phosphonic acid or salt thereof chelants.

19. The detergent composition according to any one of claims 1 to 4, further comprising a further enzyme selected from the group consisting of: lipases, cellulases, alpha-amylases, peroxidases/oxidases, pectate lyases, mannanases, and/or additional proteases.

20. A method of improving enzymatic cleaning in water having a sodium chloride content of 0.1 to 4% at 20 ℃, comprising introducing a protease having sequence identity to SEQ ID NO: 1100% into a detergent composition comprising 1 to 60 wt% of a surfactant; and subsequently treating the substrate with the composition.

21. The method of claim 20, wherein the water has a sodium chloride content of 0.25 to 3 wt.%.

22. The method of claim 20, wherein the substrate is a fabric.

23. The method of any one of claims 20 to 22, wherein the composition treating the substrate is the composition of any one of claims 5-19.

24. Use of a protease with 1100% sequence identity to SEQ ID NO for improving enzymatic cleaning in water with a sodium chloride content of 0.1 to 4% at a temperature of 15 ℃ to 45 ℃.