CN111479912A

CN111479912A - Detergent composition comprising protease

Info

Publication number: CN111479912A
Application number: CN201880077676.7A
Authority: CN
Inventors: 朱虎; 张虹; 李慧; D·A·兰; M·L·汤普森
Original assignee: Unilever NV
Current assignee: Unilever IP Holdings BV
Priority date: 2017-11-30
Filing date: 2018-10-31
Publication date: 2020-07-31
Anticipated expiration: 2038-10-31
Also published as: EP3717616A1; WO2019105675A1; EP3717616B1; BR112020010648A2; CN111479912B

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.

In another aspect, the 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 radicalEther 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 sulphate (S L ES) is particularly preferred linear alkylbenzene sulphonate 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 lipases, cellulases, α -amylases, peroxidases/oxidases, pectate lyases, mannanases, and/or additional proteases.

Preferably, the further enzyme is selected from the group consisting of lipase, cellulase, α -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 L ipolase^TMAnd L ipolase Ultra^TM、Lipex^TMAnd L ipoclean^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; phosphoric 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.

Examples of suitable protease families include aspartic proteases, cysteine proteases, glutamic proteases, asparagine (aspragine) peptide lyases, serine proteases and threonine proteases described in the MEROPS peptidase database (http:// polymers. sanger. ac. uk.). serine proteases are preferred. Subtilisin-type serine proteases are more preferred. serine proteases are a subset of proteases characterized by a serine at the active site, which forms a covalent adduct with the substrate according to Siezen et al, Protein Engng.4(1991)719-737 and Siezen et al, Protein Science 6(1997)501-523, the term "Subtilisin" refers to a subset of serine proteases which are characterized by a serine at the active site, the serine proteases form a family of covalent proteases which can be divided into 6 sub-families, namely the Subtilisin (Subtilisin) family, the thermolysin family (Therysin) family of proteases, the Subtilisin family (L) and the lantibiotic family (keisin) of proteases.

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 (α and/or β) include those of bacterial or fungal origin, including chemically modified or protein engineered mutants, amylases include, for example, α -amylase obtained from a Bacillus, e.g., a particular strain of Bacillus licheniformis as described in more detail in GB 1,296,839, or a strain of Bacillus as disclosed in WO 95/026397 or WO 00/060060^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: fungal cellulases produced by bacillus, pseudomonas, humicola, fusarium, thielavia, acremonium, e.g. from humicola insolens, thielavia terrestris, myceliophthora thermophila and fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, WO 89/09259, WO 96/029397 and WO 98/012307. 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(KaoCorporation)。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 fluorescer is preferably present in the aqueous solution used in the process in the range 0.0001 g/L to 0.1 g/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, Toiletryand Fragrance Association)1992 International layers Guide, published by CFTA Publications, and OPD 1993 Chemicals layer Directory 80th annular Edition, published by Schnell publishing Co.

Preferably, the fragrance comprises at least one note (compound) of α -isomethylionone, benzyl salicylate, citronellol, coumarin, hexylcinnamaldehyde, linalool, ethyl 2-methylpentanoate, octanal, benzyl acetate, 3, 7-dimethyl-1, 6-octadien-3-ol 3-acetate, 2- (1, 1-dimethylethyl) -cyclohexanol 1-acetate, damascone, β -ionone, tricyclodecenyl acetate, dodecanal, hexylcinnamaldehyde (hexylcinnamylamine aldehyde), cyclopentadecanolide, 2-phenylethyl phenylacetate, amyl salicylate, β -caryophyllene, ethyl undecylate, geranyl anthranilate, α -irone, β -phenylethylbenzoate, α -santalol, cedrol, cedarwood acetate, cypress formate, forwood salicylate, γ -phenyl acetate, phenyl ethyl β -phenyl 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 FlavorIngredients,1975, CRC Press; jacobs, Synthetic Food adjacents, 1947, edited by vannonstrand; 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 that facilitate encapsulation include those having a relatively low boiling point, preferably a boiling point of less than 300 ℃, preferably of 100 ℃ 250. it is also advantageous to encapsulate perfume components having a low Clog P (i.e. those that will have a higher tendency to be dispensed into water), preferably having a C L og P of less than 3.0. these materials having a relatively low boiling point and a relatively low C L og P have been referred to as "delayed release perfume"(s) and comprise one or more of allyl hexanoate, amyl acetate, amyl propionate, anisaldehyde, anisole, benzaldehyde, benzyl acetate, benzyl acetone, benzyl alcohol, benzyl formate, benzyl isovalerate, benzyl propionate, β -gamma hexenol, camphor gel, levo-carvone, d-carvone, cinnamyl alcohol, cinnamyl formate, cinnamyl alcohol, cinnamyl acetate (cinnamate, cinnamate (cinnamate), cis-fenchytrivinyl acetate, cis-fenchyl acetate, cis-3-jasmonate, cycyl alcohol, camphor gum, levo-carvone acetate, menthyl acetate, benzyl methacrylate, methyl acetate, benzyl methacrylate, methyl acetate, benzyl methacrylate, benzyl.

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, Z ü rich, 2003) and Industrial dye Chemistry, Properties Applications (K Hunger (ed), Wiley-VCH Weinheim 2003).

Hueing dyes for laundry compositions preferably have an absorption maximum 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 L and L quiitint Violet DD and 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

The microbial strains producing the protease were isolated from marine sediment samples collected from the gulf of Jiaozhou (yellow sea, 36 ° 09'N, 120 ° 32' E) by selective screening on skim milk agar plates containing (g/L sea water) tryptone 5, yeast extract 2, skim milk powder 40, agar powder 16. the plates were cultured at 20 ℃ for 48-72 hours to obtain bacterial colonies.colonies with clear hydrolysis circles of casein in the milk were evaluated as protease producers. a proteolytic L A-05 strain showing a larger hydrolysis circle was selected for further experiments.seeds and fermentation medium for L A-05 strain culture consisted of 5g tryptone, 2g yeast extract and 1L sea water, pH 7.0. the medium was autoclaved at 121 ℃ for 20 minutes.first, the isolated L A-05 strain was inoculated with 2% (v/v) seed medium and cultured at 20 ℃ and 150rpm, on a rotary shaker and then shake culture for 12 hours, the culture was transferred to conical flask containing 2-38 ml of fermentation medium and cultured at 3615 ℃ and L rpm.

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 L A-05 strain, Analytical characterization index (API) dipstick tests and 16S rRNA gene sequencing were performed, using the API dipstick to study the physiological and biochemical properties of strain L A-05, according to the manufacturer' S instructions.

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

Cloning of the amplified product in pMD18-T vector (Takara, da lian, china) and construction of recombinant plasmid pMD-16S. then, transfer of the recombinant plasmid into competent cells of escherichia coli DH5a recombinant clones of escherichia coli DH5a were cultured using L B broth medium containing ampicillin (60 μ g/ml), DNA fragments ligated to 16S rRNA in the recombinant plasmid were confirmed by commercial DNA sequencing (Sangon biotechco, &lttt translation = "L" &gtttl &l/T &gttgtt td., shanghai, china), multiple sequence alignment was performed by EzTaxon database using a recognition program, model culture strains with pairwise similarity higher than 97% were selected, and statistical evaluation of occurrence of strain by adjacent neighbor-joint genetic Analysis (molecular evolution Analysis, Analysis) by using molecular Analysis (Analysis, Analysis of phylogenies) and statistical Analysis of molecular evolution (push) using a molecular Analysis system 7.0.9.

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 (Hi L oad 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, 3ml each of the eluted fractions were collected and analyzed for protease activity, fractions showing protease activity were pooled, and then used for 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 concentration was 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 (L agzian and Asoodeh, 2012) briefly, 0.25ml aliquots of purified protease were incubated at 45 ℃ for 10 minutes with 0.75ml of 50mM Tris-HCl buffer (ph8.0) containing 1% (w/v) casein, the reaction was stopped by adding 0.5m L 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.

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)^TML RF, Bruker Daltonics, USA), by matrix assisted laser desorption ionization-time of flight mass spectrometry (MA L DI-TOF/MS) in linear forward modeMolecular mass. Data were collected by FlexControl 3.4 and analyzed with FlexAnalysis 3.4 software.

Zymogram

Briefly, the samples were mixed with β -free mercaptoethanol gel loading buffer and loaded onto the electrophoresis gel without heating, electrophoresis was performed in ice water, then the gel was immersed in 50mM Tris-HCl buffer (pH8.0) containing 2.5% Triton X-100 at 4 ℃ and shaken gently to remove SDS, the gel was washed twice with 50mM Tris-HCl buffer (pH8.0) at 4 ℃ for 20 minutes each, 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 terminate the protease reaction, stained with Coomassie Brilliant blue R-250 for 2 hours, and destained with methanol-acetic acid-water (5/1/4, v/v) overnight to reveal the bands of protease.

Isoelectric focusing

Isoelectric focusing of the purified protease 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 by ultrafiltration with 1% glycine buffer.after fixation of the IPG strips, Pre-electrophoresis (Pre-electrophoresis) was started at 700V for 20 minutes at 15 ℃ and then at 15 ℃ the sample and IEF standard were subjected to electrophoresis at 2000V for 90 minutes.finally, the bands were fixed for 30 minutes by TCA buffer treatment, stained with Coomassie blue R-250 and destained with a methanol-acetic acid-water mixture.

Determination of N-terminal amino acid sequence

According to the protocol of Matsudaira (Matsudaira, 1987), proteases purified by SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane in CAPS buffer, the PVDF membrane was lightly stained with Coomassie Brilliant blue R-250 and the band containing the enzymes was excised, then, the N-terminal amino acid sequence was analyzed by automated Edman degradation 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 B L AST homology search (NCBI, USA).

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

Protease-containing bands obtained by denaturing SDS-PAGE were carefully excised and the proteins were analyzed by tandem mass spectrometry (MA L DI MS/MS) as described in the published protocol (Marchand et al, 2009.) TOF/TOFFExpolor using the 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 test materials-Netherlands) were used for mini-bottle washing together with cotton ballast (cotton ballast) the stains were applied in triplicate in wash bottles FH26 containing 1 g/L laundry formulations (both types, labelled F1 and F2) was prepared using water, protease (control basis or salt tolerant) was added to a concentration of 5 mg/L in a total volume of 100m L, which enzyme content equaled the content of 0.5 wt% protease in the formulation.

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 stains were separated from the washing liquor, rinsed 2x in a beaker containing 1L FH26 water, then left to dry overnight.

Mathematically, Δ E is defined as:

where Δ L is a measure of the difference in darkness between the washed and white cloths, 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 E value, the whiter the cloth.

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 L AS 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
L AS acid (Indianuba)	4.500
		EPEI	2.325
SLES 3EO(Texapon N70LST)	13.500
		EPEI	0.775
BIT	0.0200
		MIT	0.0095
Soladona L A059-2012 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%, preferably 2 to 50 wt%, more preferably 4 to 50 wt% of a surfactant;

2. The detergent composition according to claim 1, wherein the protease has at least 95%, more preferably 97% sequence identity to SEQ ID No. 1.

3. The detergent composition of claim 1 or 2, wherein the protease has 1100% sequence identity to SEQ ID NO.

4. The detergent composition according to any preceding claims, wherein the detergent composition is a laundry detergent composition.

5. A laundry detergent composition according to claim 4, wherein the laundry detergent composition is a liquid or powder, preferably a liquid detergent.

6. A laundry detergent composition according to claim 4 or claim 5, wherein the laundry detergent composition comprises anionic and/or nonionic surfactants, preferably both anionic and nonionic surfactants.

7. A laundry detergent composition according to any of claims 4 to 6, 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%.

8. A laundry detergent composition according to any of claims 4 to 7, wherein the laundry detergent composition comprises a soil release polymer, preferably a polyester based soil release polymer.

9. A laundry detergent composition according to any of claims 4 to 8, wherein the level of phosphonic acid (or salt thereof) chelant is less than 0.1 wt%, preferably 0.01 wt%, more preferably the composition is free of phosphonic acid (or salt thereof) chelant.

10. The detergent composition according to any preceding claims, further comprising a further enzyme selected from lipase, cellulase, α -amylase, peroxidase/oxidase, pectate lyase, mannanase, and/or additional protease.

11. 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 ℃, 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.

12. The method of claim 11, wherein the protease has at least 95%, even more preferably 97%, sequence identity to SEQ ID No. 1; most preferably, the protease has 1100% sequence identity to SEQ ID NO.

13. The method of claim 11 or claim 12, wherein the composition treating the substrate is the composition of any one of claims 4 to 10.

14. 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 ℃.