CN101370934A - Detergent compositions - Google Patents

Abstract

The present invention relates to detergent compositions comprising a detergent ingredient and a lipase variant with reduced potential for odor generation obtained by introducing mutations in one or more regions identified in a parent lipase.

Description

Detergent composition

invention field

The present invention relates to detergent composition, especially comprise the laundry detergent of lipolytic enzyme.

background of invention

To detergent manufacturers, the especially manufacturers of clothes washing industry, improving oily dirt removal effect is its constant target.Although used the combination of many effective tensio-active agents and tensio-active agent, surfactants based product still cannot be realized the removal completely of fats/oils dirt, especially when product is used under low water temperature.From the later stage 1980s, lipase is just used in washing composition, to remove fatty dirt by fatty dirt is resolved into triglyceride level.

Until not long ago, main commercially available lipase as Lipolase (trade(brand)name, Novozymes) just can the baking stage of washing process compared with producing especially effectively effect under low moisture content.These enzymes are only tending towards producing significant cleaning effect at the second washing step, because the dirt only staying on the washed clothing of baking stage just significant steatolysis can occur, then in next washing step, remove the fat of decomposition.Yet developed recently more efficient lipase, its washing stage at cleaning course also can work effectively.Therefore the cleaning action in second washing step, because lipase can be present in first cycles of washing, so cleaning effect is significantly improved.US 6,939, described the example of these enzymes in 702 B1, WO00/60063 and Research DisclosureIP6553D.Hereinafter this kind of enzyme is called to the first washing lipase.

In addition, human consumer wishes that goods (such as clothes) are clean as much as possible.These human consumers are associated the smell of goods and the clean-up performance of these goods clean or that processed conventionally.Therefore,, from human consumer's viewpoint, validity clean and/or treatment compositions is conventionally directly given smells clean by said composition or goods that processed with said composition and is associated.Applicant has recognized that, Cucumber for example esterase and lipase can produce unpleasant lipid acid smell, especially short chain fatty acid smell, for example smell of butyric acid.Yet these materials can be especially effective cleaning agent.Regrettably, human consumer is associated the smell owing to using these reagent to produce conventionally with cleaning not.Have variant example C-end extension sequence, that reduce smell referring to WO02/062973, but these lipase Variants do not show the potent scourability of the first washing lipase, the lipase such as in those WO00/60063, comprises with trade(brand)name

the variant of selling.

Therefore, need a kind of detergent composition that comprises lipolytic enzyme to obtain excellent fats/oils dirt removal effect, and do not produce any tedious lipid acid smell.

summary of the invention

The present invention relates to the detergent composition that comprises detergent ingredients and lipase Variant, the smell that described lipase Variant has minimizing produces trend, and without C-end extension sequence.Lipase Variant is introduced sudden change by one or more regions of determining in parent lipase and is obtained.

Accompanying drawing explanation

Fig. 1 represents lipase sequence alignment.

sequence list

Sequence identification number: the DNA sequence dna of 1 presentation code thermophilic fungus lipase.

Sequence identification number: 2 represent the aminoacid sequence of thermophilic fungus lipase.

Sequence identification number: 3 represent the aminoacid sequence of the mould lipase of colter.

Sequence identification number: 4 represent the aminoacid sequence of absidia corymbifera lipase.

Sequence identification number: 5 represent the aminoacid sequence of rhizomucor miehei lipase.

Sequence identification number: 6 represent the aminoacid sequence of Rhizopus oryzae lipase.

Sequence identification number: 7 represent the aminoacid sequence of lipase from Aspergillus Niger.

Sequence identification number: 8 represent the aminoacid sequence of Tabin aspergillus lipase.

Sequence identification number: 9 represent the aminoacid sequence of Fusarium oxysporum lipase.

Sequence identification number: 10 represent the aminoacid sequence of fusarium heterosporium lipase.

Sequence identification number: 11 represent the aminoacid sequence of aspergillus oryzae lipase.

Sequence identification number: 12 represent the aminoacid sequence of penicillium camembertii lipase.

Sequence identification number: 13 represent the aminoacid sequence of smelly aspergillus niger lipase enzyme.

Sequence identification number: 14 represent the aminoacid sequence of lipase from Aspergillus Niger.

Sequence identification number: 15 represent the aminoacid sequence of aspergillus oryzae lipase.

Sequence identification number: 16 represent the aminoacid sequence of Landerina penisapora lipase.

发明详述

脂肪酶变体

parent lipase

Parent lipase can be fungal lipase, and described in " homology and sequence alignment " part, its aminoacid sequence has and sequence identification number: the homology of the thermophilic fungus lipase sequence at least 50% showing in 2.

Parent lipase can be yeast polypeptides, for example candiyeast, kluyveromyces, pichia spp, yeast, fission yeast or Ye Shi yeast polypeptides; Or filamentous fungus polypeptide more preferably, for example top spore mould, aspergillus tubigensis, short stalk mould, cryptococcus, the black powder yeast of line, sickle mycete, humic mould, Pyricularia oryzae, mucormycosis, ruin a mould, Xin Kaoma fat mould, neurospora, paecilomycerol, Penicillium notatum, Rumen Fungi, Split-gill, yellow silk aspergillus tubigensis, thermophilic ascomycete, grass roots mould, Trichoderma or Trichoderma polypeptide.

One preferred aspect, parent lipase is Ka Ersibai yeast, cereuisiae fermentum, saccharomyces diastaticus, Douglas yeast, kluyveromyces, Nuo Shi yeast or the ellipsoideus yeast polypeptide with lipase activity.

Another preferred aspect, parent lipase is microorganism Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger, aspergillus oryzae, Tabin aspergillus, sickle spore bacterioide, wheat wilt, Fusariumsp, fusarium culmorum, Fusarium graminearum, the red sickle spore of standing grain, fusarium heterosporium, five-leaved chaste tree sickle-like bacteria, Fusarium oxysporum, netted sickle-like bacteria, moniliform sickle-like bacteria, fusarium sambucinum, colour of skin sickle spore, Fusarium sporotrichioides, fusarium sulphureum, bunch capsule sickle-like bacteria, fusarium trichothecioides, fusarium, Humicola insolens, thermophilic fungus (synonym: high temperature chactomium globosum), rice black wool is mould, thermophilic fungus destroyed wire, Neurospora, penicillium purpurogenum, trichoderma harziarum, healthy and free from worry wood is mould, long shoot wood is mould, Trichodermareesei, or viride polypeptide.

Another preferred aspect, parent lipase is that thermophilic fungus belongs to lipase.

One preferred aspect, parent lipase is thermophilic fungus lipase.A preferred embodiment Zhong， parent lipase, be sequence identification number: 2 lipase.

region identification and replacement.

The region I below mentioning is sequence identification number to the site of region IV: the amino-acid residue site in 2.Use the described method of " homology and sequence alignment " part to find in different lipase corresponding (or homology) site.

replacement in the I of region

Region I is comprised of the amino-acid residue around N-terminal residue E1.In this region, preferably with the amino acid with more positive electricity, replace the amino acid in parent lipase.The amino-acid residue that region I comprises corresponding following site: 2 to 11 and 223 to 239.Following site has special significance: 4,8,11,223,227,229,231,233,234,236.Specifically, following replacement: X4V, X227G, X231R and X233R have been confirmed.

In a preferred embodiment, parent lipase and sequence identification number: 2 have at least 80%, such as 85% or 90%, the identity such as at least 95% or 96% or 97% or 98% or 99%.In a preferred embodiment Zhong， parent lipase and sequence identification number: 2 is identical.

replacement in the II of region

Region II is by forming with the amino-acid residue that contacts substrate in the one side of alcohol moiety in the one side of acyl chain.In this region, preferably with amino acid or lower hydrophobic amino acid with more positive electricity, replace the amino acid in parent lipase.The amino-acid residue that region II comprises corresponding following site: 202 to 211 and 249 to 269.Following site has special significance: 202,210,211,253,254,255,256.Specifically, confirmed following replacement: X202G, X210K, X255Y/V and X256K/R.

In a preferred embodiment, parent lipase and sequence identification number: 2 have at least 80%, such as 85% or 90%, the identity such as at least 95% or 96% or 97% or 98% or 99%.In a preferred embodiment Zhong， parent lipase and sequence identification number: 2 is identical.

replacement in the III of region

Region III is by forming flexible structure, thereby the amino-acid residue that allows substrate to enter avtive spot forms.In this region, preferably with amino acid or lower hydrophobic amino acid with more positive electricity, replace the amino acid in parent lipase.The amino-acid residue that region III comprises corresponding following site: 82 to 102.Following site has special significance: 83,86,87,90,91,95,96,99.Specifically, following replacement: X83T, X86V and X90A/R have been confirmed.

In a preferred embodiment, parent lipase and sequence identification number: 2 have at least 80%, such as 85% or 90%, the identity such as at least 95% or 96% or 97% or 98% or 99%.In a preferred embodiment Zhong， parent lipase and sequence identification number: 2 is identical.

replacement in the IV of region

Region IV is comprised of electrostatical binding to surperficial amino-acid residue.In this region, preferably with the amino acid with more positive electricity, replace the amino acid in parent lipase.The amino-acid residue that region IV comprises corresponding following site: 27 and 54 to 62.Following site has special significance: 27,56,57,58,60.Specifically, following replacement: X27R, X58N/AG/T/P and X60V/S/G/N/R/K/A/L have been confirmed.

In a preferred embodiment, parent lipase and sequence identification number: 2 have at least 80%, such as 85% or 90%, the identity such as at least 95% or 96% or 97% or 98% or 99%.In a preferred embodiment Zhong， parent lipase and sequence identification number: 2 is identical.

the amino acid in other site

Parent lipase optionally comprises other amino acid whose replacement, is especially less than 10 or be less than 5 such replacements.Example is the replacement in the one or more sites in corresponding parent lipase 24,46,74,81,83,127,131,137,147,150,203,206,211,263,264,265,267 and 269.In a specific embodiment, replace one of them site occurring in corresponding 81,147,150 and 249 sites.In a preferred embodiment, at least one replaces the group that the freely following replacement of choosing forms: X81Q/E, X147M/Y, X150G and X249R/I/L.

For example, according to rule known in the art, can more replace the replacement described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202.

parent's fat variant

In one aspect, when comparing with described parent, described variant comprises at least three replacements of total, and described replacement is selected from following one or more groups replacement:

A) at least two kinds of replacements in the I of region,

B) at least one in the II of region replaces,

C) at least one in the III of region replaces, and/or

D) at least one replacement in the IV of region.

Variant is compared and can be comprised replacement with the parent of variant, those replacements of listing in the corresponding following table 1 of described replacement.

Region I	Region II	Region III	Region IV	Outside region
					X4V+X227G+X231R+X233R	X210K+X256K	X83T+X86V	X58A+X60S	X150G
X227G+X231R+X233R	X256K	X86V	X58N+X60S	X150G
					X231R+X233R	X255Y
X231R+X233R	X202G
					X227G+X231R+X233R	X256K	X86V
X4V+X231R+X233R			X58N+X60S
					X231R+X233R		X90R	X58N+X60S
X231R+X233R	X255V	X90A
					X227G+X231R+X233R	X256K	X86V	X58N+X60S	X150G
X231R+X233R	X211L		X58N+X60S	X147M

Table 1: the variant that some are concrete.

An embodiment Zhong， parent lipase and sequence identification number more specifically: 2 is identical, thereby the variant of table 1 will be:

Region I	Region II	Region III	Region IV	Outside region
					Q4V+L227G+T231R+N233R	E210K+P256K	S83T+I86V	S58A+V60S	A150G
L227G+T231R+N233R	P256K	I86V	S58N+V60S	A150G
					T231R+N233R	1255Y
T231R+N233R	I202G
					L227G+T231R+N233R	P256K	I86V
Q4V+T231R+N233R			S58N+V60S
					T231R+N233R		I90R	S58N+V60S
T231R+N233R	1255V	I90A
					L227G+T231R+N233R	P256K	I86V	S58N+V60S	A150G
T231R+N233R	F211L		S58N+V60S	L147M

Table 2: sequence identification number: some concrete variants of 2

amino acid modified name

When describing as described in the present invention lipase Variant, use following nomenclature to be beneficial to reference: initial amino acid: site: substituted amino acid.

According to this nomenclature, for example the L-glutamic acid at 195 places, site is substituted by glycine, be expressed as G195E.Words at identical site disappearance glycine are expressed as G195 ^*, and insert additional amino-acid residue, be expressed as G195GK as Methionin.Compare with other lipase for one, comprise one in site " disappearance " amino acid of 36, the concrete lipase that simultaneously inserts an aspartic acid in this site is expressed as ^*36D.A plurality of sudden changes are separated with plus sige, that is: R170Y+G195E, is illustrated in site 170 and 195 places and respectively tyrosine and L-glutamic acid is substituted by arginine and glycine.

When the described sequence alignment method of application, X231 is illustrated in the amino acid in corresponding site 231 in parent's polypeptide.X231R represents that this amino acid replaces with R.Concerning sequence identification number: 2 X are T, be therefore illustrated in 231 places, site replaces T with R to X231R.For example, when the amino acid of a site (231) can be selected from one group of amino acid whose aminoacid replacement with another, for example, described group is comprised of R and P and Y, with X231R/P/Y, represents.

In all cases, use generally acknowledged IUPAC single-letter or trigram amino acid abbreviations.

amino acid grouping

In this manual, by amino acid, according to it, the electric charge under pH10 condition is divided into electronegative, positively charged or electroneutral amino acid.Therefore, electronegative amino acid is E, D, C (halfcystine) and Y, especially E and D.The amino acid of positively charged is R, K and H, especially R and K.When forming disulfide linkage, neutral amino acids is G, A, V, L, I, P, F, W, S, T, M, N, Q and C.By the another kind of aminoacid replacement in same group (electronegative, positively charged or electric neutrality), be called as conservative replacement.

That neutral amino acids can be divided into is hydrophobic or nonpolar (G, A, V, L, I, P, F, W and as the C of the part of disulfide linkage) and (S, T, M, N, the Q) of hydrophilic or polarity.

amino acid identity

By parameter " identity ", the dependency between two aminoacid sequences or two nucleotide sequences is described.

For purposes of the invention, by using the comparison from two aminoacid sequences of Needle program determination of EMBOSS package (http://emboss.org) 2.8.0 version.Needle program is used overall comparison algorithm, and this arthmetic statement is in Needleman, S.B. and Wunsch, C.D. (1970) J.Mol.Biol.48,443-453.The substitution matrix using is BLOSUM62, and the open point penalty in room is 10, and it is 0.5 that point penalty is expanded in room.

Aminoacid sequence of the present invention (" invention sequence "; Sequence identification number for example: the identity degree 2 amino acid/11 to 269) and between difference aminoacid sequence (" external sequence ") is calculated by the following method: the exact matching number of two sequence alignments is divided by the length of " invention sequence " or the length of " external sequence ", and the length of whichever sequence is the shortest.Result is expressed as identity per-cent.

When " invention sequence " and " external sequence " is when the same loci of lap has identical amino-acid residue, can there is exact matching.Sequence length is the number (for example sequence identification number: 2 length is 269) of the amino-acid residue in sequence.

Parent lipase and thermophilic fungus lipase (sequence identification number: 2) have at least 50%, especially at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, surpass 95% or surpass 98% amino acid identity.In a specific embodiment Zhong， parent lipase and thermophilic fungus lipase (sequence identification number: 2) identical.

Can use aforesaid method to calculate identity and homology and for sequence alignment.In situation of the present invention, homology and sequence alignment are calculated as follows.

homology and sequence alignment

For purposes of the invention, by computer program known in the art, as the GAP being provided in GCG routine package (Program Manual for the Wisconsin Package, the 8th edition, in August, 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), " Journal of Molecular Biology ", 48, 443-45), use has the GAP that following peptide sequence contrast arranges, measuring aptly homology degree: GAP generation point penalty is 3.0, and it is 0.1 that GAP extends point penalty.

In the present invention, mould, the absidia corymbifera of colter, rhizomucor miehei, Dai Shi head mold, aspergillus niger, Tabin aspergillus, Fusarium oxysporum, fusarium heterosporium, aspergillus oryzae, penicillium camembertii, smelly aspergillus, aspergillus niger, thermophilic fungus (synonym: corresponding high temperature chactomium globosum) and in Landerina penisapora lipase sequence (or homology) site is determined by the sequence alignment showing in Fig. 1.

In order finding in lipase sequence, not to be presented at the homologous site in sequence alignment, the sequence showing in paid close attention to sequence and Fig. 1 to be compared.By the highest sequence of homology that GAP program is found, carry out GAP sequence alignment, the existing sequence in new sequence and Fig. 1 is compared.(August 1994 for Program Manual for the Wisconsin Package, Version8 for GCG routine package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48,443-45) provide GAP.It is 3.0 that peptide sequence is relatively used following setting: GAP to produce point penalty, and GAP extension point penalty is 0.1.

Parent lipase and thermophilic fungus lipase (sequence identification number: 2) have at least 50%, especially at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, surpass 95% or surpass 98% amino acid identity.In a specific embodiment Zhong， parent lipase and thermophilic fungus lipase (sequence identification number: 2) identical.

hybridization

The present invention also relates to by polynucleotide encoding, the isolated polypeptide with lipase activity, described polynucleotide are under low-down preciseness condition, preferably under low preciseness condition, more preferably under middle preciseness condition, more preferably in-Gao preciseness condition under, even more preferably under high preciseness condition, most preferably under very high preciseness condition, hybridize with following nucleotide sequences: (i) Nucleotide 178 to 660, sequence identification number:: 1, (ii) be included in the cDNA sequence of Nucleotide 178 to 660, sequence identification number: 1, (iii) sequence (i) or (ii), or (iv) (i), (ii) complementary strand (J.Sambrook or (iii), E.F.Fritsch, and T.Maniatus, 1989, MolecularCloning, A Laboratory Manual, second edition, Cold Spring Harbor, New York).Sequence identification number: 1 sub-series of packets containing at least 100 in abutting connection with Nucleotide, or preferably at least 200 in abutting connection with Nucleotide.In addition, subsequence codified has the polypeptide fragment of lipase activity.

The long probe that is at least 100 Nucleotide concerning length, prehybridization and the hybridization of very high preciseness conditional definition for carrying out under the following conditions will be low to moderate very much: 42 ℃, 5X SSPE, 0.3% SDS, that 200 μ g/mL have sheared and the milt DNA of sex change, or 25% formyl ammonia is for very low and low preciseness condition, 35% formyl ammonia is for neutralization-Gao preciseness condition, or 50% formyl ammonia is for high and very high preciseness condition, and standard Southern hybridization program is hybridized most preferably 12 to 24 hours.

The long probe that is at least 100 Nucleotide concerning length, use 2X SSC, 0.2%SDS, preferably at least 45 ℃ (low-down preciseness), more preferably at least 50 ℃ (low preciseness), more preferably at least 55 ℃ (middle preciseness), more preferably at least 60 ℃ (in-Gao preciseness).Even more preferably, at least 65 ℃ (high preciseness), most preferably at least 70 ℃ (very high preciseness), finally wash vehicle material is three times, each 15 minutes.

dNA sequence dna, expression vector, host cell, lipase are produced

The transformed host cell that the invention provides the DNA sequence dna of code book invention lipase, the expression vector that comprises described DNA sequence dna and comprise described DNA sequence dna or described expression vector.They can obtain by methods known in the art.

The present invention also provides the method for producing lipase, and this method by cultivating transformant and regain lipase yielding lipase in next life from gained broth culture under being of value to the condition of producing lipase.Described method is carried out according to principle known in the art.

lipase activity

-lipase activity to tributyrin under neutral DH (LU) condition

Use Sudan Gum-arabic as emulsifying agent, emulsification tributyrin (tributyrin) is prepared lipase substrate.Then in the constant burette test of pH-, at 30 ℃, pH7 or pH9 Water Under solution tributyrin.One unit lipase activity (1LU) is equivalent to can be under pH7 condition, and per minute discharges the enzyme amount of 1 micromole's butyric acid.

-effect danger

By describe performance and smell reduce risks than effect danger Coefficient Definition be: BR=RP _avg/ R.Lipase Variant as herein described can have and is greater than 1, is greater than 1.1, or is even greater than 1 to approximately 1000 effect danger coefficient.

-average relative performance

The program of calculating average relative performance (RPavg) is present in the example 5 of this specification sheets.Lipase Variant as herein described can have at least 0.8, at least 1.1, at least 1.5, or at least 2 to approximately 1000 average relative performance (RPavg) even.

detergent ingredients

Detergent composition used herein comprises goods and clean and treatment compositions.Except as otherwise noted, term " clean and/or treatment compositions " multi-functional or " heavy duty type " washing composition, especially laundry detergent of tablet, granule type or powder-type that comprise as used herein; Liquid, gel or paste type multifunctional detergent, especially so-called heavy-filth liquid type; Liquid high-count fabric washing composition; The washing composition of detergent for washing dishware with hand or light-duty dishwashing agent, especially those high alveolitoids; Dishwashing detergent for machine washing, comprises the washing composition of multiple tablet form, granule type, liquid-type and rinse aid type, for household and enterprise.Described composition also can comprise those unit dose packagings known in the art in unit dose packaging, and those water miscible, water-insoluble and/or permeable unit dose packagings of water.

Detergent composition of the present invention can comprise one or more lipase Variants of the present invention.Except lipase Variant, detergent composition also comprises detergent ingredients.The non-limiting list of detergent ingredients described below is applicable to cleaning compositions of the present invention, and be applicable to being joined in some embodiment of the present invention, for example, to promote or to improve clean-up performance, process substrate to be cleaned or conventionally improve the aesthetic property of cleaning compositions with tinting material, dyestuff etc.The clear and definite character of these annexing ingredients and incorporation thereof by the physical form that depends on composition with and the character of the clean operation of application.Suitable detergent ingredients includes but not limited to tensio-active agent, washing assistant, sequestrant, dye transfer inhibitor, dispersion agent, enzyme and enzyme stabilizers, bleach-activating agent, hydrogen peroxide, hydrogen peroxide cource, preformed peracid, polymeric dispersant, whitening agent suds suppressor, dyestuff, corrosion inhibitor, tarnish inhibitor, spices, fabric softener, carrier, hydrotropic agent, processing aid, solvent and/or pigment.

Typical washing composition will comprise by any combination weight of following composition 5% to 30% tensio-active agent, and preferred anionic tensio-active agent is such as linear alkylbenzene sulfonate and hydroxyl epoxythio hydrochlorate; 0.005% to 0.1% protease activity albumen, wherein said proteolytic enzyme is preferably selected from Coronase ^tM, FN4FNA or Savinase ^tM, 0.001-0.1% amylase activity albumen, wherein said amylase is preferably selected from Termamyl ^tMnatalase ^tM, Stainzyme ^tMand Purastar ^tM, and 0.1% to 3% sequestrant, preferably DTPA.For granule type and tablet product, these typical washing composition will comprise 5% to 20% SYNTHETIC OPTICAL WHITNER by weight in addition, preferably SPC-D; 1% to 4% bleach-activating agent, preferably TAED and/or 0% to 30% washing assistant, preferably 5% to 30%, the washing assistant more preferably less than 10%, such as aluminosilicate zeolite A and/or tri-polyphosphate.

SYNTHETIC OPTICAL WHITNER-detergent composition of the present invention can comprise one or more SYNTHETIC OPTICAL WHITNER.

Conventionally, when using SYNTHETIC OPTICAL WHITNER, composition of the present invention can comprise by the weighing scale of this theme cleaning compositions approximately 0.1% to approximately 50%, or even approximately 0.1% to approximately 25% SYNTHETIC OPTICAL WHITNER.The example of suitable SYNTHETIC OPTICAL WHITNER comprises:

(1) hydrogen peroxide cource, inorganic over hydrogenation adduct salt for example, comprises that following an alkali metal salt is as sodium salt: perborate (being generally monohydrate or tetrahydrate), percarbonate, persulphate, superphosphate, persilicate and their mixture.In one aspect of the invention, the group that the freely following material of inorganic over hydrogenation adduct salt choosing forms: peroxyboric acid sodium salt, percarbonic acid sodium salt and their mixture; Soap; With

(2) have the bleach-activating agent of R-(C=O)-L, wherein R is alkyl, optional branched-chain alkyl.When bleach-activating agent is while being hydrophobic, it has 6 to 14 carbon atoms or 8 to 12 carbon atoms.When bleach-activating agent is while being hydrophilic, it has and is less than 6 carbon atoms or is even less than 4 carbon atoms; And L is leavings group.The example of suitable leavings group is phenylformic acid and derivative thereof, especially benzene sulfonate.Suitable bleach-activating agent comprises dodecane acyl-oxygen base benzene sulfonate, last of the ten Heavenly stems acyloxy benzene sulfonate, caprinoyl aminobenzoic acid and salt, 3 thereof; 5,5-trimethyl acetyl oxygen base benzene sulfonate, tetra acetyl ethylene diamine (TAED) and nonanoly acyloxy benzene sulfonate (NOBS).Suitable bleach-activating agent is also disclosed in WO 98/17767.Although can adopt any suitable bleach-activating agent, in one aspect of the invention, the cleaning compositions of this theme can comprise NOBS, TAED or their mixture.

(3) preformed peracid.

If existed, peracid and/or bleach-activating agent are conventionally with based on described composition approximately 0.1 to approximately 10% weight, and approximately 0.5 to approximately 60% weight or even approximately 0.6 content to approximately 40% weight are present in described composition.Its one or more hydrophilic precursor can be combined use with one or more hydrophilic peracid or its precursor.

Can select the amount of hydrogen peroxide cource and peracid or bleach-activating agent, making the mol ratio of available oxygen (from peroxide source) and peracid is 1:1 to 35:1, or 2:1 to 10:1 even.

Tensio-active agent-detergent composition can comprise tensio-active agent or surfactant system as described in the present invention, and wherein said tensio-active agent can be selected from nonionogenic tenside, anion surfactant, cats product, amphoterics, zwitterionics, semi-polar nonionic surfactants and their mixture.If existed, the content of tensio-active agent is generally approximately 0.1% to approximately 60% by the weighing scale of tested composition, approximately 0.1% to approximately 40%, approximately 0.1% to approximately 12%, approximately 1% to approximately 50%, or even approximately 5% to approximately 40%.

If existed, described washing composition is conventionally by the anion surfactant that comprises approximately 1% to approximately 40%, such as linear alkylbenzene sulfonate, alhpa olefin sulfonate, alkyl-sulphate (aliphatic alcohol sulfate), hydroxyl epoxythio hydrochlorate, secondary alkyl sulfonate, α sulfo methyl ester, alkyl or alkenyl succinic acid or soap.

Described washing composition optionally comprises approximately 0.2% to approximately 40% nonionogenic tenside, such as alcohol ethoxylate, nonyl phenol ethoxylate, alkyl polyglycoside, alkyl dimethyl amine oxide, ethoxylated fat enzyme one glycollic amide, lipid acid one glycollic amide, polyhydroxy alkyl fatty acid amide or glycosamine N-acyl group N-alkyl derivative (" glucamide ").

Washing assistant-detergent composition of the present invention can comprise one or more detergent builder or builder system.When using washing assistant, this theme composition will comprise by the weighing scale of this theme composition conventionally at least about 1%, and approximately 5% to approximately 60%, or even approximately 10% to approximately 40% washing assistant.Washing assistant includes but not limited to various an alkali metal salts, ammonium salt and the substituted ammonium salt of basic metal, ammonium and alkanol ammonium poly-phosphate, alkalimetal silicate or layered silicate, alkaline earth and alkaline carbonate, silico-aluminate washing assistant, polynary acetic acid (as ethylenediamine tetraacetic acid (EDTA) and nitrilotriacetic acid(NTA)), and polycarboxylate is (as mellitic acid, succsinic acid, citric acid, oxygen di-succsinic acid, polynary toxilic acid, benzene-1,3,5-tricarboxylic acid, carboxymethyl oxosuccinic acid) and their soluble salt.

The detergent composition of sequestrant-this paper can comprise sequestrant.Suitable sequestrant comprises copper, iron and/or manganese sequestrant and their mixture.When using sequestrant, this theme composition can comprise by the weighing scale of this theme composition approximately 0.005% to approximately 15%, or even approximately 3.0% to approximately 10% sequestrant.

Whitening agent-detergent composition of the present invention also can comprise the annexing ingredient that can change products appearance to be cleaned, as white dyes.These whitening agent absorb UV-light and send visible ray.Suitable fluorescent brightener levels comprises from approximately 0.01% weight, approximately 0.05% weight, approximately 0.1% weight, or lower aq to 0.5% weight of even approximately 0.2% weight, or the high level of 0.75% weight even.

Dispersion agent-composition of the present invention also can comprise dispersion agent.Suitable water soluble organic substance comprises homopolymerization or co-polymeric acids or its salt, and wherein polycarboxylic acid comprises at least two and is separated by and is no more than the carboxyl of two carbon atoms.

Enzyme-except lipase Variant of the present invention, detergent composition also can comprise one or more enzymes again so that clean-up performance and/or fabric care benefit effect to be provided, for example, such as proteolytic enzyme, another kind of lipase, at, amylase, carbohydrase, cellulase, polygalacturonase, mannonase arabinase, Galactanase, zytase, oxydase, laccase and/or peroxidase.

The characteristic of the enzyme of conventionally selecting should be compatible with the washing composition of selecting, and (that is, best pH and other enzyme are compatible with non-enzyme component etc.), and enzyme should be significant quantity.

Suitable proteolytic enzyme comprises those animals, plant or microbe-derived proteolytic enzyme.Preferred microorganism source.Comprise saltant type chemical modification or protein engineering.Proteolytic enzyme can be serine protease or metalloprotease, preferred microorganism Sumizyme MP or trypsin-like proteolytic enzyme.The example of Sumizyme MP is subtilisin, especially those are derived from the proteolytic enzyme of bacillus, for example subtilisin Novo, subtilysin, subtilisin 309, subtilisin 147 and subtilisin 168 (being described in WO 89/06279), sequence identification number 4 and sequence identification number 7, be described in WO 05/103244.Other suitable serine protease comprises that those are obtained from some kind of micrococci suborder, is especially disclosed in serine protease and the variant thereof of Cellulonas kind, is disclosed in WO2005052146.The example of trypsin-like proteolytic enzyme is for example trypsinase of pig or Niu Laiyuan of trypsin) and reaping hook fungi protease, be described in WO 89/06270 and WO 94/25583.

The example of available proteolytic enzyme is WO 92/19729, WO 98/20115, the variant of describing in WO 98/20116 and WO 98/34946, especially in one or more following sites, there is the variant of replacement: 27, 36, 57, 68, 76, 87, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, 245, 252 and 274, and other variant with following sudden change:: (K27R, V104Y, N123S, T124A), (N76D, S103A, V104I), or (S101G, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252K).The example of other available proteolytic enzyme is the variant of describing in WO 05/052146, especially especially in one or more following sites, has the variant of replacement: 14,16,35,65,75,76,79,123,127,159 and 179

The proteolytic enzyme of preferred commercially available acquisition comprises Alcalase ^tM, Savinase ^tM, Primase ^tM, Duralase ^tM, Esperase ^tM, Coronase ^tM, Polarzyme ^tMand Kannase ^tM(NovozymesA/S), Maxatase ^tM, Maxacal ^tM, Maxapem ^tM, Properase ^tM, Purafect ^tM, PurafectPrime ^tM, Purafect OxP ^tM, FN2, FN3 and FN4 (Genencor International Inc.).

Lipase comprises the lipase of those bacteriums or originated from fungus.Comprise saltant type chemical modification or protein engineering.The example of available lipase comprises and is obtained from humic mould (synonym: lipase thermophilic fungus), for example be obtained from high temperature chactomium globosum (synonym: lipase thermophilic fungus), be described in EP 258 068 and EP 305 216, or be obtained from the lipase of Humicola insolens, be described in WO 96/13580, Rhodopseudomonas lipase, for example be obtained from Pseudomonas alcaligenes or pseudomonas pseudoalcaligenes (EP 218 272), pseudomonas cepacia (EP 331376), (GB 1 for Si Shi pseudomonas, 372, 034), pseudomonas fluorescens, pseudomonas strain SD705 (WO 95/06720 and WO 96/27002), the lipase of prestige state fluted disc pseudomonas (WO 96/12012), bacillus lipase, such as being obtained from the bacterial strain subtilis (people such as Dartois, (1993), Biochemica et Biophysica Acta, 1131, 253-360), the lipase of bacillus stearothermophilus (JP 64/744992) or bacillus pumilus (WO 91/16422).

Other example is the lipase Variant of describing 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 such as those.

The lipase of other commercially available acquisition comprises Lipolase ^tM, Lipolase Ultra ^tMand Lipex ^tM(Novozymes A/S).

Suitable amylase (α and/or β) comprises the amylase of those bacteriums or originated from fungus.Comprise saltant type chemical modification or protein engineering.Amylase for example comprises α-the derive from amylase of bacillus, a kind of special lichem bacillus strain for example, and this bacterial strain is at GB1, has more detailed description in 296,839.

The example of available starches enzyme is the variant of describing in WO 94/02597, WO 94/18314, WO 96/23873 and WO 97/43424, especially in one or more following sites, has the variant of replacement: 15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408 and 444.

The amylase of commercially available acquisition is Duramyl ^tM, Termamyl ^tM, Stainzyme ^tM, Stainzyme Ultra ^tM, Fungamyl ^tMand BAN ^tM(Novozymes A/S), Rapidase ^tMand Purastar ^tM(from Genencor International Inc.).

Suitable cellulase comprises the cellulase of those bacteriums or originated from fungus.Comprise saltant type chemical modification or protein engineering.Suitable cellulase comprises the cellulase that is obtained from Bacillaceae, Rhodopseudomonas, the mould genus of humic, fusarium, careless Rhizopus, Acremonium, for example originate from Humicola insolens, thermophilic Huang and ruin the fungal cellulase of the mould and Fusarium oxysporum of silk, they are disclosed in US 4,435, and 307, US 5,648,263, US 5,691, and 178, US 5,776,757 and WO89/09259 in.

Especially suitable cellulase is alkalescence or the neutral cellulase with color care benefit effect.The example of these cellulases is cellulases of describing in EP 0 495 257, EP 0 531 372, WO96/11262, WO 96/29397, WO 98/08940.Other example is the cellulase variants of describing in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299 such as those.

The cellulase of commercially available acquisition comprises Renozyme ^tM, Celluclean ^tM, Endolase, ^tMcelluzyme ^tM, and Carezyme ^tM(Novozymes A/S), Clazinase ^tM, and PuradaxHA ^tM(Genencor International Inc.) and KAC-500 (B) ^tM(Kao Corporation).

peroxidase/oxydase:

Suitable peroxidase/oxydase comprises the enzyme of those plants, bacterium or originated from fungus.Comprise saltant type chemical modification or protein engineering.Available peroxidase example comprises the peroxidase that is obtained from terrible agaric, for example, be obtained from the peroxidase of Coprinus cinereus, and its variant is described in WO 93/24618, WO 95/10602 and WO 98/15257.

The peroxidase of commercially available acquisition comprises Guardzyme ^tM(Novozymes A/S).

In the time of in being present in cleaning compositions, above-mentioned enzyme can be by weight of the composition approximately 0.00001% to approximately 2%, approximately 0.0001% to approximately 1%, or the content of even approximately 0.001% to approximately 0.5% zymoprotein exists.

Enzyme stabilizers-can stablize the enzyme for washing composition by multiple technologies.The calcium that the enzyme that the present invention uses can exist in final composition and/or magnesium ion water-soluble sources are stablized, and final product offers enzyme by this ion.Also can use other conventional stablizer, polyvalent alcohol for example, such as propylene glycol or glycerine, sugar or sugar alcohol, lactic acid, boric acid or boric acid derivatives aromatic borate for example, or phenyl-boron dihydroxide derivative is such as 4-benzoyl boric acid, the method preparation that described composition can be described according to for example WO92/19709 and WO 92/19708.

Solvent-suitable solvent comprises that water and other solvent are as lipophilic fluid.The example of suitable lipophilic fluid comprises that siloxanes, other siloxanes, hydrocarbon, glycol ether, glycerol derivative are as glyceryl ether, perfluoroamine, perfluorination and hydrogen fluorine ether solvents, the floride-free organic solvent of low volatility, diol solvent, other environment amenable solvent and their mixture.

washing methods

The present invention includes a kind of a certain position of cleaning and/or process, the method of surface or fabric particularly, aforesaid method comprises the following steps: by applicant's cleaning compositions embodiment (with pure substance form or be diluted in washing liq) and surface or clothing in contact at least partly, and the then optionally above-mentioned surface of rinsing or fabric.Can before above-mentioned rinse step, to described surface or fabric, carry out washing step.For the present invention, washing includes but not limited to clean and mechanical stirring.As those skilled in the art, understand, cleaning compositions of the present invention can be ideally for the purposes of doing washing.Therefore, the present invention includes a kind of method for laundering of textile fabrics, said method comprising the steps of: fabric to be washed is contacted at least one embodiment of the cleaning compositions that described washing soln comprises applicant, cleaning additive or their mixture with described clean washing soln.Fabric can comprise any any fabric of major part that can wash under normal consumer working conditions.Described solution preferably has approximately 8 to approximately 10 pH.The strength of solution that described composition adopts is about 100ppm, and preferably 500ppm is to approximately 15,000ppm.Water temperature is generally approximately 5 ℃ to approximately 90 ℃.The present invention low water temperature as below 30 ℃ or 25 ℃ or 20 ℃ be especially useful below.The ratio of water and fabric is generally about 1:1 to about 30:1.

lipase Variant example

As the chemical substance of buffer reagent and substrate, it is the commerical prod of SILVER REAGENT at least.

-substratum and solution: LAS (Surfac PS ^tM) and Wessalith CS (Wessalith P ^tM).Other composition used is standard laboratory reagent.

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

example 1: enzyme is produced

Use the standard method of this area, build the plasmid of a gene that comprises the lipase of encoding and its transfection is entered to appropriate host cell.

Use 34 ℃ of constant temperature, the substratum that initial volume is 1.2 liters carries out fed-batch fermentation.The initial pH of substratum is made as 6.5.Once pH rises to 7.0, by adding 10% H3PO4 to keep this value constant.Dissolved oxygen levels in substratum is by changing stir speed (S.S.) and using the fixedly rate of venting of every liter of substratum per minute 1.0 litres of air to control.In the whole fed-batch fermentation stage, feeding rate maintains a constant level.Batch culture base comprises malt syrup as carbon source, and urea and yeast extract are as nitrogenous source, and the mixture of trace-metal and salt.The charging adding continuously in the fed-batch fermentation stage comprises malt syrup as carbon source, yet add urea and yeast extract, is in order to ensure sufficient nitrogenous source supply.

Use standard method known in the art to carry out the purifying of lipase, for example, for example, by filtering fermented supernatant fluid and follow-up hydrophobic chromatography and anionresin liquid, the method described in EP 0 851913 in example 3.

example 2:AMSA-automation stress detection analytical method-for calculating relative performance (RP).

Use automation stress to detect the enzyme variants of analytical method (AMSA) test present patent application.By AMSA, test, can detect the scourability of a large amount of small volume enzyme-detergent solutions.AMSA plate has a plurality of slits for test soln, and one firmly choke fabric sample, so that its capping being washed at all slotted openings place.During washing, acutely shake described plate, test soln, fabric and capping so that test soln contact fabric, and apply mechanical stress.More detailed description can be referring to WO 02/42740, especially the paragraph of the 23rd to 24 pages " Special method embodiments ".The container that comprises washing composition test soln is comprised of the cylindrical cavity in a metal sheet (diameter 6mm, dark 10mm).Dirty fabric (test substances) is positioned at the top of metal sheet, as capping and the sealing member on container.Another metal sheet is positioned at the top of dirty fabric to avoid overflowing any liquid from any one container.Two metal sheets together with dirty fabric with the frequency of 30Hz and the amplitude up-down vibration of 2mm.

Described detection analytical method is carried out under the test conditions of below appointment:

Test soln	0.5g/L LAS0.52g/L Na 2CO31.07g/L Wessalith CS 0.52g/L trisodium citrate
		Test soln volume	160 microlitres
PH value	In statu quo (≈ 9.9.)
		Washing time	20 minutes
Temperature	30℃
		Water hardness
	15°dHCa 2+/Mg 2+/NaHCO 3Ratio be 4:1:7.5
		Enzyme concn in test soln	0.125,0.25,0.50,1.0mg zymoprotein/liter (mg ep/L)
Dry	Performance: pieces of fabric is rinsing in tap water immediately after washing, and at 85 ℃ air-dry 5 minutes smells: pieces of fabric is rinsing in tap water immediately after washing, and in room temperature (20 ℃) lower dry 2 hours
		Test substances	White cream turmeric sample as described below (as the EMPA221 of cotton-spinning fabric)

Table 3

By mix 5g (Santa Maria at 50 ℃, Denmark) turmeric and 100g (38% fat, Arla, Denmark) white cream prepares white cream turmeric sample, and mixture retains approximately 20 minutes and filters (50 ℃) and removes any undissolved particle at this temperature.Mixture is cooled to 20 ℃, and by cotton spinning sample, EMPA221, immerses this white cream turmeric mixture, and then at room temperature dried overnight is also freezing until use.The preparation method of white cream turmeric sample is disclosed in the patent PA 200500775 submitting on May 27th, 2005.

The performance of enzyme variants can be measured by the colour brightness of the fabric sample with concrete enzyme variants washing.Brightness also represents from the light intensity of fabric sample reflection during available white-light illuminating.When fabric is dirty, the intensity of reflected light of its catoptrical strength ratio clean textile is low.Therefore catoptrical intensity can be used for measuring the scourability of enzyme variants.

Use a kind of plane scanner (PFU DL2400pro) of specialty to carry out color measuring, this scanning device is for obtaining the image of laundering of textile fabrics sample.Scanning resolution is 200dpi, 24 of output color depths.In order to obtain accurate result, with Kodak reflection IT8 colour atla frequent calibration scanner.

Use a specially designed application software (Novozymes Color Vector Analyzer) from scanning image, to extract light intensity value.Described program is retrieved 24 pixel values and is converted it into red, green and blue (RGB) value from image.By rgb value is added as carrier, then extracts the length of gained carrier and carry out computed strength value (Int):

$\mathrm{Int}=\sqrt{{\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="A200780002955E00611.png"\; state="\{\{state\}\}">r</figure-callout>}}^2+{\mathrm{<figure-callout\; id="2"\; label="g"\; filenames="A200780002955E00611.png"\; state="\{\{state\}\}">g</figure-callout>}}^2+{\mathrm{<figure-callout\; id="2"\; label="b"\; filenames="A200780002955E00611.png"\; state="\{\{state\}\}">b</figure-callout>}}^2}$

The scourability of variant (P) is calculated according to following formula: P=Int (v)-Int (r) wherein

Int (v) is the light intensity value with the fabric face of tested enzyme washing, and Int (r) is the light intensity value without the fabric face of tested enzyme washing.

According to definition, regulation relative performance score is the result of AMSA washing: relative performance score (RP) is performance (P) sum and the ratio of reference enzyme performance: RP=P (the tested enzyme)/P (reference enzyme) of tested enzyme variant.

RPavg refers to the average relative performance that (0.125,0.25,0.5,1.0mg ep/l) compares with reference enzyme under all four kinds of enzyme concns

RPavg＝avg(RP(0.125)，RP(0.25)RP(0.5)，RP(1.0))

If variant performance is better than reference enzyme, think that it shows the scourability of improvement.In situation of the present invention, reference enzyme is sequence identification number: 2 lipase, it has the replacement of T231R+N233R.

example 3: for calculating the dangerous GC-gas-chromatography of effect.

By solid-phase microextraction vapor-phase chromatography (SPME-GC), the butyric acid that uses following methods to measure in lipase washing sample discharges.Specify four pieces of fabric (diameter 5mm) of washing in solution to be transferred to gas-chromatography (GC) bottle in the table 3 comprising 1mg/L lipase.At a upper analytic sample of Varian3800GC of being furnished with a Stabilwax-DA w/Integra-Guard post (30m, 0.32mm internal diameter and 0.25micro-m df) and a Carboxen PDMS SPME fiber (75micro-m).Each sample, 40 ℃ of precultures 10 minutes, then samples 20 minutes with SPME fiber in the headspace in pieces of fabric.Subsequently sample is injected to chromatographic column (injector temperature=250 ℃).Post flow=2mL helium/minute.Column oven thermograde: 0 minute=40 ℃, 2 minutes=40 ℃, 22 minutes=240 ℃, 32 minutes=240 ℃.With fid detector, detect butyric acid, and based on butyric acid typical curve, calculate the amount of butyric acid.

The risk performance smell (R) of lipase Variant be the butyric acid that discharges from lipase Variant washing sample with from sequence identification number: the ratio between the butyric acid amount of 2 lipase washing sample release, sequence identification number: 2 lipase has the replacement of T231R+N233R (reference enzyme), two values are all used the butyric acid amount discharging from the washing sample of fat-free enzyme to proofread and correct.According to following formula, calculate the risk (R) of variant:

Microgram butyric acid/1 blank correction that smell=1mg zymoprotein records

α _{tested enzyme}=smell _{tested enzyme}-blank

α _{reference enzyme}=smell _{reference enzyme}-blank

R=α _{tested enzyme}/ α _{reference enzyme}

If the R coefficient of a variant is less than 1, think that it shows than the smell with reference to reducing.

example 4: with respect to the activity (LU) of the absorbancy at 280nm

Activity by following detection analytical method LU/A280 mensuration lipase with respect to the absorbancy at 280nm:

The method that the lipase activity of use above partly described is measured lipase activity.Measure lipase in the absorbancy (A280) of 280nm and calculate LU/A280 ratio.The LU/A280 of variant calculates relative LU/A280 divided by the LU/A280 of reference enzyme.In situation of the present invention, reference enzyme is sequence identification number: lipase, it has the replacement of T231R+N233R.

example 5:BR-is imitated danger

By describe performance and smell reduce risks than effect danger coefficient be therefore defined as: BR=RP _avg/ R

If the BR coefficient of a variant higher than 1, thinks that it shows the scourability of improvement and the smell of minimizing.

The result of applying above method acquisition is as follows:

Variant	In sequence identification number: 2	Average RP (RP avg)	BR	LU/A280
					1	I202G+T231R+N233R	0.84	1.41	Undetermined
2	I86V+L227G+T231R+N233R+P256K	1.08	1.52	1700
					3	Q4V+S58N+V60S+T231R+N233R	0.87	1.73	1950
4	S58N+V60S+I90R+T231R+N233R	1.06	1.27	2250
					5	I255Y+T231R+N233R	1.19	1.17	3600
6	I90A+T231R+N233R+I255V	1.13	1.14	2700
					Reference	T231R+N233R	1.00	1.00	3650
7	G91A+E99K+T231R+N233R+Q249R+270H+271T+272P+273S+274S+275G+276R+277G+278G+279H+280R	0.43	Undetermined	850
					8	G91A+E99K+T231R，N233R+Q249R+270H+271T+272P+273S+274S+275G+276R+277G+278G	0.13	Undetermined	500

Table 4

WO 2000/060063 has described reference lipase and variant 7 and 8 in table 4.

washing composition example

Confirmation for the abbreviation component of example is as follows:

LAS straight chain C _11-13sodium alkyl benzene sulfonate.

CxyAS C _1x-C _1ysodium alkyl sulfate

The C of CxyEzs and average z moles of ethylene oxide condensation _1x-C _1ysodium alkyl sulfate

CxyEy C _1x-C _1yalcohol, has average degree of ethoxylation z

QAS R ₂.N+ (CH ₃) ₂(C ₂h ₄oH), R wherein ₂=C ₁₀-C ₁₂

Silicate amorphous sodium silicate (SiO ₂: Na ₂the ratio of O is 1.6:1 to 3.2:1).

Wessalith CS molecular formula is Na ₁₂(AlO ₂siO ₂) ₁₂27H ₂the hydrated sodium aluminosilicate of O, has the primary particle sizes (weight is based on anhydrous statement) in 0.1 to 10 micrometer range.

(Na-) SKS-6 crystalline layered silicate, molecular formula is δ-Na ₂si ₂o ₅.

Citrate trianion two hydration trisodium citrates

Anhydrous citric acid citric acid.

Carbonate anhydrous sodium carbonate.

Vitriol anhydrous sodium sulphate.

The random copolymers of MA/AA 4:1 acrylate/maleic acid ester, molecular-weight average is approximately 70,000 to 80,000.

AA polymer poly sodium acrylate polymkeric substance, molecular-weight average is approximately 4,500.

PB1/PB4 anhydrous sodium perborate monohydrate/tetrahydrate.

Anhydrous SPC-D [the 2.74Na of PC3 ₂cO ₃3H ₂o ₂]

TAED tetra acetyl ethylene diamine.

NOBS nonanoyl Sodium dobesilate.

DTPA ethylidene triamine five acetic acid.

HEDP hydroxyl ethane diphosphonates

EDDS 1，2-diaminoethane-N, the sodium salt of N '-disuccinic acid, (S, S) isomer

STPP tripoly phosphate sodium STPP

Protease protein lytic enzyme, with trade(brand)name

be sold by Novozymes A/S, with trade(brand)name

purafect

and Purafect

be sold by Genencor, and proteolytic enzyme such as FNA, the FN3 and/or the FN4 that in patent WO 91/06637 and/or WO 95/10591 and/or EP 0 251 446, describe.

Amylase is with trade(brand)name purafect

be sold by Genencor;

?

?

with

be sold by the amylolytic enzyme of Novozymes A/S.

Lipase has been described any in lipase Variant 1 to 5 and their combination in the example 5 of table 4.

Mannase is sold by Novozymes's

CMC or carboxymethyl or hydroxyethyl or ester modified Mierocrystalline cellulose

HEC or

EMC

SS agglomerate suds suppressor agglomerate: 12% siloxanes/silicon-dioxide, 18% stearyl alcohol, 70% starch, is particle form.

TEPAE tetren ethoxylate

During 20 ℃ of pH, in 1% distilled water solution, measure.

example A

1. by following formula examples explanation, there is the bleach detergent compositions of granular laundry detergent form.

	A	B	C	D	E	F
							LAS	20	22	20	15	20	20
QAS	0.7	1	1	0.6	0.0	0.7
							C25E3S	0.9	0.0	0.9	0.0	0.0	0.9
C25E7	0.0	0.5	0.0	1	3	1
							STPP	23	30	23	17	12	23
Wessalith CS	0.0	0.0	0.0	0.0	10	0.0
							Silicate	7	7	7	7	7	7
Carbonate	15	14	15	18	15	15
							AA polymkeric substance	1	0.0	1	1	1.5	1
CMC	1	1	1	1	1	1
							Protease 3 2.89mg/g	0.1	0.07	0.1	0.1	0.1	0.1
Amylase 8 .65mg/g	0.1	0.1	0.1	0.0	0.1	0.1
							Lipase 18mg/g	0.03	0.07	0.3	0.1	0.07	0.1
Whitening agent-Tinopal AMS (Ciba)	0.06	0.0	0.06	0.18	0.06	0.06
							Whitening agent-Tinopal CBS-X (Ciba)	0.1	0.06	0.1	0.0	0.1	0.1
DTPA	0.6	0.3	0.6	0.25	0.6	0.6
							MgSO 4	1	1	1	0.5	1	1
PC3	0.0	5.2	0.1	0.0	0.0	0.0

PB1	4.4	0.0	3.85	2.09	0.78	3.63
							NOBS	1.9	0.0	1.66	1.77	0.33	0.75
TAED	0.58	1.2	0.51	0.0	0.015	0.28
							Vitriol/moisture	Surplus to 100%	Surplus to 100%	Surplus to 100%	Surplus to 100%	Surplus to 100%	Surplus to 100%

At 25 ℃, any composition for laundering of textile fabrics in example A is 600ppm to 10000ppm in the concentration of water, and typical intermediate conditions is 2500ppm, and the ratio of water and fabric is 25:1.Typical pH is approximately 10, but can regulate pH with the ratio of the sodium-salt form of alkyl benzene sulphonate (ABS) by changing acid.

example B

The bleach detergent compositions by following formula examples explanation with granular laundry detergent form.

	A	B	C	D
					LAS
	8	7.1	7	6.5
					C25E3S	0	4.8	0	5.2
C68S	1	0	1	0
					C25E7	2.2	0	3.2	0
QAS	0.75	0.94	0.98	0.98
					(Na-)SKS-6	4.1	0	4.8	0
Wessalith CS	20	0	17	0
					Citric acid	3	5	3	4
Carbonate	15	20	14	20
					Silicate	0.08	0	0.11	0
Stain remover	0.75	0.72	0.71	0.72
					MA/AA	1.1	3.7	1.0	3.7
CMC	0.15	1.4	0.2	1.4
					Proteolytic enzyme (56.00mg active substance/gram)	0.37	0.4	0.4	0.4
Termamyl (21.55mg active substance/g)	0.3	0.3	0.3	0.3
					Lipase (18.00mg active substance/gram)	0.05	0.15	0.1	0.5

Amylase (8.65mg active substance/gram)	0.1	0.14	0.14	0.3
					TAED	3.6	4.0	3.6	4.0
PC3	13	13.2	13	13.2
					EDDS	0.2	0.2	0.2	0.2
HEDP	0.2	0.2	0.2	0.2
					MgSO 4	0.42	0.42	0.42	0.42
Spices	0.5	0.6	0.5	0.6
					SS agglomerate	0.05	0.1	0.05	0.1
Soap	0.45	0.45	0.45	0.45
					Vitriol	22	33	24	30
Water and other composition	Surplus to 100%	Surplus to 100%	Surplus to 100%	Surplus to 100%

At 20 ℃, at 90 ℃, any above-mentioned composition for laundering of textile fabrics in example B is 10,000ppm in the concentration of water, and the ratio of water and fabric is 5:1.Typical pH is approximately 10, but can regulate pH with the ratio of the sodium-salt form of alkyl benzene sulphonate (ABS) by changing acid.

example C

	A (% by weight)	B (% by weight)	C (% by weight)	D (% by weight)	E (% by weight)	F (% by weight)
							C25E1.8S	11	10	4	6.32	6.0	8.2
LAS	4	5.1	8	3.3	4.0	3.0
							Sodium formiate	1.6	0.09	1.2	0.04	1.6	1.2
Sodium hydroxide	2.3	3.8	1.7	1.9	2.3	1.7
							Monoethanolamine	1.4	1.490	1.0	0.7	1.35	1.0
Glycol ether	5.5	0.0	4.1	0.0	5.500	4.1
							C23E9	0.4	0.6	0.3	0.3	2	0.3
DTPA	0.15	0.15	0.11	0.07	0.15	0.11
							Citric acid	2.5	3.96	1.88	1.98	2.5	1.88
C 12-14Dimethyl oxidation amine	0.3	0.73	0.23	0.37	0.3	0.225
							C 12-18Lipid acid	0.8	1.9	0.6	0.99	0.8	0.6

Borax	1.43	1.5	1.1	0.75	1.43	1.07
							Ethanol	1.54	1.77	1.15	0.89	1.54	1.15
TEPAE 1	0.3	0.33	0.23	0.17	0.0	0.0
							The hexamethylene-diamine of ethoxylation 2	0.8	0.81	0.6	0.4	0.0	0.0
1,2-PD	0.0	6.6	0.0	33	0.0	0.0
							Proteolytic enzyme *	36.4	36.4	27.3	18.2	36.4	27.3
Mannase *	1.1	1.1	0.8	0.6	1.1	0.8
							Amylase *	7.3	7.3	5.5	3.7	7.3	5.5
Lipase *	10	3.2	0.5	3.2	2.4	3.2
							Water, spices, dyestuff and other component	Surplus	Surplus	Surplus	Surplus	Surplus	Surplus

^*numerical value in mg enzyme/100g

¹as US 4,597, described in 898.

²with trade(brand)name

derive from BASF, and those described in WO 01/05874

The All Files of quoting in detailed Description Of The Invention is all introduced for your guidance in relevant portion; For quoting of any file, should not be interpreted as admitting that it is relevant prior art of the present invention.In the present invention, any implication of term or while defining contradiction in any implication of term or definition and the file of introducing for your guidance, should obey implication or the definition of giving in the present invention this term.

Although illustrated and described specific embodiment of the invention scheme, having it will be apparent to one skilled in the art that and can make a plurality of other changes and modification in the situation that not deviating from essence of the present invention and scope.Therefore all such change and modification that, claims are intended to be included in the scope of the present invention.

Sequence table

Claims (37)

1. a composition, the variant that described composition comprises detergent ingredients and parent lipase, when comparing with described parent, described variant comprises at least three kinds of replacements of total, and described replacement is selected from following one or more groups replacement:

A) at least two kinds of replacements in the I of region,

B) at least one in the II of region replaces,

C) at least one in the III of region replaces, and/or

D) at least one replacement in the IV of region.

2. detergent composition as claimed in claim 1, wherein the described replacement in the I of region is included in the replacement in the site of corresponding described site 231 and 233.

3. detergent composition as claimed in claim 2, wherein in site, 231 and 233 described replacement is replaced by R.

4. detergent composition as claimed in claim 2, wherein said variant is included in corresponding sequence identification number: the replacement in the site in 2 site 4.

5. detergent composition as claimed in claim 4, wherein in corresponding sequence identification number: the described replacement in the site in 2 site 4 is V.

6. detergent composition as claimed in claim 2, wherein said variant is included in corresponding sequence identification number: the replacement in the site in 2 site 227.

7. detergent composition as claimed in claim 6, wherein in corresponding sequence identification number: the described replacement in the site in 2 site 227 is G.

8. detergent composition as claimed in claim 1, wherein described at least one replacement in the II of region comprises the freely replacement of the group of following composition of choosing: the replacement in the site of the described site 202,211,255 of correspondence and 256.

9. detergent composition as claimed in claim 8, wherein described at least one replacement in the II of region comprises the freely replacement of the group of following composition of choosing: X202G, X211L, X255Y/V and X256K.

10. detergent composition as claimed in claim 1, wherein described at least one replacement in the II of region is included in the replacement in the site in corresponding described site 210.

11. detergent composition as claimed in claim 10, wherein the described replacement in corresponding site 210 comprises X210K.

12. detergent composition as claimed in claim 1, wherein described at least one replacement in the III of region comprises the freely replacement of the group of following composition of choosing: the replacement in the site of the described site 83,86 of correspondence and 90.

13. detergent composition as claimed in claim 11, wherein described at least one replacement in the III of region comprises the freely replacement of the group of following composition of choosing: X83T, X86V and X90A/R.

14. detergent composition as claimed in claim 1, wherein described at least one replacement in the III of region is included in the replacement in the site in corresponding described site 83.

15. detergent composition as claimed in claim 14, wherein the described replacement in corresponding site 83 comprises X83T.

16. detergent composition as claimed in claim 1, wherein described at least one replacement in the IV of region comprises the freely replacement of the group of following composition of choosing: the replacement in the site of the described site 27,58 of correspondence and 60.

17. detergent composition as claimed in claim 16, wherein described at least one replacement in the IV of region comprises the freely replacement of the group of following composition of choosing: X27R, X58N/A/G/P/T and X60S/V/G/N/R/K/A/L.

18. detergent composition as claimed in claim 1, described composition is included at least two kinds of replacements in the region IV of corresponding described site 27,58 and 60.

19. detergent composition as claimed in claim 1, described detergent composition is included at least two kinds of replacements in the IV of region, and described replacement choosing is the group of following composition freely: X27R, X58N/A/G/P/T and X60S/V/G/N/R/K/A/L.

20. detergent composition as claimed in claim 1, wherein said variant is included at least one replacement outside described localized area I to IV.

21. detergent composition as claimed in claim 20, wherein outside described localized area I to IV described at least one replace the freely group of following composition of choosing: the replacement in the site of corresponding site 81,147,150 and 249.

22. detergent composition as claimed in claim 20, wherein said at least one outside described localized area I to IV replaces the group that the freely following replacement of choosing forms: X81Q/E, X147M/Y, X150G and X249R/I/L.

23. detergent composition as claimed in claim 2, wherein said parent lipase and sequence identification number: 2 have at least 90% identity.

24. detergent composition as claimed in claim 1, wherein said parent lipase and sequence identification number: 2 is identical, and described variant comprises one group of replacement in following replacement:

a)T231R+N233R+I255Y

b)I202G+T231R+N233R

c)I86V+L227G+T231R+N233R+P256K

d)Q4V+S58N+V60S+T231R+N233R

e)S58N+V60S+I90R+T231R+N233R

f)I90A+T231R+N231R+I255V

g)S58N+V60S+I86V+A150G+L227G+T231R+N233R+P256K

h)S58N+V60S+L147M+F211L+T231R+N233R

i)Q4V+S58A+V60S+S83T+I86V+A150G+E210K+L227G+T231R+N233R+P256K

j)S58N+V60S+I86V+A150G+L227G+T231R+N233R+P256K。

25. detergent composition as claimed in claim 1, wherein said parent lipase and sequence identification number: 2 is identical, and described variant comprises one group of replacement in following replacement:

a)Q4V+S58A+V60S+S83T+I86V+A150G+E210K+L227G+T231R+N233R+P256K

b)S58N+V60S+I86V+A150G+L227G+T231R+N233R+P256K。

26. detergent composition as claimed in claim 1, the variant of wherein said lipase is characterised in that described effect danger (BR) is greater than 1 when measuring with the given method in described specification sheets.

27. detergent composition as claimed in claim 1, described detergent composition also comprises 0.1% to 40%, preferably 0.1% to 12% anion surfactant.

28. detergent composition as claimed in claim 27, wherein said anion surfactant is oxyalkylated alkyl-sulphate.

29. detergent composition as claimed in claim 1, described detergent composition also comprises 5% to 30% silico-aluminate and/or phosphate builders.

30. detergent composition as claimed in claim 1, described detergent composition also comprises peroxide source and bleach-activating agent, preferably tetra acetyl ethylene diamine.

31. washing composition as claimed in claim 1, wherein said washing composition is liquid detergent composition or solid detergent composition.

32. washing composition as claimed in claim 31, wherein said washing composition is detergent granules.

33. washing composition as claimed in claim 1, wherein said washing composition is the units dosage composition of solid tablet or the liquid sealed with dissolvable film.

34. 1 kinds of washing methodss, described method is included in the aqueous wash medium that comprises detergent composition as claimed in claim 1 and washs textiles.

35. washing methodss as claimed in claim 34, wherein said method is suitable for dirt and color spot to remove from surface, said method comprising the steps of:

A) optionally by dirt described in composition pre-treatment as claimed in claim 1 and spot to form the optionally surface of pretreated mistake;

B) composition as claimed in claim 1 of significant quantity is added to the water, to form the wash water solution that comprises the approximately 500 described compositions to about 10000ppm;

C) make described wash water solution and the Surface Contact of pretreated mistake optionally, and

D) optionally to described wash water solution and optionally the surface of pretreated mistake stirring is provided.

36. washing methodss as claimed in claim 34, the temperature of the wherein said aqueous solution is lower than 30 ℃.

37. compositions as claimed in claim 1, wherein said lipase Variant is sequence identification number: 2 variant, described variant comprises at least one in following sudden change: Q4V, S58N/A/G/P/T, I90R or Q249I/L.

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