CN101432411B - Detergent compositions - Google Patents

Abstract

The present invention relates to compositions comprising certain lipase variants and photobleaches and methods of making and using these compositions. Use of these compositions to cleanse and/or treat a site is included.

Description

detergent composition

field of invention

The present invention relates to compositions comprising a lipase and a photobleach and methods of making and using said products. the

Background of the invention

The advent of detergent-friendly lipases provides formulators with a new way to improve grease removal. These enzymes catalyze the hydrolysis of triglycerides, a major component of many common oily soils such as sebum, tallow (eg lard, ghee, butter) and vegetable oils (eg olive oil, sunflower oil, peanut oil). However these enzymes are generally not effective in the first wash cycle and are often accompanied by a malodor which is believed to be caused by the hydrolysis of fat present in dairy soils such as milk, cream, butter and yoghurt. Without being bound by theory, it is believed that these soils are prone to lipase-induced malodor because they contain functionalized triglycerides with short-chain (e.g., C4) fatty acyl units, which upon lipolysis release malodorous volatiles Sexual fatty acids. Even with improved performance of these enzymes, the malodor problem still persists. Therefore, the usefulness of this technique is severely limited. the

We have found that the combination of photobleach and certain lipase variants produces improved cleaning benefits while minimizing unpleasant malodor. Without being bound by theory, it is believed that the following mechanism may produce these beneficial effects: Improved stain removal resulting from a synergistic interaction between lipase and photobleaching agents, including carotenoids, anthocyanins, porphyrins, tannins Acid and flavin substances such as curry, pepper seasoning, tomato pasta seasoning, coffee and tea; and lipase oxidation by photobleaching after washing, for example during cleaning or drying of treated areas , resulting in a reduction in odor. the

Summary of the invention

The present invention relates to a composition comprising a photobleach and a lipase variant with reduced odor generation tendency and good relative performance and without a C-terminal extension. Lipase variants are obtained by introducing mutations in one or more defined regions of a parent lipase. The variants thus obtained must have a lipase activity not less than 80% of the parent lipase activity, expressed in relative performance. the

Description of drawings

Figure 1 shows the lipase sequence alignment. the

sequence list

SEQUENCE IDENTIFICATION NUMBER: 1 represents the DNA sequence encoding lipase from thermophilic fungi. the

SEQ ID NO: 2 represents the amino acid sequence of lipase from thermophilic fungi. the

Sequence identification number: 3 represents the amino acid sequence of Absidia lipase. the

SEQUENCE IDENTIFICATION NO.: 4 represents the amino acid sequence of Absidia coliformis lipase. the

SEQ ID NO: 5 represents the amino acid sequence of Rhizomucor miehei lipase. the

SEQ ID NO: 6 represents the amino acid sequence of Rhizopus oryzae lipase. the

SEQ ID NO: 7 represents the amino acid sequence of Aspergillus niger lipase. the

SEQ ID NO: 8 represents the amino acid sequence of Aspergillus tubingensis lipase. the

SEQ ID NO: 9 represents the amino acid sequence of the lipase from Fusarium oxysporum. the

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

X ₃ ⁺ is a group of formula

SEQ ID NO: 12 represents the amino acid sequence of Penicillium kamembert lipase. the

SEQ ID NO: 13 represents the amino acid sequence of Aspergillus foetidus lipase. the

Sequence identification number: 14 represents the amino acid sequence of Aspergillus niger lipase. the

Sequence identification number: 15 represents the amino acid sequence of Aspergillus oryzae lipase. the

SEQ ID NO: 16 represents the amino acid sequence of Landerina penisapora lipase. the

Detailed description of the invention

definition

Unless otherwise indicated, the term "cleaning composition" as used herein includes granular or powdered all-purpose or "heavy-duty" detergents, especially laundry detergents; liquid, gel or paste all-purpose detergents , especially the heavy-duty liquids commonly described; liquid delicate fabric detergents; hand dishwashing detergents or light-duty dishwashing detergents, especially those of the high sudsing type; machine dishwashing detergents, including various Tablet, granular, liquid and rinse-aid detergents; liquid cleaners and sanitizers, including antibacterial hand cleaners, laundry soaps, mouthwashes, denture cleaners, car or carpet cleaners, Bathroom cleaners; shampoos and conditioners; shower gels and foams and metal cleaners; and cleaning aids such as bleach aids and “stain remover sticks” or pre-treatment aids. the

As used herein, the phrase "independently selected from the group consisting of" means that the parts or elements selected from the reference Markush group may be the same, may be different or may be any combination of elements. the

The test methods disclosed in the Test Methods section of this application must be used to determine the value of each parameter of Applicants' invention. the

Unless otherwise indicated, all component or composition levels are based on the active level of that component or composition and do not include impurities such as residual solvents or by-products that may be present in commercially available sources. the

All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise specified. the

It should be understood that every higher numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every lower numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. the

All cited documents are, in relevant part, incorporated herein by reference. The citation of any document is not to be construed as an admission that it is available as prior art to the present invention. the

combination

The compositions of the present invention generally comprise from about 0.0001% to about 1%, from about 0.0002% to about 0.5%, or from about 0.0005% to about 0.3% of photobleach and from about 0.0005% to about 0.1%, from about 0.001% to about 0.05% , or from about 0.002% to about 0.03% lipase. the

These compositions may be in any form, eg, in the form of a cleaning composition and/or in the form of a treatment composition. the

The balance of any aspect of the above cleaning compositions consists of one or more auxiliary substances. the

Applicable lipase variants

The lipase of the composition of the present invention is a lipase variant which lacks the C-terminal extension but which has introduced mutations in certain regions of the parent lipase, thereby reducing the tendency to produce odor. the

parental lipase

The parent lipase may be a fungal lipase, as described in the "Homology and sequence alignment" section, whose amino acid sequence has at least 50% homology to the thermophilic fungal lipase sequence shown in SEQ ID NO: 2 . the

The parent lipase may be a yeast polypeptide, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide, such as Acremonium, Aspergillus Mold, Aureobasidium, Cryptococcus, Smut, Fusarium, Humicola, Magnaporthe grisea, Mucor, Myceliophthora, Neocoma, Neurospora, Paecilomyces, Penicillium, Rumen Fungus, Schizophyllum, Aspergillus flavus, Thermoascus, Rhizopus grassa, Trichoderma, or Trichoderma polypeptide. the

In a preferred aspect, the parent lipase is a Saccharomyces kalsberg, Saccharomyces cerevisiae, Saccharomyces saccharification, Saccharomyces douglas, Kluyveromyces, Knottia, or Saccharomyces ovale polypeptide having lipase activity. the

In another preferred aspect, the parent lipase is Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus tubingensis, Bacteroides fusarium, Fusarium wilt, Fusarium, Fusarium yellow, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium vitae, Fusarium oxysporum, Fusarium reticulum, Fusarium moniliforme, Fusarium elderberry, Fusarium complexion Fusarium spp., Cladosporium fusarium, Fusarium sulforaphane, Fusarium tuftum, Fusarium myceloides, Fusarium spp., Humicola insolens, Thermophilic fungi (synonym: Chaetomium thermophile), Mihei Mucor, Myceliophthora thermophila, Neurospora, Penicillium purpuragenes, Trichoderma harzianum, Trichoderma korningen, Trichoderma longibrachia, Trichoderma reesei, or Trichoderma viride polypeptide. the

In another preferred aspect, the parent lipase is a Thermomyces lipase. the

In a more preferred aspect, the parent lipase is a thermophilic fungal lipase. In a more preferred embodiment, the parent lipase is the lipase of SEQ ID NO: 2. the

Region identification and replacement.

The positions of region I to region IV mentioned below are amino acid residue positions in SEQ ID NO: 2. Corresponding (or homologous) sites in different lipases were found using the methods described in the "Homology and Sequence Alignment" section. the

Substitution in Region I

Region I consists of amino acid residues surrounding the N-terminal residue El. In this region it is preferred to replace amino acids in the parent lipase with more positively charged amino acids. Region I comprises amino acid residues corresponding to the following positions: 1 to 11 and 223-239. The following positions are of particular interest: 1, 2, 4, 8, 11, 223, 227, 229, 231, 233, 234 and 236. Specifically, the following substitutions have been identified: X1N/ ^* , X4V, X227G, X231R and X233R.

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

Substitution in Region II

Region II consists of amino acid residues that contact the substrate on one side of the acyl chain and on the side of the alcohol moiety. Amino acids in the parent lipase are preferably substituted in this region with more positively charged amino acids or less hydrophobic amino acids. Region II comprises amino acid residues corresponding to the following positions: 202 to 211 and 249 to 269. The following positions are of particular interest: 202, 210, 211, 253, 254, 255, 256, 259. Specifically, the following substitutions have been identified: X202G, X210K/W/A, X255Y/V/A, X256K/R, and X259G/M/Q/V. the

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

Substitution in Region III

Region III consists of amino acid residues that form a flexible structure allowing substrate access to the active site. Amino acids in the parent lipase are preferably substituted in this region with more positively charged amino acids or less hydrophobic amino acids. Region III comprises amino acid residues corresponding to the following positions: 82-102. The following positions are of particular interest: 83, 86, 87, 90, 91, 95, 96, 99. Specifically, the following replacements have been confirmed: X83T, X86V, and X90A/R. the

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

Substitution in Region IV

Region IV consists of amino acid residues that are electrostatically bound to the surface. In this region it is preferred to replace amino acids in the parent lipase with more positively charged amino acids. Region IV comprises amino acid residues corresponding to the following positions: 27 and 54-62. The following positions are of particular interest: 27, 56, 57, 58, 60. Specifically the following substitutions have been confirmed: X27R, X58N/AG/T/P and X60V/S/G/N/R/K/A/L. the

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

Amino acids at other positions

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

Variants may comprise substitutions outside defined regions I to IV, preferably the number of substitutions outside defined regions I to IV is less than six, or less than five, or less than four, or less than three , or less than two, such as five, or four, or three, or two or one. Alternatively, the variant does not contain any substitutions outside defined regions I to IV. the

For example, further substitutions, such as those described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202, can be made according to art-known laws. the

parental fat variant

In one aspect, when compared to the parent, the variant comprises a total of at least three substitutions selected from one or more of the group consisting of:

a) at least two, or at least three, or at least four, or at least five, or at least six, such as two, three, four, five or six substitutions in region I,

b) at least one, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six, of region II replace,

c) at least one, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six, of region III replace,

d) and/or at least one of regions IV, at least two, or at least three, or at least four, or at least five, or at least six, such as one, two, three, four, five or six substitutions. the

A variant may comprise substitutions compared to the parent of the variant corresponding to those listed in Table 1 below. the

Area I Area II Zone III Region IV outside the area X4V+X227G+ X231R+X233R X210K+X256K X83T+X86V X58A+X60S X150G X227G+X231R+ X233R X256K X86V X58N+X60S X150G X231R+X233R X255Y the the the X231R+X233R X202G the the the X227G+X231R+ X233R X256K X86V the the X4V+X231R+ X233R the the X58N+X60S the X231R+X233R the X90R X58N+X60S the X231R+X233R X255V X90A the the X227G+X231R+ X233R X256K X86V X58N+X60S X150G X231R+X233R X211L the X58N+X60S X147M X231R+X233R the the the X150K

Table 1: Some special variants

In a more specific embodiment, the parent lipase is identical to SEQ ID NO: 2, the variants of Table 1 would thus be:

Area I Area II Zone III Region IV outside the area Q4V+L227G+ T231R+N233R E210K+P256K S83T+I86V S58A+V60S A150G L227G+T231R+ N233R P256K I86V S58N+V60S A150G T231R+N233R I255Y the the the T231R+N233R I202G the the the L227G+T231R+ N233R P256K I86V the the Q4V+T231R+ N233R the the S58N+V60S the T231R+N233R the I90R S58N+V60S the T231R+N233R I255V I90A the the L227G+T231R+ N233R P256K I86V S58N+V60S A150G T231R+N233R F211L the S58N+V60S L147M T231R+N233R the the the A150K

Table 2: Some Specific Variants of Sequence Identification Number: 2

Amino Acid Modification Nomenclature

In describing lipase variants according to the present invention, the following nomenclature is used for ease of reference: Original amino acid: position: substituted amino acid

According to this nomenclature, for example, the substitution of glutamic acid at position 195 for glycine is denoted G195E. A deletion of a glycine at the same position is indicated as G195 ^* , while an insertion of an additional amino acid residue such as a lysine is indicated as G195GK. A particular lipase comprising a "missing" amino acid at position 36 compared to other lipases with an insertion of an aspartic acid at this position is denoted ^* 36D. Multiple mutations are separated by plus signs, ie: R170Y+G195E, indicating substitution of tyrosine and glutamic acid to arginine and glycine at positions 170 and 195, respectively.

X231 indicates the amino acid corresponding to position 231 in the parent polypeptide when the sequence alignment method is applied. X231R indicates that this amino acid is replaced with R. For SEQ ID NO: 2 X is T, X231R thus represents substitution of T with R at position 231. When an amino acid at a position (eg 231) can be substituted with another amino acid selected from a group of amino acids, eg, the group consisting of R and P and Y, it is indicated by X231R/P/Y. the

In all cases, the accepted IUPAC one-letter or three-letter amino acid abbreviations are used. the

amino acid grouping

In this specification, amino acids are classified as negatively charged, positively charged or neutrally charged according to their charge at pH 10. Thus, the negatively charged amino acids are E, D, C (cysteine) and Y, especially E and D. Positively charged amino acids are R, K and H, especially R and K. When forming disulfide bonds, the neutral amino acids are G, A, V, L, I, P, F, W, S, T, M, N, Q, and C. Substitution by another amino acid from the same group (negatively charged, positively charged, or neutrally charged) is called a conservative substitution. the

Neutral amino acids can be classified as hydrophobic or nonpolar (G, A, V, L, I, P, F, W, and C as part of a disulfide bond) and hydrophilic or polar (S, T, M, N, Q). the

amino acid identity

The parameter "identity" is used to describe the relatedness between two amino acid sequences or two nucleotide sequences. the

For the purposes of the present invention, the alignment of two amino acid sequences is determined by using the Needle program version 2.8.0 from the EMBOSS package (http://emboss.org). The Needle program uses the global alignment algorithm described in Needleman, S.B. and Wunsch, C.D. (1970) J.Mol.Biol.48, 443-453. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap expansion The penalty is 0.5. the

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

An exact match occurs when the "invented sequence" and the "foreign sequence" have identical amino acid residues at the positions of identity of the overlapping portions. The sequence length is the number of amino acid residues in the sequence (eg, the length of SEQ ID NO: 2 is 269). the

The parent lipase has at least 50%, especially at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, more than 95% or more than 98 % homology. In a specific embodiment, the parent lipase is identical to the thermophilic fungal lipase (SEQ ID NO: 2). the

Identity and homology can be calculated and used for sequence alignment using the methods described above. In the context of the present invention, homology and sequence alignment are calculated as follows. the

Homology and sequence alignment

For the present invention, by computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, 8th edition, 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), suitably determine the degree of homology using GAP with the following polypeptide sequence alignment settings: The GAP generation penalty is 3.0, and the GAP extension penalty is 0.1. the

In the present invention, Absidia, Absidia umbelliferum, Rhizomucor mihei, Rhizopus daishii, Aspergillus niger, Aspergillus tubingensis, Fusarium oxysporum, Fusarium heterosporum, Aspergillus oryzae, Penicillium kamembert, The corresponding (or homologous) sites in the lipase sequences of Aspergillus foetidus, Aspergillus niger, thermophilic fungi (synonym: Chaetomium thermophila), and Landerina penisapora were determined by the sequence alignment shown in FIG. 1 . the

To find homologous sites in the lipase sequence not shown in the sequence alignment, the sequence of interest was aligned with the sequence shown in Figure 1. New sequences were aligned to existing sequences in Figure 1 by performing a GAP sequence alignment of the most homologous sequences found by the GAP program. GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-45) provide GAP. Peptide sequence comparisons use the following settings: GAP generation penalty is 3.0 and GAP extension penalty is 0.1. the

The parent lipase has at least 50%, especially at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, more than 95% or more than 98% of the thermophilic fungal lipase (SEQ ID NO: 2) amino acid identity. In a specific embodiment, the parent lipase is identical to the thermophilic fungal lipase (SEQ ID NO: 2). the

hybridize

The present invention also relates to an isolated polypeptide having lipase activity encoded by a polynucleotide that operates under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably Under medium-high stringency conditions, even more preferably under high stringency conditions, most preferably under very high stringency conditions, hybridize with the following nucleotide sequence: (i) nucleotides 178 to 660, SEQ ID NO: 1, (ii) the cDNA sequence comprising nucleotides 178 to 660, SEQ ID NO: 1, the sequence of (iii) (i) or (ii), or (iv) of (i), (ii) or (iii) Complementary strands (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, New York). The subsequence of SEQ ID NO: 1 comprises at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. In addition, the subsequence may encode a polypeptide fragment having lipase activity. the

For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at the following conditions: 42°C, 5X SSPE, 0.3% SDS, 200ug /mL sheared and denatured protist DNA, or 25% formamide for very low and low stringency conditions, 35% formamide for medium-high stringency conditions, or 50% Formamide is used for high and very high stringency conditions, and the standard Southern hybridization procedure hybridizes most preferably for 12 to 24 hours. the

For long probes of at least 100 nucleotides in length, use 2X SSC, 0.2% SDS, preferably at least 45°C (very low stringency), more preferably at least 50°C (low stringency), and more preferably at least 50°C (low stringency). Preferably at least 55°C (medium stringency), more preferably at least 60°C (medium-high stringency), even more preferably at least 65°C (high stringency), most preferably at least 70°C (very high stringency ), and finally wash the carrier material three times for 15 minutes each. the

DNA sequence, expression vector, host cell, lipase production

The present invention provides a DNA sequence encoding the lipase of the present invention, an expression vector comprising the DNA sequence, and a transformed host cell comprising the DNA sequence or the expression vector. They are obtainable by methods known in the art. the

The invention also provides a method of producing lipase by culturing transformed cells under conditions favorable for lipase production and recovering lipase from the resulting broth. The methods are carried out according to principles known in the art. the

lipase activity

- Lipase activity on tributyrin at neutral pH (LU)

The lipase substrate was prepared by emulsifying tributyrin (tributyrin) using gum arabic as an emulsifier. Tributyrin was then hydrolyzed at pH 7 or pH 9 at 30°C in a pH-constant titration test. One unit of lipase activity (1 LU) corresponds to the amount of enzyme capable of releasing 1 micromole of butyrate per minute at pH 7. the

- insurance

The efficacy risk factor describing the performance versus odor reduction risk ratio is defined as: BR = RP _avg /R. The lipase variants described herein may have a risk factor greater than 1, greater than 1.1, or even greater than 1 to about 1000.

- average relative performance

The procedure for calculating the average relative performance (RPavg) is found in Example 5 of this specification. The lipase variants described herein can have an average relative performance (RPavg) of at least 0.8, at least 1.1, at least 1.5, or even at least 2 to about 1000. the

Suitable photobleaches

Suitable photobleaches include catalytic photobleaches and photoinitiators. Suitable catalytic photobleaches are selected from the group consisting of water-soluble phthalocyanines having the following chemical formula:

in:

PC is a phthalocyanine ring system;

Me is Zn; Fe(II); Ca; Mg; Na; K; Al-Z ₁ ; Si(IV); P(V); Ti(IV); Ge(IV); Cr(VI); ); Zr(IV); In(III); Sn(IV) or Hf(VI);

_Z1 is a halide; sulfate; nitrate; carboxylate; alkanol; or hydroxide ion;

Q is 0; 1 or 2;

R is 1 to 4;

Q ₁ , is a sulfo or carboxyl group; or a group of formula

_{-SO2X2 - R1} - _X3 ⁺ ;- _OR1 - _X3 ⁺ ; or-( _CH2 ),- _Y1 ⁺ ;

in

R ₁ is branched or unbranched C ₁ -C ₈ alkylene; or 1,3- or 1,4-phenylene;

X ₂ is -NH-; or -NC ₁ -C ₅ alkyl;

X ₃ ⁺ is the following formula group [0125] X ₃ ⁺ is the following formula group

Alternatively, in the case of R ₁ =C ₁ -C ₈ alkylene, it is also a group of the formula

Y ₁ ⁺ is a group of formula

t is 0 or 1

where in the above formula

R ₂ and R ₃ are independently of each other C ₁ -C ₆ alkyl

R ₄ is C ₁ -C ₅ alkyl; C ₅ -C ₇ cycloalkyl or NR ₇ R ₈ ;

R ₅ and R ₆ are C ₁ -C ₅ alkyl independently of each other;

R ₇ and R ₈ are independently hydrogen or C ₁ -C ₅ alkyl;

R ₉ and R ₁₀ are independently unsubstituted C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl, and the substituted alkyl consists of hydroxyl, cyano, carboxyl, carb-C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkoxy, phenyl, naphthyl or pyridyl substituted;

u is 1 to 6;

_A is a unit constituting an aromatic 5- to 7-membered nitrogen heterocyclic ring, where appropriate positions in the ring may further contain one or two nitrogen atoms as ring members, and

B ₁ is a unit constituting a saturated 5- to 7-membered nitrogen heterocyclic ring, and suitable positions in the ring may also contain 1 to 2 nitrogen, oxygen and/or sulfur atoms as ring members;

Q ₂ is hydroxyl; C _{1 -C 22} alkyl; branched C ₃ -C ₂₂ alkyl; _{C 2} -C ₂₂ alkenyl; branched C ₃ -C ₂₂ alkenyl and mixtures thereof; Alkoxy; sulfo or carboxyl; groups of the formula

A branched chain alkoxy group of the formula

Ethyleneoxyalkyl units of the formula

-(T ₁ ) _d -(CH ₂ ) _b (OCH ₂ CH ₂ ) _a -B ₃

or an ester of the formula

COOR ₁₈

in

C ₁ -C ₃₀ alkyl; C ₁ -C ₃₀ alkoxy; -CO ₂ H; -CH ₂ COOH; -SO ₃ -M ₁ ; -OSO ₃ -M _{1 ; 3} ^2- M ₁ ; -OPO ₃ ^2- M ₁ ; and mixtures thereof;

B ₃ is hydrogen; Hydroxy; -COOH; -SO ₃ -M ₁ ; -OSO ₃ M ₁ or C ₁ -C ₆ alkoxy;

M ₁ is a water-soluble cation;

T ₁ is -O-; or -NH-;

X ₁ and X ₄ are independently of each other -O-; -NH- or -NC ₁ -C ₅ alkyl;

R ₁₁ and R ₁₂ are hydrogen independently of each other; a sulfo group and a salt thereof; a carboxyl group and a salt thereof or a hydroxyl group; at least one of R ₁₁ and R ₁₂ is a sulfo group or a carboxyl group or a salt thereof,

Y ₂ is -O-; -S-; -NH- or -NC ₁ -C ₅ alkyl;

R ₁₃ and R ₁₄ independently of each other _are hydrogen; C ₁ -C ₆ alkyl; hydroxy-C ₁ -C ₆ alkyl; cyano-C ₁ -C 6 alkyl; sulfo-C ₁ -C ₆ alkyl ; carboxy or halogen- _{C 1} -C ₆ alkyl; unsubstituted phenyl or halogen substituted phenyl, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy; sulfo or carboxy or R ₁₃ and R ₁₄ and a nitrogen atom, which are bonded to the nitrogen atom to form a saturated 5- or 6-membered heterocyclic ring, which may additionally contain a nitrogen atom or an oxygen atom as a member of the ring;

R ₁₅ and R ₁₆ are independently of each other C ₁ -C ₆ alkyl or aryl-C ₁ -C ₆ alkyl;

R ₁₇ is hydrogen; unsubstituted C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl, substituted by halogen, hydroxyl, cyano, phenyl, carboxy, carb-C ₁ -C ₆ alkane Oxygen or C ₁ -C ₆ alkoxy substitution;

R ₁₈ is C ₁ -C ₂₂ alkyl; branched C ₃ -C ₂₂ alkyl; C ₁ -C ₂₂ alkenyl or branched C ₃ -C ₂₂ alkenyl; C ₃ -C ₂₂ ethylene glycol; C ₁ -C ₂₂ alkoxy; branched C ₃ -C ₂₂ alkoxy; and mixtures thereof;

M is hydrogen; or an alkali metal ion or an ammonium ion,

Z ₂ ^- is chlorine; bromine; alkyl sulfate or aryl sulfate ion;

a is 0 or 1;

b is 0 to 6;

c is 0 to 100;

d is 0; or 1;

e is 0 to 22;

v is an integer from 2 to 12;

w is 0 or 1; and

A ^- is an organic or inorganic ion, and

s is equal to r in the case of the monovalent anion ^A- , and less than or equal to r in the case of the polyvalent anion, A _s- is necessary for ^compensating the positive charge; where if r is not equal to 1, the _Q1 group can be the same of or different,

And wherein the phthalocyanine ring system may additionally contain a solubilizing group;

Other suitable catalytic photobleaches include xanthene dyes and mixtures thereof. In another aspect, suitable catalytic photobleaches are selected from the group consisting of sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, Eosin Y, Phoxine B, Rose Bengal, C.I. Food Red 14, and mixtures thereof. In another aspect, a suitable catalytic photobleach may be a mixture of sulfonated zinc phthalocyanine and sulfonated aluminum phthalocyanine having a weight ratio of sulfonated zinc phthalocyanine to sulfonated aluminum phthalocyanine greater than 1, greater than 1 But less than about 100, or even from about 1 to about 4. the

369)；(1-[4-(2-羟乙氧基)-苯基]-2羟基-2-甲基-1-丙-1-酮)( 

2959)；1-羟基环己基苯基-酮( 

184)；寡聚[2-羟基2-甲基-1-[4(1-甲基)-苯基]丙酮( KIP 150)；2-4-6-(三甲基苯甲酰)二苯基-氧化膦，二(2，4，6-三甲基苯甲酰)-苯基-氧化膦( 819)；(2，4，6三甲基苯甲酰)苯基磷酸乙基酯( 

TPO-L)；以及它们的混合物。  Suitable photoinitiators are selected from the group consisting of: aromatic 1,4-quinones such as anthraquinone and naphthoquinone; alpha aminoketones, especially those containing a benzoyl moiety, otherwise known as alpha-aminophenethyl Ketones; α-hydroxyketones, especially α-hydroxyacetophenones; phosphorous-containing photoinitiators, including monoacyl, bisacyl, and triacylphosphine oxides and sulfides; dialkoxyacetophenones; α-halogenated benzenes Ethanone; triacylphosphine oxides; benzoin and benzoin-based photoinitiators, and mixtures thereof. In another aspect, suitable photoinitiators are selected from the group consisting of: 2-ethylanthraquinone; vitamin K3; 2-sulfuric-anthraquinone; 2-methyl-1-[4-phenyl]-2-mol Linyl-1-one ( 907); (2-benzyl-2-dimethylamino-1-(4-morpholinylphenyl)-butan-1-one (

369); (1-[4-(2-hydroxyethoxy)-phenyl]-2 hydroxy-2-methyl-1-propan-1-one)(

2959); 1-hydroxycyclohexylphenyl-ketone (

184); Oligo[2-hydroxyl 2-methyl-1-[4(1-methyl)-phenyl]acetone ( KIP 150); 2-4-6-(trimethylbenzoyl)diphenyl-phosphine oxide, bis(2,4,6-trimethylbenzoyl)-phenyl-phosphine oxide ( 819); (2,4,6 trimethylbenzoyl) ethyl phenyl phosphate (

TPO-L); and mixtures thereof.

The above photobleaches may be used in combination (mixtures of any photobleaches may be used). Suitable photobleaches are commercially available from Aldrich, Milwaukee, Wisconsin, USA; Frontier Scientific, Logan, Utah, USA; Ciba Specialty Chemicals, Basel, Switzerland; BASF, Ludwigshafen, Germany; Lamberti S.p.A, Gallarate, Italy; , India; Organic Dyestuffs Corp., East Providence, Rhode Island, USA; and/or a photobleach made according to the examples contained herein. the

Auxiliary materials

While not essential to the invention, the non-limiting list of auxiliary substances exemplified below are suitable for use in the compositions of the invention and may be desirable to incorporate into certain embodiments of the invention, for example to aid To or enhance the cleaning performance of the substrate to be cleaned, or to adjust the aesthetics of the cleaning composition in the case of perfumes, colorants, dyes and the like. The precise nature of these additional components and their incorporation will depend upon the physical form of the composition and the nature of the cleaning operation for which it is employed. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes and enzyme stabilizers, catalytic substances, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, fabric colorants, fragrances, structural elastic agents, fabric softeners, carriers, Hydrotropes, processing aids, solvents and/or pigments. In addition to the disclosure below, suitable examples and amounts of these other adjuvants are found in US Pat. Nos. 5,576,282, 6,306,812B1 and 6,326,348B1, which are incorporated by reference. the

As stated, auxiliary ingredients are not essential to Applicants' compositions. Accordingly, certain embodiments of Applicants' compositions do not contain one or more of the following adjunct materials: surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes and enzyme stabilizers, Catalytic substances, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, fragrances, structural elastification agent, fabric softener, carrier, hydrotrope, processing aid, solvent and/or pigment. However, when one or more adjuvants are present, these one or more adjuvants may be present as detailed below:

Bleaching Agents - The cleaning compositions of the present invention may include one or more bleaching agents. Suitable bleaching agents other than bleach catalysts include photobleaches, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, and mixtures thereof. Typically, when bleaching agents are used, the compositions of the present invention may comprise from about 0.1% to about 50%, or even from about 0.1% to about 25%, by weight of the subject cleaning compositions, of bleaching agents. Examples of suitable bleaches include:

)、以及它们的混合物。合适的过羧酸包括具有分子式R-(C＝O)O-O-M的疏水和亲水过酸，其中R为烷基，任选支链烷基。当过酸为疏水时，其具有6至14个碳原子或者8至12个碳原子。当过酸为亲水时，其具有小于6个碳原子或者甚至小于4个碳原子；并且M为抗衡离子，例如钠、钾或氢；  (1) Preformed peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of percarboxylic acids and their salts, percarbonic acids and their salts, perimidic acids and their salts, permonosulfuric acid and its salt (such as

), and their mixtures. Suitable percarboxylic acids include hydrophobic and hydrophilic peracids having the formula R-(C=O)OOM, where R is an alkyl group, optionally branched. When the peracid is hydrophobic it has 6 to 14 carbon atoms or 8 to 12 carbon atoms. When the peracid is hydrophilic, it has less than 6 carbon atoms or even less than 4 carbon atoms; and M is a counterion such as sodium, potassium or hydrogen;

(2) Hydrogen peroxide sources, such as inorganic perhydrogen compound salts, including the following alkali metal salts such as sodium salts: perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, per Phosphates, persilicates and mixtures thereof. In one aspect of the invention, the inorganic perhydrogen compound salt is selected from the group consisting of sodium perborate, sodium percarbonate, and mixtures thereof. If employed, inorganic perhydroperhydrogen compound salts are generally present at levels of 0.05 to 40% by weight or 1 to 30% by weight of the total composition and are generally incorporated into such compositions as crystalline solids which may be coated. Suitable coatings include: inorganic salts such as alkali metal silicates, carbonates or borates or mixtures thereof, or organics such as water soluble or dispersible polymers, waxes, oils or fatty soaps; and

(3) Bleach activators having R-(C=O)-L, wherein R is an alkyl, optionally branched chain alkyl. When the bleach activator is hydrophobic it has 6 to 14 carbon atoms or 8 to 12 carbon atoms. When the bleach activator is hydrophilic it has less than 6 carbon atoms or even less than 4 carbon atoms; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives, especially besylate. Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid and its salts, 3,5,5-trimethylhexanoyloxybenzene Sulfonate, Tetraacetylethylenediamine (TAED) and Nonanoyloxybenzenesulfonate (NOBS). Suitable bleach activators are also disclosed in WO 98/17767. Although any suitable bleach activator may be employed, in one aspect of the invention, the subject cleaning compositions may comprise NOBS, TAED, or mixtures thereof. the

If present, peracids and/or bleach activators are typically present in the said composition at levels of about 0.1 to about 60%, about 0.5 to about 40%, or even about 0.6 to about 10% by weight of the composition. composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof. the

The amounts of hydrogen peroxide source and peracid or bleach activator can be selected such that the molar ratio of available oxygen (from peroxide source) to peracid is 1:1 to 35:1, or even 2:1 to 10:1 . the

Surfactants - The cleaning compositions according to the present invention may comprise a surfactant or surfactant system, wherein the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric Surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof. If present, surfactants are typically present at levels of from about 0.1% to about 60%, from about 1% to about 50%, or even from about 5% to about 40%, by weight of the subject compositions. the

Builders - The cleaning compositions of the present invention may contain one or more detergent builders or builder systems. When a builder is used, the subject compositions will generally comprise at least about 1%, from about 5% to about 60%, or even from about 10% to about 40%, by weight of the subject composition, of builder. Builders include, but are not limited to, alkali metal, ammonium, and alkanolammonium polyphosphates, alkali metal silicates, alkaline earth metal and alkali metal carbonates, aluminosilicate builders, and polycarboxylate compounds , ether hydroxypolycarboxylate, copolymer of maleic anhydride and ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethylmalic acid, Polyacetic acids (such as ethylenediaminetetraacetic acid and nitrilotriacetic acid) and polycarboxylic acids (such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polybasic acid, 1,3,5-tribenzoic acid , carboxymethylmalic acid), various alkali metal salts, ammonium salts and substituted ammonium salts, and their soluble salts. the

Chelating Agents - The cleaning compositions herein may contain a chelating agent. Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof. When a chelating agent is used, the subject compositions may comprise from about 0.005% to about 15%, or even from about 3.0% to about 10%, by weight of the subject composition, of a chelating agent. the

Dye Transfer Inhibiting Agents - The cleaning compositions of the present invention may also comprise one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to: polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and Polyvinylimidazole, or their mixtures. When present in the subject compositions, dye transfer inhibiting agents may be present at levels of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%, by weight of the composition exist. the

Brighteners - The cleaning compositions of the present invention may also contain additional components that can tint the article being cleaned, such as optical brighteners. Suitable fluorescer levels include lower levels of from about 0.01, about 0.05, about 0.1, or even about 0.2% by weight to higher levels of 0.5% or even 0.75% by weight. the

Dispersants - The compositions of the present invention may also contain dispersants. Suitable water-soluble organics include homo- or co-polymeric acids or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl groups not separated by more than two carbon atoms. the

Additional Enzymes - The cleaning composition may comprise one or more enzymes which provide cleaning performance and/or fabric care benefits. Examples of suitable enzymes include, but are not limited to: hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, mannan Carbohydrases, pectin lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanses Carbohydrases, arabinases, hyaluronidases, chondroitinases, laccases and amylases, or mixtures thereof. A typical combination is an enzyme combination which may comprise, for example, a protease and a lipase in combination with an amylase. When present in cleaning compositions, such additional enzymes may be present in an amount of about 0.00001% to about 2%, about 0.0001% to about 1%, or even about 0.001% to about 0.5% enzyme protein by weight of the composition exist. the

Enzyme Stabilizers - Enzymes used in detergents can be stabilized using a variety of techniques. The enzymes used in the present invention may be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the final composition, the final product providing such ions to the enzymes. In the case of aqueous compositions comprising proteases, reversible protease inhibitors such as boron compounds may be added to further improve stability. the

Catalytic Metal Complexes - Applicants' cleaning compositions may contain catalytic metal complexes. One class of metal-containing bleach catalysts are catalyst systems comprising transition metal cations with defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations; Ancillary metal cations with little or no bleach catalytic activity, such as zinc or aluminum cations; and chelating agents containing defined stability constants for catalytic and ancillary metal cations, especially ethylenediaminetetraacetic acid, ethylenediamine Tetrakis(methylenephosphonic acid) and their water-soluble salts. Such catalysts are disclosed in U.S. 4,430,243. the

The compositions of the invention can, if desired, be catalyzed by means of manganese compounds. These compounds and amounts are well known in the art and include, for example, manganese-based catalysts disclosed in US Patent No. 5,576,282. the

Cobalt bleach catalysts for use in the present invention are known and described, for example, in US Patent 5,597,936; US Patent 5,595,967. The cobalt catalysts described above are readily prepared by known methods, such as those proposed in US Patent No. 5,597,936 and US Patent No. 5,595,967. the

The compositions herein may also suitably comprise transition metal complexes of ligands such as bispidones (WO 05/042532A1 ) and/or macrocyclic rigid ligands (abbreviated "MRL"). As an example, and not limitation, the compositions and methods herein can be adjusted to provide about at least one part per billion active MRL species in the aqueous wash medium, and will typically provide about 0.005 ppm to An MRL of about 25 ppm, about 0.05 ppm to about 10 ppm, or even about 0.1 ppm to about 5 ppm. the

Suitable transition metals in the transition metal bleach catalysts of the present invention include, for example, manganese, iron and chromium. Suitable MRLs include 5,12-diyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. the

Suitable transition metal MRLs are readily prepared by known procedures, such as those set forth in WO 00/32601 and US Patent 6,225,464. the

Solvents - Suitable solvents include water and other solvents such as lipophilic fluids. Examples of suitable lipophilic fluids include silicones, other silicones, hydrocarbons, glycol ethers, glycerol derivatives such as glycerol ethers, perfluoroamines, perfluorinated and hydrofluoroether solvents, low volatility fluorine-free organic solvents, glycol solvents, other environmentally friendly solvents, and mixtures thereof. the

Method of making the composition

The compositions of the present invention may be formulated in any suitable form and prepared by any method chosen by the formulator, in US Patent 4,990,280, US Patent 20030087791A1; US Patent 20030087790A1; US Patent 20050003983A1; US Patent 20040048764A1; 6,291,412; US Patent 20050227891A1; EP 1070115A2; US Patent 5,879,584; US Patent 5,691,297; US Patent 5,574,005; Examples, these patents are all incorporated herein by reference. the

Instructions

The present invention includes a method of cleaning and/or treating a site, especially a surface or fabric. The above method comprises the steps of contacting at least a portion of a surface or fabric with an embodiment of Applicant's cleaning composition (either neat or diluted in a wash liquor) and then optionally rinsing said surface or fabric. The surface or fabric may be subjected to a washing step prior to the above rinsing step. For purposes of the present invention, washing includes, but is not limited to, scrubbing and mechanical agitation. As will be appreciated by those skilled in the art, the cleaning compositions of the present invention are ideally suited for laundry use. Accordingly, the present invention includes a method for laundering fabrics, said method comprising the steps of contacting the fabrics to be laundered with said cleaning wash solution comprising Applicants' cleaning composition, cleaning aids or at least one embodiment of a mixture thereof. Fabrics may include most any fabric capable of being laundered under normal consumer use conditions. The solution preferably has a pH of about 8 to about 10. The composition can be used at a concentration of about 500 ppm to about 15,000 ppm in solution. The water temperature is generally from about 5°C to about 90°C. The ratio of water to fabric is generally from about 1:1 to about 30:1. the

Example

Examples of lipase variants

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

- Media and solutions: LAS (Surfac PS ^™ ) and Zeolite A (Wessalith P ^™ ). The other ingredients used are standard laboratory reagents.

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

Example 1: Enzyme Production

Using standard methods in the art, a plasmid containing the gene encoding the lipase is constructed and transfected into a suitable host cell. the

Fed-batch fermentation was carried out using a culture medium with a constant temperature of 34°C and an initial volume of 1.2 liters. The initial pH of the medium was set at 6.5. Once the pH had risen to 7.0, this was kept constant by adding 10% H3PO4. Dissolved oxygen levels in the medium were controlled by varying the agitation rate and using a fixed aeration rate of 1.0 liters of air per minute per liter of medium. The feed rate was maintained at a constant level throughout the fed-batch fermentation stage. The batch medium contains malt syrup as carbon source, urea and yeast extract as nitrogen source, and a mixture of trace metals and salts. The feed added continuously during the fed-batch fermentation stage contained maltose syrup as a carbon source, whereas urea and yeast extract were added to ensure adequate nitrogen supply. the

Purification of the lipase is carried out using standard methods known in the art, for example by filtration of the fermentation supernatant and subsequent hydrophobic chromatography and anion exchange, for example as described in EP 0 851 913 in Example 3. the

Example 2: AMSA - Automated Mechanical Stress Detection Analysis - used to calculate Relative Performance (RP) .

The enzyme variants of the present patent application were tested using the Automated Mechanical Stress Assay Assay (AMSA). Through the AMSA test, the washing performance of a large number of small volume enzyme detergent solutions can be tested. The AMSA plate has multiple slots for the test solution, and a cover that firmly squeezes the fabric sample so that it is washed at all slot openings. During washing, the plate, test solution, fabric and cover are shaken vigorously so that the test solution contacts the fabric and mechanical stress is applied. A more detailed description can be found in WO 02/42740, especially the paragraph "Special method embodiments" on pages 23-24. The container containing the detergent test solution consists of a cylindrical hole (diameter 6 mm, depth 10 mm) in a metal plate. A soiled fabric (test substance) is placed on top of the metal plate, used as a lid and seal on the container. Another metal plate is placed on top of the soiled fabric to avoid spilling any liquid from either container. The two metal plates are vibrated up and down at a frequency of 30 Hz and an amplitude of 2 mm together with the soiled fabric. the

The detection assay was carried out under the test conditions specified below:

Test solution 0.5g/L LAS 0.52g/L Na2CO3 1.07g/L Zeolite A 0.52g/L Trisodium Citrate Test solution volume 160micro L PH value As is(≈9.9. washing time 20 minutes temperature 30℃ water hardness 15°dH Ca ²⁺ /Mg ²⁺ /NaHCO3 ratio: 4:1:7.5 Enzyme concentration in test solution 0.125, 0.25, 0.50, 1.0mg enzyme protein/liter Dry Performance: Fabric pieces are rinsed in tap water immediately after washing and air dried at 85°C for 5 minutes Odor: Fabric pieces are rinsed in tap water immediately after washing and dried at room temperature (20°C) for 2 hours test substance A sample of cream turmeric as described below (EMPA221 for cotton fabrics)

table 3

Cream turmeric samples were prepared by mixing 5 g (Santa Maria, Denmark) turmeric and 100 g (38% fat, Arla, Denmark) cream at 50° C., the mixture was kept at this temperature for about 20 minutes and filtered (50° C.) Remove any undissolved particles. The mixture was cooled to 20 °C and a cotton spun sample, EMPA221, was dipped into this cream-turmeric mixture, then dried overnight at room temperature and frozen until use. In the patent application PA200500775 filed on May 27, 2005, a method for the preparation of a cream turmeric sample is disclosed. the

The performance of an enzyme variant can be measured by the color brightness of a fabric sample washed with a particular enzyme variant. Brightness can also be expressed in terms of the intensity of light reflected from a fabric sample when illuminated with white light. When the fabric is soiled, the intensity of the reflected light is lower than that of a clean fabric, so the intensity of the reflected light can be used to measure the wash performance of the enzyme variant. the

Color measurements were performed using a professional flatbed scanner (PFU DL2400pro) which was used to acquire images of laundered fabric samples. The scanning resolution is 200dpi, and the output color depth is 24 bits. For accurate results, frequently calibrate the scanner with the Kodak reflective IT8 color chart. the

A specially designed application software (Novozymes Color Vector Analyzer) was used to extract light intensity values from the scanned images. The program retrieves 24-bit pixel values from the image and converts them to red, green and blue (RGB) values. Calculate the intensity value (Int) by adding the RGB values as a vector, then extracting the length of the resulting vector:

$\mathrm{<span\; class="notranslate">\; Int</span>}<span\; class="notranslate">\; =</span>\sqrt{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="r"\; filenames="S2007800028202E00011.png,S2007800028202E00021.png"\; state="\{\{state\}\}">r</figure-callout></span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="g"\; filenames="S2007800028202E00011.png,S2007800028202E00021.png"\; state="\{\{state\}\}">g</figure-callout></span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="b"\; filenames="S2007800028202E00011.png,S2007800028202E00021.png"\; state="\{\{state\}\}">b</figure-callout></span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}$

The wash performance (P) of the variants was calculated according to the following formula:

P＝Int(v)-Int(r) where

Int(v) is the light intensity value of the fabric surface washed with the test enzyme, Int(r) is the light intensity value of the fabric surface washed without the test enzyme. the

By definition, it is stated that the relative performance score is the result of AMSA washing: the relative performance score (RP) is the ratio of the sum of the performance (P) of the test enzyme variants to the performance of the reference enzyme: RP = P(test enzyme)/P(reference enzyme ). the

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

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

A variant is considered to exhibit improved wash performance if it performs better than the reference enzyme. In the context of the present invention, the reference enzyme is lipase of SEQ ID NO: 2, which has the substitution T231R+N233R. the

Example 3: GC-Gas Chromatography for Efficacy Calculation .

Butyric acid release in lipase washed samples was measured by solid phase microextraction gas chromatography (SPME-GC) using the following method. Four fabric pieces (5 mm in diameter) washed in the solutions specified in Table 3 containing 1 mg/L lipase were transferred to gas chromatography (GC) bottles. Samples were analyzed on a Varian 3800GC equipped with a Stabilwax-DA w/Integra-Guard column (30m, 0.32mm id and 0.25micro-m df) and a Carboxen PDMS SPME fiber (75micro-m). Each sample was pre-incubated at 40°C for 10 minutes and then sampled with SPME fibers in the headspace on the fabric piece for 20 minutes. The sample was then injected into the chromatographic column (injection port temperature = 250°C). Column flow = 2 mL Helium/min. Temperature gradient of the column oven: 0 minutes = 40°C, 2 minutes = 40°C, 22 minutes = 240°C, 32 minutes = 240°C. Butyric acid was detected with an FID detector, and the amount of butyric acid was calculated based on the butyric acid standard curve. the

The risk performance odor of a lipase variant, R, is the ratio between the amount of butyrate released from a lipase variant washed sample and the amount of butyrate released from a lipase washed sample with Sequence ID: 2, Sequence ID: 2 The lipase in has a substitution of T231R+N233R (reference enzyme), both values are corrected using the amount of butyric acid released from the lipase-free washed samples. The risk (R) of a variant is calculated according to the following formula:

Odor = micrograms of butyric acid measured by 1mg of enzyme protein/l blank correction

Alpha _{Test Enzyme} = Odor _{Test Enzyme} - Blank

Alpha _{Reference Enzyme} = Odor _{Reference Enzyme} - Blank

R = alpha _{test enzyme} /alpha _{reference enzyme}

A variant was considered to exhibit reduced odor compared to the reference if its R-factor was less than 1. the

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

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

Lipase activity was determined using the method described in the lipase activity section above. The absorbance (A280) of lipase at 280 nm was measured and the LU/A280 ratio was calculated. Relative LU/A280 was calculated by dividing the LU/A280 of the variant by the LU/A280 of the reference enzyme. In the context of the present invention, the reference enzyme is lipase of SEQ ID NO: 2, which has the substitution T231R+N233R. the

Example 5: BR-Benefit Risk

The effectiveness risk factor which will describe the performance to odor reduction risk ratio is thus defined as: BR = RP _avg /R

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

The results obtained by applying the above method are as follows:

Variants In serial identification number: 2 Mean RP (RP _avg ) BR LU/A280 1 I202G+T231R+N233R 0.84 1.41 undecided 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 references 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 undecided 850 8 G91A+E99K+T231R, N233R+ Q249R+270H+271T+272P+273S+ 274S+275G+276R+277G+278G 0.13 undecided 500 the the the the the

Table 4

WO 2000/060063 describes reference lipases and variants 7 and 8 in Table 4. the

Example 6

BR-Benefit Risk

The efficacy risk of the variants listed in Table 5 was measured. Efficacy coefficients were measured in the same way as described in Example 5 and all listed variants had an efficacy risk coefficient greater than 1. the

Variants Mutation in SEQ ID NO: 2 references T231R+N233R 9 L97V+T231R+N233R 10 A150G+T231R+N233R 11 I90R+T231R+N233R 12 I202V+T231R+N233R 13 L227G+T231R+N233R+P256K 14 I90A+T231R+N233R 15 T231R+N233R+I255P 16 I90V+I255V+T231R+N233R 17 F211L+L227G+T231R+N233R+I255L+P256K 18 S58N+V60S+T231R+N233R+Q249L 19 S58N+V60S+T231R+N233R+Q249I

20 A150G+L227G+T231R+N233R+P256K twenty one K46L+S58N+V60S+T231R+N233R+Q249L+D254I twenty two Q4L+E43T+K46I+S58N+V60S+T231R+N233R+ Q249L+D254I twenty three Q4L+S58N+V60S+T231R+N233R+Q249L+D254I twenty four K46I+S58N+V60S+T231R+N233R+Q249L+D254L 25 K46L+S58N+V60S+K223I+T231R+N233R+D254I 26 E43T+K46I+S58N+V60S+T231R+N233R+Q249L+ D254I 27 S58N+V60S+I86V+A150G+L227G+T231R+N233R+ P256K 28 K24R+K46R+K74R+I86V+K98R+K127R+D137K+ A150G+K223R+T231R+N233R 29 S58A+V60A+I86V+T231R+N233R 30 K24R+K46R+S58N+V60S+K74R+I86V+K98R+ K127R+D137K+K223R+T231R+N233R 31 S58A+V60A+I86V+A150G+T231R+N233R 32 S58N+V60V+D62G+T231R+N233R 33 Q4V+S58N+V60S+I86V+T231R+N233R+Q249L 34 Q4V+S58N+V60S+I86V+A150G+T231R+N233R+ I255V 35 Q4V+S58N+V60S+I90A+A150G+T231R+N233R+ I255V 36 Y53A+S58N+V60S+T231R+N233R+P256L 37 I202L+T231R+N233R+I255A 38 S58A+V60S+I86V+A150G+L227G+T231R+N233R+ P256K 39 D27R+S58N+V60S+I86V+A150G+L227G+T231R+ N233R+P256K 40 V60K+I86V+A150G+L227G+T231R+N233R+P256K 41 Q4V+S58A+V60S+S83T+I86V+A150G+E210K+

the L227G+T231R+N233R+P256K 42 Q4V+V60K+S83T+I86V+A150G+L227G+T231R+ N233R+P256K 43 D27R+V60K+I86V+A150G+L227G+T231R+N233R+ P256K 44 Q4N+L6S+S58N+V60S+I86V+A150G+L227G+ T231R+N233R+P256K 45 E1N+V60K+I86V+A150G+L227G+T231R+N233R+ P256K 46 V60K+I86V+A150G+K223N+G225S+T231R+N233R+ P256K 47 E210V+T231R+N233R+Q249R 48 S58N+V60S+E210V+T231R+N233R+Q249R 49 Q4V+V60K+I90R+T231R+N233R+I255V 50 Q4V+V60K+A150G+T231R+N233R 51 V60K+S83T+T231R+N233R 52 V60K+A150G+T231R+N233R+I255V 53 T231R+N233G+D234G 54 S58N+V60S+I86V+A150G+E210K+L227G+T231R+ N233R+Q249R+P256K 55 S58N+V60S+I86V+A150G+E210K+L227G+T231R+ N233R+I255A+P256K 56 S58N+V60S+I86V+A150G+G156R+E210K+L227G+ T231R+N233R+I255A+P256K 57 S58T+V60K+I86V+N94K+A150G+E210V+L227G+ T231R+N233R+P256K 58 S58T+V60K+I86V+D102A+A150G+L227G+T231R+ N233R+P256K 59 S58T+V60K+I86V+D102A+A150G+E210V+L227G+ T231R+N233R+P256K 60 S58T+V60K+S83T+I86V+N94K+A150G+E210V+

the L227G+T231R+N233R+P256K 61 S58A+V60S+I86V+T143S+A150G+L227G+T231R+ N233R+P256K 62 G91S+D96V+D254R 63 V60L+G91M+T231W+Q249L 64 T37A+D96A+T231R+N233R+Q249G 65 E56G+E87D+T231R+N233R+D254A 66 E210K+T231R+N233R 67 D27H+E87Q+D96N+T231R+N233R+D254V 68 F181L+E210V+T231R+N233R 69 D27N+D96G+T231R+N233R 70 D96N+T231R+N233R 71 T231R+N233I+D234G 72 S58K+V60L+E210V+Q249R 73 S58H+V60L+E210V+Q249R 74 Q4V+F55V+I86V+T231R+N233R+I255V 75 Q4V+S58T+V60K+T199L+N200A+E210K+T231R+ N233R+I255A+P256K 76 Q4V+D27N+V60K+T231R+N233R 77 I90F+I202P+T231R+N233R+I255L 78 S58N+V60S+D158N+T231R+N233R 79 S58N+V60S+S115K+T231R+N233R 80 S58N+V60S+L147M+A150G+F211L+T231R+N233R 81 V60K+A150G+T231R+N233R 82 I90V+L227G+T231R+N233R+P256K 83 T231R+N233R+I255S 84 I86G+T231R+N233R 85 V60K+I202V+E210K+T231R+N233R+I255A+P256K 86 I90G+I202L+T231R+N233R+I255S 87 S58G+V60G+T231R+N233R

table 5

Reference lipases are described in WO 2000/060063. the

composition example

Unless otherwise indicated, starting materials were obtained from Aldrich, P.O. Box 2060, Milwaukee, WI 53201, USA. the

Examples 1 to 6

The granular laundry detergent composition is designed for use in hand wash or top load washing machines. the

The concentration of any of the above compositions for laundering fabrics in water is from 600 ppm to 10,000 ppm at 25°C, typical intermediate conditions are 2500 ppm, and the ratio of water to fabric is 25:1. the

Examples 7 to 10

A granular laundry detergent composition designed for use in front-loading automatic washing machines. the

The concentration of any of the above compositions for laundering fabrics in water is 10,000 ppm at a temperature of 20°C to 90°C and the ratio of water to fabric is 5:1. Typical pH is about 10. the

Examples 11 to 16 Heavy Duty Liquid Laundry Detergent Compositions

Raw Materials and Instructions for Composition Examples 1 to 16

Linear alkylbenzene sulfonate with an average aliphatic carbon chain length of C ₁₁ -C ₁₂ supplied by Stepan, Northfield, Illinois, USA

C _12-14 Dimethylhydroxyethylammonium chloride supplied by Clariant GmbH, Sulzbach, Germany

AE3S is a _C12-15 alkyl ethoxy(3) sulfate supplied by Stepan, Northfield, Illinois, USA

AE7 is a C _12-15 alcohol ethoxylate with an average degree of ethoxylation of 7, supplied by Huntsman, Salt Lake City, Utah, USA

Sodium tripolyphosphate supplied by Rhodia, Paris, France

Zeolite A, supplied by Industrial Zeolite (UK) Ltd, Grays, Essex, UK

1.6R silicate supplied by Koma, Nestemica, Czech Republic

Sodium carbonate, supplied by Solvay, Houston, Texas, USA

Polyacrylate with molecular weight 4500 supplied by BASF, Ludwigshafen, Germany

Hydroxymethylcellulose is BDA, supplied by CPKelco, Arnhem, Netherlands

Supplied by Novozymes, Bagsvaerd, Denmark

Lipase variants 1 to 5, and combinations thereof are described in Example 5 of Table 4. the

AMS，荧光增白剂2是 

CBS-X，磺化酞菁锌和直接紫9是 

Violet BN-Z，它们都由Ciba Specialty Chemicals，Basel，Switzerland供应  Fluorescent whitening agent 1 is

AMS, optical brightener 2 is

CBS-X, zinc sulfonated phthalocyanine and direct violet 9 are

Violet BN-Z, both supplied by Ciba Specialty Chemicals, Basel, Switzerland

Diethylenetriaminepentaacetic acid supplied by Dow Chemical, Midland, Michigan, USA

Sodium percarbonate supplied by Solvay, Houston, Texas, USA

Sodium perborate supplied by Degussa, Hanau, Germany

NOBS is sodium nonanoyloxybenzenesulfonate supplied by Eastman, Batesville, Arkansas, USA

由Clariant GmbH， Sulzbach，Germany供应  TAED is tetraacetylethylenediamine, trade name

Supplied by Clariant GmbH, Sulzbach, Germany

Stain remover is Repel-o- PF, provided by Rhodia, Paris, France

Acrylic/maleic acid copolymer with a molecular weight of 70,000 and a ratio of acrylate to maleate of 70:30 was supplied by BASF, Ludwigshafen, Germany

The protease was FN3, supplied by Genencor International, Palo Alto, California, USA

1,2-Ethylenediamine-N,N′-disuccinic acid, sodium salt, (S,S) isomer (EDDS) supplied by Octel, Ellesmere Port, UK

Hydroxyethane diphosphonate (HEDP) supplied by Dow Chemical, Midland, Michigan, USA

Foam suppressor aggregate supplied by Dow Corning, Midland, Michigan, USA

HSAS is a mid-branched alkyl sulfate disclosed in US 6,020,303 and US 6,060,443

C _12-14 dimethylamine oxide supplied by Procter & Gamble Chemicals, Cincinnati, Ohio, USA

The nonionic material is preferably a C ₁₂ -C ₁₃ ethoxylate, preferably with an average degree of ethoxylation of 9.

Protease, supplied by Genencor International, Palo Alto, California, USA

^* Value in mg enzyme/100g

¹ As described in US 4,597,898.

购自BASF，以及如WO 01/05874中所述的那些  ² by product name

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

脂肪酶。  described in this manual

Lipase.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. the

Claims (9)

1. a composition, the variant that described composition comprises optical white and parent lipase, when comparing with described parent, described variant comprises at least three kinds of replacements of total, wherein said parent lipase and sequence identification number: 2 identical and described variants have one of following replacement group:

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+N233R+I255V。

2. composition as claimed in claim 1, wherein said lipase Variant is characterised in that described effect danger is greater than 1 when measuring with following method, wherein, describe performance and smell reduce risks than the dangerous coefficient of effect be: BR=RP _avg/ R, wherein, RP _avgrepresent average relative performance, R represents risk.

3. as right, will remove the composition as described in 1, wherein said composition comprises 0.1% to 40% anion surfactant.

4. composition as claimed in claim 3, wherein said composition is a kind of clean and/or treatment compositions.

5. composition as claimed in claim 1, wherein said composition comprises sulfonation phthalocyanine phthalocyanine zinc.

6. composition as claimed in claim 1, the mixture that wherein said composition comprises sulfonation phthalocyanine phthalocyanine zinc and aluminum phthalocyanine, the sulfonation phthalocyanine phthalocyanine zinc that described mixture has and the weight ratio of aluminum phthalocyanine are greater than 1.

7. composition as claimed in claim 1, wherein said composition comprises aluminum phthalocyanine.

8. composition as claimed in claim 1, wherein said optical white comprises xanthene dyestuff, anthraquinone or naphthoquinones.

9. a method for clean and/or treat surface or fabric, said method comprising the steps of: described surface or fabric are contacted with the composition of claim 1, then optionally wash and/or rinsing described in surface or fabric.

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