CN1303450A - Enhancers such as N-hydroxyacetanilide - Google Patents

Abstract

The invention provides a method for bleaching dyes in solution, which comprises contacting dyes with laccase and an enhancer with structure -NOH-CO-, especially N-hydroxyacetanilide enhancer, in aqueous solution.

Description

N-hydroxyacetanilide enhancer

technical field

The present invention relates to a very efficient method of bleaching dyes using laccases and enhancers. The invention also relates to a detergent composition.

technical background

It has long been found that colored substances rinsed from dyed fabrics can be bleached by a phenoloxidase enzyme. WO 91/05839 describes the use of peroxidases or oxidases to inhibit dye transfer in this way. Some oxidizable substances are also shown, such as metal ions as accelerators, enhancers or mediators, phenolic compounds such as 7-hydroxycoumarin, vanillin, and p-hydroxybenzenesulfonate (or ester), Can promote enzymatic bleaching reaction (see WO9105839).

Other types of enhancers are also disclosed, such as phenothiazines, phenoxazines, methylsyringate and acetosyringone (see WO94/12621, WO95/01426 and WO95/01426).

Yet another class of enhancers is the aliphatic, cycloaliphatic, heterocyclic and aromatic compounds containing NO, NOH or H-N(R)-OH disclosed in WO94/29425 and WO97/48786. In WO94/29425 and WO97/48786 benzotriazoles are preferred as dye bleaching enhancers.

It is an object of the present invention to provide a selected group of -NOH compounds which, in combination with laccases, are very effective in bleaching dyes in solution.

Summary of the invention

It has been surprisingly found that selected groups of -NOH compounds work well as enhancers for dye bleaching in solution with laccases.

其中A是

Accordingly, the present invention provides a method for bleaching a dye in solution, comprising contacting the dye with laccase and an enhancer having the following structure in an aqueous solution:

where A is

B is H, or C ₁ -C ₄ unbranched alkyl, wherein said alkyl may contain ether groups, R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, NH ₂ , COOH, CN, SO ₃ H, CH ₃ , COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ₉ , or NOH-CO-R ₁₀ , wherein R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are C ₁ -C ₂ Unbranched alkyl groups.

Detailed Description of the Invention Enhancer

其中A是

B是H，或C₁-C₄不分支烷基，其中所述的烷基可以含有醚基，R²、R³、R⁴、R⁵和R⁶是H、NH₂、COOH、SO₃H、CN、CH₃、COCH₃、NO₂、OCH₃、NR₇R₈、COOR₉，或NOH-COR₁₀，其中R⁷、R⁸、R⁹和R¹⁰是C₁-C₂不分支烷基；特别地R²、R³、R⁴、R⁵和R⁶是H、COOH、SO₃H、CH₃、COCH₃、OCH₃、NR₇R₈，或NOH-CO-R₉，其中R⁷、R⁸和R⁹是C₁-C₂不分支烷基；尤其R²、R³、R⁴、R⁵和R⁶中至少三个应是H。The present invention relates to a method of bleaching dyes in solution, comprising contacting the dyes with laccase and an enhancer having the structure in aqueous solution:

where A is

B is H, or C ₁ -C ₄ unbranched alkyl, wherein said alkyl can contain ether groups, R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, NH ₂ , COOH, SO ₃ H, CN, CH ₃ , COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ₉ , or NOH-COR ₁₀ , wherein R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are C ₁ -C ₂ unbranched Alkyl; in particular R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, COOH, SO ₃ H, CH ₃ , COCH ₃ , OCH ₃ , NR ₇ R ₈ , or NOH-CO-R ₉ , wherein R ⁷ , R ⁸ and R ⁹ are C ₁ -C ₂ unbranched alkyl groups; especially at least three of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ should be H.

In a preferred embodiment, B is H, or C ₁ -C ₂ unbranched alkyl, R ² , R ³ , R ⁴ , R5 and R ⁶ are H, NH ₂ , COOH, SO ₃ H, CN, CH _3. COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ⁹ , or NOH-CO-R _1O , wherein R ⁷ , R ⁸ , R ⁹ and R ^1O are C ₁ -C ₂ unbranched alkyl groups; Especially R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, COOH, SO ₃ H, CH ₃ , COCH ₃ , OCH ₃ , NR ₇ R ₈ , or NOH-CO-R ₉ , wherein R ⁷ , R ⁸ and R ⁹ are C ₁ -C ₂ unbranched alkyl groups; especially at least three of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ should be H.

In another preferred embodiment B is H, or CH ₃ , and R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, NH ₂ , COOH, SO ₃ H, CN, CH ₃ , COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ₉ , or NOH-CO-R ₁₀ , wherein R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are C ₁ -C ₂ unbranched alkyl groups; especially R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, COOH, SO ₃ H, CH ₃ , COCH ₃ , OCH ₃ , NR ₇ R ₈ , or NOH-CO-R ₉ , wherein R ⁷ , R ⁸ and R ⁹ is C ₁ -C ₂ unbranched alkyl; especially at least three of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ should be H.

In a particularly preferred embodiment, the enhancer is N-hydroxyacetanilide.

The concentration of the enhancer of the present invention may be 1-1000 μM, preferably 5-500 μM, more preferably 10-200 μM.

Preparation of enhancer

The enhancers described in this application can be prepared by methods known in the art; general schemes are shown in Organic Synthesis 67, 1989, p. 187-192. Some enhancers are also commercially available.

N-hydroxyacetanilide was prepared according to the method described in Organic Synthesis 67, 1989, p.187-192. enzyme

The general concentration of the laccase according to the invention is 1-10000 μg enzyme protein per liter of aqueous solution, especially 5-2000 μg enzyme protein per liter of aqueous solution, especially 5-1000 μg enzyme protein per liter of aqueous solution.

The atmosphere usually contains sufficient molecular oxygen for the requirement. Additional oxygen can be added if more _O2 is desired. Laccase and laccase-related enzymes

Preferably, laccases or laccase-related enzymes are used in combination with a selected group of -NOH compounds as described above for dye bleaching in solution.

In the present specification, laccases and laccase-related enzymes include any laccases included in the enzyme classification (EC 1.10.3.2), including any catechols included in the enzyme classification (EC 1.10.3.1) Oxidases, including any bilirubin oxidase included in the enzyme class (EC 1.3.3.5), or any monophenol monooxygenase included in the enzyme class (EC 1.14.18.1).

The above-mentioned enzymes may be derived from plants, bacteria or fungi (including filamentous bacteria and yeast), suitable examples include laccases, which may be derived from strains of the following genera: Aspergillus, Neurospora, such as Neurospora crassa Fungi; Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes , such as T.villosa and T.versicolor, Rhizoctonia (Rhizoctonia), such as Rhizoctonia solani; Coprinus, such as Cinereus, C.comatus, C. friesii, and C. plicatilis; Psathyrella, such as P. condelleana; Panaeolus, such as P. (P.papilionaceus); Myceliophthora, such as M.thermophila (M.thermophila); Schytalidium, such as S.thermophilum, Polyporus, such as P. pinsitus; Coriolus spp., such as Coriolus radiata (WO92/01046); or Coriolus spp., such as Coriolus rugosa (JP 2-238885).

Preference is given to laccases derived from the genus Coprinus, Myceliophthora, Polyporus, Mypoporia, Schytalidium or Rhizoctonia; especially preferred are those derived from Coprinus cinerea, Myceliophthora thermophila, Polyporus pinsitus, Vermilion Laccase from Poria, Schytalidium thermophilum or Rhizoctonia solani.

The laccase or enzymes related to laccase can also be prepared in this way: under the conditions that allow the expression of laccase, the host cells transformed with the recombinant DNA vector carrying the encoding The DNA sequence of the laccase and a plurality of DNA sequences capable of expressing the DNA sequence are obtained, and the laccase is recovered from the culture medium. Laccase Activity Assay (LACU)

Laccase activity was determined by oxidation of syringaldazine under aerobic conditions. The purple color produced was measured spectrophotometrically at 530 nm. The analysis conditions are 19 mM syringaldazine, 23 mM acetate buffer, pH 5.5, 30° C., and the reaction condition is 1 minute.

One laccase unit (LACU) is the amount of enzyme that catalyzes the conversion of 1.0 μmol of syringaldazine per minute under these conditions. Laccase activity assay (LAMU)

Laccase activity was determined by oxidation of syringaldazine under aerobic conditions. The purple color produced was measured spectrophotometrically at 530 nm. The analysis conditions were 19 mM syringaldazine, 23 mM Tris/maleate buffer, pH 7.5, 30° C., and the reaction conditions were 1 minute.

One laccase unit (LAMU) is the amount of enzyme that catalyzes the conversion of 1.0 μmol of syringaldazine per minute under these conditions. dye or colorant

In a preferred embodiment, the method of the present invention finds application in the bleaching of textile dyes or colorants, or in the bleaching of textile dyes or colorants in solution.

Colorants and dyes are a large class of natural and synthetic compounds. The following description of dyes/colorants does not limit the scope of the claimed invention.

Synthetic textile dyes are generally azo compounds (one or more azo groups or diazene groups), such as acid red 151, direct blue 1, direct brown 44, and orange II, or anthraquinone compounds, such as acid blue 45.

Reactive blue 19 can be found in other structure types.

Some dyes also carry groups capable of coupling to the fabric surface (reactive dyes), some complex with metal ions. These changes do not affect the utility of the invention.

A different structure is the indigo moiety, such as the soluble indigo carmine dye.

Other dyes and colorants may be natural, or synthetically identical or similar in structure to natural ones. The types of colored substances that can be extracted from vegetables are polyphenols, anthocyanins and carotenoid compounds. industrial application

A particular embodiment of the invention is the home and public laundry process.

During this washing and rinsing process, dyes and colorants on fabrics can be leached into the wash or rinse liquid, causing the wash to bleach. The present invention provides methods for bleaching colored compounds in solution to reduce this undesirable effect.

The invention can also be used for the bleaching of stains on fabrics: these stains are usually caused by red wine, fruits such as black currant, cherries, strawberries and tomatoes (especially ketchup and pasta sauce ( spaghetti sauce)), vegetables such as carrots, beetroot, etc., tea, coffee, spices such as curry and paprika, grass, or ballpoint pen/ink.

In another specific embodiment, dyes rinsed into treatment water during textile processing may be bleached by the method of the invention to prevent poor adhesion.

In another particular embodiment, it is found that the method of the present invention is used in the treatment of waste water discharged from chemical or pharmaceutical factories, dye manufacturing, printing and dyeing factories, or textile factories, the method comprising the presence of the enhancer of the present invention Treatment of wastewater with laccase under certain conditions.

In the methods described above and in other uses of the invention, the enhancer may be added in one or more additions at the start of the process or after the start of the process. detergent composition

The enhancer of the present invention can be incorporated into detergent compositions as an ingredient therein.

The detergent composition of the present invention may be formulated as a hand or machine laundry detergent composition comprising a laundry additive composition suitable for pretreatment of soiled fabrics and a rinse added fabric softener composition, or formulated as Detergent compositions for general household hard surface cleaning treatments, or compositions formulated for hand or machine dishwashing.

In a particular aspect, the invention provides a detergent additive comprising a booster according to the invention and a laccase. Detergent additives and detergent compositions may contain one or more other enzymes such as proteases, lipases, cutinases, amylases, carbolytic enzymes, cellulases, pectinases, mannanases, arabinases Enzymes (arabinase), galactanase, xylanase, oxidase, such as laccase and/or peroxidase.

Generally the properties of the selected enzyme should be compatible with the selected detergent (ie pH optimum, compatibility with other enzyme and non-enzyme ingredients) and the enzyme should be present in an effective amount.

Protease: Suitable proteases include those derived from animals, plants or microorganisms. Those derived from microorganisms are preferred. Chemically modified or protein engineered mutants are also included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from the genus Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279 ). Examples of trypsin-like proteases are trypsin (eg of porcine or bovine origin) and the Fusarium proteases described in WO89/06270 and WO94/25583.

Examples of useful proteases are the variants described in WO92/19729, WO98/20115, WO98/20116 and WO98/34946, especially variants with substitutions at one or more of the following positions: 27, 36, 57, 76 , 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available proteases include Alcalase ^™ , Savinase ^™ , Primase ^™ , Duralase ^™ , Esperase ^™ and Kannase ^™ (Novo Nordisk A/S), Maxatase ^™ , Maxacal ^™ , Maxapem ^™ , Properase ^™ , Purafect ^™ , Purafect OxP ^™ , FN2 ^™ and FN3 ^™ (Genencor International Inc.).

Lipase: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include Humicola (synonym Thermomyces), those derived from H. lanuginosa (T. lanuginosus) as described in EP258068 and EP305216, or those derived from H. insolens as described in WO96/13580, pseudo Lipase of the genus Monas, such as Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP218 272), Pseudomonas allium (EP 331 376), Pseudomonas stutzeri (GB 1,372,034), Pseudomonas fluorescens, a Pseudomonas strain SD705 (WO95/06720 and WO96/27002), P.wisconsinensis (WO96/12012), Bacillus lipase, such as from Bacillus subtilis (Dartois (1993) Acta Biochemistry and Biophysics, 1311, 253-360), Bacillus stearothermophilus (JP 64/744992), or Bacillus pumilus (WO91/16422).

Examples of other lipase variants are WO92/05249, WO94/01541, EP 407 225, EP260 105, WO95/35381, WO96/Q0292, WO95/30744, WO94-/25578, WO95/14783, WO95/22615, WO97/ Lipases described in 04079 and WO97/07202.

Preferred commercially available lipases include Lipolase ^™ , Lipolase Ultra ^™ and LipoPrime ^™ (Novo Nordisk A/S).

Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are also included. Amylases include, for example, alpha-amylases obtained from Bacillus, such as the alpha-amylase from a particular strain of Bacillus licheniformis as detailed in GB 1,296,839.

有用的淀粉酶的例子是WO94/02597、WO94/18314、WO96/23873和WO97/43424记载的各种，尤其是在以下一或多个位点有取代的变体：15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408和444。

Commercially available amylases are Duramyl ^™ , Termamyl ^™ , Fungamyl ^™ and BAN ^™ (NovoNordisk A/S), Rapidase ^™ and Purastar ^™ (Genencor International Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are also included. Suitable cellulases include cellulases derived from the following genera: Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, such as US 4,435,307, US5,648,263, US5,691,178, US5,776,757 and WO89/09259 disclose fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum.

Suitable cellulases are in particular alkaline or neutral cellulases with a color care effect. Examples of these cellulases are the cellulases described in EP0495257, EP0531372, WO96/11262, WO96/29397, WO98/08940. Other examples are cellulase variants as described in WO94/07998, EP0531315, US5,457,046, US5,686,593, US5,763,254, WO95/24471, WO98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme ^™ and Carezyme ^™ (Novo Nordisk A/S), Clazinase ^™ , and Puradax HA ^™ (Genencor International Inc.), and KAC-500(B) ^™ (Kao Corporation).

Peroxidases/oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are also included. Useful peroxidases include those derived from Coprinus, such as described in WO93/24618, WO95/10602 and WO98/15257, peroxidases from Coprinus cinereus and variants thereof.

Commercially available peroxidases include Guardzyme ^™ (Novo Nordisk A/S).

Detergent enzymes can be added to a detergent composition as an additive containing one or more enzymes separately, or by adding a composite additive containing all of these enzymes. The detergent additives of the present invention, that is, individual additives or composite additives, can be formulated as granules, liquids, slurries and the like. Preferred detergent additive formulations are granules, especially non-dusting granules; liquids, especially stabilized liquids, or slurries.

Dust-free granules can be prepared as disclosed in US 4,106,991 and 4,661,452 and optionally coated as known in the art. Examples of waxy coating materials are polyoxyethylene products (polyethylene glycol, PEG) with an average molecular weight of 1,000-20,000; ethoxylated nonylphenols with 16-50 ethylene oxide units; ethoxylated Fatty alcohols, wherein the alcohol contains 12-20 carbon atoms and has 15-80 ethylene oxide units; fatty alcohols; fatty acids; mono-, di- and triglycerides of fatty acids. GBl483591 provides film-forming coating materials suitable for fluidized bed technology. Liquid enzyme preparations can be stabilized by the addition of polyols such as propylene glycol, sugar or sugar alcohols, lactic acid or boric acid according to established methods. Protected enzymes can be prepared as disclosed in EP238,216.

The detergent composition of the present invention can be in any simple dosage form, such as bar, tablet, powder, granule, ointment or liquid. Liquid detergents can be aqueous, typically containing up to 70% water and 0-30% organic solvents, or non-aqueous.

Detergent compositions contain one or more surfactants, which may be nonionic, including semi-polar, and/or anionic and/or cationic and/or zwitterionic. Surfactants are generally used in amounts of 0.1% to 60% by weight.

If the detergent contains anionic surfactants, the amount is usually about 1% to about 40%, which can be linear alkylbenzene sulfonate, α-olefin sulfonate, alkyl sulfate (fatty alcohol sulfate) , fatty alcohol ethoxysulfates, secondary alkanesulfonates, alpha-sulfo fatty acid methyl esters, alkyl- or alkenyl succinic acids or soaps.

Detergents, if they contain nonionic surfactants, usually in amounts of about 0.2% to about 40%, can be fatty alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglucosides, alkyl dimethyl Amine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").

Detergents can contain 0-65% detergent builders or complexing agents such as zeolites, diphosphates, triphosphates, phosphonates, carbonates, citrates, nitrilotriacetic acid, ethylenediaminetetraacetic acid , diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acids, soluble silicates or layered silicates (eg Hoechst's SKS-6).

Detergents may contain one or more polymers. Such as carboxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, polyvinylpyridine-N-oxide, polyvinylimidazole, polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymer.

Detergents may contain a bleaching system which may contain a source of hydrogen peroxide, such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzene Sulfonate compound. Alternatively the bleaching system may contain peroxyacids such as amides, imides, or sulfones.

The enzymes of the detergent compositions of the present invention may be stabilized with conventional stabilizers, such as polyols, such as propylene glycol or glycerol, sugar or sugar alcohols, lactic acid, boric acid, or boric acid derivatives, such as aryl borates, or Phenylboronic acid (phenylboronic acid) derivatives, such as 4-formylphenylboronic acid, can also be prepared according to the method described in WO92/19709 and WO92/19708.

The detergent may also contain other conventional detergent ingredients such as fabric conditioners, which include clays, suds boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners agents, solubilizers, tarnish inhibitors or fragrances.

It is now contemplated that in detergent compositions, any enzyme may be added in an amount equivalent to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, more preferably 0.05-5 mg of enzyme protein per liter of wash liquor 0.1-1 mg enzyme protein.

The boosters and laccases of the present invention may additionally be added to the detergent compositions disclosed in WO 97/07202, which is incorporated herein by reference.

The following examples further illustrate the invention and they do not limit the scope of the claims of the invention. Example 1 Methyl orange bleaching materials and methods with various laccases and various NOH-type enhancers

The recombinant Coprinus cinereus laccase (rCcL) was prepared as described in WO97/08325, and dialyzed in borate at pH 9, 57.5 LAMU/ml.

The recombinant Myceliophthora thermophila laccase (rMtL) was prepared according to WO95/33836, 1419 LAMU/ml.

According to the record of WO96/00290, prepare recombinant Polyporus pinsitus laccase, 1330LAMU/ml. buffer

The composition of the Britton-Robinson buffer is as follows:

0.1M Phosphate

0.1M borate

0.1M acetate dye

Methyl Orange (Merck, Germany) Enhancer N-Hydroxyacetanilide 1-Hydroxybenzotriazole 2-Hydroxypyridine-N-oxide Indol-1-yl] ester (PFC-202) cyanuric acid 3-hydroxy-1,2,3-benzotriazin-4-(3H)-one N-hydroxyphthalimide 2-B Base-5-phenylisoxazolium-4'-sulfonate, H20p,p'-oxydiphenetole (p,p'-Azoxydiphenetole) quinoline-N-oxide isoquinoline-N- Determination of the bleaching action of di-2-pyridylketone oxime

Final concentration 50μl methyl orange, AbsU _464nm ~7 1.4AbsU125μl 0.1M Britton-Robinson buffer 50mM 25μl 2mM mediator (or water) 0.2mM 50μl laccase dilution

Enzymes are added to initiate the reaction. Absorbance at 464 nm was measured on a Cobas Fara at 30°C for 3 minutes (reading every 5 seconds). Initial bleaching was determined from the first linear part of the response curve. Results and discussion

The effect of the N-hydroxy compounds described above as laccase mediators on the bleaching of methyl orange with three recombinant laccases (rPpL, rMtL and rCcL) at various pH values was determined. All kinetic data are summarized in Tables 1-3. The larger the negative number, the better the effect.

It can be seen from the table that compared with other N-OH type enhancers, bleaching with O=CN-OH type enhancers (see N-hydroxyacetanilide) has an exceptionally good effect. pH4 pH5 pH6 pH7 pH8 No enhancer (control) -0.004 -0.006 -0.011 -0.012 -0.005 N-Hydroxyacetanilide -0.521 -0.719 -0.674 -0.498 -0.136 1-Hydroxybenzotriazole -0.016 -0.018 -0.019 -0.017 -0.005 2-Hydroxypyridine-N-oxide -0.008 -0.013 -0.025 -0.020 -0.007 N-Benzoyl-N-Benzene -0.163 -0.247 -0.529 -0.592 -0.124 PFC-202 -0.005 -0.010 -0.016 -0.000 0.011 Cyanuric acid -0.005 -0.008 -0.012 -0.012 -0.005 3-Hydroxy-1,2,3-benzotriazin-4-(3H)-one 0.004 -0.004 -0.011 -0.008 -0.002 N-Hydroxyphthalimide -0.006 -0.007 -0.014 -0.011 -0.004 2-Ethyl-5-phenylisoxazolium-4'-sulfonate, H20 0.005 0.000 -0.006 -0.004 -0.001 p,p'-Azodiphenethyl ether 0.116 0.196 0.002 0.008 0.019 Quinoline-N-oxide -0.004 -0.006 -0.010 -0.008 -0.004 Isoquinoline-N-oxide -0.002 -0.005 -0.008 -0.010 -0.004 Bis-2-pyridylketoxime -0.004 -0.006 -0.008 -0.006 -0.001 Table 1. The initial change of methyl orange at A464nm per minute when using rCcL (the dilution of rCcL solution is 1:200) pH4 pH5 pH6 pH7 No enhancer (control) -0.018 -0.011 -0.002 -0.002 N-Hydroxyacetanilide -1.306 -1.847 -0.630 -0.101 1-Hydroxybenzotriazole -0.050 -0.019 -0.007 -0.002 2-Hydroxypyridine-N-oxide -0.020 -0.016 -0.010 -0.005 N-Benzoyl-N-Benzene -0.269 -0.296 -0.082 -0.012 PFC-202 -0.020 -0.017 0.001 0.005 Cyanuric acid -0.013 -0.010 -0.002 0.000 3-Hydroxy-1,2,3-benzotriazin-4-(3H)-one -0.016 -0.011 -0.001 0.002 N-Hydroxyphthalimide -0.019 -0.016 -0.005 0.000 2-Ethyl-5-phenylisoxazolium-4'-sulfonate, H2O -0.012 -0.004 -0.002 0.000 p,p'-Azodiphenethyl ether 0.019 0.086 0.012 0.020 Quinoline-N-oxide -0.016 -0.011 -0.002 0.000 Isoquinoline-N-oxide -0.017 -0.010 -0.004 0.000 Bis-2-pyridylketoxime -0.026 -0.010 -0.001 0.001 Table 2. The initial change of methyl orange at A464nm per minute when using rMtL (dilution of rMtL solution is 1:300) pH4 pH5 pH6 pH7 No enhancer (control) -0.073 -0.038 -0.017 -0.002 N-Hydroxyacetanilide -4.743 -6.528 -1.837 -0.055 1-Hydroxybenzotriazole -0.206 -0.077 -0.017 -0.005 2-Hydroxypyridine-N-oxide -0.084 -0.052 -0.024 -0.006 N-Benzoyl-N-Benzene -0.732 -1.402 -0.420 -0.012 PFC-202 -0.047 -0.035 -0.001 0.000 Cyanuric acid -0.037 -0.022 -0.011 -0.002 3-Hydroxy-1,2,3-benzotriazin-4-(3H)-one -0.020 -0.020 -0.008 0.000 N-Hydroxyphthalimide -0.050 -0.035 -0.011 -0.001 2-Ethyl-5-phenylisoxazolium-4-sulfonate, H2O -0.017 -0.017 -0.006 0.001 p,p'-Azodiphenethyl ether 0.121 0.013 0.017 0.018 Quinoline-N-oxide -0.074 -0.041 -0.016 -0.002 Isoquinoline-N-oxide -0.074 -0.037 -0.014 -0.002 Bis-2-pyridylketoxime -0.124 -0.107 -0.053 -0.005 table 3. Initial change of methyl orange at A464nm per minute with rPpL (1:200 dilution of rPpL solution) Example 2 Methyl orange bleaching with laccase and new -NOH-CO-type media Materials and methods Enzymes such as Polyporus pinsitus laccase dye as described in Example 1

Determination of the bleaching effect of methyl orange (Merck, Germany) enhancer N-hydroxyacetanilide N-(4-cyanophenyl)-N-hydroxyacetamide (CAS 80584-65-2) 50μl 0.1mg/ml formazan Base orange 125 μl 0.1M Britton-Robinson buffer (see example 1) 25 μl 2mM mediator or water (as a control) 50 μl laccase dilution (1:400)

Methyl orange, buffer and medium are mixed in a microtiter plate and the enzyme is added to initiate the reaction. Absorption at 450 nm was measured after 15 minutes. Results and discussion

The effects of N-hydroxyacetanilide and N-(4-cyanophenyl)-N-hydroxyacetamide as mediators on the bleaching of methyl orange with laccase at pH 4-6 were determined. All kinetic data are summarized in Table 4. Low values represent good results.

The data show that N-(4-cyanophenyl)-N-hydroxyacetamide is as effective in bleaching dyes as N-hydroxyacetanilide in solution. pH4 pH5 pH6 No enhancer (control) 0.920 1.104 1.183 N-Hydroxyacetanilide 0.077 0.068 0.087 N-(4-cyanophenyl)-N-hydroxyacetamide 0.065 0.055 0.083 Table 4. Determination of absorption at 450 nm after 15 minutes Example 3 Bleaching of various dyes with laccase and N-hydroxyacetanilide Materials and methods The enzyme is Polyporus pinsitus laccase as described in Example 1, except that the concentration is 1726 LACU/ml . Dye Acid Blue 113 (Aldrich) CSB (Aldrich) Acid Blue 45 (Aldrich) Cibachron Marine (Ciba Specialty Chemicals) Enhancer N-hydroxyacetanilide Determination of bleaching effect 50 μl dye solution 125 μl 0.1M Britton-Robinson buffer, pH5 (see Example 1) 25 μl 2mM mediator or water (as a control) 50 μl laccase dilution (1:400) or water (as a control)

The dye, buffer and mediator are mixed in a microtiter plate and the reaction is initiated by adding laccase. The absorbance at 595 nm was measured after 5 minutes. Results and discussion

N-hydroxyacetanilide as a mediator in the bleaching of various dyes with laccase. All kinetic data are summarized in Table 5. Low values represent good results.

The data show that the mediator promotes bleaching of various dyes. dye -enzyme-mediator +enzyme-mediator +enzyme+mediator Acid Blue 113 0.474 0.265 0.062 CSB 0.948 0.459 0.207 Acid Blue 45 0.941 0.490 0.123 Cibachron Marine 0.981 0.438 0.235 Table 5. Absorbance values measured at 595 nm after 5 minutes.

Claims (13)

1. A method for bleaching dyes in solution, characterized in that it comprises contacting the dye with laccase and an enhancer having the following structure in an aqueous solution: where A is

B is H, or a C ₁ -C ₄ unbranched alkyl group, wherein said alkyl group may contain an ether group, R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, NH ₂ , COOH, SO ₃ H, CN, CH ₃ , COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ₉ , or NOH-CO-R ₁₀ , wherein R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are C ₁ -C ₂ Unbranched alkyl groups. 2. The method according to claim 1, characterized in that the enhancing agent is N-hydroxyacetanilide. 3. The method according to claim 1, characterized in that the laccase is a microbial laccase. 4. The method according to claim 3, characterized in that the laccase is derived from the genus Coprinus, Myceliophthora, Polyporus, Mypoporia, Scytalidium or Rhizoctonia. 5. The method according to claim 4, wherein the laccase is derived from Coprinus cinerea, Myceliophthora thermophila, Polyporus pinsitus, Mycoporus vermilion, Scytalidiumthermophilum, or Rhizoctonia solani. 6. 5. A method according to any one of claims 1-5, characterized in that it is a method of inhibiting the transfer of textile dyes from one dyed fabric to another when the fabrics are co-washed in the same wash liquor. 7. A method according to claim 6, characterized in that an enhancing agent is added thereto at the beginning or during the treatment. 8. The method according to claim 1, characterized in that the concentration of the enhancer is in the range of 1-1000 μM. 9. A detergent composition characterized by containing laccase, a surfactant and a booster having the formula: where A is B is H, or a C ₁ -C ₄ unbranched alkyl group, wherein said alkyl group may contain an ether group, R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are H, NH ₂ , COOH, SO ₃ H, CN, CH ₃ , COCH ₃ , NO ₂ , OCH ₃ , NR ₇ R ₈ , COOR ₉ , or NOH-CO-R ₁₀ , wherein R ⁷ , R ⁸ , R ⁹ and R10 are C ₁ -C ₂ Unbranched alkyl groups. 10. A detergent composition according to claim 9, wherein the enhancer is N-hydroxyacetanilide. 11. The detergent composition according to claim 9, wherein the laccase is derived from Coprinus, Myceliophthora, Polyporus, Mypoporia, Scytalidium or Rhizoctonia. 12. The detergent composition according to claim 11, wherein the laccase is derived from Coprinus cinerea, Myceliophthora thermophila, Polyporus pinsitus, Mycoporus vermilion, Scytalidiumthermophilum, or Rhizoctonia solani. 13. The detergent composition according to any one of claims 9-12, characterized in that it also contains one or more other enzymes, especially protease, lipase, amylase, cellulase, and/or cutin enzyme.