FIELD OF INVENTION

The invention relates to a method of oxidizing a compound with a phenol oxidizing enzyme and an enhancing agent. The invention also relates to a detergent additive and to a detergent composition.

BACKGROUND ART

By a phenol oxidizing enzyme is meant an enzyme which by using hydrogen peroxide or molecular oxygen, is capable of oxidizing organic compounds containing phenolic groups. Examples of such enzymes are peroxidases and oxidases.

It has earlier been found that coloured substances leached from dyed fabrics could be bleached by means of a phenol oxidizing enzyme. The use of peroxidases or oxidases for inhibiting dye transfer in this way is described in WO

91/05839.

Certain oxidizable substances, e.g. , metal ions and phenolic compounds such as 7-hydroxycoumarin, vanillin, and p-hydroxybenzenesulfonate, have been described as accelerators or enhancing agents able to enhance enzymatic bleaching reactions (cf. e.g. WO 92/18683, WO 92/18687, and

Kato M and Shimizu S. Plant Cell Physiol. 1985 26 (7), pp.

1291-1301 (cf. Table 1 in particular)). In WO 94/12621 other types of enhancing agents are disclosed, e.g., phenothiazines and phenoxazines.

It is the object of this invention to provide a new group of enhancing agents which are effective for enhancing phenol oxidizing enzymes.

SUMMARY OF THE INVENTION

It has now surprisingly been found that a new group of organic chemical substances performs excellently as enhancers of phenol oxidizing enzymes. This new group of organic chemical substances not only make the bleaching reactions faster compared with using the phenol oxidizing enzyme alone, but many compounds which could not be bleached at all, may now be bleached by using the method of the invention. Accordingly, the invention provides a method of oxidizing a compound with a phenol oxidizing enzyme, characterized by the presence of an enhancing agent of the following formula:

in which formula A is a group such as -D, -CH=CH-D, -CH=CH- CH=CH-D, -CH=N-D, -N=N-D, or -N=CH-D, in which D is selected from the group consisting of -CO-E, -S0 ₂ -E, -N-XY, and -N ^* -XYZ, in which E may be -H, -OH, -R, or -OR, and X and Y and Z may be identical or different and selected from -H and -R; R being a C,-C ₁₆ alkyl, preferably a C^C _g alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C may be the same or different and selected

BRIEF DESCRIPTION OF THE DRAWING

The present invention is further illustrated by reference to Fig. 1 which shows the bleaching of gradually added Acid Blue 45 in phosphate/borate buffer pH 10 at 35^;

(I) : Only dye addition; (II) : Dye addition in the presence of Laccase; (III) : Dye addition in the presence of Laccase + Acetosyringone; the experiment conducted as described in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of oxidizing a compound with a phenol oxidizing enzyme, characterized by the presence of an enhancing agent of the following formula:

in which formula A is a group such as -D, -CH=CH-D, -CH=CH- CH=CH-D, -CH=N-D, -N=N-D, or -N=CH-D, in which D is selected from the group consisting of -CO-E, -S0 ₂ -E, -N-XY, and -N*-XYZ, in which E may be -H, -OH, -R, or -OR, and X and Y and Z may be identical or different and selected from -H and -R; R being a C^-C^ alkyl, preferably a ^-C ₈ alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C may be the same or different and selected ^from <V 1 ≤ m < 5.

In a preferred embodiment A in the above mentioned formula is -CO-E, in which E may be -H, -OH, -R, or -OR; R being a 0,-C^ alkyl, preferably a 0,-C ₈ alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C may be the same or different and selected from

^C m ^H 2m+1 1 < m < 5.

In the above mentioned formula A may be placed me to the hydroxy group instead of being placed in the paraposition as shown.

In particular embodiments, the enhancing agent is acetosyringone, syringaldehyde, methylsyringate, syringic acid, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate, octylsyringate or ethyl 3-(4-hydroxy-3,5- dimethoxyphenyl)acrylate.

The enhancing agent of the invention may be prese in concentrations of from 0.01 to 1000 μM, more preferred 0. to 250 μM, most preferred 1 to 100 μM.

Preparation of Enhancing Agents

The enhancing agents described in the present application may be prepared using methods well known to tho skilled in the art; some of the enhancing agents are also commercially available.

We produced methylsyringate, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate and octylsyringate by using the method disclosed in Che . Ber. 67, 1934, p. 67.

Ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate w synthesised from syringaldehyde and triethyl phosphonoaceta in ethanol/sodium ethanolate. The product was after purification characterised by ¹ H-NMR and ¹³ C-NMR (showing spectra as expected) and the melting point was 68-70 ^β C.

Hydrogen peroxide/Oxygen

If the phenol oxidizing enzyme requires a source o hydrogen peroxide, the source may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide, e.g., percarbonate or perborate, or a hydrogen peroxide generating enzyme system, e.g., an oxidase and a substrate for the oxidase, e.g., an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof. Hydrogen peroxide may be added at the beginnin

or during the process, e.g., in an amount corresponding to levels of from 0.001-25 mM, particularly to levels of from 0.01-1 mM.

If the phenol oxidizing enzyme requires molecular oxygen, molecular oxygen from the atmosphere will usually be present in sufficient quantity. If more 0 ₂ is needed, additional oxygen may be added.

Phenol Oxidizing Enzyme

In the context of the present invention the enzyme of the phenol oxidizing enzyme may be an enzyme possessing peroxidase activity or a laccase or a laccase related enzyme as described below.

Peroxidases and Compounds possessing Peroxidase Activity

Compounds possessing peroxidase activity may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7), or any fragment derived therefrom, exhibiting peroxidase activity, or synthetic or semisynthetic derivatives thereof (e.g. porphyrin ring systems or microperoxidases, cf. e.g. US Patent 4,077,768, EP Patent Application 537,381, International Patent Applications WO 91/05858 and WO 92/16634).

Preferably, the peroxidase employed in the method of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g. Fusarium. Humicola. Tricoderma. Myrothecium. Verticilium. Arthromvces. Caldariomvces. Ulocladium. Embellisia. Cladosporium or Dreschlera. in particular Fusarium oxysporu (DSM 2672) , Humicola insolens. Trichoderma resii. Myrothecium verrucaria (IFO 6113) , Verticillu alboatrum. Verticillum dahlie. Arthromyces ra osus (FERM P-7754) , Caldariomvces fumago. Ulocladium chartarum. Embellisia allior Dreschlera halodes.

Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, e.g. Coprinus. Phanerochaete. Coriolus or Trametes. in particular Coprinus cinereus f. microsporus (IFO

58371) , Coprinus macrorhizus. Phanerochaete chrvsosporium (e.g. NA-12) or Trametes (previously called Polvporus) . e.g. T. versicolor (e.g. PR4 28-A) .

Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, o e.g. Rhizopus or Mucor, in particular Mucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomvces spheroides (ATTC 23965) , Streptomvces thermoviolaceus (IFO 12382) or Streptoverticillu verticillium ssp. verticillium. s Other preferred bacteria include Bacillus pumilus (ATCC 12905) , Bacillus stearothermophilus. Rhodobacter sphaeroides. Rhodomonas palustri. Streptococcus lactis. Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-ll) . 0 Further preferred bacteria include strains belonging to Myxococcus. e.g. M. virescens.

The peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA 5 sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture. 0 Particularly, a recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp. , in particular C. macrorhizus or C. cinereus according to WO 92/16634.

In the context of this invention, compounds 5 possessing peroxidase activity comprise peroxidase enzymes and peroxidase active fragments derived from cytochromes, haemoglobin or peroxidase enzymes, and synthetic or

semisynthetic derivatives thereof, e.g. , iron porphyrins, and iron phthalocyanines and derivatives thereof.

Determination of Peroxidase Activity fPODU)

1 peroxidase unit (PODU) is the amount of enzyme 5 that catalyzes the conversion of 1 μmole hydrogen peroxide per minute at the following analytical conditions: 0.88 mM hydrogen peroxide, 1.67 mM 2,2'-azinobis(3-ethylbenzothia- zoline-6-sulfonate) , 0.1 M phosphate buffer, pH 7.0, incubated at 30°C, photometrically followed at 418 nm.

ιo Laccase and Laccase Related Enzymes

In the context of this invention, laccases and laccase related enzymes comprise any laccase enzyme comprised by the enzyme classification (EC 1.10.3.2), any catechol oxidase enzyme comprised by the enzyme classification (EC

151.10.3.1), any bilirubin oxidase enzyme comprised by the enzyme classification (EC 1.3.3.5) or any monophenol monooxygenase enzyme comprised by the enzyme classification (EC 1.14.99.1) .

The above mentioned enzymes may be derived from

20 plants, bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of Aspergillus. Neurospora. e.g., N. crassa. Podospora. Botrytis. Collybia. Fomes. Lentinus. Pleurotus, Trametes. e.g., T. villosa and T. versicolor. Rhizoctonia.

25 e.g., R. solani. Coprinus _f e.g., C. cinereus. C. comatus. C. friesii. and C. plicatilis. Psathyrella. e.g., P. condelleana. Panaeolus. e.g., P. papilionaceus, Myceliophthora. e.g., M. thermophila. Schvtalidium. Polyporus. e.g., P. pinsitus. Phlebia, e.g., P. radita (WO

3092/01046), or Coriolus. e.g., C. hirsutus (JP 2-238885). The laccase or the laccase related enzyme may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA sequence encoding said laccase as

35 well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the laccase, in a

culture medium under conditions permitting the expression of the laccase enzyme, and recovering the laccase from the culture.

Determination of Laccase Activity CLACU) Laccase activity is determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetate buffer, pH 5.5,

30 ^* C, 1 min. reaction time. 1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 μmole syringaldazin per minute at these conditions.

Industrial Applications

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

Colorants and dyes are broad classes of natural and synthetic compounds. The following description and examples of dyes/colorants are not intended to be in any way limiting to the scope of the invention as claimed:

Synthetic textile dyes bleachable by the method of the invention are typically azo compounds (with one or several azo, or diazenediyl, groups), as exemplified by Acid Red 151, Direct Blue 1, Direct Brown 44, and Orange II, or anthraquinone compounds, as exemplified by Acid Blue 45:

Acid Red 151

Direct Blue 1

Direct brown 44

Orange II

Acid Blue 45

Other structural motifs may occur together with these, as exemplified in the formula of Reactive Blue 19:

Reactive Blue 19

Some dyes furthermore carry groups capable of coupling to fabric surfaces (reactive dyes) , and some dyes are complexed to metal ions. These modifications will often not influence the applicability of the present invention.

A different structure bleachable by the method of the invention is the indigo moiety, here exemplified by the soluble dye indigo carmine:

Indigo Carmine

Other dyes and colorants may be of natural origin or may be synthesized as identical to or resembling natural structures. Examples of categories of coloured substances extractable from vegetable sources are polyphenolic, anthocyanine and carotenoid compounds. A specific embodiment of the present invention is provided by household and institutional laundering processes. In such washing and rinsing processes, dyes and colorants present on fabrics may leach into the washing or rinsing liquor and discoloration of the laundry may result. Bleaching

of the coloured compounds in solution by the method of the invention may counteract this undesirable effect. Other systems for dye transfer inhibition are known in the art (e.g. WO 91/05839) . In another specific embodiment, dyes leached into process water during textile processing may be bleached by the method of the invention to prevent undesirable deposition. Other systems are known in the art (e.g. WO 92/18697) . In a third embodiment, the method of the invention finds application in bleaching of pulp for paper production.

Accordingly, the invention provides a method for bleaching of lignin-containing material, in particular bleaching of pulp for paper production, which method comprises treatment of the lignin or lignin containing material with a phenol oxidizing enzyme and an enhancing agent as described in the present invention.

In a fourth embodiment, the method of the invention finds application for lignin modification, e.g., in the manufacture of wood composites, e.g., wood fibre materials such as chipboards, fibre boards, or particle boards, or in the manufacture of laminated wood products, such as laminated beams and plywood. In a fifth embodiment, the method of the invention finds application in treatment of waste water, e.g., waste water from the chemical or pharmaceutical industry, from dye manufacturing, from dye-works, from the textile industry, or from pulp production (cf. e.g. US 4,623,465, or JP-A-2-31887) .

In a more specific aspect, the invention provides a method for treatment of waste water from dye manufacturing, from dye-works, from textile industry, or from pulp manufacturing, the method comprising treatment of the waste water with a phenol oxidizing enzyme in the presence of an enhancing agent of the invention.

In the above mentioned processes and in other applications of the invention, the enhancing agent may be

added at the beginning of the process or later, in one or several additions.

According to the invention the phenol oxidizing enzyme may be present in concentrations of from 0.001-100 mg enzyme protein per liter.

Detergent Compositions

According to the invention, the enhancing agent and the phenol oxidizing enzyme may typically be a component of a detergent composition. As such, it may be included in the detergent composition in the form of a detergent additive. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo

Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonyl- phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film- forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenient form, e.g. as powder, granules, paste or liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. The detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzene- sulfonate (LAS) , alpha-olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE) , carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanol- amide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154) . The detergent composition may additionally comprise one or more other enzymes, such as amylases, lipases, cutinases, proteases, and cellulases.

The detergent may contain 1-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA) , ethylenediaminetetraacetic acid (EDTA) , diethylenetri- aminepentaacetic acid (DTPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) . The detergent may also be unbuilt, i.e. essentially free of detergent builder.

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose (CMC) , poly(vinyl- pyrrolidone) (PVP) , polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA) , polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/- acrylic acid copolymers.

The detergent may additionally contain other bleaching systems which may comprise a H ₂ 0 ₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) . Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzymes of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e.g. , WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corro¬ sion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, bactericides, optical brighteners, or perfume.

The pH (measured in aqueous solution at use con¬ centration) will usually be neutral or alkaline, e.g., in the range of 7-11.

Particular forms of detergent compositions within the scope of the invention include:

1) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 0 - 3%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. suds suppressors, perfume, optical 0 - 5% brightener, photobleach)

2) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

303) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ 5 - 9% culated as acid)

Alcohol ethoxylate (e.g. C _l2 . ₁₅ alco¬

35 hol, 7 EO) 7 - 14%

Soap as fatty acid (e.g. C ₁₆ . ₂₂ fatty 1 - 3% acid)

Sodium carbonate (as Na-jCO,) 10 - 17%

Soluble silicate (as Na,0,2Si0 ₇ ) 3 - 9%

Zeolite (as NaAlSiOJ 23 - 33%

Sodium sulfate (as Na ₇ S04) 0 - 4%

Sodium perborate (as NaBO,.H _? 0) 8 - 16%

TAED 2 8%

Phosphonate (e.g. EDTMPA) 0 - 1%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 0 - 3%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. suds suppressors, perfume, optical 0 - 5% brightener)

4) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ 8 - 12% culated as acid)

Alcohol ethoxylate (e.g. C ₁₂ . ₁₅ alco¬ hol, 7 EO) 10 - 25%

Sodium carbonate (as Na _? C0,) 14 - 22%

Soluble silicate (as Na _? 0,2SiO _? ) 1 - 5%

Zeolite (as NaAlSiOJ 25 - 35%

Sodium sulfate (as Na _? S0J 0 - 10%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 1 - 3%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. suds 0 - 5% suppressors, perfume)

5) An aqueous liquid detergent composition comprising

Linear alkylbenzenesulfonate (cal¬ 15 - 21% culated as acid)

Alcohol ethoxylate (e.g. C ₁₂ . ₁₅ alco¬ hol, 7 EO or C _1? _ _ις alcohol, 5 EO) 12 - 18%

Soap as fatty acid (e.g. oleic 3 - 13% acid)

Alkenylsuccinic acid (C„. _1Δ ) 0 - 13%

Aminoethanol 8 - 18%

Citric acid 2 - 8%

Phosphonate 0 - 3%

Polymers (e.g. PVP, PEG) 0 - 3%

Borate (as B _Δ 0 ₇ ) 0 - 2%

Ethanol 0 - 3%

Propylene glycol 8 - 14%

Enzymes (calculated as pure enzyme 0. 0001 - 0.1% protein)

Minor ingredients (e.g. dispersants, suds suppressors, per¬ 0 - 5% fume, optical brightener)

6) An aqueous structured liquid detergent composition comprising

257) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Fatty alcohol sulfate 5 - 10%

Ethoxylated fatty acid monoethanol- 3 - 9% amide

30 Soap as fatty acid 0 - 3%

Sodium carbonate (as Na _? C0,) 5 - 10%

Soluble silicate (as Na ₇ 0,2SiO _? ) 1 - 4%

Zeolite (as NaAlSiOJ 20 - 40%

Sodium sulfate (as Na.,SOJ 2 - 8%

35 Sodium perborate (as NaBO,.H _? 0) 12 - 18%

TAED 2 - 7%

Polymers (e.g. maleic/acrylic acid 1 - 5% copolymer, PEG)

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. optical brightener, suds suppressors, per¬ 0 - 5% fume)

8) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid) 8 - 14%

Ethoxylated fatty acid monoethanol- 5 - 11% amide

Soap as fatty acid 0 - 3%

Sodium carbonate (as Na _? C0J 4 - 10%

Soluble silicate (as Na ₇ 0,2SiO _? ) 1 - 4%

Zeolite (as NaAlSiOJ 30 - 50%

Sodium sulfate (as Na _? SOJ 3 - 11%

Sodium citrate (as C _A H _ς Na-,0 ₇ ) 5 - 12%

Polymers (e.g. PVP, maleic/acrylic 1 - 5% acid copolymer, PEG)

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. suds 0 - 5% suppressors, perfume)

9) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid) 6 - 12%

Nonionic surfactant 1 - 4%

Soap as fatty acid 2 - 6%

Sodium carbonate (as Na _P CO,) 14 - 22%

Zeolite (as NaAlSiOJ 18 - 32%

Sodium sulfate (as Na.,S0J 5 - 20%

Sodium citrate (as C _Λ H _ς Na _τ 0 ₇ ) 3 - 8%

Sodium perborate (as NaBO,.H ₇ 0) 4 9%

Bleach activator (e.g. NOBS or 1 5% TAED)

Carboxymethylcellulose 0 2%

Polymers (e.g. polycarboxylate or 1 5% PEG)

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. optical 0 5% brightener, perfume)

10) An aqueous liquid detergent composition comprising

Linear alkylbenzenesulfonate (calculated as acid) 15 - 23%

Alcohol ethoxysulfate (e.g. C ₁₂ . ₁₅ alcohol, 2-3 EO) 8 - 15%

Alcohol ethoxylate (e.g. C ₁₂ _ ₁₅ al¬ cohol, 7 EO, or 3 - 9% C _1? . _1S alcohol, 5 EO)

Soap as fatty acid (e.g. lauric 0 - 3% acid)

Aminoethanol 1 - 5%

Sodium citrate 5 - 10%

Hydrotrope (e.g. sodium 2 - 6% toluensulfonate)

Borate (as B _Δ 0 ₇ ) 0 - 2%

Carboxymethylcellulose 0 - 1%

Ethanol 1 - 3%

Propylene glycol 2 - 5%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. polymers, dispersants, perfume, optical 0 - 5% brighteners)

11) An aqueous liquid detergent composition comprising

Linear alkylbenzenesulfonate (calculated as acid) 20 - 32%

Alcohol ethoxylate (e.g. C ₁₂₁₅ alco¬ hol, 6 - 12% 7 EO, or C _1? . _1S alcohol, 5 EO)

Aminoethanol 2 - 6%

Citric acid 8 - 14%

Borate (as B _Δ 0 ₇ ) 1 - 3%

Polymer (e.g. maleic/acrylic acid copolymer, anchoring polymer such as, e.g. , lauryl 0 - 3% methacrylate/acrylic acid copolymer)

Glycerol 3 - 8%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. hydro- tropes, dispersants, perfume, 0 - 5% optical brighteners)

12) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Anionic surfactant (linear alkylbenzenesulfonate, alkyl sulfa¬ te, alpha-olefinsulfonate, alpha- 25 - 40% sulfo fatty acid methyl esters, alkanesulfonates, soap)

Nonionic surfactant (e.g. alcohol 1 - 10% ethoxylate)

Sodium carbonate (as Na ₇ COJ 8 - 25%

Soluble silicates (as Na _? 0, 2SiO _? ) 5 - 15%

Sodium sulfate (as Na _? SOJ 0 - 5%

Zeolite (as NaAlSiOJ 15 - 28%

Sodium perborate (as NaB0,.4H _? 0) 0 - 20%

Bleach activator (TAED or NOBS) 0 - 5%

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. perfume, 0 - 3% optical brighteners)

13) Detergent formulations as described in 1) - 12) wherein all or part of the linear alkylbenzenesulfonate is replaced by (C ₁₂ -C ₁₈ ) alkyl sulfate.

14) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

(C _1? -C _1R ) alkyl sulfate 9 - 15%

Alcohol ethoxylate 3 - 6%

Polyhydroxy alkyl fatty acid amide 1 - 5%

Zeolite (as NaAlSiOJ 10 - 20%

Layered disilicate (e.g. SK56 from Hoechst) 10 - 20%

Sodium carbonate (as Na-,COJ 3 - 12%

Soluble silicate (as Na _? 0,2SiO _? ) 0 - 6%

Sodium citrate 4 - 8%

Sodium percarbonate 13 - 22%

TAED 3 - 8%

Polymers (e.g. polycarboxylates and 0 - 5% PVP=

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. optical brightener, photo bleach, perfume, 0 - 5% suds suppressors)

15) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

(C _1? -C _1Λ ) alkyl sulfate 4 - 8%

Alcohol ethoxylate 11 - 15%

Soap 1 - 4%

Zeolite MAP or zeolite A 35 - 45%

Sodium carbonate (as Na _? C0,) 2 - 8%

Soluble silicate (as Na _? 0,2Si0 ₇ ) 0 - 4%

Sodium percarbonate 13 - 22%

TAED 1 - 8%

Carboxymethyl cellulose 0 - 3%

Polymers (e.g. polycarboxylates and 0 - 3% PVP)

Enzymes (calculated as pure enzyme 0.0001 - 0.1% protein)

Minor ingredients (e.g. optical 0 - 3% brightener, phosphonate, perfume)

16) Detergent formulations as described in 1) - 15) which contain a stabilized or encapsulated peracid, either as an ιo additional component or as a substitute for already specified bleach systems.

17) Detergent compositions as described in 1) , 3) , 7) , 9) and 12) wherein perborate is replaced by percarbonate.

18) Detergent compositions as described in 1) , 3) , 7) , 9) , 1512) , 14) and 15) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369. 1994, pp. 637-639.

19) Detergent composition formulated as a nonaqueous

20 detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali. The detergent may also comprise anionic surfactant and/or a bleach system.

The following examples further illustrate the 25 present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed.

EXAMPLE 1

Bleaching of Direct Blue 1 with soybean peroxidase with and without acetosyringone

A crude soy bean peroxidase (SBP) , obtained from Mead Corp., Dayton, Ohio, was purified by anion and cation chroma- tography followed by gelfiltration to a single protein on SDS-PAGE with an Revalue (A^^) of 2.2:

125 ml of crude SBP were adjusted to pH 7, diluted to 2.3 mS and filtered through 0.8 μ filter. The sample was applied to 300 ml DEAE column equilibrated with 20 mM phosphate pH 7.0 and the peroxidase eluted with a 1 M NaCl linear gradient in the same buffer. Fractions with peroxidase activity were pooled.

Pooled fractions from anion exchange chromatography (190 ml) were concentrated and washed by ultrafiltration (GR61PP membrane from Dow, Denmark). pH was adjusted to 5.3 ionic strength to 2.3 mS in the sample before application to a 200 ml S-Sepharose column previously equilibrated with 50 mM acetate pH 5.3. The effluent containing the peroxidase activity was concentrated and washed by ultrafiltration to a final volume of approx. 10 ml.

A 5 ml concentrated sample from cation exchange chroma¬ tography was applied to a 90 cm Sephacryl S-200 column equilibrated and eluted with 0.1 M acetate pH 6.1. Fractions with peroxidase activity giving only one band on SDS-PAGE were pooled.

The bleaching rate of Direct Blue 1 (DB1) by the purified SBP was determined using an enhancer according to the invention. The following conditions were used:

Final concentration 200 μl 50 mM Britton-Robinson buffer* pH 6, 8 and 10, respectively 10 mM 200 μl DBl - 3.0 Abs. Units (610 nm) °' ⁶ ( ^A 610r 5200 μl SBP with A _404nm = 0.0005 at pH 6 and 8 or with A 404nm = 0.005 at pH 10 0.0001 or

200 μl 50 μM enhancer 10 μM 200 μl 100 μM H ₂ 0 ₂ 20 μM

o * (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) . ** corresponding to approximately to 0.04 mg/1 and 0.4 mg/1.

Reagents were mixed in a thermostated cuvette at 30"C and the bleaching was started by addition of hydrogen 5 peroxide. The bleaching was detected spectrophotometrically at 610 nm, which is the wavelength of the absorption peak of DBl. Bleaching was followed for 4 minutes, and the reduction in absorbance (lOOx(A _6l0nmstart -A _610nm4mjn .)/A _{610rτι start} %) was determined. ^{0 A} ₆ i _{θrm start} ^{was determ} i ^{ned b} Y replacement of hydrogen peroxide with water.

Table 1

Bleaching of Direct Blue 1 with SBP in 4 Minutes

From the results presented in Table 1 above, it appears that by adding an enhancer of the invention a much faster bleaching of the dye is obtained compared to the 0 experiment without enhancer.

EXAMPLE 2

Bleaching of Direct Blue 1 with Coprinus cinereus peroxidase with and without enhancers

A Coprinus cinereus peroxidase (CiP) obtained as 5 described in WO 9412621 was used.

Dilutions of CiP were made in a solution of 0.15 gram/1 of Triton X-405.

The bleaching rate of Direct Blue 1 (DBl) by purified CiP was determined using the following conditions:

10 Final concentration

200 μl 50 mM Britton-Robinson buffer* 10 mM

200 μl DBl - 3.0 Abs. Units (610 nm) 0.6 (A _61fJn J

200 μl 0.40 mg/1 CiP (pH 8.5) 0.08 mg/1 (pH 8.5) or

0.80 mg/1 CiP (pH 10.5) 0.16 mg/1 (pH 10.5)

15200 μl 25 μM enhancer 5 μM

200 μl 100 μM H ₂ 0 ₂ 20 μM

* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) .

Reagents were mixed in a thermostated cuvette at 2030 ^β C and the bleaching was started by addition of hydrogen peroxide. The bleaching was detected spectrophotometrically at 610 nm, which is the wavelength of the absorption peak of DBl. Bleaching was followed for 1 minute, and the initial reduction in absorbance, -ΔmAbs/minute, was determined.

Table 2

Initial Bleaching of Direct Blue 1 with CiP

Enhancer -ΔmAbs/minute pH: 8.5 10.5

Acetosyringone 239 1

Syringaldehyde 151 4

Methylsyringate 245 8

No enhancer 2 0

From the results presented in Table 2 above it appears that by adding an enhancer of the invention a much faster bleaching of the dye compared to the experiment without enhancer is obtained. Even at pH 10.5 a significant bleaching with an enhancer of the invention is obtained, whereas no bleaching at all can be seen without the addition of an enhancer.

EXAMPLE 3

Bleaching of Chicago Sky Blue 6B fCSB) with Coprinus cinereus peroxidase and enhancers

Bleaching tests were performed in exactly the same way as described in Example 2 except that instead of using DBl

Chigaco Sky Blue (CSB) (obtainable from Aldrich) was used, and the following enhancers were tested: methylsyringate ethylsyringate propylsyringate butylsyringate hexylsyringate octylsyringate ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate.

The following results were obtained:

Table 3

Initial Bleaching of CSB with CiP

Enhancer -ΔmAbs/minute pH: 8.5 10.5 methylsyringate 211 42 ethy1syringate 240 52 propy1syringate 228 60 butylsyringate 228 48 hexylsyringate 276 36 octylsyringate 192 15 ethyl 3-(4-hydroxy-3,5- dimethoxyphenyl)acrylate 48 48

No enhancer 8 6

EXAMPLE 4

Bleaching of Direct Blue 1 fDBl) using various Coprinaceae laccases and methylsyringate at PH 5.5-8.5.

Bleaching of the dye Direct Blue 1 at various pH values was conducted using a laccase obtained from Coprinus comatus. Coprinus friesii. Coprinus plicatilis. Panaeolus papilionaceus or Psathyrella condolleana and methylsyringate.

The above mentioned strains were fermented in the following way:

The strains were inoculated on PDA agar plates (PDA: 39 g/1 potato dextrose agar) and grown at 26°C for 3 days. Shake flasks were then inoculated with 6-8 small squares ("0.5 cm x 0.5 cm) of agar containing mycelium and fermented for 3-10 days at 26"C and 200 rpm using the following medium:

Deposit no. Medium Growth

Coprinus comatus* CBS 631.95 A 10 days Coprinus friesii CBS 629.95 A 3 days Panaeolus papilionaceus CBS 630.95 A 10 days Psathyrella condolleana CBS 628.95 B 7 days Coprinus plicatilis CBS 627.95 A 8 days

* All the strains mentioned in this Example have been deposited according to the Budapest Treaty on the Inter¬ national Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, on 16 August 1995, at Centraalbureau voor Schimmelcultures, Oosterstraat 1, Postbus 273, NL-3740 AG Baam, Netherlands, under the above mentioned Accession numbers.

Media: A: soja meal 30 g/1 maltodextrin 15 g/1 bacto peptone 5 g/1 pluronic 0.2 g/1

B: potato meal 50 g/1 barley meal 25 g/1

BAN 800MG* 0.025 g/1

Na-caseinate 5 g/1 crushed soja 10 g/1

Na2HP04, 12 H20 4.5 g/1

Pluronic 0.05 ml/1

* BAN 800MG obtainable from Novo Nordisk A/S.

After fermentation the culture broths were centrifugated and the supernatants were used in the tests described below.

The bleaching rate of DBl was determined using the following conditions:

Final concentration 400 μl 50 mM Britton-Robinson buffer*,

(pH 5.5, 7.0, and 8.5 respectively), 20 mM

200 μl DBl - 3.0 Abs. Units (610 nm) 0.6 (A _610n 200 μl 50 μM methylsyringate 10 μM 200 μl laccase at pH 5 and 7: 4 LACU/1 at pH 8.5: 20 LACU/1

* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) .

Reagents were mixed in a l ml thermostated cuvette at

30"C and the bleaching was started by addition of the laccase.

The bleaching was followed spectrophotometrically at

610 nm, which is the wavelength of the absorption peak of DBl, with readings every 5 sec. for a period of 5 minutes.

The initial bleaching rate was determined from the first linear part of the absorbance curve.

The following results were obtained with methyl¬ syringate:

-ΔmAbs minute

Laccase: pH: 5.5 7.0 8.5

C. comatus 33 23 2 C. friesii 40 55 61

Pan. papilionaceus 16 19 18

Ps. condolleana 45 54 43

C. plicalitis 42 39 14

The following results were obtained with no enhancer:

-ΔmAbs/minute Laccase: pH: 5.5 7.0 8.5

5 C. comatus 0 0 0

C. friesii 0 0 0

Ps. condolleana 0 0 0

C. plicalitis 0 0 0

EXAMPLE 5

ιo Bleaching of Direct Blue 1 fDBl) using Coprinus cinereus laccase with/without enhancing agents at pH 5.5-8.5.

Bleaching of the dye Direct Blue 1 at various pH values was conducted using Coprinus cinereus laccase and one of the following enhancing agents: 15 None acetosyringone syringaldehyde methylsyringate.

The laccase was obtained in the following way: 20 Coprinus cinereus (IFO 30116 - freely available to the public from Institute of Fermentation, Osaka (IFO) under the indicated deposit number) was inoculated from a PDA agar slant (PDA: 39 g/1 potato dextrose agar) into a 100 ml shake flask containing medium A (Medium A is described in Example 253) . The culture was cultivated for 6 days at 26"C and 100 rpm. A 10-liter fermentor containing medium A was inoculated with the 100 ml culture broth. The fermentation ran for 6 days at 26"C and 100 rpm. The culture broth was filtrated and concentrated by ultrafiltration. Further purification was 30 carried out using hydrophobic interaction chromatography followed by anionic exchange chromatography. This process resultated in a preparation with a laccase activity of 3.6 LACU/ml. The estimated purity was >80% on a protein basis.

The bleaching rate of DBl was determined using the following conditions:

Final concentration 400 μl 50 mM Britton-Robinson buffer*, (pH 5.5, 7.0 and 8.5 respectively), 20 mM

200 μl DBl - 3.0 Abs. Units (610 nm) 0.6 (A _610nm )

200 μl 50 μM enhancing agent 10 μM

200 μl C. cinereus laccase 1 mg/1

* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) .

Reagents were mixed in a 1 ml thermostated cuvette at 30"C and the bleaching was started by addition of the laccase.

The bleaching was followed spectrophotometrically at 610 n , which is the wavelength of the absorption peak of DBl, with readings every 5 sec. for a period of 5 minutes. The initial bleaching rate was determined from the first linear part of the absorbance curve.

The following results were obtained:

Enhancing agent -ΔmAbs/minute

pH: 5.5 7.0 8.5

none 13 5 3 aceto- syringone 28 94 50 syring¬ aldehyde 29 79 28 methyl¬ syringate 20 94 57

33

EXAMPLE 6

Bleaching of Direct Blue 1 (DBl) using Coprinus cinereus laccase and acetosyringone

Bleaching of the dye Direct Blue 1 at various pH values was conducted using Coprinus cinereus laccase and the enhancing agent acetosyringone.

The laccase was obtained as described in Example 5. The bleaching rate of DBl was determined using the following conditions: Final concentration

400 μl 50 mM Britton-Robinson buffer*, (pH 4, 5, 6, 7, and 8 respectively), 20 mM

200 μl DBl - 3.0 Abs. Units (610 nm) 0.6 (A _610n

200 μl 50 μM acetosyringone 10 μM 200 μl C. cinereus laccase 3.2 mg/1

* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) .

Reagents were mixed in a 1 cm thermostated cuvette at 30°C and the bleaching was started by addition of the laccase.

The bleaching was detected spectrophotometrically at 610 nm, which is the wavelength of the absorption peak of DBl. After 5 sec. bleaching was followed for 4 minutes.

The following results were obtained:

Initial DBl bleaching

(-ΔmAbs/ min) pH (% of pH 7-value)

4 18 % 5 13 %

6 35 %

7 100 %

8 69 %

It can be seen from the results given above that the optimum bleaching is achieved at pH around 7, but the system also shows an effective bleaching at pH 8.

EXAMPLE 7

Bleaching of Direct Blue 1 with Trametes villosa laccase with and without enhancing agents

Laccase obtained from Trametes villosa: 800 ml culture broth of Trametes villosa. CBS 678.70, was filtered with filter aid to give a clear filtrate, which was concentrated and washed by ultrafiltration on a membrane with a cut-off of 6-8 kDa. One ml samples of concentrated preparation was applied onto a Q-Sepharose HP column (Pharmacia, Sweden) equilibrated with 0.1 M fosfate pH 7, and the laccase was eluted with a flat NaCl gradient around 0.25 M. Fractions with laccase activity from 10 runs were pooled and concentrated by ultrafiltration to an activity of 500 LACU/ml.

The following conditions were used:

Final concentration 400 μl 50 mM Britton-Robinson buffer*, pH 5.5 and pH 7.0 respectively, 20 mM

200 μl DBl - 3.0 Abs. Units (610 nm) 0.6 (A _610nm ) 200 μl 50 μM enhancer 10 μM

200 μl Enzyme dilution

* (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH) .

Reagents were mixed in a 1 cm thermostated cuvette at 30°C and the bleaching was started by addition of enzyme.

The bleaching was detected spectrophotometrically at 610 nm, which is the absorption peak of DBl. After 5 sec. bleaching was followed for 4 minutes.

From the results presented below, it appears that adding enhancers of the invention a much faster bleaching of

the dye can be obtained compared to the experiment without enhancer. Enzyme dosages given are in the final incubation mixture.

Bleaching of Direct Blue 1 with Trametes villosa laccase, obtained as described above, at pH 5.5 (1.6 mg/1) and pH 7.0 (16 mg/1) :

Enhancer DBl bleaching in 4 minutes

(-ΔmAbs/4 min) pH 5.5 pH 7.0

No enhancer 0 0

Acetosyringone 447 242

Syringaldehyde 438 112

EXAMPLE 8

Bleaching of gradually added Acid Blue 45 with Coprinus cine¬ reus laccase with and without enhancing agent

Ideally, dye transfer inhibition systems for laundry applications should be tested in a real wash where dyed fabrics give off dyes to the wash solution as a result of the combined action of the detergent, temperature and mechanical agitation taking place.

To simulate such a process, however, a magnetically stirred beaker was used as the reaction vessel and dye was added gradually from a stock solution (using a Metrohm 725 dosimat) . The solution was monitored spectrophotometrically using a Zeiss multichannel spectrometer (MCS) equipped with a fibre-optics immersion probe.

Stock solutions of acetosyringone was prepared in a suitable water/ethanol mixture. Stock solutions of the anthraquinone dye Acid Blue 45 were made with water.

The laccase was recovered from a 10-liter fermentation of Coprinus cinereus (IFO 30116) as described in Example 4.

The following conditions were used in the experiment: Temperature: 35 ^β C

Medium and pH: 50 mM/50 mM phosphate/borate buffer at pH 10 Acetosyringone (when applicable) : 10 μM

Laccase: 10 mg/1 Dye addition program: linear addition at a rate of ca 0.34 abs/40 min, referring to the absorbance of Acid Blue 45 at its maximum absorbance wavelength (590 nm for Acid Blue 45) .

Fig. 1 shows the results of the bleaching tests. The following symbols are used: (I) ; Only dye addition; (II) : Dye addition in the presence of Laccase; (III) : Dye addition in the presence of Laccase + acetosyringone.

It can be seen from Fig. 1 that the bleaching effect is enhanced by acetosyringone.

EXAMPLE 9

Dve Transfer Inhibition Using Coprinus cinereus Laccase

A small-scale experiment was carried out in which clean cotton test pieces were washed together with dyed fabrics bleeding dye into the wash solution, the experiment conducted in the absence and in the presence of laccase and enhancing agent.

After wash, the Hunter colour difference between the above mentioned cotton pieces and clean cotton pieces (washed in the absence of bleeding fabrics) was measured and taken as a measure of the degree of dye transfer resulting from the wash.

Materials used:

Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DBl) .

Clean white cotton (bleached, no optical brightener added) .

Liquid detergent and powder detergent as typically met in the North American market place; both detergents contained no bleaching system.

Coprinus cinereus laccase, obtained as described in Example 4.

Washing procedure:

The washing processes were carried out in beakers with magnetical stirring at 35°C for 15 min., after which the test fabrics were rinsed thoroughly in tap water and air- dried overnight in the dark before the Hunter readings were taken by using a Datacolor Elrephometer 2000 reflectance spectrometer.

Laccase system: Laccase at a level of 10 mg/1 with the enhancing agent acetosyringone at a level of 10 μM.

The following results were obtained:

Wash in liguid detergent solution ( 2 g/1. water hardness

Hunter colour difference (delta E) with respect to white, washed cotton

Cotton washed Cotton washed with AR 151 with DB 1 bleeders bleeders

Wash with no laccase system 12 26

Wash with laccase system 1 7

Wash in powder detergent solution (1 g/1. water hardness 6"dH at PH 10.0:

Hunter colour difference (delta E) with respect to white, washed cotton

Cotton washed Cotton washed with AR 151 with DB 1 bleeders bleeders

Wash with no laccase system 21 29

Wash with laccase system

Typical significant differences in the delta E readings are 2-3 units, so the data reflect significant reduction of dye transfer with the laccase treatments relative to the treat¬ ment with no laccase system.

EXAMPLE 10

Dye Transfer Inhibition Using Mvceliophthora thermophila Laccase

A small-scale experiment was carried out in which clean cotton test pieces were washed together with dyed fabrics bleeding dye into the wash solution, the experiment conducted in the absence and in the presence of laccase and enhancing agent.

After wash, the Hunter colour difference between the above mentioned cotton pieces and clean cotton pieces (washed in the absence of bleeding fabrics) was measured and taken as a measure of the degree of dye transfer resulting from the wash.

Materials used:

Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DBl) .

Clean white cotton (bleached, no optical brightener added) .

Liquid detergent (No. 1) as typically met in the European market place; liquid detergent (No. 2) as typically met in the North American market place.

Myceliophthora thermophila laccase, produced as described in PCT/US95/06815) .

Washing procedure:

The washing processes were carried out in beakers with magnetical stirring at 35 ^β C for 15 min., after which the test fabrics were rinsed thoroughly in tap water and air- dried overnight in the dark before the Hunter readings were taken by using a Datacolor Elrephometer 2000 reflectance spectrometer.

Laccase systems: M. thermophila laccase at a level of 0.87 mg/1 with the enhancing agent acetosyringone (AS) or the enhancing agent methylsyringate (MS) at a level of 10 μM.

The following results were obtained:

Wash in solution of liquid detergent No. 1 (l g/1. water hardness 12'dH) at an initial pH of 7.0:

Hunter colour difference (delta E) with respect to white, washed cotton

Cotton washed Cotton washed with AR 151 with DB 1 bleeders bleeders

Wash with no laccase system 7 27

Wash with AS-based laccase system 13

Wash with MS-based laccase system 12

Wash in solution of liquid detergent No. 2 (2 g/1. water hardness 6'dH) at pH 8.5:

Hunter colour difference (delta E) with respect to white, washed cotton

Cotton washed Cotton washed with AR 151 with DB 1 bleeders bleeders

Wash with no laccase system 14 29

Wash with AS-based laccase system 10

Wash with MS-based laccase system

Typical significant differences in the delta E readings are 2-3 units, so the data reflect significant reduction of dye transfer with the laccase treatments relative to the treat¬ ment with no laccase system.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 23 • ^lineS _

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | χ |

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Barn, Nether- land

Date of deposit Accession Number

16 A u g u st 1995 C B S 631 . 95

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | [

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if tlte indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specif the general ature o) 'the indications eg., "Accession Number of Deposit")

For International Bureau use only

I I This sheet was received by the International Bureau on:

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Form PCT/RO/134 (July 1992)

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 29 ine S 3

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet |χ |

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Barn, Nether- land

Date of deposit Accession Number

16 A u g u s t 199 5 C B S 629 . 95

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Slates)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., 'Accession Number of Deposit")

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule \3bis)

A. The indications made below relate to the microorganism referred to in the description on page S _ 29 . line 4 - 5

B. IDENTD7ICATION OF DEPOSIT Further deposits are identified on an additional sheet [X |

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Barn, Nether- land

Date of deposit Accession Number

16 August 1995 C B S 630 . 95

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | |

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 o _f Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftlie indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemaiional Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

For International Bureau use only

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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bιs)

For Internationa] Bureau use only

I I This sheet was received by the International Bureau on.

Authorized officer

Form PCT/RO/134 (July 1992)

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3b )

The indications made below relate to the microorganism referred to in the description on page 9 , lineS g

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Barn, Nether- land

Date of deposit Accession Number

16 Aug ust 1995 CBS 627 . 95

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftlie indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

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