CA2201334C - Enhancers such as acetosyringone - Google Patents

Abstract

The present invention relates to a method of oxidizing a compound with a phenol oxidizing enzyme (e.g. a peroxidase or a laccase) and an enhancing agent (e.g. acetosyringone). The invention also relates to a detergent additive and to a detergent composition.

Description

~ WO 96/10079 PCT/DK95100384 ENHANCERS SUCH AS ACETOSYRINGONE
= 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 lo 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. 5 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:

OH
Bo oc A
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, -SOZ-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 C1-C16 alkyl, preferably a C1-C$ 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 CmH2,+1 ; 1 5 m S 5. =
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 C;

= WO 96/10079 22~/ ii 13 3/, PCT/DK95/00384 3 "~
,,.

(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:

1o OH -BO oe #
A
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, -S02-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 C1-C16 alkyl, preferably a C1-C$ 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 f rom CmH2,+1 ; 15 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 C1-C16 alkyl, preferably a C1-C$ alkyl, which alkyl may 3o 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 CmH2m+1 ; 1 - m <- 5.

WO 96/10079 220 13 34 PCT/DK95/00384 ~

In the above mentioned formula A may be placed meta 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 present 1o in concentrations of from 0.01 to 1000 M, more preferred 0.1 to 250 M, most preferred 1 to 100 M.

Preparation of Enhancing Agents The enhancing agents described in the present application may be prepared using methods well known to those 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 Chem. Ber.
2o 67, 1934, p. 67.
Ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate was synthesised from syringaldehyde and triethyl phosphonoacetate in ethanol/sodium ethanolate. The product was after purification characterised by 1H-NMR and 13C-NMR (showing spectra as expected) and the melting point was 68-70 C.
Hydrogen peroxide/Oxygen If the phenol oxidizing enzyme requires a source of hydrogen peroxide, the source may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of 3o 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 beginning W O 96110079 22ii 13 3 4 PCT/DK95100384 ~,! 't 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 5 oxygen, molecular oxygen from the atmosphere will usually be present in sufficient quantity. If more 02 is needed, additional oxygen may be added.
Phenol Oxidizing Enzyme In the context of the present invention the enzyme lo 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 possessinct 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, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumaao, Ulocladium chartarum, Embellisia allior Dreschlera halodes.

WO 96/10079 220 13 34 PCT/DK95/00384 ~

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
8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus), e.g.
T. versicolor (e.g. PR4 28-A).
Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, 1o e.g. Rhizopus or Mucor, in particular Mucor hiemalis.
Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC
23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
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-11).
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
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.
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 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 (PODU) 1 peroxidase unit (PODU) is the amount of enzyme that catalyzes the conversion of 1 mole 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.
lo 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
1.10.3.1), 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 plants, bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of Asperaillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinus, 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
92/01046), 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 well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the laccase, in a WO 96/10079 22 13 34 PCT/DK95/00384 ~

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

Determination of Laccase Activity (LACU) 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 M 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 pmole 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 HO
NaO3S N=N -N ~ ~ .

WO 96110079 2,20 13 34 PCT/DK95100384 Direct Blue 1 NH OH
NaO3S 2 HO NHZ
N=N SO3Na = I / / -O o SO3Na CH3 H3C SO3Na Direct brown 44 N
=N N=N N=N S03Na NaO3S N=N I:Z' Orange II

/ I \

OH
N=N

S O3Na Acid Blue 45 NaO3S

S03Na other structural motifs may occur together with these, as exemplified in the formula of Reactive Blue 19:

Reactive Blue 19 O H'-/ S03Na \ I \ I .
O NH

SO2CH2CH,OSO3Na 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 O H
O;S N

O

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 WO 96/10079 .PCYKDK95100384 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 15.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 2o 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.
25 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
30 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 35 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.

Deteraent 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 1o 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.

WO 96110079 22 0 13 3 4 PCT1nK95100384 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 lo 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 H202 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 1o 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/l comprising 2o Linear alkylbenzenesulfonate (cal- 7 - 12%
culated as acid) Alcohol ethoxysulfate (e.g. C1-18 alcohol, 1-2 EO) or alkyl sulate 1 - 4%
(e.g. C _ ) Alcohol ethoxylate (e.g. C14-15 alco-hol, 5 - 9%
7 EO) Sodium carbonate (as Na CO ) 14 - 20%
Soluble silicate (as Na 0,2Si0 ) 2 - 6%
Zeolite (as NaAlSi0 ) 15 - 22%
Sodium sulfate (as Na SO ) 0 - 6% =
Sodium citrate/citric acid (as CHNaO/CHO ) 0 - 15%
Sodium perborate (as NaBO.H O) 11 - 18%
TAED 2 - 6%
Carboxymethylcellulose 0 - 2% 11 Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 0 - 3%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein) = 5 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/l comprising 1o Linear alkylbenzenesulfonate (cal-culated as acid) 6 - 11%
Alcohol ethoxysulfate (e.g. C12_1a alcohol, 1-2 EO or alkyl sulfate 1 - 3%
(e.g. C _ ) 15 Alcohol ethoxylate (e.g. C14-15 alco-hol, 7 EO) 5 - 9%
Sodium carbonate (as Na CO ) 15 - 21%
Soluble silicate (as Na O,2Si0 ) 1 - 4%
Zeolite (as NaA1SiO) 24 - 34%
Sodium sulfate (as Na SO ) 4 - 10%
Sodium citrate/citric acid (as CHNaO/CHO ) 0 - 15%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 1 - 6%
Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein) Minor ingredients (e.g. suds 0 - 5%
suppressors, perfume) 3o 3) A detergent composition formulated as a granulate having = a bulk density of at least 600 g/l comprising Linear alkylbenzenesulfonate (cal- 5 - 9%
culated as acid) Alcohol ethoxylate (e.g. C1z-15 alco-hol, 7 EO) 7 - 14%
Soap as fatty acid (e.g. C16-Z2 fatty 1 - 3%
acid) WO 96/10079 2 0 3 3 4 PCT/DK95/00384 ~

Sodium carbonate (as Na CO ) 10 - 17%
Soluble silicate (as Na 0,2Si0 ) 3 - 9%
Zeolite (as NaAlSiO ) 23 - 33%
Sodium sulfate (as Na SO4) 0 - 4%
Sodium perborate (as NaBO.H O) 8 - 16%
TAED 2 - 8%
Phosphonate (e.g. EDTMPA) 0 - 1%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. maleic/acrylic acid 1o 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) 2o Alcohol ethoxylate (e.g. C12-15 alco-hol, 7 EO) 10 - 25%
Sodium carbonate (as Na CO ) 14 - 22%
Soluble silicate (as Na 0,2Si0 ) 1 - 5%
Zeolite (as NaAlSiO) 25 - 35%
Sodium sulfate (as Na SO ) 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) Wo 96110079 .34 PCT/DK95/00384 5) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (cal- 15 - 21%
culated as acid) Alcohol ethoxylate (e.g. C12_15 alco-hol, 7 EO or C alcohol, 5 EO) 12 - 18%
Soap as fatty acid (e.g. oleic 3 - 13%
acid) Alkenylsuccinic acid (C _ ) o - 13%
Aminoethanol 8 - 18%
1o Citric acid 2 - 8%
Phosphonate 0 - 3%
Polymers (e.g. PVP, PEG) 0 - 3%
Borate (as B O) 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 Linear alkylbenzenesulfonate (calculated as acid) 15 - 21%
Alcohol ethoxylate (e.g. C12_15 alcohol, 7 EO, or 3 - 9%
C alcohol, 5 EO) Soap as fatty acid (e.g. oleic 3 - 10%
acid) to Zeolite (as NaAlSiO ) 14 - 22%
Potassium citrate 9 - 18%
Borate (as B O) 0 - 2%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. PEG, PVP) 0 - 3%
Anchoring polymers such as, e.g., lauryl methacrylate/acrylic acid 0 - 3%
copolymer; molar ratio 25:1; MW

Glycerol 0 - 5%
2o Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein) Minor ingredients (e.g.
dispersants, suds suppressors, 0 - 5%
perfume, optical brighteners) 7) A detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising Fatty alcohol sulfate 5 - 10%
Ethoxylated fatty acid monoethanol- 3 - 9%
amide Soap as fatty acid 0 - 3%
Sodium carbonate (as Na CO ) 5 - 10%
Soluble silicate (as Na 0,2Si0 ) 1 - 4%
Zeolite (as NaAlSiO ) 20 - 40%
Sodium sulfate (as Na SO ) 2 - 8%
Sodium perborate (as NaBO.H O) 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 lo Linear alkylbenzenesulfonate (calculated as acid) 8 - 14%
Ethoxylated fatty acid monoethanol- 5 - 11%
amide Soap as fatty acid 0 - 3%
Sodium carbonate (as Na CO ) 4 - 10%
Soluble silicate (as Na O,2Si0 ) 1 - 4%
Zeolite (as NaAlSi0 ) 30 - 50%
Sodium sulfate (as Na SO ) 3 - 11%
Sodium citrate (as C H Na O) 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%
3o Nonionic surfactant 1 - 4%
Soap as fatty acid 2 - 6%
Sodium carbonate (as Na CO ) 14 - 22%
Zeolite (as NaAlSiO ) 18 - 32%
Sodium sulfate (as Na SO) 5 - 20%
Sodium citrate (as C H Na O) 3 - 8%

Sodium perborate (as NaBO.H O) 4 - 9%
Bleach activator (e.g. NOBS or 1 - 5%
TAED) Carboxymethylcellulose 0 - 2%
5 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%
lo brightener, perfume) 10) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as acid) 15 - 23%
Alcohol ethoxysulfate (e.g. C12_15 15 alcohol, 2-3 EO) 8 - 15%
Alcohol ethoxylate (e.g. C12_15 al-cohol, 7 EO, or 3 - 9%
C alcohol, 5 EO) Soap as fatty acid (e.g. lauric 0 - 3%
2o acid) Aminoethanol 1 - 5%
Sodium citrate 5 - 10%
Hydrotrope (e.g. sodium 2 - 6%
toluensulfonate) Borate (as B O) 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) VVO 96110079 '~ ~ 3 4 11) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as acid) 20 - 32%
Alcohol ethoxylate (e.g. C12_15 alco-hol, 6 - 12%
7 EO, or C alcohol, 5 EO) Aminoethanol 2 - 6%
Citric acid 8 - 14%
Borate (as B O) 1 - 3%
lo 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/l 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 CO) 8 - 25%
Soluble silicates (as Na O, 2Si0 ) 5 - 15%
Sodium sulfate (as Na SO ) 0 - 5%
Zeolite (as NaAlSi0) 15 - 28%
Sodium perborate (as NaBO .4H O) 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 (C12-C18) alkyl sulfate.

14) A detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising (C -C ) alkyl sulfate 9 - 15%
Alcohol ethoxylate 3 - 6%
Polyhydroxy alkyl fatty acid amide 1 - 5%
Zeolite (as NaAlSi0 ) 10 - 20%
1o Layered disilicate (e.g. SK56 from Hoechst) 10 - 20%
Sodium carbonate (as Na CO ) 3 - 12%
Soluble silicate (as Na 0,2Si0 ) 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/l comprising (C -C ) alkyl sulfate 4 - 8%
Alcohol ethoxylate 11 - 15%
Soap 1 - 4%
Zeolite MAP or zeolite A 35 - 45%
Sodium carbonate (as Na CO ) 2 - 8%
Soluble silicate (as Na O,2Si0 ) 0 - 4%
P Sodium percarbonate 13 - 22%
TAED 1 - 8%

W O 96110079 ~ 2 0 4 PCT/DK95100384 Carboxymethyl cellulose 0 - 3%
Polymers (e.g. polycarboxylates and 0 - 35%
PVP) Enzymes (calculated as pure enzyme 0.0001 - 0.1%
protein) Minor ingredients (e.g. optical o - 3%
brightener, phosphonate, perfume) 16) Detergent formulations as described in 1) - 15) which contain a stabilized or encapsulated peracid, either as an io 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), 12), 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 2o 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 present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed.

PCT/DK95/00384 ~
WO 96/10079 ~ ~ "~ ~ ~ 3 4 Bleaching of Direct Blue 1 with soybean peroxidase with and without acetosyrinaone 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 RZ-value (A404~A2$0n) of 2.2:
125 ml of crude SBP were adjusted to pH 7, diluted to 2.3 mS and filtered through 0.8 f.c filter. The sample was 1o 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 2o 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:

W O 96110079 220 13 .3/ PCT/DK95/00384 ~i Final concentration 200 .l 50 mM Britton-Robinson buffer*
pH 6, 8 and 10, respectively 10 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A610nm) 5 200 E.cl SBP with A4o4rm = 0.0005 at pH 6 and 8 or with A4o4rm = 0.005 at pH 10 0.0001 or 0. 001 (A404nm) 200 l 50 M enhancer 10 M
200 fa, l 10 0/..cM H202 2 0 /.cM

1o * (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/l.
Reagents were mixed in a thermostated cuvette at 30 C
and the bleaching was started by addition of hydrogen 15 peroxide. The bleaching was detected spectrophotometrically at 610 nm, which is the wavelength of the absorption peak of DB1. Bleaching was followed for 4 minutes, and the reduction in absorbance (100x (A610nm,start-A610nm,4min. ) /A610nm,start ) was determined.

20 A61orn-,start was determined by replacement of hydrogen peroxide with water.

Table 1 Bleaching of Direct Blue 1 with SBP in 4 Minutes Enhancer % DBi bleaching in 4 min.
pH 6 pH 8 pH 10 10x[SBP]
25 No 0.7 <0.7 <0.7 acetosyringone 19.8 20.0 3.3 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 3o experiment without enhancer.

WO 96/10079 220 ' 3 3 4 PCT/DK95/00384 Bleaching of Direct Blue 1 with Coprinus cinereus peroxidase with and without enhancers A Coprinus cinereus peroxidase (CiP) obtained as 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 (DB1) by purified CiP was determined using the following conditions:

Final concentration 200 l 50 mM Britton-Robinson buffer* 10 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61onm) 200 l 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/i (pH 10.5) 200 l 25 M enhancer 5 M
200 1 10 0AM H202 20 f.t,M

* (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 2o 30 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 DB1. Bleaching was followed for 1 minute, and the initial reduction in absorbance, -AmAbs/minute, was determined.

WO 96110079 ~ ~ O PCT/DK95100384 2 7 ~!

Table 2 Initial Bleaching of Direct Blue 1 with CiP
Enhancer -AmAbs/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 1o 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.

Bleaching of Chicago Sky Blue 6B (CSB) with Coprinus cinereus peroxidase and enhancers Bleaching tests were performed in exactly the same way 2o as described in Example 2 except that instead of using DB1 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 -AmAbs/minute pH: 8.5 10.5 methylsyringate 211 42 ethylsyringate 240 52 propylsyringate 228 60 butylsyringate 228 48 hexylsyringate 276 36 1o octylsyringate 192 15 ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate 48 48 No enhancer 8 6 Bleaching of Direct Blue 1(DB1) using various Coprinaceae laccases and methvlsyringate 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/l 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:

WO 96/10079 220 13 34 pCT)DK95100384 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 1o 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 Baarn, Netherlands, under the above mentioned Accession numbers.

Media:
A: soja meal 30 g/1 maltodextrin 15 g/1 bacto peptone 5 g/l pluronic 0.2 g/l B: potato meal 50 g/l barley meal 25 g/1 BAN 800MG* 0.025 g/1 Na-caseinate 5 g/1 crushed soja 10 g/1 Na2HPO4, 12 H20 4.5 g/1 Pluronic 0.05 ml/l * BAN 800MG obtainable from Novo Nordisk A/S.
After fermentation the culture broths were centrifugated so and the supernatants were used in the tests described below.

The bleaching rate of DB1 was determined using the following conditions:

Final concentration 400 l 50 mM Britton-Robinson buffer*, (pH 5.5, 7.0, and 8.5 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0. 6 (A67orm) 5 200 l 50 M methylsyringate 10 M
200 l 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).

10 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 nm, which is the wavelength of the absorption peak of 15 DB1, 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:

20 -AmAbs/minute Laccase:
pH: 5.5 7.0 8.5 C. comatus 33 23 2 25 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 30 enhancer:

-AmAbs/minute Laccase:
pH: 5.5 7.0 8.5 s C. comatus 0 0 0 C. friesii .0 0 0 Ps. condolleana 0 0 0 C. plicalitis 0 0 0 Bleaching of Direct Blue 1 (DB1) 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 Conrinus cinereus laccase and one of the following enhancing agents:
None acetosyringone syringaldehyde methylsyringate.

The laccase was obtained in the following way:
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 3). 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 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 DB1 was determined using the following conditions:

Final concentration 400 l 50 mM Britton-Robinson buffer*, 5(pH 5.5, 7.0 and 8.5 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61orm) 200 l 50 M enhancing agent 10 M
200 l C. cinereus laccase 1 mg/1 * (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric 1o 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 15 610 nm, which is the wavelength of the absorption peak of DB1, 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:
2o Enhancing agent -AmAbs/minute pH: 5.5 7.01 8.5 none 13 5 3 aceto-25 syringone 28 94 50 syring-aldehyde 29 79 28 methyl-syringate 20 94 57 WO 96110079 220 13 34 PCTmK95/00384 Bleaching of Direct Blue 1 (DB1) using Coprinus cinereus laccase and acetosvringone Bleaching of the dye Direct Blue 1 at various pH
values was conducted using Coorinus cinereus laccase and the enhancing agent acetosyringone.
The laccase was obtained as described in Example 5.
The bleaching rate of DB1 was determined using the following conditions:
Final concentration 400 l 50 mM Britton-Robinson buffer*, (pH 4, 5, 6, 7, and 8 respectively), 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61 on.) 200 l 50 M acetosyringone 10 M
200 l C. cinereus laccase 3.2 mg/l * (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 DB1. After 5 sec. bleaching was followed for 4 minutes.
The.following results were obtained:
Initial DB1 bleaching (-AmAbs/ min) pH (% of pH 7-value) 4 18 ~
3o 5 13 0 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.

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 lo 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 NaC1 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 l 50 mM Britton-Robinson buffer*, pH 5.5 and pH 7.0 respectively, 20 mM
200 l DB1 - 3.0 Abs. Units (610 nm) 0.6 (A61orm) 200 l 50 M enhancer 10 M
200 l 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 DB1. After 5 sec.
3o bleaching was followed for 4 minutes.
From the results presented below, it appears that adding enhancers of the invention a much faster bleaching of WQ 96(10079 220 13 34 PCT/DK95/00384 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, 5 obtained as described above, at pH 5.5 (1.6 mg/1) and pH 7.0 (16 mg/1):

Enhancer DB1 bleaching in 4 minutes (-AmAbs/4 min) pH 5.5 pH 7.0 No enhancer 0 0 Acetosyringone 447 242 Syringaldehyde 438 112 Bleachina 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 3o anthraquinone dye Acid Blue 45 were made with water.

WO 96/10079 220 13 34 PCT/DK95/00384 ~

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 C
Medium and pH: 50 mM/50 mM phosphate/borate buffer at pH 10 Acetosyringone (when applicable): 10 M
Laccase: 10 mg/1 io 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.

2o Dye 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 f rom the wash.

Materials used:
Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DB1).

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 tUrocedure :
The washing processes were carried out in beakers 1o 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 M.
The following results were obtained:

Wash in liquid detergent solution (2 g/l, 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 12 26 Wash with laccase system 1 7 Wash in powder detergent solution (1 g/l, 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 lo system 21 29 Wash with laccase system 4 8 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.

Dye Transfer Inhibition Using MYceliophthora 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.

WO 96/10079 220 13 3 4 PC7Y7)K95100384 Materials used:
Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DB1).
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.
Mviceliophthora thermonhila laccase, produced as io described in PCT/US95/06815).

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

The following results were obtained:

Wash in solution of liquid detergent No. 1 (7 g/l, 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 -------------------------------------------------------------so Wash with no laccase system 7 27 Wash with AS-based laccase system 5 13 Wash with MS-based laccase system 4 12 5 Wash in solution of liquid detergent No. 2 (2 g/l, water hardness 6 dH) at nH 8.5:

Hunter colour difference (delta E) with respect to white, washed cotton Cotton washed Cotton washed 10 with AR 151 with DB 1 bleeders bleeders ------------------------------------------------------------Wash with no laccase system 14 29 15 Wash with AS-based laccase system 5 10 Wash with MS-based laccase system 3 8 Typical significant differences in the delta E readings are 2o 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.

Claims (26)

1. A method for bleaching dyes or colorants in solution with a phenol oxidizing enzyme, characterized by the presence of an enhancing agent of the following formula:
wherein A is -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, -SO2-E, -N-XY, and -N+-XYZ; E is -H, -OH, -R, or -OR; and X and Y and Z are identical or different and selected from -H and -R, wherein R is a C1-C16 alkyl, which alkyl is saturated or unsaturated, branched or unbranched, and optionally substituted with a carboxy, sulfo, or amino group; and B and C are the same or different and selected from C m H2m+1, wherein 1 <= m <= 5.

2. A method according to claim 1, in which the enhancing agent is selected from the group consisting of acetosyringone, syringaldehyde, methylsyringate, and syringic acid.

3. A method according to any one of claims 1-2, in which the phenol oxidizing enzyme is a peroxidase and in which a hydrogen peroxide source is also present.

4. A method according to claim 3, wherein the peroxidase is horseradish peroxidase, soybean peroxidase, or a peroxidase enzyme derived from Coprinus, or Bacillus, or Myxococcus.

5. A method according to any one of claims 3-4, wherein the hydrogen peroxide source is hydrogen peroxide or a hydrogen peroxide precursor, or a hydrogen peroxide generating enzyme system, or a peroxycarboxylic acid or a salt thereof.

6. A method according to claim 1, in which the phenol oxidizing enzyme is a laccase or a laccase related enzyme together with oxygen.

7. A method according to claim 6, wherein the laccase or laccase related enzyme is a laccase which is derived from Trametes or Coprinus, or a bilirubin oxidase which is derived from Myrothecium.

8. A method according to any one of claims 1-7, in which said method is a method for inhibiting the transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor.

9. A method according to claim 8, in which the enhancing agent is added at the beginning of, or during, the process.

10. A method according to any one of claims 8-9, in which the concentration of the enhancing agent is in the range of from 0.01-1000 µM.

11. A detergent additive comprising a phenol oxidizing enzyme and an enhancing agent of the formula wherein A is -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, -SO2-E, -N-XY, and -N+-XYZ; E is -H, -OH, -R, or -OR; and X and Y and Z are identical or different and selected from -H and -R, wherein R is a C1-C16 alkyl, which alkyl is saturated or unsaturated, branched or unbranched, and optionally substituted with a carboxy, sulfo, or amino group; and B and C are the same or different and selected from C m H2m+1, wherein 1 <= m <= 5.

12. A detergent additive according to claim 11, in which the enhancing agent is selected from the group consisting of acetosyringone, syringaldehyde, methylsyringate, and syringic acid.

13. A detergent additive according to any one of claims 11-12, in which the phenol oxidizing enzyme is a peroxidase and in which a hydrogen peroxide source is also present.

14. A detergent additive according to claim 13, wherein the peroxidase is horseradish peroxidase, soybean peroxidase, or a peroxidase enzyme derived from Coprinus, or Bacillus, or Myxococcus.

15. A detergent additive according to any one of claims 13-14, wherein the hydrogen peroxide source is hydrogen peroxide or a hydrogen peroxide precursor, or a hydrogen peroxide generating enzyme system, or a peroxycarboxylic acid or a salt thereof.

16. A detergent additive according to claim 11, in which the phenol oxidizing enzyme is a laccase or a laccase related enzyme together with oxygen.

17. A detergent additive according to claim 16, wherein the laccase or laccase related enzyme is a laccase which is derived from Trametes or Coprinus, or bilirubin oxidase which is derived from Myrothecium.

18. A detergent additive according to any one of claims 11-17, provided in the form of a granulate, a liquid, a slurry, or a protected enzyme.

19. A detergent composition comprising a phenol oxidizing enzyme, a surfactant and an enhancing agent of the formula:

wherein A is -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, -SO2-E, -N-XY, and -N+-XYZ; E is -H, -OH, -R, or -OR; and X and Y and Z are identical or different and selected from -H and -R, wherein R is a C1-C16 alkyl, which alkyl is saturated or unsaturated, branched or unbranched, and optionally substituted with a carboxy, sulfo, or amino group;
and B and C are the same or different and selected from C m H2m+1, wherein 1<= m<= 5.

20. A detergent composition according to claim 19, in which the enhancing agent is selected from the group consisting of acetosyringone, syringaldehyde, methylsyringate, and syringic acid.

21. A detergent composition according to claim 20, in which the phenol oxidizing enzyme is a peroxidase and in which a hydrogen peroxide source is also present.

22. A detergent composition according to claim 21, wherein the peroxidase is horseradish peroxidase, soybean peroxidase, or a peroxidase enzyme derived from Coprinus, or Bacillus, or Myxococcus.

23. A detergent composition according to any one of claims 21-22, wherein the hydrogen peroxide source is hydrogen peroxide or a hydrogen peroxide precursor, or a hydrogen peroxide generating enzyme system, or a peroxycarboxylic acid or a salt thereof.

24. A detergent composition according to claim 19, in which the phenol oxidizing enzyme is a laccase or a laccase related enzyme together with oxygen.

25. A detergent composition according to claim 24, wherein the laccase or laccase related enzyme is a laccase which is derived from Trametes or Coprinus, or bilirubin oxidase which is derived from Myrothecium.

26. A detergent composition according to any one of claims 19-25, which further comprises one or more other enzymes.