AN ENZYME FOR DYING KERATINOUS FIBRES

Abstract: The present invention relates to a dyeing composition, a method for dying keratinous fibres, in particular hair, fur, hide and wool, and the use of a Scytalidium laccase for dyeing.

Description: Title: An enzyme for dying keratmous fibres FIELD OF THE INVENTION The present invention relates to a dyeing composition for keratmous fibres, in particular hair, fur, hide and wool, a method for dying and the use of a Scytalidi um laccase for dyeing. BACKGROUND OF THE INVENTION It has been used for many years to dye the hair to cover appearing grey hair. The need to do so arises from the fact that grey hair is the first sign of having past adolescence, which can be hard to accept for many people. For instance, in certain parts of Asia it is widely used by both men and women to dye the hair with dyes often referred to by humorous people as "black shoe polish" . During the last few decades hair dyeing has become more and more popular m the western world. At first Punk Rockers and other society critical groups dyed their hair in extreme colours as a part of their protest against the established society, but today especially many young people also uses hair dyes (in more soft tints than the Punk Rockers) as a sort of "cosmetic" to change or freshen up their "look". In general hair dyeing compositions on the market today can be divided into three mam groups: - temporary hair dyes, - semi-permanent hair dyes, and - permanent oxidative hair dyes. The temporary hair dyes are only intended to change the natural hair colour for a short period of time and usually functions by depositing dyes on the surface of the hair. Such hair dyes are easy to remove with normal shampooing. When using semi-permanent hair dyes the colour of the dyed hair can survive for five or more shampoo gs . This is achieved by using dyes havmg a high affinity for hair keratin and which is able to penetrate mto the interior of the hair shaft. Permanent hair dyes are very durable to sunlight, shampooing and other hair treatments and need only to be refreshed once a month as new hair grows out. With these dye g systems the dyes are created directly in and on the hair. Small aromatic colour¬ less dye precursors (e.g. p-phenylene-diamme and o- ammophenol) penetrate deep mto the hair where said dye precursors are oxidised by an oxidising agent mto coloured polymeric compounds. These coloured compounds are larger than the dye precursors and can not be washed out of the hair. By including compounds referred to as modifiers (or couplers) the hair dyemg composition a number of hair colour tints can be obtained. Cathecol and Resorcmol are examples of such modifiers. Traditionally H 2 0 2 is used as the oxidizing agent (colour builder), but also as a bleaching agent. Dyemg compositions comprising H 2 0_ are often referred to as "lightening dyes" due to this lightening effect of H 2 0 2 . The use of H 2 0 2 dye compositions have some disadvantages as H 2 0 damages the hair. Further, oxidative dyemg often demands high pH (normally around pH 9-10) , which also inflicts damage on the hair. Consequently, if usmg dye compositions comprising H 2 C_ it is not recommendable to dye the hair often. To overcome the disadvantages of using H?0 t has been sug¬ gested to use oxidation enzymes to replace H 2 0 2 . US patent no. 3,251,742 (Revlon) describes a method for dye g human hair by dye formation in si tu { i . e . on the hair) . An oxidative enzyme is used to the colour formation reactions at a substantially neutral pH (pH 7-8.5) . Laccases, tyrosmases, polyphenolases and catacolases are mentioned as the suitable oxidation enzymes. EP patent no. 504.005 (Perma S.A.) concerns compositions for dying hair which do not require the presence of H 2 0 (hydrogen peroxide) . The composition comprises an enzyme capable of catalyzing the formation of the polymeric dyes and also dye precursors, such as bases and couplers, a buffer solution wherem the pH of said composition is between 6.5 and 8 and said enzyme has an optimal activity in the pH range between 6.5 and 8. Rhi zoctonia praticola laccase and Rhus vermcifera laccase have a pH-optimum between 6.5 and 8 and can be used to form the polymeric dyes accordmg to this patent. Abstract of Papers American Chemical Society vol. 209, no. 1-2, 1995 discloses the cloning of a laccase from a Scytalidi um thermophilum. The abstract does not mention the use of said laccase for dye g hair. SUMMARY OF THE INVENTION The obiect of the present invention is to provide improved permanent dyemg compositions for keratmous fibres, m parti¬ cular hair, fur, hide and wool, which is less damaging to the keratmous fibres than e.g. dyemg compositions for hair using H 2 0 2 . It has now surprisingly been found that it is possible to provide such an improved dyemg composition by using an enzyme derived from a strain of the filamentous fungus genus Scytali - dium as the oxidation enzyme. In the first aspect the mvention relates to a permanent dyemg composition for keratmous fibres, in particular hair, fur, hide and wool, comprising an oxidation enzyme comprising 1) one or more oxidation enzymes derived from a strain of the genus Scytalidium, 2 ) one or more dye precursors, and optionally 3) one or more modifiers. In a preferred embodiment of the invention the oxidation en¬ zyme is a laccase derived from a strain of the genus Scytali - dium, m particular from a strain of the species Scytalidi um thermophilum . Secondly, it is the ob3ect of the invention to provide a method for dying keratmous fibres, comprising contacting a laccase derived from a strain of the genus Scytalidium with the keratmous fibres and at least one dye precursor m the pre¬ sence or absence of at least one modifier for a suitable period of time and under conditions sufficient to permit oxidation of the dye precursor mto a coloured compound. SU TESHEET Finally the invention relates to the use of an oxidation enzyme derived from a strain of the genus Scytalidi um for oxidative dyemg of keratmous fibres, m particular hair, fur, hide and wool. BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows the dyeing effect of the Scytalidi um thermophil um laccase (rStL-FXu-1) DETAILED DESCRIPTION OF THE INVENTION The object of the present invention is to provide improved permanent dyemg compositions for keratmous fibres, m parti¬ cular hair, fur, hide and wool, which is less damaging to the keratmous fibres than e.g. hair dyemg compositions using H 2 0 2 . It has surprisingly be found that it is possible to provide such an improved dyemg composition by using an oxidation enzyme derived from a strain of the filamentous fungus genus Scytalidium. When using said oxidation enzyme derived from a strain of the genus Scytalidi um the colour developed is as wash stable as oxidative dyemg of e.g. hair using H 2 0 2 and the light fastness is as good as when dyemg chemically. Consequently, m the first aspect the present invention re¬ lates to a permanent dye composition for keratmous fibres , in particular hair, fur, hide and wool, comprising 1) one or more oxidation enzymes derived from a strain of the genus Scytalidi um, 2 ) one or more dye precursors, and optionally 3) one or more modifiers. In an embodiment of the invention the oxidation enzyme is a laccase derived from a strain of genus Scytalidi um, such as a strain of Scytalidi um thermophil um e . g. the purified laccase described m WO 95/33837 (PCT/US95/06816) from Novo Nordisk, which is hereby incorporated. SEQ ID No 1 shows a DNA sequence encoding a suitable laccase derivable from a strain of the spe¬ cies Scytalidi um thermophilum . E. coli JM101 containing the expression vector pShThl5 comprising SEQ ID NO 1 has been deposited under the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoπa, Illinois, 61604. The vector have been given the Accession Number NRRL B-21262. Also contemplated according to the invention are laccases derived from other microorganisms being more than 80° homo¬ logous to SEQ ID NO 1 derived from a strain of the species Scytalidi um thermophil um. In addition, Scytalidi um laccases also encompass alternative forms of laccases which may be found m S. thermophil um and as well as laccases which may be found in other fungi which are synonyms of fall within the definition of S. thermophil um as defined by Straatsma and Samson, (1993), Mycol . Res. 97, 321- 328) . These mclude S. mdonesiacum, Torula thermophila, Humi - cola brevi s var. thermoidea, Humi cola brevi spora, H. grisea var. thermoidea, Humi cola msol ens, and Humi cola lanugmosa (also known as Thermomyces lanugmosus) . It is to be understood that the Scytalidi um laccase may be produced homologously, or heterologously using filamentous fungus, yeast or bacteria as the host cell. Examples of filamentous fungi host cells mclude strains of the species of Trichoderma, preferably a strain of Tri choderma harzianum or Trichoderma reesei , or a species of Aspergill us, most preferably Aspergill us oryzae or Aspergill us niger, or yeast cells, such as e.g. a strain of Saccharomyces, in parti¬ cular Saccharomyces cerevisiae, Saccharomyces kl uyveri or Saccharomyces uvaru , a strain of Schizosaccharomyces sp. , such as Schizosaccharomyces pombe, a strain of Hansenula sp. , Pichia sp., Yarrowia sp., such as Yarrowia l polytica, or Kluyveromy- ces sp., such as _luyveromyces lactis, or a bacteria, such as gram-positive bacteria such as strains of Bacill us, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkal ophilus, B. amyl oliquefaciens, B. coagulans, B. circulans, B . lautus, B. megateri um or B. thuringiensis, or strains of Streptomyces, such as S . l vidans or S. murmus, or gram-negative bacteria such as Escherichia coli . Laccases (benzenediol:oxygen oxidoreductases) (E.C. class TESHEET 1.10.3.2 accordmg to Enzyme Nomenclature (1992) Academic Press, Inc) are multi-copper containing enzymes that catalyze the oxidation of phenols. Laccase-mediated oxidations result in the production of aryloxy-radical intermediates from suitable phenolic substrates; the ultimate coupling of the intermediates so produced provides a combination of dimeπc, oligomeric, and polymeric reaction products. Certain reaction products can be used to form dyes suitable for dyemg hair (see below) . In an embodiment of the invention the Scytalidi um laccase is neutral. In the context of laccases of the present mvention this means that the pH optimum lies m the range from between 6.0 and 8.0. To obtain dyemg of the keratmous fibres, such as hair, the dyemg composition of the invention also comprises a dye precursor which is converted mto a coloured compound (i.e. a dye) by the oxidation agent which according to the invention is an oxidation enzyme derived from a strain of the species Scytalidi um, such as a strain of Scytalidi um thermophil um . Without being limited thereto the dye precursor (s) may be (an) aromatic compound(s) belonging to one of three major che¬ mical families: the diammes, am ophenols (or ammonaphtols) and the phenols. Examples of isatin derivative dye precursors can be found m DE 4, 314, 317-A1. Further, a number of mdole or mdolme derivative dye precursors are disclosed m WO 94/00100. Said dye precursors mentioned these documents are hereby incorporated herein by reference. Examples of such suitable dye precursors include compounds from the group comprising p-phenylene-diamme (pPD) , p-tolu- ylene-diamme, chloro-p-phenylenediamme, p-ammophenol, o-ami- nophenol and 3, 4-dιammotoluene, 2-methyl-l, 4-dιammobenzene, 4-methyl-o-phenylenedιamιne, 2-methoxy-p-phenylenedιamme, 2- chloro-1, 4-dιammo-benzene, 4-ammo diphenylamme, l-ammo-4-β- methoxyethylamino-benzene, l-ammo-4-bιs- (β-hydroxyethyl) -amo- nibenzene, 1-3-dιammo-benzene, 2-methyl-l, 3-dιammo-benzene, 2, -dιammotoluene, 2, 6-dιammopyrιdme, l-hydroxy-2-ammo-ben- zene, l-hydroxy-3-ammo-benzene, l-methyl-2-hydroxy-4-ammo- benzene, l-methyl-2-hydroxy-4-β-hydroxyethylammo-benzene, 1- hydroxy-4-ammo-ebnzene, l-hydroxy-4-methylammo-benzene , 1-me- thoxy-2 , -dιammo-benzene, l-ethoxy-2 , 3-dιammo-benzene, 1-β- hydroxyethyloxy-2 , -dιammo-benzene, phenazmes , such as 4 , 7- phenaz edicarboxylic acid, 2 , 7-phenazmedιcarboxylιc acid, 2- phenazmecarboxylic acid, 2 , 7-dιammophenazme , 2 , 8-dιammo- phenazme , 2 , 7-dιammo-3 , 8-dιmethoxyphenazιne , 2 , 7-dιammo-3- methoxyphenazme, 2 , 7-dιammo 3-methoxyphenazme, 3-dιmethyl 2 , 8-phenazmedιamme, 2 , 2 ' - [ ( 8-ammo-7-methyl-2-phenazmyl ) l i- no] bιs-ethanol, 2 , 2 ' - [ ( 8-ammo-7-methoxy-2-phenazιnyl ) l i- no] bιs-ethanol, 2 , 2 ' - [ ( 8-amιno-7-chloro-2-phenazmyl ) immo ] bιs- ethanol , 2- [ ( 8-amιno-7-methyl-2-phenazmyl ) ammo] -ethanol , 2,2'- [ (8-amιno-2-phenazmyl) lmmo]bis-ethanol, 3-ammo-7- (dimethylammo) -2, 8-dιmethyl-5-phenyl-chloπde, 9- (diethylami- no)- benzo[ajphenazme-1, 5-dιol, N- [8- (diethylam o) -2-phenazι- nyl]- methanesulfonamide, N- (8-methoxy-2-phenazmyl) - Methane- sulfonamide, N,N,N' ,N' -tetramethyl-2, 7-phenazmedιamme, 3,7- dιmethyl-2-phenazmamme, p-ammo benzoic acids, such as p- amino benzoic acid ethyl, p-ammo benzoic acid glycerid, p- amino benzoic acid isobutyl, p-dimethylammo benzoic acid amil, p-dimethylammo benzoic acid octyl, p-diethoxy am o benzoic amil, p- dipropoxy ammo benzoic acid ethyl, acetylsalicylic acid, lsatm derivatives, such as 2,3-dιammo benzoic acid. In an embodiment the laccase is used with the dye precursor directly to oxidise it mto a coloured compound. The dye precursor may be used alone or m combination with other dye precursors. However, it is believed that when using a diamine or an a mophenol as the dye precursor at least one of the intermediate m the copolymerisation must be an ortho- or para - diamine or ammophenol. Examples of such are described in US patent no. 3,251,742 (Revlon) , the contents of which are incorporated herein by reference. Optionally the dyemg composition of the invention (especially hair dyemg compositions) also comprises a modifier (coupler) by which a number of colour tints can be obtained. In general modifiers are used in hair dyemg compositions, as the colours resulting from hair dyemg compositions without modifier (s) are usually unacceptable for most people. Modifiers are typically m-diamines, m-ammophenols, or polyphenols. The modifier (coupler) reacts with the dye precur¬ sor (s) m the presence of the oxidative enzyme, converting it into a coloured compound. Examples of modifiers (couplers) mclude m-phenylene- diamme, 2, 4-dιammoanιsole, 1-hydroxynaphthalene ( -naphthol) , 1, 4-dιhydroxybenzene (hydroqumone) , 1, 5-dιhydroxynapthalene, 1,2-dιhydroxybenzene (pyrocatechol) , 1, 3-dιhydroxybenzene (re- sorcmol), 1, 3-dιhydroxy-2-methylbenzene, 1, 3-dιhydroxy-4- chlorobenzene (4-chlororesorcmol) , 1, 2, 3, trihydroxybenzene, 1,2, 4-trιhydroxybenzene, 1,2, 4-tπhydroxy-5-methylbenzene, 1,2, 4-trihydroxytoluene. In the second aspect the invention relates to a method for dying keratmous fibres, in particular hair, fur, hide and wool, comprising contacting a laccase derived from a strain of the genus Scytalidi um with the keratmous fibres and at least one dye precursor in the presence or absence of at least one modifier, for a period of time and under conditions sufficient to permit oxidation of the dye precursor mto coloured compounds ( .e. a dye) . The dyemg method can be conducted with one or more dye precursors, either alone or m combination with one or more modifiers. The amount of dye precursor (s) and other ingredients used in the composition of the invention are in accordance with usual commercial amounts. When using a Scytalidi um laccase, such as the Scytalidium thermophil um laccase mentioned above, the method for dye g keratmous fibres of the mvention may be carried out at room temperature, preferably around the optimum temperature of the enzyme, at a pH m the range from 3.0 to 9.0, preferably 4.0 to 8.0, especially pH 6.0 to 8.0. Suitable dye precursors and optional modifiers are described above. The use of this Scytalidium laccase is an improvement over the more traditional use of H 2 0 as the latter can damage the keratmous fibres, such as hair. Further, normally prior art methods requires a high pH, which is also damaging to the keratmous fibres. In contrast hereto, the reaction with laccase can be conducted at acidic or neutral pH, and the oxygen needed for oxidation comes from the air, rather than via harsh chemical oxidation. The result provided by the use of the Scytalidi um laccase is comparable to that achieved with use of H 2 0 2 , not only in colour development, but also wash stability and light fastness. An additional commercial advantage is that a single container package can be made containing both the laccase and the precursor, an oxygen free atmosphere, which arrangement is not possible with the use of H 2 0 2 . MATERIALS AND METHODS Materials: Ha r: 6" De Meo Virgin Natural White Hair (De Meo Brothers Inc. US) Enzymes: Laccase from Scytalidi um thermophilum described WO 95/33837 (PCT/US95/06816) from Novo Nordisk Deposit of Biological Material The following biological material has been deposited on the 25 th May 1994 under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoπa, Illinois, 61604 and given the following accession number. Deposit Accession Number E. coli JM101 containing pShThl5 NRRL B-21262. Dye precursors: 0 . 1 % w/w p-phenylene-diamme m 0. 1 M K-phosphate buf fer , pH 7 . 0 . (pPD) 0 . 1 % w/w p-toluylene-diamme in 0 . 1 M K-phosphate buffer, pH 7 . 0 . 0 . 1 % w/w chloro-p-phenylenediamme 0 . 1 M K-phosphate SUBSTITUTE SHEET buffer, pH 7.0. 0.1 % w/w p-aminophenol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w o-aminophenol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w 3, 4-diaminotoluene in 0.1 M K-phosphate, buffer pH 7.0. Modifiers: 0.1 % w/w m-phenylene-diamine in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w 2, 4-diaminoanisole in 0,1 M K-phosphate buffer, pH 7.0. 0.1 % w/w a-naphthol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w hydroquinone in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w pyrocatechol in 0.1 M K-phosphate buffer, pH 7.0. 0.1% w/w resorcinol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % w/w 4-chlororesorcmol in 0.1 M K-phosphate buffer, pH 7.0. The dye precursor is combined with one of the above indicated modifiers so that the final concentration in the dyeing solution is 0.1 % w/w with respect to precursor and 0.1 % w/w with respect to modifier. Other solutions: 3% H 2 0 2 (in the final dye solution) Commercial shampoo Equipment: Minolta CR200 Chroma Meter Day light bulb: 1000 LUX (D65) Determination of Laccase Activity (LACU) Laccase activity is determined from the oxidation of syrin- galdazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 mM 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 micromole syrmgaldazm per mmute at these conditions. Assessment of the hair colour The quantitative colour of the hair tresses are determined on a Minolta CR200 Chroma Meter by the use the parameters L* ("0"=black and "100"=whιte) , a* ("-"=green and "+"=red) and b* ("-" blue and *'+" yellow) . DL*, Da* and Db* are the delta values of L*, a* and b* respect¬ ively compared to L*, a* and b* of untreated hair (e.g. DL* = I* sample - Li untreated hair - DE* is calculated as DE*=0(DL* 2 +Da* 2 +Db*) and is an expression for the total quantitative colour change. EXAMPLES Example 1 Dyemg effect The dyemg effect of a Scytalidi um thermophilum laccase was tested using the dye precursor o-ammophenol and the modifier m-phenylenediamine. Hair dye g 1 gram De Meo white hair tresses were used. 4 ml dye precursor solution (including modifier) is mixed with 1 ml laccase on a Whirley mixer, applied to the hair tresses and incubated at 30°C for 60 minutes. The hair tresses are then rinsed with running water, washed with shampoo, rinsed with running water, combed, and air dried. The a*, b* and L* was determined on the Chroma Meter and the DE* values were then calculated. A hair tress sample treated without enzyme was used as a blind. The result of the hair dyeing test is shown m figure 1. T Example 2 Wash stability Tresses of white De Meo hair (1 gram) is used for testing the wash stability of hair dyed using Scytalidi um thermophil um laccase, compared with hair dyed using H 2 0 2 , and p-phenylene- diamme (pPD) as the dye precursor. Further the wash stability is compared with a commercial oxidative dye. The oxidative hair dyemg is carried out as described in Example 1. Hair wash The dyed hair tresses are wetted and washed for 15 seconds with 50 ml of commercial shampoo, and rinsed with water for 1 mmute and air dried. The hair tresses are washed up to 18 times. The a*, b* and L* is determined o the Chroma Meter and the ΔE* values are then calculated. Example 3 The light fastness Tresses of blond European hair are used for testing the light fastness of hair dyed usmg Scytalidi um thermophil um laccase in comparison to hair dyed using H ^ 0 2 . p-phenylene- diamme was used as dye precursor. The dyemg of the hair was carried out as described in Example 1. One hair tress is kept dark, while an other is kept at day light (i.e. under a day light bulb (D65) ) , at approximately 1000 LUX) for up to 275 hours. The a*, b* and L* parameters are determined immediately after the dyeing of the hair, and further during exposure to day light. DE* then calculated from the determined a*, b* and L* values. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Novo Nordisk A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (D) COUNTRY: Denmark (E) POSTAL CODE (ZIP): DK-2880 (F) TELEPHONE: +45 4444 8888 (G) TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: An enzyme for dying hair (iϋ) NUMBER OF SEQUENCES: 2 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2476 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Scytalidium thermophilum (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 349..411 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 502..559 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 632..686 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 1739..1804 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join (106..348, 412..501, 560..631, 687..1738, 1805..2194) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGAATTTAA ATACAGGAAG ATCGCATTCA ATCCAGCCTA GACTGCACAA TGGTTCTGCA 60 CGACCGTCGC ACACCTGCCA ATAGTGTTAA TAACGGNCTA ATACC ATG AAG CGC TTC 117 Met Lys Arg Phe 1 TTC ATT AAT AGC CTT CTG CTT CTC GCA GGG CTC CTC AAC TCA GGG GCC 165 Phe Ile Asn Ser Leu Leu Leu Leu Ala Gly Leu Leu Asn Ser Gly Ala 5 10 15 20 CTC GCG GCT CCG TCT ACA CAT CCC AGA TCA AAC CCC GAC ATA CTG CTT 213 Leu Ala Ala Pro Ser Thr His Pro Arg Ser Asn Pro Asp Ile Leu Leu 25 30 35 GAA AGA GAT GAC CAC TCC CTT ACG TCT CGG CAA GGT AGC TGT CAT TCT 261 Glu Arg Asp Asp His Ser Leu Thr Ser Arg Gin Gly Ser Cys His Ser 40 45 50 CCA AGC AAC CGC GCC TGT TGG TGC TCT GGC TTC GAT ATC AAC ACG GAT 309 Pro Ser Asn Arg Ala Cys Trp Cys Ser Gly Phe Asp Ile Asn Thr Asp 55 60 65 TAT GAG ACC AAG ACT CCA AAC ACC GGA GTG GTG CGG CGG GTTAGTATCC 358 Tyr Glu Thr Lys Thr Pro Asn Thr Gly Val Val Arg Arg 70 75 80 CAAGTTACGT TTGACCAAGA AATGGACGTG AAGTGTGCTG ACTCTCCCGC TAG 411 TAC ACC TTT GAT ATC ACC GAA GTC GAC AAC CGC CCC GGT CCC GAT GGG 459 Tyr Thr Phe Asp Ile Thr Glu Val Asp Asn Arg Pro Gly Pro Asp Gly 85 90 95 GTC ATC AAG GAG AAG CTC ATG CTT ATC AAC GAC AAA CTC CTG GTAGG 506 Val Ile Lys Glu Lys Leu Met Leu Ile Asn Asp Lys Leu Leu 100 105 110 GTCCTCTCGA ACGCCTGCGT CTGCCACACA GCGTAAAACT AACGAACCGC TAG 559 GGC CCG ACA GTC TTC GCA AAC TGG GGC GAC ACC ATC GAG GTG ACC GTC 607 Gly Pro Thr Val Phe Ala Asn Trp Gly Asp Thr Ile Glu Val Thr Val 115 120 125 AAC AAC CAC CTG AGA ACC AAC GGA GTAAGCGTTC GGACACAAAG CCCAGCAACC 661 Asn Asn His Leu Arg Thr Asn Gly 130 135 TAGACACACT CAACTGACCA AGTAG ACC TCC ATC CAC TGG CAC GGC TTG CAC CAA 716 Thr Ser Ile His Trp His Gly Leu His Gin 140 145 AAA GGA ACC AAC TAC CAC GAC GGC GCC AAC GGC GTG ACC GAG TGT CCC 764 Lys Gly Thr Asn Tyr His Asp Gly Ala Asn Gly Val Thr Glu Cys Pro 150 155 160 ATC CCG CCC GGT GGC TCC CGA GTC TAC AGC TTC CGA GCG CGC CAA TAT 812 Ile Pro Pro Gly Gly Ser Arg Val Tyr Ser Phe Arg Ala Arg Gin Tyr 165 170 175 GGA ACG TCA TGG TAC CAC TCC CAC TTC TCC GCC CAG TAT GGC AAC GGC 860 Gly Thr Ser Trp Tyr His Ser His Phe Ser Ala Gin Tyr Gly Asn Gly 180 185 190 GTG AGC GGC GCC ATC CAG ATC AAC GGA CCC GCC TCC CTG CCC TAC GAC 908 Val Ser Gly Ala Ile Gin Ile Asn Gly Pro Ala Ser Leu Pro Tyr Asp 195 200 205 ATC GAC CTC GGC GTC CTC CCG CTG CAG GAC TGG TAC TAC AAG TCC GCC 956 Ile Asp Leu Gly Val Leu Pro Leu Xaa Asp Trp Tyr Tyr Lys Ser Ala 210 215 220 225 GAC CAG CTC GTC ATC GAG ACC CTG GCC AAG GGC AAC GCT CCG TTC AGC 1004 Asp Gin Leu Val Ile Glu Thr Leu Xaa Lys Gly Asn Ala Pro Phe Ser 230 235 240 GAC AAC GTC CTC ATC AAC GGC ACC GCA AAG CAC CCC ACC ACT GGC GAA 1052 Asp Asn Val Leu Ile Asn Gly Thr Ala Lys His Pro Thr Thr Gly Glu 245 250 255 GGG GAG TAC GCC ATC GTG AAG CTC ACC CCG GGC AAA CGC CAT CGC CTG 1100 Gly Glu Tyr Ala Ile Val Lys Leu Thr Pro Asp Lys Arg His Arg Leu 260 265 270 CGG CTC ATC AAC ATG TCG GTG GAG AAC CAC TTC CAG GTC TCG CTG GCG 1148 Arg Leu Ile Asn Met Ser Val Glu Asn His Phe Gin Val Ser Leu Ala 275 280 285 AAG CAC ACC ATG ACG GTC ATC GCG GCG GAC ATG GTC CCC GTC AAC GCC 1196 Lys His Thr Met Thr Val Ile Ala Ala Asp Met Val Pro Val Asn Ala 290 295 300 305 ATG ACC GTC GAC AGC CTG TTT ATG GCC GNC GGG CAG CGG TAT GAT GTT 1244 Met Thr Val Asp Ser Leu Phe Met Ala Val Gly Gin Arg Tyr Asp Val 310 315 320 ACC ATC GAC GCG AGC CAG GCG GTG GGG AAT TAC TGG TTC AAC ATC ACC 1292 Thr Ile Asp Ala Ser Gin Ala Val Gly Asn Tyr Trp Phe Asn lie Thr 325 330 335 TTT GGA GGG CAG CAG AAG TGC GGC TTC TCG CAC AAT CCG GCG CCG GCA 1340 Phe Gly Gly Gin Gin Lys Cys Gly Phe Ser His Asn Pro Ala Pro Ala 340 345 350 GCC ATC TTT CGC TAC GAG GGC GCT CCT GAC GCT CTG CCG ACG GAT CCT 1388 Ala Ile Phe Arg Tyr Glu Gly Ala Pro Asp Ala Leu Pro Thr Asp Pro 355 360 365 GGC GCT GCG CCA AAG GAT CAT CAG TGC CTG GAC ACT TTG GAT CTT TCA 1436 Gly Ala Ala Pro Lys Asp His Gin Cys Leu Asp Thr Leu Asp Leu Ser 370 375 380 385 CCG GTG GTG CAA AAG AAC GTG CCG GTT GAC GGG TTC GTC AAA GAG CCT 1484 Pro Val Val Gin Lys Asn Val Pro Val Asp Gly Phe Val Lys Glu Pro 390 395 400 GGC AAT ACG CTG CCG GTG ACG CTC CAT GTT GAC CAG GCC GCG GCT CCA 1532 Gly Asn Thr Leu Pro Val Thr Leu His Val Asp Gin Ala Ala Ala Pro 405 410 415 CAC GTG TTT ACG TGG AAG ATC AAC GGG AGC GCT GCG GAC GTG GAC TGG 1580 His Val Phe Thr Trp Lys Ile Asn Gly Ser Ala Ala Asp Val Asp Trp 420 425 430 GAC AGG CCG GTG CTG GAG TAT GTC ATG AAC AAT GAC CTG TCT AGC ATT 1628 Asp Arg Pro Val Leu Glu Tyr Val Met Asn Asn Asp Leu Ser Ser Ile 435 440 445 CCG GTC AAG AAC AAC ATT GTG AGG GTG GAC GGA GTC AAC GAG TGG ACG 1676 Pro Val Lys Asn Asn Ile Val Arg Val Asp Gly Val Asn Glu Trp Thr 450 455 460 465 TAC TGG CTC GTC GAA AAC GAC CCG GAG GGC CGC CTC AGT TTG CCG CAT 1724 Tyr Trp Leu Val Glu Asn Asp Pro Glu Gly Arg Leu Ser Leu Pro His 470 475 470 CCG ATG CAT CTA CAC GTAAGTCACA TCCCCCACTA CCATTCGGAA TGACCACCAG 1779 Pro Met His Leu His 475 GTACTGACAC CCTCCTCCTC AATAG GGA CAC GAT TTC TTT GTC CTA GGC CGC 1831 Gly His Asp Phe Phe Val Leu Gly Arg 480 485 TCC CCC GAC GTC TCG CCC GAT TCA GAA ACC CGC TTC GTC TTT GAC CCG 1879 Ser Pro Asp Val Ser Pro Asp Ser Glu Thr Arg Phe Val Phe Asp Pro 490 495 500 GCC GTC GAC CTC CCC CGT CTG CGC GGA CAC AAC CCC GTC CGG CGC GAC 1927 Ala Val Asp Leu Pro Arg Leu Arg Gly His Asn Pro Val Arg Arg Asp 505 510 515 SUBSTITUTE SHEET GTC ACC ATG CTT CCC GCG CGC GGC TGG CTG CTG CTG GCC TTC CGC ACG 1975 Val Thr Met Leu Pro Ala Arg Glu Trp Leu Leu Leu Ala Phe Arg Thr 520 525 530 GAC AAC CCG GGC GCG TGG TTG TTC CAC TGC CAC ATC GCG TGR CAC GTG 2023 Asp Asn Pro Gly Ala Trp Leu Phe His Cys His Ile Ala Trp His Val 535 540 545 TCG GGC GGG TTA AGC GTC GAC TTT CTG GAG CGG CCG GAC GAG CTG CGC 2071 Ser Gly Gly Leu Ser Val Asp Phe Leu Glu Arg Pro Asp Glu Leu Arg 550 555 560 565 GGG CAG CTG ACG GGA GAG AGC AAG GCG GAG TTG GAG CGT GTT TGT CGC 2119 Gly Gin Leu Thr Gly Glu Ser Lys Ala Glu Leu Glu Arg Val Cys Arg 570 575 580 GAG TGG AAG GAT TGG GAG GCG AAG AGC CCG CAT GGG AAG ATC GAT TCG 2167 Glu Trp Lys Asp Trp Glu Ala Lys Ser Pro His Gly Lys Ile Asp Ser 585 590 595 GGG TTG AAG CAG CGG CGA TGG GAT GCG TGAGGTAGTT GGGCGGATTG 2214 Gly Leu Lys Gin Arg Arg Trp Asp Ala 600 605 TTTAACACGT AGTGGGTAAG GTTGGGGCGG GTTTGTTTGG CGTTTTCAGG GGTTGGGGTG 2274 CGGATGCTGG TCATCCGGGA AACGGCTCTA CAACTGGTGT CAATAGACTA ATATAGAGTG 2334 ATCAAAGAAC TGAGGTTCTG AAAGAGGCGT GGAAGTCGCG TTGTGACTCC CTTTGCCATG 2394 TTGGGAAGTG TGGCTCAACA TTGTGTTCAG GTTTGCTCAG GGTGATNTCG AACTGACGTN 2454 TTGATGAGGG TTATTGCNTA GA 2476 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 616 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Scytalidium thermophilum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Arg Phe Phe Ile Asn Ser Leu Leu Leu Leu Ala Gly Leu Leu 1 5 10 15 Asn Ser Gly Ala Leu Ala Ala Pro Ser Thr His Pro Arg Ser Asn Pro 20 25 30 Asp Ile Leu Leu Glu Arg Asp Asp His Ser Leu Thr Ser Arg Gin Gly 35 40 45 Ser Cys His Ser Pro Ser Asn Arg Ala Cys Trp Cys Ser Gly Phe Asp 50 55 60 Ile Asn Thr Asp Tyr Glu Thr Lys Thr Pro Asn Thr Gly Val Val Arg 65 70 75 80 Arg Tyr Thr Phe Asp Ile Thr Glu Val Asp Asn Arg Pro Gly Pro Asp 85 90 95 Gly Val Ile Lys Glu Lys Leu Met Leu lie Asn Asp Lys Leu Leu Gly 100 105 110 Pro Thr Val Phe Ala Asn Trp Gly Asp Thr Ile Glu Val Thr Val Asn 115 120 125 Asn His Leu Arg Thr Asn Gly Thr Ser Ile His Trp His Gly Leu His 130 135 140 Gin Lys Gly Thr Asn Tyr His Asp Gly Ala Asn Gly Val Thr Glu Cys 145 150 155 160 Pro Ile Pro Pro Gly Gly Ser Arg Val Tyr Ser Phe Arg Ala Arg Gin 165 170 175 Tyr Gly Thr Ser Trp Tyr His Ser His Phe Ser Ala Gin Tyr Gly Asn 180 185 190 Gly Val Ser Gly Ala Ile Gin Ile Asn Gly Pro Ala Ser Leu Pro Tyr 195 200 205 Asp Ile Asp Leu Gly Val Leu Pro Leu Gin Asp Trp Tyr Tyr Lys Ser 210 215 220 Ala Asp Gin Leu Val Ile Glu Thr Leu Ala Lys Gly Asn Ala Pro Phe 225 230 235 240 Ser Asp Asn Val Leu Ile Asn Gly Thr Ala Lys His Pro Thr Thr Gly 245 250 255 Glu Gly Glu Tyr Ala Ile Val Lys Leu Thr Pro Asp Lys Arg His Arg 260 265 270 Leu Arg Leu Ile Asn Met Ser Val Glu Asn His Phe Gin Val Ser Leu 275 280 285 Ala Lys His Thr Met Thr Val Ile Ala Ala Asp Met Val Pro Val Asn 290 295 300 Ala Met Thr Val Asp Ser Leu Phe Met Ala Xaa Gly Gin Arg Tyr Asp 305 310 315 320 Val Thr Ile Asp Ala Ser Gin Ala Val Gly Asn Tyr Trp Phe Asn Ile 325 330 335 Thr Phe Gly Gly Gin Gin Lys Cys Gly Phe Ser His Asn Pro Ala Pro 340 345 350 Ala Ala Ile Phe Arg Tyr Glu Gly Ala Pro Asp Ala Leu Pro Thr Asp 355 360 365 Pro Gly Ala Ala Pro Lys Asp His Gin Cys Leu Asp Thr Leu Asp Leu 370 375 380 Ser Pro Val Val Gin Lys Asn Val Pro Val Asp Gly Phe Val Lys Glu 385 390 395 400 Pro Gly Asn Thr Leu Pro Val Thr Leu His Val Asp Gin Ala Ala Ala 405 410 415 Pro His Val Phe Thr Trp Lys Ile Asn Gly Ser Ala Ala Asp Val Asp 420 425 430 Trp Asp Arg Pro Val Leu Glu Tyr Val Met Asn Asn Asp Leu Ser Ser 435 440 445 Ile Pro Val Lys Asn Asn Ile Val Arg Val Asp Gly Val Asn Glu Trp 450 455 460 SUBSTITUTE SHEET Thr Tyr Trp Leu Val Glu Asn Asp Pro Glu Gly Arg Leu Ser Leu Pro 465 470 475 480 His Pro Met His Leu His Gly His Asp Phe Phe Val Leu Gly Arg Ser 485 490 495 Pro Asp Val Ser Pro Asp Ser Glu Thr Arg Phe Val Phe Asp Pro Ala 500 505 510 Val Asp Leu Pro Arg Leu Arg Gly His Asn Pro Val Arg Arg Asp Val 515 520 525 Thr Met Leu Pro Ala Arg Gly Trp Leu Leu Leu Ala Phe Arg Thr Asp 530 535 540 Asn Pro Gly Ala Trp Leu Phe His Cys His Ile Ala Trp His Val Ser 545 550 555 560 Gly Gly Leu Ser Val Asp Phe Leu Glu Arg Pro Asp Glu Leu Arg Gly 565 570 575 Gin Leu Thr Gly Glu Ser Lys Ala Glu Leu Glu Arg Val Cys Arg Glu 580 585 590 Trp Lys Asp Trp Glu Ala Lys Ser Pro His Gly Lys Ile Asp Ser Gly 595 600 605 Leu Lys Gin Arg Arg Trp Asp Ala 610 615 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bts) For receiving Office use only For International Bureau use only α This sheet was received with the international application I I This sheet was received with the International Bureau on Authorized officer Authorized officer form . CT / ..U/ U4 (.July 1V- SUBSTIT TE SHEET

Claims: PATENT CLAIMS 1. A dyemg composition comprising an oxidation enzyme characterised in that the composition comprises: 1) one or more oxidation enzymes derived from a strain of the genus Scytalidium, 2 ) one or more dye precursors, and optionally 3) one or more modifiers. 2. The dyeing composition according to claim 1, wherein the oxidation enzyme is derived from a strain of the genus Scytalidi um laccase 3. The dyeing composition according to claim 2 , wherein the laccase is derived from a strain of the species Scytalidi um th ermophil urn . 4. The dyeing composition according to claims 2 and 3, wherein the laccase is neutral. 5. The dyeing composition according to claim 3, having the sequence shown in SEQ ID No 1. 6. The dyeing composition according to claim 5, wherein the sequence encoding the laccase is homologous to the SEQ ID NO 1. 7. The dyeing composition according to claim 6, wherein the sequence encoding the laccase is more than 80% homologous to SEQ ID NO 1. 8. The dyemg composition accordmg to any of claims 1 to 7, comprising a dye precursor selected from the group comprising p-phenylene-diamme (pPD) , p-toluylene-diamme, chloro-p-phe- nylenediamine, p-aminophenol, o-aminophenol and 3, 4-dιammoto- luene, 2-methyl-l, 4-diam obenzene, 4-methyl-o-phenylenedιamι- ne, 2-methoxy-p-phenylenedιamme, 2-chloro-l, 4-dιamιno-benzene, 4-amιno diphenylamine, l-ammo-4-β-methoxyethylammo-benzene, l-ammo-4-bιs- (β-hydroxyethyl) -amonibenzene, 1-3-dιammo-benze- ne, 2-methyl-l, 3-dιammo-benzene, 2, 4-dιammotoluene, 2, 6-dι- aminopyridine, l-hydroxy-2-amιno-benzene, l-hydroxy-3-amιno- benzene, l-methyl-2-hydroxy-4-ammo-benzene, l-methyl-2-hydro- xy-4-β-hydroxyethylammo-benzene, l-hydroxy-4-amιno-benzene, 1- hydroxy-4-methylamιno-benzene, l-methoxy-2, -dιammo-benzene, l-ethoxy-2, 3-dιamιno-benzene, l-β-hydroxyethyloxy-2, 4-dιamιno- benzene, phenazmes, such as 4, 7-phenaz edιcarboxylιc acid, 2, 7-phenazmedιcarboxylιc acid, 2-phenaz ecarboxylιc acid, 2, 7-dιammophenazme, 2 , 8-dιammophenazme, 2, 7-di mmo-3, 8- dimethoxyphenaz e, 2, 7-dιammo-3-methoxyphenazιne, 2,7-dιamιno 3-methoxyphenazme, 3-dιmethyl 2, 8-phenazmedιamme, 2,2'-[(8- ammo-7-methyl-2-phenazmy1) ιmmo]bιs-ethanol, 2, 2 '- [ (8-ammo- 7-methoxy-2-phenazmyl) ιmmo]bιs-ethanol, 2, 2 '- [ (8-ammo-7- chloro-2-phenazmyl) lm ojbis-ethanol, 2-[ (8-ammo-7-methyl-2- phenazmyl) ammo] -ethanol, 2,2 '- [ (8-ammo-2-phenazmyl) l i- no]bιs-ethanol, 3-ammo-7- (dimethylammo) -2, 8-dιmethyl-5-phe- nyl-chloride, 9- (diethylam o) - benzo [a]phenazme-l, 5-dιol, N- [8- (diethylammo) -2-phenazιnyl] - methanesulfonamide, N-(8- methoxy-2-phenazmyl) - Methanesulfonamide, N,N,N' ,N' -tetra- methyl-2, 7-phenazmedιamme, 3, 7-dιmethyl-2-phenazmamme, p- ammo benzoic acids, such as p-ammo benzoic acid ethyl, p- ammo benzoic acid glycerid, p-ammo benzoic acid isobutyl, p- dimethylammo benzoic acid amil, p-dimethylammo benzoic acid octyl, p-diethoxy am o benzoic amil, p- dipropoxy am o benzoic acid ethyl, acetylsalicylic acid, isatm derivatives, such as 2,3-dιammo benzoic acid. 9. The dyeing composition according to claims 8, comprising a dye modifier selected from the group comprising m-phenylene- diamine, 2, 4-dιammoanιsole, 1-hydroxynaphthalene (α-naphthol) , 1, 4-dιhydroxybenzene (hydroqumone) , 1, 5-dιhydroxynapthalene, 1,2-dιhydroxybenzene (pyrocatechol) , 1, 3-dιhydroxybenzene (re- sorcmol), 1, 3-dιhydroxy-2-methylbenzene, 1, 3-dιhydroxy-4-chlo- robenzene (4-chlororesorcmol) , 1,2, 3, trihydroxybenzene, 1,2,4- trihydroxybenzene, 1,2, -tnhydroxy-5-methylbenzene, 1,2,4-trι- hydroxytoluene. 10. A method for dying comprising contacting a laccase derived from a strain of the genus Scytalidi um with the kera¬ tmous fibres and at least one dye precursor in the presence or absence of at least one modifier for a period of time and under conditions sufficient to permit oxidation of the dye precursor mto a coloured compound. 11. The method accordmg to claim 10, wherein the dyemg is carried out at a pH m the range from 3.0 to 9.0, preferably 4.0 to 8.0, especially 6.0 to 8.0. T SHEET 12. Use of an oxidation enzyme derived from a strain of the genus Scytalidium for oxidative dyeing keratinous fibres, particular hair, fur, hide and wool. 13. The use according to claim 14, wherem the oxidation enzyme is derived from a strain of the species Scytalidi um th ermoph Hum.

Inventors: AASLYNG DORRIT, ROERBAEK KAREN, SOERENSEN NIELS HENRIK, AASLYNG, DORRIT, ROERBAEK, KAREN, SOERENSEN, NIELS, HENRIK
Applicants: AASLYNG DORRIT, NOVONORDISK AS, ROERBAEK KAREN, SOERENSEN NIELS HENRIK, AASLYNG, DORRIT, NOVO NORDISK A/S, ROERBAEK, KAREN, SOERENSEN, NIELS, HENRIK
Application year: 1996
Main classification: C09B67
IPC codes: A61K8, A61Q5, C09B67, C12N9, C12N15, C12R1, D06P1, D06P3