WO2007054034A1 - Uses of laccase - Google Patents

Abstract

The present invention provides methods for enzymatic oxidation of a substrate with a laccase from Streptomyces coelicolor. The substrate thereof can be a dye or a colorant or a textile. The invention also provides compositions comprising the laccase. An enhancing agent can be further included.

Description

USES OF LACCASE

FIELD OF THE INVENTION

The present invention relates to methods for enzymatic oxidation of a substrate, such as bleaching methods, with a^ laccase. The present invention further relates to a composition comprising a laccase having 60% identity with SEQ ID NO: 2.

BACKGROUND

A new laccase from Streptomyces coelicolor was disclosed in Machczynski et al., "Characterization of SLAC: A small laccase from Streptomyces coelicolor with unprecedented activity", Protein Science, 13:2388-2397 (2004).

SUMMARY

The present invention provides as a first aspect a method for enzymatic oxidation of a substrate, comprising contacting the substrate preferably at pH 4-12, more preferably 9-12 with a polypeptide which has laccase activity and at least 60% identity with SEQ ID NO:2. The method can be carried out at 25-100 degrees Celsius, preferably 50-100 degrees Celsius, with a polypeptide which has laccase activity and at least 60% identity with SEQ ID NO:2; wherein the polypeptide has at least 50% residual activity at 50-100 degrees Celsius.

In a second aspect is provided a detergent composition comprising a surfactant and a polypeptide, which has laccase activity and at least 60% identity with SEQ ID NO:2.

In a third aspect is provided a composition comprising a textile material and a polypeptide, which has laccase activity and at least 60% identity with SEQ ID NO:2. In embodiments, the methods and compositions also include an enhancing agent.

DEFINITIONS

Laccase activity: The term "laccase activity" is defined herein by the enzyme classification EC 1.10.3.2 (laccase) as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB); or enzyme classification EC 1.10.3.1 (catechol oxidase), enzyme classification EC 1.10.3.4 (o- aminophenol oxidase), or enzyme classification EC 1.3.3.5 (bilirubin oxidase). The polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the laccase activity of the polypeptide consisting of the amino acid sequence shown as amino acids 1 to 343 of SEQ ID NO: 2.

Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE. Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation which contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at ^■ least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% , pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation.

The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptides are in "essentially pure form", i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods.

Herein, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "polypeptide in isolated form."

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

The degree of identity between an amino acid sequence of the present invention

("invention sequence"; e.g. amino acids 1 to 343 of SEQ ID NO:2) and a different amino acid sequence ("foreign sequence") is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence" or the length of the

"foreign sequence", whichever is the shortest. The result is expressed in percent identity.

An exact match occurs when the "invention sequence" and the "foreign sequence" have identical amino acid residues in the same positions of the overlap (in the alignment example below this is represented by "|"). The length of a sequence is the number of amino acid residues in the sequence (e.g. the length of SEQ ID NO:2 is 343).

In the alignment example below, the overlap is the amino acid sequence "HTWGER.NL" of Sequence 1; or the amino acid sequence "HGWGEDANL" of Sequence 2. A gap is indicated by a "-".

Alignment example

Sequence 1: ACMSHTWGER-NL

1 M ! M

Sequence 2: HGWGEDANLAMNPS

DETAILED DESCRIPTION

Polypeptides having laccase activity

The polypeptides of the invention comprise amino acid sequences which has a degree of identity to amino acids 1 to 343 of SEQ ID NO:2 (i.e., the mature polypeptide) of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 97%, which have laccase activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides have an amino acid sequence which differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from amino acids l to 343 of SEQ ID NO:2. A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has laccase activity. In a preferred aspect, a polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, a polypeptide comprises amino acids 1 to 343 of SEQ ID NO:2, or an allelic variant thereof; or a fragment thereof that has laccase activity. In another preferred aspect, a polypeptide comprises amino acids 1 to 343 of SEQ ID NO:2. In another preferred aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has laccase activity. In another preferred aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2. In another preferred aspect, a polypeptide consists of amino acids 1 to 343 of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has laccase activity. In another preferred aspect, a polypeptide consists of amino acids 1 to 343 of SEQ ID NO:2.

Preferably the polypeptides of the invention are obtainable from a species of Streptomyces, such as Streptomyces coelicolor. In a second aspect, the present invention relates to polypeptides having laccase activity which are encoded by polynucleotides which hybridize under low stringency conditions, preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 1 to 1029 of SEQ ID NO:1, or (ii) a complementary strand of (i) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

Low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% fonnamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS preferably at least at 50°C (low stringency), preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70⁰C (very high stringency).

The polypeptides of the invention also include artificial variants comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO:2. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- , hydroxyproline, 6-iV-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

The total number of amino acid substitutions, deletions and/or insertions of amino acids 1 to 343 of SEQ ID NO:2 is 10, preferably 9, more preferably 8, more preferably 7, more

^' preferably at most 6, more preferably at most 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

The source of oxygen required by the polypeptides having laccase activity may be oxygen from the atmosphere or an oxygen precursor for in situ production of oxygen. Oxygen from the atmosphere will usually be present in sufficient quantity. If more O₂ is needed, additional oxygen may be added, e.g. as pressurized atmospheric air or as pure pressurized O₂.

Determination of Laccase Activity (LAMU) Laccase activity may be determined from the oxidation of syringaldazine under aerobic conditions. The violet colour produced is measured at 530 nm. The analytical conditions are 19 mM syringaldazine, 23 mM Tris/maleate buffer, pH 7.5, 30°C, 1 min. reaction time.

1 laccase unit (LAMU) is the amount of enzyme that catalyses the conversion of 1.0 mmole syringaldazine per minute at these conditions.

Enhancing agent

An enhancing agent may be used with the polypeptides having laccase activity to improve the effect of the methods of the invention. Enhancing agents acting as electron donors for the polypeptides having laccase activity include both inorganic and organic compounds known in the art. Several examples of such enhancing agents are shown below.

The enhancing agent may be selected from the group consisting of aliphatic, cyclo- aliphatic, heterocyclic or aromatic compounds containing the moiety >N-OH. In a preferred embodiment of the invention the enhancing agent is a compound of the general formula I:

wherein R¹, R², R³, R⁴ are individually selected from the group consisting of hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C_1-12-alkyl, C_1-6- alkoxy, carbonyl(C_1-12-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and salts and esters thereof, wherein the R¹, R², R³, R⁴ may be substituted with R⁵, wherein R⁵ represents hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C_1-12-alkyl, C_1-6-alkoxy, carbonyl(C_1-12-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and salts and esters thereof;

[X] represents a group selected from (-N=N-), (-N=CR⁶-)_m, (-CR⁶=N-)_m, (-CR⁷=CR⁸-)_m, (- CR⁶=N-NR⁷-), (-N=N-CHR⁶-), (-N=CR⁶-NR⁷-), (-N=CR⁶-CHR⁷-), (-CR⁶=N-CHR⁷-), (- CR⁶=CR⁷-NR⁸-), and (-CR⁶=CR⁷-CHR⁸-), wherein R⁶, R⁷, and R⁸ independently of each other are selected from H, OH, NH₂, COOH, SO₃H₅ C_1-6-alkyl, NO₂, CN, Cl, Br, F, CH₂OCH₃, OCH₃, and COOCH₃; and m is 1 or 2.

The term "C_1-n-alkyl" wherein n can be from 2 through 12, as used herein, represent a branched or straight alkyl group having from one to the specified number of carbon atoms. Typical C_1-6-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl and the like.

In a more preferred embodiment of the invention the enhancing agent is a compound of the general formula II:

wherein R¹, R², R³, R⁴ are individually selected from the group consisting of hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C_1-12-alkyl, C_1-6- alkoxy, carbonyl(C_1-12-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and salts and esters thereof, wherein the R¹, R², R³, R⁴ may be substituted with R⁵, wherein R⁵ represents hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C_1-12-alkyl, C_1-6-alkoxy, carbonyl(C_1-12-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl, carbamoyl, phosphono, phosphonooxy, and salts and esters thereof.

The enhancing agent may also be a salt or an ester of formula I or II. Further preferred enhancing agents are oxoderivatives and N-hydroxy derivatives of heterocyclic compounds and oximes of oxo- and formyl-derivatives of heterocyclic compounds, said heterocyclic compounds including five-membered nitrogen-containing heterocycles, in particular pyrrol, pyrazole and imidazole and their hydrogenated counterparts (e.g. pyrrolidine) as well as triazoles, such as 1,2,4-triazole; six-membered nitrogen-containing heterocycles, in particular mono-, di- and triazinanes (such as piperidine and piperazine), morpholine and their unsaturated counterparts (e.g. pyridine and pyrimidine); and condensed heterocycles containing the above heterocycles as substructures, e.g. indole, benzothiazole, quinoline and benzoazepine. Examples of preferred enhancing agent from these classes of compounds are pyridine aldoximes; N-hydroxypyrrolidinediones such as N-hydroxysuccinimide and N- hydroxyphthalimide; 3,4-dihydro-3-hydroxybenzo[l,2,3]triazine-4-one; formaldoxime trimer

(N,N',N"-trihydroxy-l,3,5-triazinane); and violuric acid (l,3-diazinane-2,4,5,6-tetrone-5- oxime).

Still further enhancing agents which may be applied in the invention include oximes of oxo- and formyl-derivatives of aromatic compounds, such as benzoquinone dioxime and salicylaldoxime (2-hydroxybenzaldehyde oxime), and N-hydroxyamides and N- hydroxyanilides, such as N-hydroxyacetanilide. Preferred enhancing agents are selected from the group consisting of 1- hydroxybenzotriazole; 1-hydroxybenzotriazole hydrate; 1-hydroxybenzotriazole sodium salt; 1-hydroxybenzotriazole potassium salt; 1-hydroxybenzotriazole lithium salt; 1- hydroxybenzotriazole ammonium salt; 1-hydroxybenzotriazole calcium salt; 1- hydroxybenzotriazole magnesium salt; and l-hydroxybenzotriazole-6-sulphonic acid. A particularly preferred enhancing agent is 1 -hydroxybenzotriazole.

All the specifications of N-hydroxy compounds above are understood to include tautomeric forms such as N-oxides whenever relevant.

Another preferred group of enhancing agents comprises a -CO-NOH- group and has the general formula III:

in which A is:

and B is the same as A; or B is H or Ci-₁₂-alkyl, said alkyl may contain hydroxy, ester or ether groups (e.g. wherein the ether oxygen is directly attached to A-N(OH)C=O-, thus including N- hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6 independently of each other are H, OH, NH₂, COOH, SO₃H, C_1-8-alkyl, acyl, NO₂, CN₅ Cl, Br₅ F₅ CF₃, NOH-CO- phenyl, C0-N0H-phenyl₅ C_1-6-CO-NOH-A₅ CO-NOH-A, C0R12, phenyl-CO-NOH-A₅ 0R7, NR8R9, COORlO, or NOH-CO-Rl I₅ wherein R7, R8, R9₅ RlO, Rl 1 and R12 are d-n-alkyl or acyl. R2, R3, R4₅ R5 and R6 of A are preferably H₅ OH₅ NH₂, COOH₅ SO₃H₅ C_1-3-alkyl, acyl,

NO₂, CN, Cl₅ Br₅ F₅ CF₃, NOH-CO-phenyl, CO-NOH-phenyl, COR12, 0R7, NR8R9, COORlO₅ or NOH-CO-RI l₅ wherein R7₅ R8 and R9 are C_1-3-alkyl or acyl, and RlO₅ RIl and R12 are C_1-3-alkyl; more preferably R2, R3₅ R4₅ R5 and R6 of A are H₅ OH₅ NH₂, COOH₅ SO₃H₅ CH₃, acyl, NO₂, CN, Cl, Br, F, CF₃, CO-NOH-phenyl, COCH₃, 0R7, NR8R9, or COOCH₃, wherein R7, R8 and R9 are CH₃ or COCH₃; even more preferably R2, R3, R4, R5 and R6 of A are H₅ OH₅ COOH, SO₃H₅ CH₃, acyl, NO₂, CN₅ Cl, Br, F, CO-NOH-phenyl, OCH₃, COCH₃, or COOCH₃; and in particular R2, R3, R4, R5 and R6 of A are H₅ OH₅ COOH₅ SO₃H₅ CH₃, NO₂, CN, Cl₅ Br, CO-NOH-phenyl, or OCH₃.

R2, R3, R4, R5 and R6 of B are preferably H₅ OH₅ NH₂, COOH₅ SO₃H₅ C_1-3-alkyl, acyl, NO₂, CN₅ Cl, Br₅ F₅ CF₃, NOH-CO-phenyl, CO-NOH-phenyl, CORl 2, OR7₅ NR8R9, COORlO, or NOH-CO-RI l₅ wherein R7₅ R8 and R9 are C_1-3-alkyl or acyl, and RlO, RI l and R12 are C_1-3-alkyl; more preferably R2, R3, R4, R5 and R6 of B are H₅ OH, NH₂, COOH₅ SO₃H₅ CH₃, acyl, NO₂, CN, Cl, Br₅ F, CF₃, CO-NOH-phenyl, COCH₃, 0R7, NR8R9, or COOCH₃, wherein R7, R8 and R9 are CH₃ or COCH₃; even more preferably R2, R3, R4, R5 and R6 of B are H₅ OH₅ COOH, SO₃H₅ CH₃, acyl, NO₂, CN, Cl₅ Br₅ F, CO-NOH-phenyl, OCH₃, COCH₃, or COOCH₃; and in particular R2, R3₅ R4₅ R5 and R6 of B are H₅ OH₅ COOH, SO₃H, CH₃, NO₂, CN, Cl, Br₅ CO-NOH-phenyl, or OCH₃.

B is preferably H or C_1-3-alkyl, said alkyl may contain hydroxy, ester or ether groups; preferably said alkyl may contain ester or ether groups; more preferably said alkyl may contain ether groups.

In an embodiment, A and B independently of each other are:

or B is H or C_1-3-alkyl, said alkyl may contain hydroxy, ester or ether groups (e.g. wherein the ether oxygen is directly attached to A-N(OH)C=O-, thus including N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6 independently of each other are H, OH, NH₂, COOH, SO₃H, Ci-₃-alkyl, acyl, NO₂, CN, Cl, Br, F, CF₃, NOH-CO-phenyl, CO-NOH-phenyl, C0R12, 0R7, NR8R9, COORlO, or NOH-CO-RI l, wherein R7, R8 and R9 are d.₃-alkyl or acyl, and RlO, Rl 1 and R12 are C_1-3-alkyl.

In another embodiment, A and B independently of each other are:

or B is H or C_1-3-alkyl, said alkyl may contain hydroxy or ether groups (e.g. wherein the ether oxygen is directly attached to A-N(OH)C=O-, thus including N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6 independently of each other are H, OH, NH₂, COOH, SO₃H, CH₃, acyl, NO₂, CN₅ Cl, Br, F, CF₃, CO-NOH-phenyl, COCH₃, 0R7, NR8R9, or

COOCH₃, wherein R7, R8 and R9 are CH₃ or COCH₃.

In another embodiment, A and B independently of each other are:

or B is H or C_1-3-alkyl, said alkyl may contain hydroxy or ether groups (e.g. wherein the ether oxygen is directly attached to A-N(OH)C=O-, thus including N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6 independently of each other are H, OH, COOH, SO₃H, CH₃, acyl, NO₂, CN, Cl, Br, F, CO-NOH-phenyl, OCH₃, COCH₃, or COOCH₃.

In another embodiment, A and B independently of each other are:

or B is C_1-3-alkyl, said alkyl may contain ether groups (e.g. wherein the ether oxygen is directly attached to A-N(OH)C=O-, thus including N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6 independently of each other are H, OH, COOH, SO₃H, CH₃, NO₂, CN, Cl, Br, CO-NOH-phenyl, or OCH₃. The terms "C_1-n-alkyP wherein n can be from 2 through 12, as used herein, represent a branched or straight alkyl group having from one to the specified number of carbon atoms.

Typical C_1-6-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl and the like. The term "acyl" as used herein refers to a monovalent substituent comprising a d-₆-alkyl group linked through a carbonyl group; such as e.g. acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and the like.

In an embodiment at least one of the substituents R2, R3, R4, R5 and R6 of A are H, preferably at least two of the substituents R2, R3, R4, R5 and R6 of A are H, more preferably at least three of the substituents R2, R3, R4, R5 and R6 of A are H, most preferably at least four of the substituents R2, R3, R4, R5 and R6 of A are H, in particular all of R2, R3, R4, R5 and R6 of A are H.

In another embodiment at least one of the substituents R2, R3, R4, R5 and R6 of B are H, preferably at least two of the substituents R2, R3, R4, R5 and R6 of B are H, more preferably at least three of the substituents R2, R3, R4, R5 and R6 of B are H, most preferably at least four of the substituents R2, R3, R4, R5 and R6 of B are H, in particular all of R2, R3, R4, R5 and Rό ofB are H.

In particular embodiments according to the invention the enhancing agent is selected from the group consisting of 4-nitrobenzoic acid-N-hydroxyanilide;

4-methoxybenzoic acid-N-hydroxyanilide;

N,N'-dihydroxy-N,N'-diphenylterephthalamide; decanoic acid-N-hydroxyanilide;

N-hydroxy-4-cyanoacetanilide; N-hydroxy-4-acetylacetanilide;

N-hydroxy-4-hydroxyacetanilide ;

N-hydroxy-3-(N'-hydroxyacetamide)acetanilide;

4-cyanobenzoic acid-N-hydroxyanilide;

N-hydroxy-4-nitroacetanilide; N-hydroxyacetanilide;

N-hydroxy-N-phenyl-carbamic acid isopropyl ester;

N-hydroxy-N-phenyl-carbamic acid methyl ester;

N-hydroxy-N-phenyl-carbamic acid phenyl ester; N-hydroxy-N-phenyl-carbamic acid ethyl ester; and N-hydroxy-N-(4-cyanophenyl)-carbamic acid methyl ester.

Another group of preferred enhancing agents is phenolic compounds (alkylsyringates) of the general formula IV:

wherein the letter A in said formula denotes be 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, -SO₂-E, -N-XY, and -N⁺-XYZ₅ in which E may be -H, -OH, -R₅ or -OR₅ 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-C₈ alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulpho or amino group; and B and C may be the same or different and selected from C_mH_2m+1, where m = I₅ 2, 3₅ 4 or 5.

In the above mentioned general formula IV₅ A may be placed meta to the hydroxy group instead of being placed in the para-position as shown.

In particular embodiments of the invention the enhancing agent is selected from the group having the general formula V: OMe

OMe

in which A is a group such as -H₅ -OH₅ -CH₃, -OCH₃, -O(CH₂)_nCH₃, where n = 1, 2, 3, 4, 5, 6, 7 or 8.

Yet another group of preferred enhancing agents are the compounds as described in general formula VI:

in which general formula A represents a single bond, or one of the following groups: (- CH₂-), (-CH=CH-), (-NRl 1-), (-CH=N-), (-N=N-), (-CH=N-N=CH-), or (>C=O); and in which general formula the substituent groups Rl-RI l₅ which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, Ci-g-alkyl; which carbamoyl, sulfamoyl, phenyl, and amino groups may furthermore be unsubstituted or substituted once or twice with a substituent group R12; and which C_1-8-alkyl group may be saturated or unsaturated, branched or unbranched, and may furthermore be unsubstituted or substituted with one or more substituent groups Rl 2; which substituent group R12 represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, or C_1-8-alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with hydroxy or methyl. and in which general formula R5 and R6 may together form a group -B-, in which B represents a single bond, one of the following groups (-CH₂-), (-CH=CH-), (-CH=N-); or B represents sulfur, or oxygen.

In particular embodiments of the invention the enhancing agent is selected from the group having the general formula VII:

in which general formula X represents a single bond, oxygen, or sulphur; and in which general formula the substituent groups R1-R9, which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, C^-alkyl; which carbamoyl, sulfamoyl, phenyl, and amino groups may furthermore be unsubstituted or substituted once or twice with a substituent group RlO; and which Cμs-alkyl group may be saturated or unsaturated, branched or unbranched, and may furthermore be unsubstituted or substituted with one or more substituent groups RlO; which substituent group RlO represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy and esters and salts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, or Q-s-alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted once or twice with hydroxy or methyl.

According to the invention, the enhancing agent may be present in a concentration in the range of from 0.01 mM to 1000 mM, preferably in the range of from 0.05 mM to 500 mM, more preferably in the range of from 0.1 mM to 100 mM, and most preferably in the range of from 0.1 mM to 50 mM.

Compositions

The present invention provides a composition comprising a polypeptide having laccase activity, a textile material and a dye or colorant, wherein the dye or colorant may be attached to the textile. In an embodiment, the composition also includes an enhancing agent.

The polypeptide having laccase activity, and optionally also the enhancing agent, may be formulated as a liquid (e.g. aqueous), a solid, a gel, a paste or a dry product formulation. The dry product formulation may subsequently be re-hydrated to form an active liquid or semi- liquid formulation usable in the method of the invention.

When the polypeptide having laccase activity and the enhancing agent are formulated as a dry formulation, the components may be mixed, arranged in discrete layers or packaged separately.

When other than dry form formulations are used and even in that case, it is preferred to use a two-part formulation system having the polypeptide having laccase activity separate from the enhancing agent. The composition of the invention may further comprise auxiliary agents such as wetting agents, thickening agents, buffer(s) for pH control, stabilisers, perfume, colourants, fillers and the like.

Useful wetting agents are surfactants, i.e. non-ionic, anionic, amphoteric or zwitterionic surfactants. Surfactants are further described above.

When the composition of the invention includes water, the pH of such aqueous composition is in the range of from pH 2 to 12, preferably in the range of from pH 4 to 12, more preferably in the range of from pH 6 to 12, most preferably in the range of from pH 8 to 12, and in particular in the range of from pH 9 to 11.

Detergent composition

The polypeptides having laccase activity of the invention may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the polypeptides having laccase activity of the invention and a surfactant. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase (such as a laccase), and/or a peroxidase (such as a haloperoxidase). In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from

Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331

376), P. stutzeri (GB 1,372,034), P. fluorescent, Pseudomonas sp. strain SD 705 (WO

95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO , 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such 'cellulases are cellulases described in EP 0 495 257, EP 0 531 372,

WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. 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 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 molar weights of 1000 to 20000; ethoxylated nonylphenols having froni 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 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. 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., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous. The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight. When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefmsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap. When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides"). The detergent may contain 0.5-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, polyvinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacry late/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a hydrogen peroxide source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.

Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) 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, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, and the polypeptides having laccase activity of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liqour, preferably 0.05-10 mg of enzyme protein per liter of wash liqour, more preferably 0.1-5 mg of enzyme protein per liter of wash liqour, and most preferably 0.1-1 mg of enzyme protein per liter of wash liqour.

The polypeptides having laccase activity of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

Textile

In context of the invention the term "textile" includes fabrics, garments, and yarns. Fabric can be constructed from fibers by weaving, knitting or non- woven operations.

Weaving and knitting require yarn as the input whereas the non-woven fabric is the result of random bonding of fibers (paper can be thought of as non-woven). In the present context, the term "fabric" is also intended to include fibers and other types of processed fabrics.

Woven fabric is constructed by weaving "filling" or weft yarns between wrap yarns stretched in the longitudinal direction on the loom. The wrap yarns must be sized before weaving in order to lubricate and protect them from abrasion at the high speed insertion of the filling yarns during weaving. The filling yarn can be woven through the warp yarns in a "over one - under the next" fashion (plain weave) or by "over one - under two" (twill) or any other myriad of permutations. Strength, texture and pattern are related not only to the type/quality of the yarn but also the type of weave. Generally, dresses, shirts, pants, sheeting's, towels, draperies, etc. are produced from woven fabric.

Knitting is forming a fabric by joining together interlocking loops of yarn. As opposed to weaving which is constructed from two types of yarn and has many "ends", knitted fabric is produced from a single continuous strand of yarn. As with weaving, there are many different ways to loop yarn together and the final fabric properties are dependent both upon the yarn and the type of knit. Underwear, sweaters, socks, sport shirts, sweat shirts, etc. are derived from knit fabrics.

Non-woven fabrics are sheets of fabric made by bonding and/or interlocking fibers and filaments by mechanical, thermal, chemical or solvent mediated processes. The resultant fabric can be in the form of web-like structures, laminates or films. Typical examples are disposable baby diapers, towels, wipes, surgical gowns, fibers for the "environmental friendly" fashion, filter media, bedding, roofing materials, backing for two-dimensional fabrics and many others. According to the invention, the method of the invention may be applied to any fabric known in the art (woven, knitted, or non-woven). In particular the oxidation (bleaching) process may be applied to cellulose-containing or cellulosic fabrics, such as cotton, viscose, rayon, ramie, linen, lyocell (e.g. Tencel, produced by Courtaulds Fibers), or mixtures thereof, or mixtures of any of these fibers together with synthetic fibres (e.g., polyester, polyamid, nylon) or other natural fibers such as wool and silk., such as viscose/cotton blends, lyocell/cotton blends, viscose/wool blends, lyocell/wool blends, cotton/wool blends; flax (linen), ramie and other fabrics based on cellulose fibers, including all blends of cellulosic fibers with other fibers such as wool, polyamide, acrylic and polyester fibers, e.g., viscose/cotton/polyester blends, wool/cotton/polyester blends, flax/cotton blends etc. The term "wool," means any commercially useful animal hair product, for example, wool from sheep, camel, rabbit, goat, llama, and known as merino wool, Shetland wool, cashmere wool, alpaca wool, mohair, etc. and includes wool fiber and animal hair. The method of the invention can be used with wool or animal hair material in the form of top, fiber, yarn, or woven or knitted fabric. The enzymatic treatment can also be carried out on loose flock or on fibers made from wool or animal hair material. The treatment can be performed at many different stages of processing. The fabric to be bleached may be dyed or undyed. According to the invention textile may be desized, scoured and/or bleached in aqueous medium in the presence of a polypeptide having laccase activity of the invention.

Methods and Uses

The present invention provides methods for enzymatic oxidation of a substrate, comprising contacting the substrate at pH 9-12 with a polypeptide which has laccase activity and at least 60% identity with SEQ ID NO:2. In an embodiment, the polypeptide retains at least 50% (preferably at least 60%, more preferably at least 70%, most preferably at least 80%) activity after exposure to pH 12 at 40 degrees Celsius for one hour.

The invention also provides methods for enzymatic oxidation of a substrate, comprising contacting the substrate at 50-100 degrees Celsius with a polypeptide which has laccase activity and at least 60% identity with SEQ ID NO:2.

In an embodiment, the polypeptide retains at least 50% (preferably at least 55%, more preferably at least 60%) residual activity after exposure to 90 degrees Celsius for one hour. A substrate according to the invention may be a dye or colorant, a textile material, or lignin or a lignin containing material. Oxidation of the substrate occurs when it acts as an electron donor for the polypeptide having laccase activity or - when an enhancing agent is used

- the enhancing agent. In a preferred embodiment, the method for oxidizing a substrate is a method for bleaching a substrate.

The following embodiments illustrate various uses of the invention.

In an embodiment, the method of the invention finds application for bleaching of a textile dye or colorant in solution or attached to textile. Bleaching includes changing the color of the dye/textile, such as whitening or fading the color of the dye/textile. 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. Other structural motifs may occur together with these, as exemplified by

Reactive Blue 19.

Another example of bleachable dyes is disperse dyes, which are characterized by being nonionic and have a very limited solubility in water. Disperse dyes include azo, nitroarylamine, and anthraquinone based dyes. Examples of disperse dyes include Disperse Red 60, Disperse Yellow 3, Disperse Blue 3, Disperse Blue 27, Disperse Blue 56, and Disperse Violet 1.

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, exemplified by the soluble dye indigo carmine.

Other dyes and colorants may be of natural origin or may be synthesized as identical to or resembling natural structures. Examples of categories of coloured substances extractable from vegetable sources are polyphenolic, anthocyanine and carotenoid compounds. An embodiment of the present invention is provided by household and institutional laundering processes. In such washing and rinsing processes, dyes and colorants present on fabrics may leach into the washing or rinsing liquor and discoloration of the laundry may result. Bleaching of the coloured compounds in solution by the method of the invention may counteract this undesirable effect. Other systems for dye transfer inhibition are known in the art (e.g. WO 91/05839).

In another 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 yet another embodiment, the method of the invention finds application in bleaching of pulp for paper production.

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

In yet another embodiment, the method of the invention finds application for lignin modification, e.g., in the manufacture of wood composites, e.g., wood fibre materials such as chipboards, fibre boards, or particle boards, or in the manufacture of laminated wood products, such as laminated beams and plywood.

In yet another embodiment, the method of the invention finds application in treatment of waste water, e.g., waste water from the chemical or pharmaceutical industry, from dye manufacturing, from dye-works, from the textile industry, or from pulp production (cf. e.g. US 4,623,465, or JP-A-2-31887). In a more specific embodiment, the invention provides a method for treatment of waste water from dye manufacturing, from dye-works, from textile industry, or from pulp manufacturing, the method comprising treatment of the waste water with a polypeptide having laccase activity according to the invention.

In the above mentioned processes and in other applications of the invention, an enhancing agent may be added at the beginning of the process or later, in one or several additions.

According to the invention the polypeptide having laccase activity may be present in concentrations of 0.001-100 mg enzyme protein per liter, preferably 0.005-50 mg enzyme protein per liter, more preferably 0.01-50 mg enzyme protein per liter, most preferably 0.05-10 mg enzyme protein per liter, and in particular 0.1-10 mg enzyme protein per liter. Bleaching

According to the invention bleaching may be carried out using any known process conditions in the art. In an embodiment the bleaching may be carried out at a temperature in the range of from about 3O⁰C to about 100°C, more preferably from about 40°C to about 90°C. The pH range may be from about pH 5 to about pH 12, preferably from about pH 7 to about pH 12, more preferably from about pH 7 to about pH 11, even more preferably from about pH 8 to about pH 11, and most preferably from about pH 9 to about pH 11. The reaction time may preferably be in the range of from about 15 minutes to about 3 hours.

As mentioned above bleaching may result in a whitening of the textile. The value of whiteness index (WI) is measured using a MacBeth Color Eye equipped with Optiview 7000 software. The Whiteness index is calculated from the following equation:

WI = Y + 800(X_n-X) + 1700(y_n-y) where Y, x and y are chromaticity coordinates of the sample, and X_n and y_n are those of illuminant using the standard illuminant D65 (imitating daylight). The bleached materials may also be subject to additional processes. For example, for textile materials, the preparation may include the application of finishing techniques such as desizing and scouring, and other treatment processes, such as imparting antimicrobial properties (e.g., using quaternary ammonium salts), flame retardancy (e.g., by phosphorylation with phosphoric acid or urea), increasing absorbency (by coating or laminating with polyacrylic acid), providing an antistatic finish (e.g., using amphoteric surfactants (N-oleyl- N,N-dimethylglycine)), providing a soil release finish (e.g., using NaOH), providing an antisoiling finish (e.g., using a fluorochemical agent), and providing an antipilling finish (e.g., using NaOH, alcohol).

The method of the invention may be carried out in the presence of conventional fabric, fiber, or yarn finishing agents, including wetting agents, polymeric agents, dispersing agents, etc.

A conventional wetting agent may be used to improve the contact between the substrate and the enzyme used in the method. The wetting agent may be a nonionic surfactant, e.g. an ethoxylated fatty alcohol. A preferred wetting agent is an ethoxylated and propoxylated fatty acid ester such as Berol 087 (product of Akzo Nobel, Sweden).

Examples of suitable polymeris agents include proteins (e.g. bovine serum albumin, whey, casein or legume proteins), protein hydrolysates (e.g. whey, casein or soy protein hydrolysate), polypeptides, lignosulfonates, polysaccharides and derivatives thereof, polyethylene glycol, polypropylene glycol, polyvinyl pyrrolidone, ethylene diamine condensed with ethylene or propylene oxide, ethoxylated polyamines, or ethoxylated amine polymers.

The dispersing agent may preferably be selected from nonionic, anionic, cationic, ampholytic or zwitterionic surfactants. More specifically, the dispersing agent may be selected from carboxymethylcellulose, hydroxypropylcellulose, alkyl aryl sulphonates, long-chain alcohol sulphates (primary and secondary alkyl sulphates), sulphonated olefins, sulphated monoglycerides, sulphated ethers, sulphosuccinates, sulphonated methyl ethers, alkane sulphonates, phosphate esters, alkyl isothionates, acylsarcosides, alkyltaurides, fluorosurfactants, fatty alcohol and alkylphenol condensates, fatty acid condensates, condensates of ethylene oxide with an amine, condensates of ethylene oxide with an amide, sucrose esters, sorbitan esters, alkyloamides, fatty amine oxides, ethoxylated monoamines, ethoxylated diamines, alcohol ethoxylate and mixtures thereof. A preferred dispersing agent is an alcohol ethoxylate such as Berol 08 (product of Akzo Nobel, Sweden).

The bleaching processing may be performed using any machinery known in the art. The fabric may be further finished by one or more of the following treatments as are known in the art: dyeing, biopolishing, brightening, softening, and/or anti- wrinkling treatment(s).

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

EXAMPLES

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

EXAMPLE 1

Cloning of recombinant laccase

The laccase gene was obtained from the Ml 45 derivative of Streptomyces coelicolor

A3(2). Genomic DNA was used as template in a PCR reaction. The sequence of the forward primer was AGG ATT CAC CAT GGA CAG GCG AGG CTT TAA C (SEQ ID NO:3) and the sequence of the reverse primer was ACT CGA GTC AGT GCT CGT GTT CGT GTG

CGG C (SEQ ID NO:4). The genomic DNA was prepared using FastDNA® SPIN Kit for Soil from Qbiogene Inc. The Phusion DNA polymerase from Finnzymes was used for the PCR in combination with the GC buffer due to the GC rich nature of the template DNA.

PCR mixture: 10 μL Phusion buffer GC

5 μL dNTPs (2.5 mM) 0.5 μL forward primer (100 μM) 0.5 μL reverse primer (100 μM) l μL gDNA (10x diluted) 0.5 μL Phusion polymerase

H₂O to 50 μL

PCR cycle:

Step 1: 30 seconds at 98°C Step 2: 10 seconds at 98⁰C; 20 seconds at 6O⁰C; 45 seconds at 72⁰C; repeat 35 times

Step 3: 10 minutes at 72⁰C Step 4: Hold at 4⁰C

The PCR reaction resulted in a single band of approximately 1100 basepair size visible on an agarose gel. The band was extracted from the gel by Qiagen's QIAquick Gel Extraction Kit. The recovered DNA was subsequently subjected to restriction at 37⁰C for 3 hours: 15 μL DNA 2 μL NEB buffer 2 0.5 μL XhoI 0.5 μL BamHI

The gel-cleaned DNA was ligated into pENI2516 as a BamHI-XhoI fragment to create pLAQ029. The resulting plasmid was initially transformed into E. coli strain DHlOB and the insert was sequenced to confirm its nucleotide sequence. The plasmid was subsequently transformed into Aspergillus oryzae strain ToC 1512 for expression.

EXAMPLE 2

Fermentation The transformed strain of A. oryzae was grown for expression of laccase enzyme. Typically, a 100 mL of YP media was inoculated with spores from a stock in 50% glycerol stored at -8O⁰C. The starter culture was grown in a baffled 250 mL flask for 3-4 days at 37⁰C and 180 rpm. 20 mL of this culture was then used to inoculate 500 mL MY51 medium added 2% maltose and 500 μM CuSO₄ in a 2 L flask with baffles. The flask was placed in an orbital shaker at 180 rpm and grown for one week at 37⁰C before being harvested. The enzymatic activity in the broth was monitored daily using the assay.

EXAMPLE 3 Purification

The fermentation broth was filtered using Mira cloth to remove fungal hyphae. This filtrate was subsequently sterile filtered using a Corning 0.45 μm filter with pre-fϊlter. The resulting filtrate was made 1.2 M in ammonium sulphate and loaded onto a XK 16 column with 15 mL Source 15Phe media pre-equilibrated with 1.2 M ammonium sulphate. The column was then washed with 1.2 M ammonium sulphate at 10 mL/min until a stable baseline was reached. The bound protein was eluted with 10 niM Tris-HCl (pH 8.0). Fractions of 5 mL were collected. The fractions of a clear blue colour, indicative of laccase enzyme, were pooled. The pooled fractions were washed using an Amicon cell with a 10 kDa cut-off filter to remove ammonium sulphate and to exchange the buffer to 50 mM borate buffer (pH 9.0). The washed protein solution was then loaded onto a XK 16 column with 15 mL Source 15Q media pre- , equilibrated with 50 mM borate buffer (pH 9.0). The column was washed with the same buffer until a stable baseline was reached. The bound protein was eluted with a linear gradient from 0 to 0.5 M NaCl in 50 mM borate buffer (pH 9.0) over 20 column volumes. Fractions of 10 mL were collected. The fractions containing pure laccase, as estimated by SDS-PAGE, spectroscopy and activity assay, were pooled and concentrated. The concentrated solution of enzyme was stored at -2O⁰C until use. All purification steps were carried out at room temperature

EXAMPLE 4 Laccase activity assay

The enzymatic activity was determined spectrophotometrically by oxidation of syringaldazine at 30⁰C: 10 μL enzyme (0.8 mg/mL) was added to a 1 cm quartz cuvette containing 1 mL 25 mM Tris-malate buffer (pH 7.5) and 75 μL 0.28 mM syringaldazine. The change in absorbance at 530 nm was monitored for 90 seconds. The slope of the progress curve was used to calculate the initial rate of the reaction.

EXAMPLE 5 pH profile

The pH dependence of the enzymatic activity was determined using the laccase substrate, syringaldazine. The activity assays were performed as described for the standard assay. However, the Tris-malate buffer normally used in the assay was substituted with Britton- Robinson buffers. The buffers were 100 mM in phosphoric acid, 100 mM in acetic acid, and 100 mM in boric acid. The pH was adjusted to the desired value by addition of sodium hydroxide.

EXAMPLE 6 Temperature profile

The temperature dependence of the enzymatic activity was determined using the assay in the range 3O⁰C - 9O⁰C. All solutions, apart from the enzyme, were pre-equilibrated at the desired temperature. The assay itself was performed in spectrophotometer with a temperature controlled cuvette holder.

EXAMPLE 7 pH stability

The pH stability was determined by incubation of the enzyme in 25 mM Britton- Robinson buffers at 40⁰C for one hour. The concentration of enzyme in the buffered solutions was 0.8 mg/mL. The activity was determined using 10 μL of the treated sample in the assay.

EXAMPLE 8

Temperature stability

The temperature stability was determined by incubation of the enzyme in 25 mM Britton- Robinson buffers at 4O⁰C for one hour. The concentration of enzyme in the buffered solutions was 0.8 mg/mL. The activity was determined using 10 μL of the treated sample in the assay.

EXAMPLE 9

Bleaching of curcumin in solution

The bleaching of curcumin in solution was studied in the pH range 2-12. The microplate- based spectrophotometric bleaching assay has been described previously. Briefly, wells in a 96-well microplate was added 200 μL solution of 50 niM Britton-Robinson buffer and 26.7 μM curcumin. The bleaching reaction was started by addition of 10 μL enzyme (stock solution: 0.16 g/L) to each well and the progress of the reaction was monitored at 440 nm for 5 minutes in a microplate reader at 25⁰C.

EXAMPLE 10

Bleaching of curcumin on fabric

A stock solution of curcumin was prepared by dissolving 10 mg curcumin in 10 mL 96% ethanol. 1 mL stock solution was added to 49 mL water to generate the working solution. Cotton swatches (06 mm) placed in wells of a 96 well microplate was soaked with 100 μL working solution and allowed to dry at 6O⁰C. Each well was then added 100 μL 100 mM Britton-Robinson buffer (pH 2-12). The bleaching reaction was started by addition of 100 μL enzyme solution. The plates were incubated at room temperature on an orbital shaker. The results were documented by scanning the plates in a flat-bed scanner and measuring the whiteness.

EXAMPLE 11

Streptomyces coelicolor laccase for denim bleaching - pH profile

Preparation of buffer system at different pH ' s :

5OmM pH4.5 Acetate Buffer (per liter): 1.764g of Sodium Acetate and 1.71g Acetic Acid were dissolved in 1 L of de-ionized water; 5OmM pH7.0 Phosphate Buffer (per liter): 10.926g Na₂HPO₄, 12H₂O and 3.043g NaH₂PO₄, 2H₂O were dissolved in 1 L of de-ionized water;

5OmM pH9.0 Borate Buffer (per liter): 15.26g Borax and 2.47g Boric Acid were dissolved in 1 L of de-ionized water;

5OmM pHIO Glycine-NaOH Buffer (per liter): 3.753g Glycine and 1.28g NaOH were dissolved in 1 L of de-ionized water;

5OmM pHl l Carbonate Buffer (per liter): 12.88g Na₂CO₃, 10H₂O and 0.42g NaHCO₃ were dissolved in 1 L of de-ionized water.

Raw denim fabrics were desized with Aquazyme Ultra 1200L (commercial available from Novozymes A/S, Denmark) at dosage of 0.54% owg under 6O⁰C for 15min, then treated with Denimax Acid XCL (commercial available from Novozymes A/S, Denmark) at dosage of 0.5g/L with liquid garment ratio of 15:1 under 55⁰C, pH4.5. The fabric was then washed with 0.25g/L Novasol P (commercial available from Novozymes A/S, Denmark) at 55⁰C for lOmin, followed by 2 washes with cold water. The treated fabric was tumble dried and cut to 12.5 cm tall and 23 cm long, and sewn to form a tube with height of 12.5cm. In a 500ml beaker, added one piece of the above mentioned denim tube and 50ml buffer with specified pH, then add SLAC to the beaker at 4.8LAMU/kg denim and Methyl Syringate at 0.62g/kg. The beaker was loaded in Launder-O-Meter (Model: LP2, SDL-Atlas) and temperature was increased to 65⁰C and kept there for bleaching. After 60min, the denim tube was taken out and dipped into 2g/L Na₂CO₃ solution at 8O⁰C for lOmin. The fabric was further rinsed with hot water and cold water and then dried in a tumble dryer. Another beaker with no addition of laccase and Methyl Syringate was set as Control. L* value on the treated fabrics was measured with DataColor SF450 (DataColor Cooperate, US). The results indicated a broad pH range for the combination of SLAC and Methyl Syringate to provide bleaching effects on denim (Table 1).

Table 1 : Bleaching effect of SLAC and Methyl syringate at different pH's

EXAMPLE 12

Streptomyces coelicolor laccase for denim bleaching - pH 11

The buffer preparation and pretreatment of denim fabrics are with the same procedures described in 1). The sewn denim tubes were bleached in LOM with 50ml buffer. SLAC at 96LAMU/kg and 1-Hydroxybenzotiazole (HOBT) at 12.4g/kg were dosed to the beaker filled with buffer at pHl l. Control samples were washed at pH4.5 and pHl l with no addition of SLAC and HOBT. The L* values are given in Table 2.

Table 2 Bleaching effect on denim fabrics at pHl 1

EXAMPLE 13

Streptomyces coelicolor laccase for denim bleaching — denim fabrics abraded with neutral cellulase

The buffer preparation and pretreatment of denim fabrics are with the same procedures described in 1) except that Denimax 399S (commercial available from Novozymes AJS, Denmark) was applied at 0.3g/L, 55⁰C, pH6.5 during abrasion. The sewn denim tubes were bleached in LOM with 150ml buffer. SLAC at 48LAMU/kg and Methyl Syringate or Violuric acid at 6.2g/kg were dosed to the beaker filled with buffer at pH9. Control samples were washed at pH9 with no addition of SLAC and Methyl Syringate or Violuric acid. The L* values are given in Table 3.

Table 3 Bleaching effect on denim fabrics abraded with neutral cellulase

SLAC + Violuric acid, ρH9 22.79

EXAMPLE 14

Streptomyces coelicolor laccase for cotton bleaching

Woven cotton fabric was desized with Aquazym 120L (commercial available from Novozymes A/S, Denmark) and washed. 6 g pre-treated fabric was added to a beaker for Lab- O-Mat with 60ml buffer solution at pH9. SLAC and Methyl Syringate were dosed to the bath as specified in Table 4. Another sample with no addition of SLAC and Methyl Syringate was set as control. The beaker was loaded to the Lab-O-Mat and heated up 60⁰C and kept there for 60 min. Afterwards the fabric was subjected to one hot rinse and one cold rinse. The washed fabric was dried and conditioned under 21°C/65%RH for 24 hours before testing. DataColor SF450 was used for CIE Whiteness reading.

Claims

1. A method for enzymatic oxidation of a substrate, comprising contacting the substrate with a polypeptide which has laccase activity and at least 60% identity with SEQ ID NO:2.

2. The method of claim 1, wherein the polypeptide contacts the substrate at pH 4-12.

3. The method of claim 2, wherein the substrate contacts the polypeptide at pH 9-12.

4. The method of any one of claims 1-3, wherein the polypeptide contacts the substrate at 25- 100 degrees Celsius.

5. The method of claim 4, wherein the polypeptide contacts the substrate at 50-100 degrees

Celsius.

6. The method of any one of claims 1 to 5, wherein the substrate is a dye or colorant, or a textile.

7. The method of claim 6, wherein the dye is indigo.

8. The method of any one of claims 1-5, wherein the substrate is lignin or a lignin containing material.

9. The method of any one of claims 1-8, which further comprises contacting the substrate with an enhancing agent.

10. A detergent composition comprising a surfactant and a polypeptide, which has laccase activity and at least 60% identity with SEQ ID NO:2.

11. A composition comprising a textile material and a polypeptide, which has laccase activity and at least 60% identity with SEQ ID NO:2.

12. The compositions of claims 10 or 11, which further comprise an enhancing agent.