WO2012054485A1 - Local color modification of dyed fabrics using a laccase system - Google Patents

Local color modification of dyed fabrics using a laccase system Download PDF

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Publication number
WO2012054485A1
WO2012054485A1 PCT/US2011/056717 US2011056717W WO2012054485A1 WO 2012054485 A1 WO2012054485 A1 WO 2012054485A1 US 2011056717 W US2011056717 W US 2011056717W WO 2012054485 A1 WO2012054485 A1 WO 2012054485A1
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method
laccase
textile
preceding
embodiments
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PCT/US2011/056717
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French (fr)
Inventor
Andreas Krouwer
Piera M. Pericu
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Danisco Us Inc.
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Priority to US39431210P priority Critical
Priority to US61/394,312 priority
Application filed by Danisco Us Inc. filed Critical Danisco Us Inc.
Publication of WO2012054485A1 publication Critical patent/WO2012054485A1/en

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Classifications

    • DTEXTILES; PAPER
    • D06TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06MTREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS, OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • DTEXTILES; PAPER
    • D06TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06PDYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS, OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/02After-treatment
    • DTEXTILES; PAPER
    • D06TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06PDYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS, OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/15Locally discharging the dyes
    • DTEXTILES; PAPER
    • D06TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06PDYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS, OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/15Locally discharging the dyes
    • D06P5/158Locally discharging the dyes with other compounds

Abstract

The present systems, compositions, and methods relate to local color modification of dyed fabrics using a laccase enzyme system.

Description

LOCAL COLOR MODIFICATION OF DYED FABRICS

USING A LACCASE SYSTEM

PRIORITY

[01] The present application claims priority to U.S. Provisional Patent Application Serial No. 61/394,312, filed on October 18, 2010, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[02] The present systems, compositions, and methods relate to local color modification of dyed fabrics using a laccase enzyme system.

BACKGROUND

[03] Laccases are copper-containing phenol oxidizing enzymes that are known to be good oxidizing agents in the presence of oxygen. Laccases are found in microbes, fungi, and higher organisms. Laccase enzymes are used for many applications, including pulp and paper bleaching, treatment of pulp waste water, de-inking, industrial color removal, bleaching in laundry detergents, oral care teeth whiteners, as catalysts or facilitators for polymerization and oxidation reactions, in the textiles industry, and in the food industry.

[04] Laccases are known to be produced by a wide variety of fungi, including species of the genii Aspergillus, Neurospora, Podospora, Botrytis, Pleurotus, Fornes, Phlebia, Trametes, Polyporus, Stachybotrys, Rhizoctonia, Bipolaris, Curvularia, Amerosporium, Lentinus, Myceliophtora, Coprinus, Thielavia, Cerrena, Streptomyces, and Melanocarpus. For many applications, the oxidizing efficiency of a laccase can be improved through the use of a mediator, also known as an enhancing agent.

SUMMARY

[05] Described are systems, compositions, and methods relating to local color modification of dyed fabrics using a laccase enzyme system. In one aspect, a textile processing method is provided, comprising contacting a portion of dyed textile with a laccase enzyme system for a length of time and under conditions sufficient to cause a localized color modification to the portion of the textile.

[06] In some embodiments, the method is performed by wetting but not submerging the portion of the dyed textile with a composition comprising the laccase enzyme system. In some embodiments, the method is performed by applying a composition comprising the laccase enzyme system to the portion of the dyed textile, and then wetting but not submerging the portion of the dyed textile.

[07] In some embodiments, the laccase enzyme system is provided in a single composition. In some embodiments, the laccase enzyme system is provided as an aqueous, gel, semi-solid, or solid formulation.

[08] In some embodiments, the color modification is selected from lightening of color, change of color, change in color cast, and bleaching.

[09] In some embodiments, the textile is indigo-dyed denim. In some embodiments, the textile is indigo and sulfur-dyed denim.

[10] In some embodiments, the textile is a pair of jeans. In some embodiments, the portion of the textile is a pant leg, sleeve, cuff, collar, pocket, or belt loop. In some embodiments, the portion of the textile is in the form of a predetermined shape. In some embodiments, the portion of the textile is in the form of a letter, word, logo, or trademark.

[11] In some embodiments, the laccase enzyme system comprises a laccase enzyme and a mediator. In some embodiments, the laccase is a microbial laccase. In some embodiments, the laccase is from a Cerrena species. In some embodiments, the laccase is from Cerrena unicolor. In some embodiments, the laccase is laccase D from C. unicolor. In some embodiments, the mediator is syringonitrile.

[12] In some embodiments, the method is performed at a temperature of from about 20°C to about 40°C. In some embodiments, the method is performed at a temperature of from about 20°C to about 30°C. In some embodiments, the method is performed at the ambient temperature of tap water. In some embodiments, the method is performed at ambient air temperature.

[13] In another aspect, a locally color-modified dyed textile produced by any of these methods is provided.

[14] These and other aspects and embodiments of the present system, compositions, and methods will be apparent from the description and accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[15] Figure 1 is an image of indigo-dyed jeans that were subjected to localized color modification in the shape of a heart. DETAILED DESCRIPTION

[16] Described are systems, compositions, and methods relating to local color modification of dyed fabrics using laccase enzymes and associated reagents. The systems, compositions, and methods are useful, for example, for processing textiles to affect local color modification on fabrics and garments, including complete, consumer-ready garments. Various aspects and embodiments of the systems, compositions, and methods are to be described.

Definitions

[17] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Singleton et al. ,

DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide a general dictionary of many of the terms used herein. The following terms are defined for additional clarity.

[18] As used herein, the term "enzyme" refers to a protein that catalyzes a chemical reaction. The catalytic function of an enzyme constitutes its "enzymatic activity" or "activity." An enzyme is typically classified according to the type of reaction it catalyzes, e.g. , oxidation of phenols, hydrolysis of peptide bonds, incorporation of nucleotides, etc.

[19] As used herein, the term "substrate" refers to a substance (e.g., a chemical compound) on which an enzyme performs its catalytic activity to generate a product.

[20] As used herein, a "laccase" is a multi-copper containing oxidase (EC 1.10.3.2) that catalyzes the oxidation of phenols, polyphenols, and anilines by single-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process.

[21] As used herein, "laccase activity" is measured in units/gram (U/g), wherein one unit is defined as the amount of laccase activity required to oxidize 1 nmol of 2,2'-azinobis(3- ethylbenzthiazoline-6-sulfonate; ABTS) substrate per second under conditions of an assay based on the ability of laccase enzyme to oxidize ABTS into its corresponding stable cation radical,

+

i.e. , ABTS . Unlike the initial form of ABST, the radical form is dark green in color with increased absorbance at 420 nm. The amount of green color formation is proportional to the amount of laccase activity, and can be compared to a laccase standard curve to determine the absolute amount of laccase activity.

[22] As used herein, "variant" proteins encompass related and derivative proteins that differ from a parent/reference protein by a small number of amino acid substitutions, insertions, and/or deletions. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids residues. In some embodiments, variant proteins have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity to a parent/reference protein.

[23] As used herein, the term "analogous sequence" refers to a polypeptide sequence within a protein that provides a similar function, tertiary structure, and/or conserved residues with respect to a sequence within a parent/reference protein. For example, in structural regions that contain an alpha helix or a beta sheet structure, replacement amino acid residues in an analogous sequence maintain the same structural feature. In some embodiments, analogous sequences result in a variant protein that exhibits a similar or improved function with respect to the parent protein from which the variant is derived.

[24] As used herein, a "homologous protein" or "homolog" refers to a protein (e.g. , a laccase enzyme) that has a similar function (e.g. , enzymatic activity) and/or structure as a reference protein (e.g. , a laccase enzyme from a different source). Homologs may be from evolutionarily related or unrelated species. In some embodiments, a homolog has a quaternary, tertiary and/or primary structure similar to that of a reference protein, thereby potentially allowing for replacement of a segment or fragment in the reference protein with an analogous segment or fragment from the homolog, with reduced disruptiveness of structure and/or function of the reference protein in comparison with replacement of the segment or fragment with a sequence from a non-homologous protein.

[25] As used herein, "wild-type," "native," and "naturally-occurring" proteins are those found in nature. The terms "wild-type sequence" refers to an amino acid or nucleic acid sequence that is found in nature or naturally occurring. In some embodiments, a wild- type sequence is the starting point of a protein engineering project, for example, production of variant proteins.

[26] As used herein, a "signal sequence" refers to a sequence of amino acids bound to the N- terminal portion of a protein, and which facilitates the secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.

[27] As used herein, the term "derivative" refers to a protein that is derived from a parent/reference protein by addition of one or more amino acids to either or both the N- and C- terminal end(s), substitution of one or more amino acid residues at one or a number of different sites in the amino acid sequence, deletion of one or more amino acid residues at either or both ends of the protein or at one or more sites in the amino acid sequence, and/or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a protein derivative is often achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative protein.

[28] As used herein, the terms "polypeptide, "protein," and "peptide," refer to a composition comprised of amino acids (i.e., amino acid residues). The conventional one-letter or three-letter codes for amino acid residues are used. A polypeptide may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[29] As used herein, the term "textile" refers to fibers, yarns, fabrics, garments, and non- woven materials. The term encompasses textiles made from natural and synthetic (e.g., manufactured) materials, as well as natural and synthetic blends. The term "textile" refers to both unprocessed and processed fibers, yarns, woven or knit fabrics, non-wovens, and garments. In some embodiments, a textile contains cellulose.

[30] As used herein, the term "fabric" refers to a manufactured assembly of fibers and/or yarns that has substantial surface area in relation to its thickness and sufficient cohesion to give the assembly useful mechanical strength.

[31] As used herein, the term "garment" refers to a clothing item made from one or more fabrics. Garments typically include fabrics that are already cut to size and sewn or stitched together. Garments may or may not include buttons, eyelets, straps, zippers, hook-and-loop closures, and the like, which can be attached before or after localized color modification.

[32] As used herein, the term "color modification" refers to a change in the chroma, saturation, intensity, luminance, and/or tint of a color associated with a fiber, yarn, fabric, garment, or non- woven material, collectively referred to as textile materials. Color modification encompasses chemical modification to a chromophore as well as chemical modification to the material to which a chromophore is attached. Examples of color modification include fading, bleaching, and altering tint. A particular color modification to indigo-dyed denim is fading to a "vintage look," which has a less intense blue/violet tint and more subdued grey appearance than the freshly-dyed denim. [33] As used herein, the term "local color modification" refers to color modification, as defined, above, that is performed on only a portion of a fabric or garment. Unlike generalized textile color modification, which is typically performed in a bath, i.e. , in a submerged environment, local color modification is performed using a wetted but not submerged fabric or garment, typically on a table, work bench, or other hard surface, on a hanging or otherwise suspended fabric or garment, or using rollers or other processing equipment that do not subject the fabric or garment to a submerged environment, such that only a portion of the garment can be subjected to color modification without affecting the remainder of the fabric or garment.

[34] As used herein, "a portion of a fabric or garment" refers to anything less than the whole fabric or garment. Where specified, a portion of a fabric or garment may refer to an indicated structural or decorative feature a fabric or garment, such as a pant leg, a sleeve, a pocket, a belt loop, a cuff, a hem, and the like.

[35] As used herein, the term "bleaching" refers to the process of treating a textile material such as a fiber, yarn, fabric, garment or non- woven material to produce a lighter color. This term includes the production of a brighter and/or whiter textile, e.g. , in the context of a textile processing application, as well as lightening of the color of a stain, e.g., in the context of a cleaning application.

[36] As used herein, the terms "size" and "sizing" refer to compounds used in the textile industry to improve weaving performance by increasing the abrasion resistance and strength of a yarn. Size is usually made of starch or starch-like compounds.

[37] As used herein, the terms "desize" and "desizing" refer to the process of

eliminating/removing size (generally starch) from a textile, usually prior to applying special finishes, dyes or bleaches.

[38] As used herein, the term "desizing enzyme" refers to an enzyme used to remove size. Exemplary enzymes are amylases, cellulases, and mannanases.

[39] As used herein, the term "% identity" refers to the level of nucleic acid sequence identity between a nucleic acid sequence that encodes a laccase as described herein and another nucleic acid sequence, or the level of amino acid sequence identity between a laccase enzyme as described herein and another amino aid sequence. Alignments may be performed using a conventional sequence alignment program. Exemplary levels of nucleic acid and amino acid sequence identity include, but are not limited to, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, or more, sequence identity to a given sequence, e.g. , the coding sequence for a laccase or the amino acid sequence of a laccase, as described herein.

[40] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at www.ncbi.nlm.nih.gov/BLAST. See also, Altschul, et ah, 1990 and Altschul, et ah , 1997.

[41] Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA

Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, et al , 1997.)

[42] An alignment of selected sequences in order to determine " identity" between two or more sequences, may be performed using, for example, the CLUSTAL-W program in

Mac Vector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[43] As used herein, the terms "chemical mediator" and "mediator" are used interchangeably to refer to a chemical compound that functions as a redox mediator to shuttle electrons between an enzyme exhibiting oxidase activity {e.g. , a laccase) and a secondary substrate or electron donor. Such chemical mediators are also known in the art as "enhancers" and "accelerators."

[44] As used herein, the terms "secondary substrate" and "electron donor" are used interchangeably to refer to a dye, pigment {e.g. , indigo), chromophore {e.g. , polyphenolic, anthocyanin, or carotenoid), or other secondary substrate to and from which electrons can be shuttled by an enzyme exhibiting oxidase activity.

[45] The following abbreviations/acronyms have the following meanings unless otherwise specified:

EC enzyme commission

EDTA ethylenediaminetetraacetic acid

kDa kiloDalton

MW molecular weight

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt weight percent

°C degrees Centigrade

H20 water

d¾0 or DI deionized water dIH20 deionized water, Milli-Q filtration

g gram

μ microgram

mg milligram

kg kilogram

μΐ^ and μΐ microliter

mL and ml milliliter

mm millimeter

μιη micrometer

M molar

mM millimolar

μΜ micromolar

U unit

sec and " second

min and ' minute

hr hour

eq. equivalent

N normal

RTU ready-to-use

U Unit

owg on weight of goods

CIE International Commission on Illumination

[46] Numeric ranges are inclusive of the numbers defining the range. The singular articles "a," "an," "the," and the like, include the plural referents unless otherwise clear from context. Unless otherwise specified, polypeptides are written in the standard N-terminal to C-terminal direction and polynucleotides are written in the standard 5' to 3' direction. It is to be understood that the particular methodologies, protocols, and reagents described, are not intended to be limiting, as equivalent methods and materials can be used in the practice or testing of the present compositions and methods. Although the description is divided into sections to assist the reader, section heading should not be construed as limiting and the description in one section may apply to another. All publications cited herein are expressly incorporated by reference.

Laccase and Laccase Related Enzymes

[47] The present laccase enzyme systems, compositions, and methods include one or more laccases or laccase-related enzymes, herein collectively referred to as "laccases" or "laccase enzymes." Such laccases include any laccase enzyme encompassed by EC 1.10.3.2, according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Laccase enzymes from microbial and plant origin are known in the art. A microbial laccase enzyme may be derived from bacteria or fungi (including filamentous fungi and yeasts). Suitable examples include a laccase derived or derivable from a strain of

Aspergillus, Neurospora {e.g. , N. crassa), Podospora, Botrytis, Collybia, Cerrena {e.g. , C. unicolor), Stachybotrys, Panus (e.g. , P. rudis), Thielavia, Fomes, Lentinus, Pleurotus, Trametes {e.g. , T. villosa, and T. versicolor), Rhizoctonia {e.g. , R. solani), Coprinus {e.g. , C. plicatilis and C. cinereus), Psatyrella, Myceliophthora {e.g. , M. thermonhila), Schytalidium, Phlebia {e.g., P. radita (WO 92/01046)), or Coriolus {e.g. , C. hirsutus (JP 2238885)), Spongipellis, Polyporus, Ceriporiopsis subvermispora, Ganoderma tsunodae, and Trichoderma.

[48] A laccase may be produced by culturing a host cell transformed with a recombinant DNA vector that includes nucleotide sequences encoding the laccase. The DNA vector may further include nucleotide sequences permitting the expression of the laccase in a culture medium, and optionally allowing the recovery of the laccase from the culture.

[49] An expression vector containing a polynucleotide sequence encoding a laccase enzyme may be transformed into a suitable host cell. The host cell may be a fungal cell, such as a filamentous fungal cell, examples of which include but are not limited to species of Trichoderma [e.g. , T. reesei (previously classified as T. longibrachiatum and currently also known as Hypocrea jecorina], T. viride, T. koningii, and T. harzianum), Aspergillus {e.g., A. niger, A. nidulans, A. oryzae, and A. awamori), Penicillium, Humicola {e.g. , H. insolens and H. grisea), Fusarium {e.g., F. graminum and F. venenatum), Neurospora, Hypocrea, and Mucor. A host cell for expression of a laccase enzyme may also be from a species of Cerrena {e.g., C.

unicolor). Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall using techniques known in the art.

[50] Alternatively, the host organism may from a species of bacterium, such as Bacillus [e.g. , B. subtilis, B. licheniformis, B. lentus, B. (now Geobacillus) stearothermophilus, and fi. brevis], Pseudomonas, Streptomyces {e.g. , S. coelicolor, S. lividans), or E. coli. The transformation of bacterial cells may be performed according to conventional methods, e.g. , as described in Maniatis, T. et al , "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, 1982. The screening of appropriate DNA sequences and construction of vectors may also be carried out by standard procedures {cf. supra).

[51] The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells. In some embodiments, the expressed enzyme is secreted into the culture medium and may be recovered therefrom by well-known procedures. For example, laccases may be recovered from a culture medium as described in U.S. Patent Publication No. 2008/0196173. In some embodiments, the enzyme is expressed intracellularly and is recovered following disruption of the cell membrane. [52] In particular embodiments, the expression host may be Trichoderma reesei with the laccase coding region under the control of a CBHl promoter and terminator (see, e.g. , U.S. Patent No. 5,861,271). The expression vector may be, e.g. , pTrex3g, as disclosed in U.S. Patent No. 7,413,887. In some embodiments, laccases are expressed as described in U.S. Patent Publication Nos. 2008/0196173 or 2009/0221030.

[53] The following laccase genes and laccases are described in U.S. Publication No.

2008/0196173, and may be used as described:

A. Cerrena laccase Al from CBS115.075 strain (SEQ ID NO: 1):

MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50

TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100

AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150

KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG 200

PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250

SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDSGMNSAI 300

LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350

DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400

VVELVIPAGV VGGPHPFHLH GHNFWWRSA GTDQYNFNDA ILRDVVSIGG 450

TGDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNLTPQ 500

AWDELCPKYN ALSAQKKLNP STT 523

B. Cerrena laccase A2 from CBS154.29 strain (SEQ ID NO: 2):

MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50

TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100

AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150

KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG 200

PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250

SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDNGMNSAI 300

LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350

DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400

VVELVIPAGV VGGPHPFHLH GHNFWWRSA GTDQYNFNDA ILRDVVSIGG 450

TEDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNPTPQ 500

AWDELCPKYN ALNAQKKLNP STT 523

C. Cerrena laccase Bl from CBS115.075 strain (SEQ ID NO: 3):

MSLLRSLTSL IVLVIGAFAA IGPVTDLHIV NQNLDPDGFN RPTVLAGGTF 50

PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100

VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150

PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200

NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250

TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300

VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350

FNAGRFTING ASLTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400

IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDWSIGGAN 450

DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500

NQLCPLYDAL SPGDT 515 D. Cerrena laccase B2 from CBS154.29 strain (SEQ ID NO: 4):

MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGLN RPTVLAGGTF 50

PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100

VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150

PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200

NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250

TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300

VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350

FNAGRFTING TSFTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400

IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDWSIGGAN 450

DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500

NQLCPLYDAL SPGDT 515

E. Cerrena laccase B3 (partial) from ATCC20013 strain (SEQ ID NO: 5):

MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGFN RPTVLAGGTF 50

PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100

VTQCPITSGN SFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150

PNKQLYDVDN GKTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200

NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250

TVDSIQIFAG QRYSFVMQAN QPVGNYWI 278

F. Cerrena laccase C (partial) from CBS154.29 strain (SEQ ID NO: 6):

AIGPVADLHI TDDTIAPDGF SRPAVLAGGG FPGPLITGNK GDAFKLNVID 50

ELTDASMLKX TSIHWHGFFQ KGTNWADGPA FVNQCPITTG NSFLYDFQVP 100

DQAGTYWYHS HLSTQYCDGL RGAFVVYDPS DPHKDLYDVD DESTVITLAD 150

WYHTLARQIV GVAISDTTLI NGLGRNTNGP ADAALAVINV DAGKRYRFRL 200

VSISCDPNWV FSIDNHDFTV IEVDGVNSQP LNVDSVQIFA GQRYSF 246

G. Cerrena laccase Dl from CBS154.29 strain (SEQ ID NO: 7):

MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50

TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100

AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150

NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200

PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250

PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300

LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350

NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400

VVELVIPPLA VGGPHPFHLH GHNFWWRSA GSDEYNFDDA ILRDVVSIGA 450

GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500

AWDELCPKYN GLSASQKVKP KKGTAI 526

H. Cerrena laccase D2 from CBS115.075 strain (SEQ ID NO: 8):

MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50

TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100

AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150

NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200

PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250

PLTVDSIQIF AGQRYSFVLN ANQPDDNYWI RAMPNIGRNT TTLDGKNAAI 300

LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350

NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400

VVELVIPPLA VGGPHPFHLH GHNFWWRSA GSDEYNFDDA ILRDVVSIGA 450 GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ AWDELCPKYN GLSASQKVKP KKGTAI

I. Cerrena laccase E (partial) from CBS154.29 strain (SEQ ID NO: 9):

AIGPVADLKI VNRDIAPDGF IRPAVLAGGS FPGPLITGQK GNEFKINVVN 50

QLTDGSMLKS TSIHWHGFFQ KGTNWADGPA FVNQCPIATN NSFLYQFTSQ 100

EQPGTFWYHS HLSTQYCDGL RGPLVVYDPQ DPHAVLYDVD DESTI ITLAD 150

WYHTLARQVK GPAVPGTTLI NGLGRHNNGP LDAELAVISV QAGKRQVQFT 200

LFTLYRFRLI SISCDPNYVF SIDGHDMTVI EVDSVNSQPL KVDSIQIFAG 250

QRYSFVLNAN QP 262

In some embodiments, a laccase D enzyme having the following amino acid sequence (SEQ ID NO: 10; signal sequence in italics) may be used:

MGLNSAITSL AILALSVGSY AAIGPVADLH IVNKDLAPDG VQRPTVLAGG 50

TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100

AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150

NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200

PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250

PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300

LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350

NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400

VVELVIPPLA VGGPHPFHLH GHNFWWRSA GSDEYNFDDA ILRDVVSIGA 450

GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500

AWDELCPKYN GLSASQKVKP KKGTAI 526

The mature processed form of this polypeptide is as follows (SEQ ID NO: 11):

AIGPVADLHIVNKDLAPDGVQRPTVLAGGTFPGTLITGQKGDNFQLNVIDDLTDDRMLTPTS IHWHGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVPDQAGTFWYHSHLSTQYCDGLRGAF VVYDPNDPHKDLYDVDDGGTVITLADWYHVLAQTVVGAATPDSTLINGLGRSQTGPADAELA VISVEHNKRYRFRLVSISCDPNFTFSVDGHNMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVL NANQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSPLNEADLRPLV PAP PGNAVPGGADINHRLNLTFSNGLFS INNASFTNPS PALLQILSGAQNAQDLLPTGSY IGLELGKVVELVIPPLAVGGPHPFHLHGHNFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEV TIRFVTDNPGPWFLHCHIDWHLEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQK VKPKKGTAI

[54] In some embodiments, laccase enzymes suitable for use in the present compositions and methods are mature polypeptides that lack a signal sequence that may be used to direct secretion of a full-length polypeptide from a cell. A suitable mature polypeptide may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, or more, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. Preferably, such polypeptides have enzymatic laccase activity, as determined using the assays and procedures described, herein.

[55] In some embodiments, laccase enzymes suitable for use in the present compositions and methods are truncated with respect to a full-length or mature parent/reference sequence. Such truncated polypeptides may be generated by the proteolytic degradation of a full-length or mature polypeptide sequence or by engineering a polynucleotide to encode a truncated polypeptide. Exemplary polypeptides are truncated at the amino and/or carboxyl-terminus with respect to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. The truncation may be of a small number, e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, or of entire structural or functional domains. A suitable truncated polypeptide may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, or more, amino acid sequence identity to the corresponding portion of one or more of the above-references amino acid sequences. Preferably, such polypeptides have enzymatic laccase activity, as determined using the assays and procedures described, herein.

Mediators

[56] In some embodiments, the present laccase enzyme systems, compositions, and methods, further include one or more chemical mediator agents that enhance the activity of the laccase enzyme. A mediator (also called an enhancer or accelerator) is a chemical that acts as a redox mediator to effectively shuttle electrons between the enzyme exhibiting oxidase activity and a dye, pigment (e.g. , indigo), chromophore (e.g. , polyphenolic, anthocyanin, or carotenoid, for example, in a colored stain), or other secondary substrate or electron donor.

[57] In some embodiments the chemical mediator is a phenolic compound, for example, methyl syringate, or a related compound, as described in, e.g. , PCT Application Nos. WO 95/01426 and WO 96/12845. The mediator may also be an N-hydroxy compound, an N-oxime compound, or an N-oxide compound, for example, N-hydroxybenzotriazole, violuric acid, or N- hydroxyacetanilide. The mediator may also be a phenoxazine/phenothiazine compound, for example, phenothiazine-10-propionate. The mediator may further be 2,2'-azinobis-(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS). Other chemical mediators are well known in the art, for example, the compounds disclosed in PCT Application No. WO 95/01426, which are known to enhance the activity of a laccase. The mediator may also be acetosyringone, methyl syringate, ethyl syringate, propyl syringate, butyl syringate, hexyl syringate, or octyl syringate.

[58] In some embodiments, the mediator is 4-cyano-2,6-dimethoxyphenol, 4-carboxamido- 2,6-dimethoxyphenol or an N-substituted derivative thereof such as, for example, 4-(N-methyl carboxamido)-2,6-dimethoxyphenol, 4-[N-(2-hydroxyethyl) carboxamido]-2,6- dimethoxyphenol, or 4-(N,N-dimethyl carboxamido)-2,6-dimethoxyphenol.

[59] In some embodiments, the mediator is described by the following formula:

Figure imgf000015_0001

in which A is a group such as -R, -D, -CH=CH-D, -CH=CH-CH=CH-D, -CH=N-D, -N=N- D, or -N=CH-D, D is selected from the group consisting of -CO-E, -SO2-E, -CN, -NXY, and

-N+XYZ, E is -H, -OH, -R, -OR, or -NXY, and X,Y, and Z are independently selected from - H, -OH, -OR, and -R; where R is a Ci - Ci6 alkyl, preferably a Ci -C8 alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C are independently selected from Cm H2m+i ; 1 < m < 5.

[60] In some embodiments, A in the above mentioned formula is -CN or -CO-E, wherein E may be -H, -OH, -R, -OR, or -NXY, where X and Y are independently selected from -H, -OH, -OR, and -R, where R is a Ci -C16 alkyl, preferably a Ci -C8 alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C are independently selected from Cm H2m+i; 1 < m < 5. In some embodiments, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as "syringonitrile" or "SN").

[61] Note that in the above mentioned formula, A may be placed meta to the hydroxy group, instead of being placed in the para position as shown.

[62] For applications such as textile processing, the mediator may be present in a

concentration of about 0.005 to about 1,000 μιηοΐβ per g denim, about 0.05 to about 500 μιηοΐβ per g denim, about 0.1 to about 100 μιηοΐβ per g denim, about 1 to about 50 μιηοΐβ per g denim, or about 2 to about 20 μιηοΐβ per g denim.

[63] The mediators may be prepared by methods known to the skilled artisan, such as those disclosed in PCT Application Nos. WO 97/11217 and WO 96/12845 and U.S. Patent No. 5,752,980. Other suitable mediators are described in, e.g., U.S. Patent Publication No.

2008/0189871.

Methods of Use

[64] Generally, the methods involve locally contacting a textile or garment with a laccase enzyme system for a length of time, and under conditions, sufficient to produce at least one measurable or visual local effect to the fabric or textile. Exemplary effects are, e.g. , a change in color, a change in color cast, lightening, bleaching, and fading. The present laccase enzyme systems, compositions, and methods can be use in applications where localized color modification of dyed fabrics is desirable. Examples of localized color modification include color modification the fronts or backs of jeans, to sleeves, to collars, to cuffs, to belt loops, and the like.

[65] The method may be performed by wetting (but not submerging) a portion of the dyed textile with a composition comprising the laccase enzyme system, or performed by applying a composition comprising the laccase enzyme system to a portion of the dyed textile, and then wetting (but not submerging) the portion of the dyed textile. An important feature of the method is that less than the entire fabric or garment is contacted with the laccase enzyme system, and that color modification occurs on less than the entire fabric or garment.

[66] The laccase enzyme system may be provided in a single composition, which may be aqueous, gel, semi-solid, or solid in form. Alternatively, the laccase enzyme system may be provided in a plurality of compositions, which may be aqueous, gel, semi-solid, or solid, or a combination, thereof. Where the laccase system, or components, thereof, are provided in liquid form, they can be applied to fabrics or garments by pouring, dripping, brushing, blotting, spraying, dipping, and the like. Where the laccase system, or components, thereof, are provided in gel, paste, or other semi-solid form, they can be applied using a stick applicator, by dispensing from a tube or syringe, drawn with a crayon, or the like. Where the laccase system, or components, thereof, are provided in dry or solid form, they can be applied by shaking from a container, as a talc, or the like.

[67] In many embodiments, the fabric or garment may be wetted before and/or after application of the laccase; however, the fabric or garment is generally not submerged in an aqueous medium (i.e. , in a "bath"), since this procedure would allow the color modification to become generalized. Thus, particular embodiments of the methods are expressly not performed under submerged conditions.

[68] In some embodiments, the localized color modification may be in the form of a predetermined pattern or shape, as exemplified by the heart shape illustrated in Figure 1. In further embodiments, the pattern or shape may in the form of letters (including numbers and symbols), words, logos, trademarks (including tradedress), or the like. In some embodiments, the pattern or shape may be designed to mimic the appearance of conventional textile processing methods, such as sandblasting.

[69] In some embodiments, the predetermined pattern or shape is selected by the textile manufacturer. In other embodiments, the predetermined pattern or shape is selected by a retailer or consumer. In some cases, the term predetermined may apply to the overall aesthetic desired, rather than an exact pattern. For example, local color variant can be in the form of unique and artistic designs, even designs with a random element, which are still considered to be predetermined as used herein.

[70] In some embodiments, different levels of local color modification are produced by applying different amounts of a laccase enzyme system, applying a laccase enzyme system for different amounts of time, or applying laccase enzyme systems that have been preincubated for different amounts of time to dyed fabric. In this manner, a dyed fabric can be modified to have more than one color modification, e.g. , a "light-colored portion" and a "lighter colored portion." The ability to produce different levels of color modification greatly increases the complexity of patterns and shapes that can be produced.

[71] Textiles that can be subjected to color modification as described include cellulosic and non-cellulosis textiles, for example, cotton, linen, flax, hemp, jute wool, silk, nylon, polyester, acrylic, and blends, thereof.

[72] The textile may be dyed with any dye that may be decolorized using a laccase enzyme. Examples of dyes include, but are not limited to, azo, monoazo, disazo, nitro, xanthene, quinoline, anthroquinone, triarylmethane, paraazoanyline, azineoxazine, stilbene, aniline, and phthalocyanine dyes, or mixtures thereof. In some embodiments, the dye is an azo dye (e.g. , Reactive Black 5 (2,7-naphthalenedisulfonic acid, 4-amino-5- hydroxy-3,6-bis((4-((2-

(sulfooxy)ethyl)sulfonyl)phenyl)azo)-tetrasodium salt), Reactive Violet 5, methyl yellow, congo red). In some embodiments, the dye is an anthraquinone dye (e.g. , remazol blue), indigo (indigo carmine), or a triarylmethane/paraazoanyline dye (e.g. , crystal violet, malachite green). In various embodiments, the dye is a reactive, direct, disperse, or pigment dye. In some embodiments, the dye is comprised within an ink. In some embodiments, the dye is indigo and/or a sulfur-based dye. In some embodiments, the textile is denim dyed with indigo and/or a sulfur-based dye. In a particular embodiment, the textile is dyed with indigo, and the laccase enzyme and mediator are used to oxidize the indigo to isatin. [73] The methods contemplate the use of one or more of the laccases, many of which are described herein. In some embodiments, the laccase is from a Cerrena species, such as C.

unicolor. In some embodiments, the laccase comprises, consists of, or consists essentially of the amino acid sequence of any of the C. unicolor laccase enzymes described herein, or an amino acid sequence having any of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% identity to any of the C. unicolor laccase enzymes described herein, and having laccase enzymatic activity. In a particular embodiment, the laccases is C. unicolor laccase D, or a closely related laccase.

[74] In some embodiments, such methods include localized incubation of a laccase enzyme with a dyed fabric at a low temperature, for example, about 50°C or less, about 45°C or less, or even about 40°C or less. In some embodiments, the temperature is between about 10°C and about 50°C. In some embodiments, the temperature is between about 15°C and about 45°C. In some embodiments, the temperature is between about 20°C and about 40°C. In some embodiments, the temperature is between about 25° to about 35 °C. In some embodiments, the temperature is about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C. In some embodiments, the temperature is ambient air temperature.

[75] In some embodiments, one or more laccase enzymes are used at a concentration of about 0.005 to about 5,000 mg/liter, about 0.05 to about 500 mg/liter, about 0.1 to about 100 mg/liter, or about 0.5 to about 10 mg/liter. In some processing embodiments, a laccase is used at a concentration of about 0.005 to about 5,000 mg/kg of fabric (such as denim), e.g., about 0.05 to about 500 mg/kg of fabric, about 0.1 to about 100 mg/kg of fabric, or about 0.5 to about 10 mg/kg of fabric. In some embodiments, a laccase is used at a pH of about 5 to about 8, about 5 to about 7.5, about 5 to about 7, about 5.5 to about 6.5, about 5 to about 6, or about 6.

Exemplary pH values are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[76] The present localized color modification systems, compositions, and methods, can be utilized following various textile processing steps applied to whole garments or fabrics, for example, desizing, abrading (physical or enzymatic), scouring, dyeing, bleach clean-up, biopolishing, enzymatic or chemical color modification, and the like.

[77] Abrading and/or biopolishing may be performed using a suitable cellulase, such as those derived from microorganisms which are known to be capable of producing cellulolytic enzymes, e.g. , species of Humicola, Thermomyces, Bacillus, Trichoderma, Fusarium, Myceliophthora, Phanerochaete, Irpex, Scytalidium, Schizophyllum, Penicillium, Aspergillus or Geotricum. Known species capable for producing celluloytic enzymes include Humicola insolens, Fusarium oxysporum or Trichoderma reesei. Non- limiting examples of suitable cellulases are disclosed in U.S. Patent No. 4,435,307; European patent application No. 0 495 257; PCT Patent Application No. WO 91/17244; and European Patent Application No. EP-A2-271 004, all of which are incorporated herein by reference.

[78] In some embodiments, the present systems, compositions, and methods, are using before or after "stonewashing" using a cellulase, bleaching using an aryl esterase, and/or color modification to the entire textile or garment using a laccase, can be combined to provide a comprehensive enzymatic textile processing system. Such a system allows a textile processor to produce textiles with a wide variety of finishes without the need to use conventional textile processing chemical.

[79] In some embodiments, the present systems, compositions, and methods, are using before or after "desizing," e.g. , using an amylase, "scouring", e.g. , using a pectate lyase, and/or bleach- clean-up," using catalase. Compositions and Kits for Local Color Modification

[80] The present systems and compositions can be provided in one of more aqueous, gel, semi-solid, or solid compositions comprising, consisting of, or consisting essentially of a laccase enzyme and, optionally, a mediator suitable for localized fabric application. In some embodiments, the present systems and compositions can be provided in the form of a single "just add water" or "ready to use" (RTU) composition. Such compositions may further contain one or more buffers, dispersants, surfactants, blockers, polymers, preservatives, and the like. An exemplary buffer is a monosodium phosphate/adipic acid system. In particular embodiments, the compositions are in granular form for ease of storage and transportation. Such compositions are diluted with water prior to use.

[81] Once reconstituted for use from a convenient storage form, the laccase enzyme system may be allowed to preincubate for a period of time, e.g., 5 minutes to 2 hours, 10 minutes to 1 hour, 15 minutes to 45 minutes, 20 minutes to 30 minutes, and the like. Exemplary preincubation times are about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, and 120 minutes.

[82] The laccase compositions may then be applied to a fabric or garment by pouring, dripping, brushing, blotting, spraying, using a stick applicator, by dispensing from a tube or syringe, drawn with a crayon, shaking from a container, as a talc, or the like. Where the laccase enzyme system is in the form of a liquid, it is preferably applied by spraying (e.g. , using an air brush or aerosol bottle), or using a free-hand brush, roller, or the like. A stencil may be used to help define the pattern or shape.

[83] The present systems, compositions, and methods are further described in the following numbered paragraphs.

1. A textile processing method is provided, comprising contacting a portion of dyed textile with a laccase enzyme system for a length of time and under conditions sufficient to cause a localized color modification to the portion of the textile.

2. In some embodiments, the method of paragraph 1 is performed by wetting but not submerging the portion of the dyed textile with a composition comprising