CA2747813A1 - Laccases and methods of use thereof at low temperature - Google Patents

Abstract

Laccase enzymes and nucleic acid sequences encoding such laccase enzymes are described. The laccase enzymes may be employed in conjunction with mediators in improved methods for modifying the color of denim fabrics. Low temperature and single-bath textile processing using laccase enzymes are also described.

Description

LACCASES AND METHODS OF USE THEREOF AT LOW TEMPERATURE
PRIORITY
[01] The present application claims priority to U.S. Provisional Patent Application Serial Nos.
61/140,724, filed on December 24, 2008, 61/154,882, filed on February 24, 2009, and 61/237,532, filed on August 27, 2009, each of which is incorporated by reference in its entirety.

TECHNICAL FIELD
[02] The present systems, compositions, and methods relate to laccase enzymes and nucleic acid sequences encoding such laccase enzymes. The laccase enzymes may be employed in conjunction with mediators in improved methods for modifying the color of denim fabrics.

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, and as catalysts or facilitators for polymerization and oxidation reactions.

[04] Laccases can be utilized for a wide variety of applications in a number of industries, including the detergent industry, the paper and pulp industry, the textile industry and the food industry. In one application, phenol oxidizing enzymes are used as an aid in the removal of stains, such as food stains, from clothes during detergent washing. Most laccases exhibit pH
optima in the acidic pH range while being inactive in neutral or alkaline pHs.

[05] 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.
However, there remains a need for laccases having different performance profiles in various applications.

[06] For many applications, the oxidizing efficiency of a laccase can be improved through the use of a mediator, also known as an enhancing agent. Systems that include a laccase and a mediator are known in the art as laccase-mediator systems (LMS). The same compounds can also be used to activate or initiate the action of laccase.

[07] There are several known mediators for use in a laccase-mediator system.
These include HBT (1-hydroxybenzotriazole), ABTS [2,2'- azinobis(3- ethylbenzothiazoline-6-sulfinic acid)], NHA (N-hydroxyacetanilide), NEIAA (N-acetyl-N-phenylhydroxylamine), HBTO (3-hydroxy 1,2,3-benzotriazin-4(3H)-one), and VIO (violuric acid). In addition, there are several compounds containing NH-OH or N-O groups that have been found to be useful as mediators.

[08] Functional groups and substituents have large effects on mediator efficiency. Even within the same class of compounds, a substituent can change the laccase specificity towards a substrate, thereby increasing or decreasing mediator efficacy greatly. In addition, a mediator may be effective for one particular application but unsuitable for another application. Another drawback for current mediators is their tendency to polymerize during use.
Thus, there is a need to discover efficient mediators for specific applications. One such application is the bleaching of textiles, wherein it is also important that the mediators are not unduly expensive or hazardous.
Other applications of the laccase-mediator system are given below.

[09] Methods of use for laccases at low temperatures would provide a benefit in terms of energy savings, for example, in textile processing methods where energy input for heating of processing baths could be reduced. Development of methods in which laccase enzymes are used at low temperatures for applications such as enzymatic bleaching would be desirable.
SUMMARY

[10] Described are enzymatic oxidation systems, compositions, and methods, involving laccases. In one aspect, a textile processing method is provided, comprising contacting a textile with a laccase enzyme and, optionally, a mediator at a temperature less than 40 C, for a length of time and under conditions sufficient to cause a color modification of the textile. In some embodiments, the color modification is selected from lightening of color, change of color, change in color cast, reduction of redeposition/backstaining, and bleaching.
In some embodiments, the temperature is from about 20 C to less than 40 C. In some embodiments, the temperature is from about 20 to about 35 C. In some embodiments, the temperature is from about 20 C to about 30 C. In some embodiments, the temperature is from about 20 C to about 23 C. In some embodiments, the temperature is 20 C, 21 C9 22 C9 23 C9 24 C9 25 27 C9 28 C9 29 C9 30 C9 31 C9 32 C9 33 C9 34 C, or 35 C. In some embodiments, the temperature is the ambient temperature of tap water.

[11] In some embodiments, the textile is indigo-dyed denim. In some embodiments, the textile is sulfur-dyed denim. In some embodiments, the denim is desized and/or stonewashed prior to or simultaneously with contacting the textile with the laccase enzyme and the mediator.
In some embodiments, the stonewashing and contacting the textile with the laccase enzyme and the mediator occur in the same bath.

[12] In some embodiments, the method further comprises contacting the textile with a cellulase enzyme, simultaneously or sequentially with contacting the textile with the laccase enzyme and the mediator. In some embodiments, contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed sequentially, and wherein contacting the textile with the cellulase enzyme is performed prior to contacting the textile with the laccase enzyme and the mediator. In some embodiments, contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed sequentially in the same bath without draining the bath between contacting the textile with a cellulase enzyme and contacting the textile with the laccase enzyme and the mediator.

[13] In some embodiments, contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed a temperature less than 40 C. In some embodiments, the temperature is from about 20 C to less than 40 C. In some embodiments, the temperature is from about 20 to about 35 C. In some embodiments, the temperature is from about 20 C to about 30 C. In some embodiments, the temperature is from about 20 C to about 23 C. In some embodiments, the temperature is 20 C, 21 C9 24 C9 25 C9 26 C9 27 C9 28 C9 29 C9 30 C9 31 C9 32 C9 33 C9 34 C, or 35 C. In some embodiments, the temperature is the ambient temperature of tap water.

[14] In some embodiments, the cellulase enzyme acts synergistically with the laccase enzyme to produce a textile with a greater degree of lightening of color of the textile, change in color, change in color cast, reduction of redoposition/backstaining, and/or bleaching. In some embodiments, the cellulase enzyme acts additively with the laccase enzyme to produce a textile with a greater degree of lightening of color of the textile, change in color, change in color cast, reduction of redoposition/backstaining, and/or bleaching in comparison to an identical method in which cellulase is not included.

[15] In some embodiments, the laccase is a microbial laccase. In some embodiments, 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.

[16] In some embodiments, the laccase has an amino acid sequence that is at least 70%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the laccase has an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the laccase has an amino acid sequence that is at least 90%, or even at least 95%, identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID
NO: 19, and SEQ ID NO: 20.

[17] In some embodiments, the laccase has an amino acid sequence that is at least 70%
identical to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the laccase has an amino acid sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID
NO: 20. In some embodiments, the laccase has an amino acid sequence that is at least 90%
identical to SEQ
ID NO: 19 or SEQ ID NO: 20. In some embodiments, the laccase has an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

[18] In some embodiments, the laccase enzyme and the mediator are provided together in a ready-to-use composition. In some embodiments, the laccase enzyme and the mediator are provided in a solid form. In some embodiments, the laccase enzyme and the mediator are provided as granules. In particular embodiments, the mediator is syringonitrile.

[19] In another aspect, laccases, nucleic acid sequences encoding such laccases, and vectors and host cells for expressing the laccases are provided. The laccases can be used at low temperatures in methods in which a reduction of energy input would be desirable, such as textile processing. In some embodiments, the laccase enzyme comprises, consists of, or consists essentially of the amino acid sequence depicted in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, or 20, or an amino acid sequence having at least about 60%, 65%, 70%, 80%, 90%, 91%, 92%,93%,94%, 95%, 96%, 97%,98%,99%, or even 99.5%, identical to any of SEQ ID
NOs:
2, 4, 6, 8, 12, 14, 16, 18, 19, or 20. In particular embodiments, the laccase has an amino acid sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5%, identical to SEQ ID NO: 19 or SEQ ID NO: 20. In still more particular embodiments, the laccase has the amino acid sequence SEQ ID NO: 19 or SEQ ID
NO: 20.

Preferably, such polypeptides have laccase enzymatic activity, which can be determined, e.g., using the assays described, herein.

[20] In another aspect, a composition comprising a laccase enzyme comprising, consisting of, or consisting essentially of any of the aforementioned amino acid sequences is provided. In 5 some embodiments, the composition further comprises a buffering system to maintain the pH of the composition at about 5.5 to about 6.5 in solution. In some embodiments, the composition further comprises a mediator. The mediator may be selected from, e.g., acetosyringone, syringaldehyde, syringamide, methyl syringamide, 2-hydroxyehyl syringamide, methyl syringate, dimethylsyringamide, shrine acid, and 4-hydroxy-3,5-dimethoxybenzonitrile (syringonitrile). In one embodiment, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile. In some embodiments, the composition is in a solid form. In some embodiments, the laccase enzyme and the mediator are provided together in a ready-to-use composition.
In some embodiments, the laccase enzyme and the mediator are provided in a solid form.
In some embodiments, the laccase enzyme and the mediator are provided as granules. In particular embodiments, the mediator is syringonitrile.

[21] In some embodiments, the laccase enzyme is used at a pH of about 5 to about 7, a temperature of about 20 C to about 30 C, a liquor ratio of about 5:1 to about 10:1, and for a time period of about 15 to about 60 minutes.

[22] 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

[23] Figure 1 shows the effects of modifying the color of stonewashed denim with laccase enzymes at different temperatures, as described in Example 1.

[24] Figure 2 shows the effects of laccase and mediator ratios on modifying the color of stonewashed denim, as described in Example 2.

[25] Figure 3 shows the effect of temperature on modifying the color of stonewashed denim with a "ready to use" laccase composition, as described in Example 3.

[26] Figure 4 shows the effect of temperature on color-modifying performance of laccase enzymes on stonewashed denim, as described in Example 3.

[27] Figure 5 shows the effect of cellulase treatment in combination with laccase-mediated color modification, as described in Examples 4-6.

DETAILED DESCRIPTION

[28] Described are enzymatic oxidation systems, compositions, and methods, involving laccases. The systems, compositions, and methods are useful, for example, for low-temperature processing of textiles to affect color modification. Such processing uses less energy than conventional textile processing technologies, and involves more environmentally-friendly chemical reagents. Various aspects and embodiments of the systems, compositions, and methods are to be described.

Definitions [29] 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.

[30] 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.

[31] 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.

[32] 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.

[33] 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.

[34] 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.

[35] 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.

[36] 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.

[37] 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.

[38] As used herein, the term "culturing" refers to growing a population of microbial cells under suitable conditions in a liquid, semi-solid, or solid medium for expressing a polypeptide of interest. In some embodiments, culturing is conducted in a vessel or reactor, as known in the art.

[39] 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.

[40] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[41] As used herein, the term "expression vector" refers to a DNA construct containing a DNA coding sequence (e.g., gene sequence) that is operably linked to one or more suitable control sequence(s) capable of effecting expression of the coding sequence in a host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

[42] As used herein, the term "host cell" refers to a cell or cell line into which a recombinant expression vector for production of a polypeptide may be transfected, transformed, or otherwise introduced for expression of a polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in total genomic DNA
complement) to the parent cell due to natural, accidental, or deliberate mutation. A host cell may be bacterial or fungal. A host cell includes a cell transfected or transformed in vivo with an expression vector.

[43] As used herein, the term "introduced," in the context of inserting a nucleic acid sequence into a cell includes "transfection," "transformation," and "transduction," and refers to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, wherein the nucleic acid sequence is incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[44] As used herein, "cleaning compositions" and "cleaning formulations" refer to compositions that may be used for the removal of undesired compounds from items to be cleaned, such as fabrics, dishes, contact lenses, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes), and other solid and surfaces. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the enzyme(s) used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.

[45] The terms further refer to any composition that is suitable for cleaning, bleaching, disinfecting, and/or sterilizing a object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners;
fabric softeners;
and textile and laundry pre-spotters, as well as dish detergents).

[46] Indeed, the terms "cleaning compositions" and "cleaning formulations"
include (unless otherwise indicated) granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents;
hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and "stain-stick" or pre-treat types.

[47] As used herein, the terms "detergent composition" and "detergent formulation" are used in reference to mixtures that are intended for use in a wash medium for the cleaning of soiled objects. In some embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., "dishwashing detergents"). In addition to enzyme(s), "detergent compositions" and "detergent formulations"
encompasses detergents that contain surfactants, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.

[48] As used herein, the phrase "detergent stability" refers to the stability of an enzyme, and optionally an associated substrate or mediator, in a detergent composition. In some embodiments, the stability is assessed during the use of the detergent, while in other embodiments, the term refers to the stability of a detergent composition during storage.

[49] As used herein the term "hard surface cleaning composition," refers to detergent compositions for cleaning hard surfaces such as floors, walls, tiles, stainless steel vessels (e.g., fermentation tanks), bath and kitchen fixtures, and the like. Such compositions may be provided in any form, including but not limited to solids, liquids, emulsions, and the like.

[50] As used herein, the term "dishwashing composition" refers to all forms of compositions for cleaning dishes, including but not limited to granular and liquid forms.

[51] As used herein, the term "disinfecting" refers to the removal or killing of microbes, including fungi, bacteria, spores, and the like.
5 [52] As used herein, the term "fabric cleaning composition" refers a form of detergent composition for cleaning fabrics, including but not limited to, granular, liquid and bar forms.
[53] As used herein, the terms "polynucleotide," "nucleic acid," and "oligonucleotide," are used interchangeably to refers to a polymeric form of nucleotides of any length and any three-dimensional structure, whether single- or multi-stranded (e.g., single-stranded, double-stranded, 10 triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin.
The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. For example, phosphate may be replaced by P(O)S ("thioate"), P(S)S
("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R
or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.
[54] 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.
[55] As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally, e.g., as in a purified restriction fragment, or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when incubated with a complementary nucleic acid in the presence of nucleotides and polymerase at a suitable temperature and pH.
The primer is preferably single stranded but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
[56] As used herein, the terms "recovered," "isolated," "purified," and "separated" refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally-associated, or associated as the result of heterologous expression.
[57] As used herein, the term "textile(s)" refers to fibers, yams, 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.
[58] As used herein, the phrase "textile(s) in need of processing" refers to a textile that needs to be desized, scoured, bleached, and/or biopolished to produce a desired effect.
[59] As used herein, the phrase "textile(s) in need of color modification"
refers to a textile that needs to be altered with respect to it color. These textiles may or may not have been already subjected to other treatments. Similarly, these textiles may or may not need subsequent treatments.
[60] 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.
[61] 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.
Without being limited to a theory, it is proposed that color modification results from the modification of chromaphores associated with a textile material, thereby changing its visual appearance. The chromophores may be naturally-associated with the material used to manufacture a textile (e.g., the white color of cotton) or associated with special finishes, such as dying or printing. 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.
[62] 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.
[63] 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.
[64] 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.
[65] As used herein, the term "desizing enzyme(s)" refers to an enzyme used to remove size.
Exemplary enzymes are amylases, cellulases, and mannanases.
[66] 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.
[67] 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 al., 1990 and Altschul, et al., 1997.

[68] 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.) [69] 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.
[70] 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."
[71] As used herein, the terms "draining" or "dropping" with respect to a bath in which textile materials are present refers to fully or partially releasing/emptying the solvent and reagents present in a bath. Draining a bath is typically performed between process steps such that the solvent and reagents present in one processing step do not interfere with a subsequent processing step. Draining may be accompanied by one or more rinse steps to further remove such the solvent and reagents.
[72] 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.
[73] 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 H2O water dH2O or DI deionized water dIH2O deionized water, Milli-Q filtration g or gm gram g microgram mg milligram kg kilogram L and l microliter mL and ml milliliter mm millimeter m micrometer M molar mm millimolar M 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 [74] 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 [75] The enzymatic oxidation 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 5 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.
[76] 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 10 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.
[77] 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 15 [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.
[78] 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 B. 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).
[79] 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.
[80] In particular embodiments, the expression host may be Trichoderma reesei with the laccase coding region under the control of a CBH1 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.
[81] The following laccase genes and laccases are described in U.S.
Publication No.
2008/0196173:
A. Cerrena laccase Al gene from CBS 115.075 strain Polynucleotide sequence (SEQ ID NO: 1):

Translated protein sequence (SEQ ID NO: 2):

B. Cerrena laccase A2 gene from CBS 154.29 strain Polynucleotide sequence (SEQ ID NO: 3):

Translated protein sequence (SEQ ID NO: 4):

C. Cerrena laccase B 1 gene from CBS 115.075 strain Polynucleotide sequence (SEQ ID NO: 5):

Translated protein sequence (SEQ ID NO: 6):

D. Cerrena laccase B2 gene from CBS 154.29 strain Polynucleotide sequence (SEQ ID NO: 7):

Translated protein sequence (SEQ ID NO: 8):

E. Cerrena laccase B3 gene (partial) from ATCC20013 strain Polynucleotide sequence (SEQ ID NO: 9):

Translated protein sequence (SEQ ID NO: 10):

F. Cerrena laccase C gene (partial) from CBS 154.29 strain Polynucleotide sequence (SEQ ID NO: 11):

Translated protein sequence (SEQ ID NO: 12):

G. Cerrena laccase D1 gene from CBS154.29 strain Polynucleotide sequence (SEQ ID NO: 13):

Translated protein sequence (SEQ ID NO: 14):

H. Cerrena laccase D2 gene from CBS115.075 strain Polynucleotide sequence (SEQ ID NO: 15):

Translated protein sequence (SEQ ID NO: 16):

1. Cerrena laccase E gene (partial) from CBS154.29 strain Polynucleotide sequence (SEQ ID NO: 17):

Translated protein sequence (SEQ ID NO: 18):

A Laccase D enzyme having the following amino acid sequence (SEQ ID NO: 19;
signal sequence in italics) may be used in the methods described herein:

The mature processed form of this polypeptide is as follows (SEQ ID NO: 20):
AIGPVADLEIVNKDLAPDGVQRPTVLAGGTFPGTLITGQKGDNFQLNVIDDLTDDRMLTPTS
IEWEGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVPDQAGTFWYESELSTQYCDGLRGAF
VVYDPNDPEKDLYDVDDGGTVITLADWYEVLAQTVVGAATPDSTLINGLGRSQTGPADAELA
VISVEENKRYRFRLVSISCDPNFTFSVDGENMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVL
NANQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSPLNEADLRPLV
PAPVPGNAVPGGADINHRLNLTFSNGLFSINNASFTNPSVPALLQILSGAQNAQDLLPTGSY
IGLELGKVVELVIPPLAVGGPEPFELEGENFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEV
TIRFVTDNPGPWFLECHIDWELEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQK
VKPKKGTAI

[82] 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: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID
NO: 19, and SEQ ID NO: 20. Preferably, such polypeptides have enzymatic laccase activity, as determined using the assays and procedures described, herein.
[83] 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: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. 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 [84] In some embodiments, the enzymatic oxidation 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.
[85] 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.
[86] 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) carboxamidol-2,6-dimethoxyphenol, or 4-(N,N-dimethyl carboxamido)-2,6-dimethoxyphenol.
[87] In some embodiments, the mediator is described by the following formula:
B-O
HO
A
_P7 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, -S02-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 C1 - C16 alkyl, preferably a Cl -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 Hem+1 ; 1 < m < 5.

[88] 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 C1 -C16 alkyl, preferably a C1 -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 Hem+1; 1 < m < 5.
In some embodiments, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as "syringonitrile" or "SN").
[89] 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.
[90] For applications such as textile processing, the mediator may be present in a concentration of about 0.005 to about 1,000 pmole per g denim, about 0.05 to about 500 pmole per g denim, about 0.1 to about 100 pmole per g denim, about 1 to about 50 mole per g denim, or about 2 to about 20 mole per g denim.
[91] 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 [92] The present systems and compositions can be use in applications where enzymatic laccase activity is useful or desirable. Among these applications/methods is color modification of a substrate, which may be associated with a textile. In some embodiments, such methods include incubation of a laccase enzyme with a suitable substrate at a low temperature, for example, about 40 C or less. In some embodiments, the temperature is between about 20 C and about 40 C. In some embodiments, the temperature is between about 20 to about 35 C. In some embodiments, the temperature is about 20 C, 25 C, 30 C, or 35 C. In some embodiments, the temperature is the ambient temperature of tap water, for example, about 20 C to about 23 C.
The temperature may be maintained within a narrow range or allowed to fluctuate without significantly affecting the performance of the system and compositions.
[93] The methods contemplate the use of one or more of the laccases 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 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% identity to any of the C. unicolor laccase enzymes described herein, and having laccase enzymatic activity.
[94] In some embodiments, the systems and methods are used in a textile processing method, for example a method for modifying the color of a textile product, including, e.g., fibers, yarns, cloth, or complete garments. Generally, the methods involve contacting the textile with a laccase and a mediator for a length of time, and under conditions, sufficient to result in at least one (i.e., one or more) measurable effects selected from, e.g., a change in color, a change in color cast, lightening, bleaching, fading, and/or a reduction of redeposition/backstaining. In some embodiments, the methods are used to impart a "vintage look" to dyed denim products. In the case of indigo-dyed denim, the vintage look has a less intense blue/violet tint and more subdued grey appearance than the freshly-dyed denim. In the case of sulfur-dyed denim, the vintage look is faded without the brown tint that can result from hypochlorite treatment.
Accordingly, while an aspect of the color modification obtained using laccases can be characterized as a "bleaching" affect, this term does not fully describe the color modifications possible using laccases.
[95] Textiles provided for color modification may be a cellulosic textiles or blends of cellulosic and synthetic fibers. 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. The denim may optionally be desized and/or stonewashed prior to color modification with the laccase enzyme.
[96] Generally, given the same amount of abrasion in a textile processing method, denim strength is reduced to a greater degree at a higher temperature, compared to a lower temperature.
Because the present methods can be performed at lower temperatures compared to conventional methods, they have the advantage of reducing the damage to textiles during processing compared to conventional methods. Moreover, laccase enzymes generally do not react with cellulosic textile fibers to reduce their strength during processing.
Accordingly, in some embodiments, the present methods do not affect the physical strength of the denim, or reduce the loss of physical strength compared to conventional methods. Where the denim is stretch denim comprising, e.g., elastane or spandex, and the present methods do not affect the stretch performance of the fabric, or reduce the loss of stretch performance compared to conventional methods.
[97] In some embodiments, the laccase is used in a textile processing method in combination with at least one other enzyme. Where such processing is simultaneous, enzymatic treatment may be performed at a low temperature as described herein. Where the processing is sequential, the laccase may be used at a low temperature as described herein, and the other enzyme(s) may optionally also be used at a low temperature. In some embodiments, the laccase is used in combination with a cellulase enzyme, either simultaneously or sequentially. In one embodiment, the textile is contacted with the laccase and cellulase simultaneously. In another 5 embodiment, the textile is contacted with the laccase and cellulase sequentially. In one embodiment, the textile is contacted with the cellulase first to effect "stonewashing," and then with the laccase to affect color modification. In another embodiment, the textile is contacted with the laccase first, and then with the cellulase. Where cellulase and laccase treatments are sequential, the two processing steps can be performed in the same bath, and without draining the 10 bath between treatments. Such methods are referred to as "single-bath"
methods.
[98] Suitable cellulases may be derived from microorganisms which are known to be capable of producing cellulolytic enzymes, such as, e.g., species of Humicola, Thermomyces, Bacillus, Trichoderma, Fusarium, Myceliophthora, Phanerochaete, Irpex, Scytalidium, Schizophyllum, Penicillium, Aspergillus or Geotricum. Known species capable for producing celluloytic 15 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.
[99] In some embodiments, enzymatic "stonewashing" using a cellulase, bleaching using an 20 aryl esterase, and color modification 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.
[100] Laccases can also be used in other aspects of textile manufacturing, generally including 25 aspects of treatment, processing, finishing, polishing, production of fibers, or the like. In addition to modifying the color of dyed denim, laccases can be used in de-coloring dyed waste (including indigo-dyed waste), in fabric dyeing, in textile bleaching work-up, in fiber modification; in achieving enhanced fiber or fabric properties, and the like.
[101] In further embodiments, the present systems and compositions may also be used in a 30 method for modifying the color of wool. For example, European Patent No. EP

discloses that laccases can be used for dyeing wool. Laccases can also be used in the leather industry. For example, laccases can be used in the processing of animal hides including but not limited to de-hairing, liming, bating and/or tanning of hides.

[102] The present systems and compositions may also be used in a method for modifying the color of pulp or paper products. Such methods involve contacting the pulp or paper product in need of color modification with a laccase as described, herein, for a length of time and under conditions sufficient for color modification to occur. In particular embodiments, the color modification is bleaching.
[103] The present systems and compositions may also be used in a method for hair color modification. Laccases have reportedly been found to be useful for hair dyeing (see, e.g., WO
95/33836 and WO 95/33837). Such methods involve contacting the hair having a color to be modified with the laccase for a length of time and under conditions suitable for changing the color of the hair.
[104] The present systems and compositions may also be used in the field of waste-water treatment. For example, laccases can be used in decolorization of colored compounds; in detoxification of phenolic components; for anti-microbial activity (e.g., in water recycling); in bio-remediation; etc.
[105] The present systems and compositions may also be used in the depolymerization of high-molecular-weight aggregates, deinking waste paper, the polymerization of aromatic compounds, radical-mediated polymerization and cross-linking reactions (e.g., paints, coatings, biomaterials), the activation of dyes, and coupling organic compounds.
[106] The present systems and compositions may also be used in a cleaning composition or component thereof, or in a detergent for use in a cleaning method. For example, laccases can be used in the cleaning, treatment or care of laundry items such as clothing or fabric; in the cleaning of household hard surfaces; in dish care, including machine dishwashing applications; and in soap bars and liquids and/or synthetic surfactant bars and liquids. The enzymes presented herein can be useful, for example, in stain removal/de-colorization, and/or in the removal of odors, and/or in sanitization, etc. Laccase mediators can be used as sanitization and antimicrobial agents (e.g., wood protection, detergents), independently of or in conjunction with laccase enzymes.
[107] Laccases can be used in other aspects of field of personal care. For example, laccases can be used in the preparation of personal products for humans such as fragrances, and products for skin care, hair care, oral hygiene, personal washing and deodorant and/or antiperspirants, for humans. Laccases can be useful, for example, in hair dyeing and/or bleaching, nails dyeing and/or bleaching; skin dyeing and/or bleaching; surface modification (e.g., as coupling reagent);
as an anti-microbial agent; in odor removal; teeth whitening; etc. Laccases can be used in the field of contact lens cleaning. For example, laccases can be used in the cleaning, storage, disinfecting, and/or preservation of contact lenses.
[108] Laccases can be used in the field of bio-materials. For example, laccases can be used as bio-catalysts for various organic reactions; and/or in connection with biopolymers; in connection with packaging; in connection with adhesives; in surface modification (activation and coupling agent); in production of primary alcohols; in connection with biosensors and/or organic syntheses; etc. Laccases are capable of oxidizing a wide variety of colored compounds having different chemical structures, using oxygen as the electron acceptor.
[109] The present systems and compositions may also be used for the removal of lignin from lignocellulose-containing material (e.g., the delignification of pulp), the bleaching of lignocellulose-containing material (i.e. the enzymatic de-inking of recycled paper) and/or the treatment of waste water arising from the manufacture of paper or cellulose.
Such processes may use a laccase enzyme in combination with adding or metering-in non-aromatic redox agents plus phenolic and/or non-phenolic aromatic redox compounds, the phenolic and non-phenolic units of the lignin either being oxidized directly by the action of these phenolic and/or non-phenolic aromatic compounds, or the lignin being oxidized by other phenolic and/or non-phenolic compounds produced by the oxidizing action of these compounds.
[110] Laccases can be used in other aspects relating to pulp and paper. For example, laccases can be used in the manufacture of paper pulps and fluff pulps from raw materials such as wood, bamboo, and cereal rice straw; the manufacture of paper and boards for printing and writing, packaging, sanitary and other technical uses; recycling of cellulose fiber for the purpose of making paper and boards; and the treatment of waste products generated by and treated at pulp or paper mills and other facilities specifically dedicated to the manufacture of paper, pulp, or fluff. Laccases can be useful, for example, in wood processing; in pulp bleaching; in wood fiber modification; in bio-glue (lignin activation) for MDF manufacturing; for enhanced paper properties; in ink removal; in paper dyeing; in adhesives (e.g. lignin based glue for particle- or fiber boards); etc.
[111] Laccases can be used in the field of feed. For example, the laccases can be used as a feed additive alone or as part of a feed additive with the aim to increase the nutritional value of feed for any kind of animals such as chicken, cows, pigs, fish and pets;
and/or as a processing aid to process plant materials and food industry by products with the aim to produce materials/products suitable as feed raw materials.
[112] Laccases can be used in the field of starch processing. For example, laccases can be used in the processing of a substrate including starch and/or grain to glucose (dextrose) syrup, fructose syrup or any other syrup, alcohol (potable or fuel) or sugar. Such starch processing may include processing steps such as liquefaction, saccharification, isomerization, and de-branching of a substrate.
[113] Laccases can be used in the field of food. For example, laccases can be used in the preparation, processing, or as an active ingredient in foods such as yellow fat, tea based beverages, culinary products, bakery, and frozen foods for human consumption.
Laccases can be used, for example, as a bread improver, in food preservation, as an oxygen scavenger, etc.
Laccases can be used for reducing or eliminating the microbial load of various foods (e.g., meats) or feed.
[114] Any of the methods or uses for laccases described herein may be performed at a low temperature, e.g., at a temperature lower than about 40 C, e.g., less than about 40 C, less than about 37 C, less than about 35 C, less than about 32 C, less than about 30 C, less than about 27 C, less than about 25 C, and less than about 22 C. Exemplary temperature ranges are from about 20 C to less than about 40 C. Exemplary temperatures are 20 C, 21 C9 22 24 C9 25 C9 26 C9 27 C9 28 C9 29 C9 30 C9 31 C9 32 C9 33 C9 34 C, or 35 C. In some embodiments, the temperature is at room temperature or the ambient temperature of tap water, for example, about 20 C to about 23 C.
[115] Any of the methods or uses for laccases described herein may be performed using any of the laccase enzymes described herein, e.g., laccases from Cerrena unicolor. In some embodiments, laccases are used at a concentration of about 0.005 to about 5000 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 denim processing embodiments, a laccase is used at a concentration of about 0.005 to about 5000 mg/kg of denim, about 0.05 to about 500 mg/kg of denim, about 0.1 to about 100 mg/kg of denim, or about 0.5 to about 10 mg/kg of denim. In some embodiments, a laccase is used at a pH of 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, and 7Ø

Ready to Use Compositions and Kits [116] As described above, the present compositions include one or more laccases, and optionally one or more mediators. In some embodiments, the compositions comprise a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
ID

NO: 20, or a variant or fragment, thereof. In particular embodiments the compositions comprise a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from SEQ ID NO: 19 and 20, or a variant or fragment, thereof.
Preferably, such polypeptides have enzymatic laccase activity, which can be determined using the assays and procedures described, herein [117] Such composition can also be provided in the form of a "ready to use"
(RTU) composition comprising, consisting of, or consisting essentially of a laccase enzyme and a mediator. In some embodiments, the mediator is selected from acetosyringone, syringaldehyde, syringamide, methyl syringamide, 2-hydroxyethyl syringamide, methyl syringate, syringonitrile, dimethylsyringamide, and syringic acid. In one embodiment, the mediator is syringonitrile (4-hydroxy-3,5-dimethoxybenzonitrile). The RTU composition may further contain one or more compounds to provide a pH buffer when the composition is in solution. For example, in some embodiments, the composition contains monosodium phosphate and adipic acid as a buffering system. The RTU composition may be in a solid, granular form for ease of storage and transportation. The composition is then diluted with water to provide an aqueous solution for use, e.g., as described. RTU compositions may also include any number of additional reagents, such as dispersants, surfactant, blockers, polymers, preservatives, and the like.

[118] The following examples are provided to illustrate the systems, compositions, and methods, and should in no way be construed as limiting. Other aspects and embodiments will be apparent to the skilled person in view of the description.

EXAMPLES
[119] The following enzyme nomenclature is used in the Examples:
Trade name Description PRIMAGREENR EcoWhite 1 Mycobacterium smegmatis perhydrolase, S54V variant of SEQ ID NO: 1 PRIMAGREENR EcoFade LT Cerrena unicolor laccase and syringonitrile in a dry formulation OPTISIZE 160 amylase Amylase from Bacillus amyloliquefaciens INDIAGE Neutra L Endoglucanase from Streptomyces sp. 1 1AG8 INDIAGE 2XL Cellulase from Trichoderma reesei INDIAGE SUPER GX Cellulase from Trichoderma reesei NOVOPRIME 268 Laccase from Aspergillus oryzae NOVOPRIME F258 Methyl syringate DENILITE II S Laccase from Aspergillus oryzae and methyl syringate Example 1 - Effect of temperature on laccase-mediated color modification of stonewashed denim Enzyme 5 [120] Granular Laccase D enzyme from Cerrena unicolor (38,000 U/g) was used in this experiment. One laccase unit is defined as the amount of laccase activity that oxidizes 1 nmol of ABTS substrate per second under conditions of an assay based on the ability of laccase enzyme to oxidize ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate)) into its corresponding stable cation radical, ABTS . Accumulation of the radical causes the ABTS to turn a dark green color 10 and an increase in absorbance at 420 nm. The color formation is proportional to laccase activity and is monitored against a laccase standard.

Mediator [121] 4-hydroxy-3,5-dimethoxybenzonitrile (syringonitrile, SN) was purchased from Punjab Chemicals & Crop Protection Limited (Mumbai, India).

15 Procedure [122] 12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine under the following conditions:
= Desizing for 15 minutes at 10:1liquor ratio 50 C with 0.5 g/1(15 g) of amylase (Genencor) and 0.5 g/1(15 g) of a non-ionic surfactant [e.g., Rucogen BFA
20 (Rudolf Chemie) or Ultravon RW (Huntsman)].
= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
[123] Following desizing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:

= Cold rinse for 5 minutes at 10:1 liquor ratio.

25 = Stonewashing for 60 minutes at 10:1 liquor ratio 55 C with 1 kg of pumice stone, pH 4.5 (1 g/1 tri-sodium citrate dihydrate and 1 g/1 citric acid monohydrate) and 1.2 g/1 INDIAGE 2XL cellulase (Genencor).

= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
[124] After stonewashing, laccase treatment was performed in a Unimac UF 50 washing 30 machine according to the following process:

= 30 minutes at 10:1 liquor ratio, with either (i) C. unicolor laccase D and syringonitrile at pH 6 (0.7 g/1 monosodium phosphate and 0.17 g/1 adipic acid) and temperatures of 40 C, 30 C, or 23 C or (ii) NOVOPRIME Base 268 and NOVOPRIME F258 at pH 4.8 (0.29 g/1 monosodium phosphate and 0.56 g/1 of adipic acid) and temperatures of 40 or 30 C.
= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
Evaluation of denim legs [125] The amount of color modification, reported as "bleaching," of denim legs was evaluated after laccase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. The CIE color space, also known as the CIELUV color space, was adopted by the International Commission on Illumination (CIE) in 1976, and involves the values L*, u*, and v* calculated as follows:

t* = 116 ( ` 16 wl Een 0 008Ã 56 yo- ypi v* i3L*'(v --Vo) wh era Y: TE EsIEniuhis iral lit! ~' i IrEstEE1'Euhis, va uo ~i' i C= can a'so he use_ ) u', s/ : Ghrornaticity ooordinates from the CIE 1976 UGS diaglain Ye, ikv, Vo: Tristirnulus valEie Y (or Yi c and chromatic.:ity coordinates u', Vof the perfect E-etlo-Ainq difluser.

[126] For each denim leg, 8 measurements were taken and the results from the 12 legs (96 measurements total) were averaged. The results are shown in Tables 1 and 2 and in Figure 1.

Table 1. Results using C. unicolor laccase and syringonitrile C. unicolor laccase Syringonitrile Bleaching Standard concentration, g/l concentration, Temp., C level, deviation (U/ml) g/l (mM) CIE* Lab 0.54 0.07 40 38.3/-1.2/-12.0 0.5/0.1/0.1 (20.5) (0.39) 0.3 0.07 40 37.6/-0.5/-12.3 0.6/0.1/0.1 (11.4) (0.39) 0.15 0.07 40 36.4/-0.2/-12.8 0.5/0.1/0.1 (5.7) (0.39) 0.54 0.07 (20.5) (0.39) 30 36.2/-0.2/-12.8 0.5/0.1/0.1 0.3 0.07 (11.4) (0.39) 30 36.1/-0.2/-13.0 0.5/0.1/0.1 0.15 0.07 30 35.3/0.0/-13.3 0.5/0.1/0.1 (5.7) (0.39) 0.15 0.07 23 (no steam) 34.0/0.3/-13.5 0.6/0.1/0.1 (5.7) (0.39) Table 2. Results using A. oryzae laccase from and methyl syringate NOVOPRIME NOVOPRIME Bleaching Standard Base 268 conc., g/1 F258 conc., g/l Temp., C level, deviation (mM) CIE* Lab 0.47 0.07 40 36.2/-0.5/-11.2 0.6/0.1/0.2 (0.33) 0.27 0.07 40 36.5/-0.4/-11.4 0.6/0.1/0.2 (0.33) 0.15 0.07 40 35.7/-1.0/-11.9 0.5/0.1/0.2 (0.33) 0.15 0.07 30 33.9/0.1/-12.6 0.5/0.1/0.2 (0.33) [127] The results show the effectiveness of C. unicolor laccase and syringonitrile in affecting a color change of stonewashed denim.

Example 2 - Effect of the laccase:mediator ratio on color modification of stonewashed denim Procedure [128] 12 denim legs weighing approximately 3 kg (total) were desized and stonewashed as described in Example 1. After stonewashing, laccase treatment was performed in a Unimac UF
50 washing machine according to the following process:

= C. unicolor laccase D and syringonitrile, 30 minutes at 10:1 liquor ratio, pH 6 (0.7 g/l monosodium phosphate and 0.17 g/l adipic acid) at 40 C.

= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
Evaluation of denim legs [129] Color modification of denim legs was evaluated as described in Example 1. The results are shown in Table 3 and Figure 2.

Table 3. Results using C. unicolor laccase and syringonitrile in different ratios C. unicolor laccase Mediator Bleaching Standard concentration, g/l concentration, Temp., C level, (U/ml) g/l (mM) CIE* Lab deviation 0.15 0.07 40 36.4/-0.2/-12.8 0.5/0.1/0.1 (5.7) (0.39) 0.15 0.08 40 37.0/-0.4/-12.7 0.5/0.1/0.1 (5.7) (0.44) 0.15 0.1 (5.7) (0.55) 40 37.1/-0.4/-12.7 0.6/0.1/0.1 [130] The results show that the ratio of laccase enzyme to mediator can be manipulated to alter color modification.

Example 3 - Effect of temperature on color modification performance of composition containing laccase and mediator on stonewashed denim [131] For the purpose of investigating laccase-mediated color modification performance at low temperature, a "ready-to-use" (RTU) composition was prepared as shown in Table 4. The monosodium phosphate and adipic acid provide a buffering function at about pH
6 in an application of use as described below.

Table 4. Ready-to-use formulation Component % w/w Monosodium phosphate (anhydrous) 70 Adipic acid 7 C. unicolor laccase D granules (38,000 U/g) 15 Syringonitrile 8 Procedure [132] 12 denim legs weighing approximately 3 kg (total) were desized and stonewashed as described in Example 1. After stonewashing, laccase treatment was performed in a Unimac UF
50 washing machine according to the following process:
= 30 minutes at 10:1 liquor ratio at 30 C or without incoming steam (i.e., temperature of 21-22 C) with the RTU laccase composition described above or DENILITE II S
(Novozymes) at concentrations and temperatures as described in the Tables 5 and 6, below.]
= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
Evaluation of denim legs [133] Color modification of denim legs was evaluated as described in Example 1. The results are shown in Tables 5 and 6 and in Figures 3 and 4.

Table 5. Results using C. unicolor RTU composition RTU laccase, Temp., 'C Bleaching level, Standard % owg* CIE* Lab deviation 1 30 35.1/-0.7/-13.5 0.6/0.1/0.2 3 30 38.5/-1.3/-12.6 0.7/0.1/0.2 1 21-22 33.3/-0.4/-13.6 0.6/0.1/0.1 3 21-22 37.2/-1.013.3 0.7/0.1/0.1 * "owg" = on weight of goods Table 6. Results using an A. oryzae laccase RTU composition DENILITE II S, Temp., 'C Bleaching level, Standard % owg CIE* Lab deviation 3 30 36.1/-1.3/-10.9 0.6/0.1/0.2 3 21-22 33.8/-0.8/-12.1 0.5/0.1/0.2 [134] The results show that a C. unicolor laccase RTU composition provides superior color modification at low temperature compared to conventional commercial laccase compositions.
Example 4 - One-step stonewashing and color modification at 30 C

5 [135] 12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.
[136] Following desizing, the denim was stonewashed and bleached in a Unimac washing machine under the following conditions:

= 30 minutes, 30 C at 10:1 liquor ratio, pH 6, (i) 0.4 % owg INDIAGE Super GX
10 cellulase (Genencor) + 3% owg RTU laccase composition described in Example 3 (i.e., "stonewashing + bleaching 1-step") or (ii) INDIAGE Super GX cellulase, alone (i.e., "stonewashing only").

= 2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice stones were used. The results are shown in Table 7 and Figure 5.
Table 7. Results of one-step stonewashing and color modification Temp. Bleaching level, Standard ( C) CIE*Lab deviation Stonewashing only 30 25.4/1.3/-12 0.3/0.1/0.3 Stonewashing+ bleaching, 1 step 30 27.3/0.6/-12.2 0.5/0.2/0.2 [137] The results show that color modification can be achieved using laccase and cellulase simultaneously.

Example 5 - Two-step stonewashing and color modification at 30 C

[138] 12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.
[139] Following desizing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:
= 30 minutes, 30 C at 10:1 liquor ratio, pH 5.5, 0.4% owg INDIAGE Super GX
cellulase (Genencor) [140] Following stonewashing, the denim was bleached in a Unimac UF 50 washing machine under the following conditions:

= 30 minutes, 30 C at 10:1 liquor ratio, pH 6, 3% owg RTU laccase composition described in Example 3.

= 2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice stones were used.
[141] The results are shown in Table 8 and Figure 5. The two-step stonewashing and color modification results were compared to the results for stonewashing alone as described in Example 4.

Table 8. Results of two-step stonewashing and color modification Temp. Bleaching level, ( C) CIE* L a b Standard deviation Stonewashing only 30 25.4/1.3/-12 0.3/0.1/0.3 Stonewashing + bleaching, 2 steps 30 31.9/-0.3/-12.9 0.6/0.2/0.1 [142] The results show that color modification by laccase treatment can be achieved following stonewashing.

Example 6 - Laccase-mediated color modification of denim at 300 without stonewashing [143] 12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.
[144] Following desizing, the denim was bleached in a Unimac UF 50 washing machine under the following conditions:

= 30 minutes, 30 C at 10:1 liquor ratio, pH 6, 3% owg RTU laccase composition described in example 3.

= 2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice stones were used.
[145] The results are shown in Table 9 and Figure 5. The color modification results were compared to the results for stonewashing alone as described in Example 4.

Table 9. Results of color modification without stonewashing Temp. ( C) Bleaching level, Standard deviation CIE* Lab Stonewashing only 30 25.4/1.3/-12 0.3/0.1/0.3 Bleaching, no stonewashing 30 26.9/0.7/12.1 0.5/0.1/0.2 [146] The results show that the amount of color modification produced by laccase treatment without stonewashing is higher than with stonewashing alone.

Example 7 - Stonewashing and color modification with cellulase and laccase in a single-bath bath process without pumice stones [147] This Example shows that effective stonewashing and color modification can be obtained using laccase and cellulase in a single-bath process.

Enzyme [148] PRIMAGREEN EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).
Procedure [149] Starting material was desized denim weighing approximately 3 kg (ballast + 2 legs for evaluation).
[150] The denim was stonewashed in a Renzacci LX 22 washing machine under the following conditions:
= 40 minutes, 50 C at 10:1 liquor ratio, pH 6.5 0.4 % owg of INDIAGE Neutra L
cellulase (Batch No. 40105358001 activity 5197 NPCNU/g) (Genencor).

= After stonewashing 1 leg was taken out and dried for evaluation.
= Following stonewashing, and without draining (i.e., dropping) the bath, the second denim leg was subjected to color modification under the following conditions:
= 40 minutes, 40 C at 10:1 liquor ratio and 1 % owg of RTU PRIMAGREEN EcoFade LT

= 2 cold rinses for 3 minutes = The denim was dried in an industrial dryer Evaluation of denim legs [151] Color modification and stonewashing on denim legs were evaluated after laccase treatment and after cellulase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. Six measurements were taken for each leg, and the results were averaged.
[152] The results are summarized in Table 10. The amount of color modification obtained with sequential (i.e., two-step) addition of cellulase and laccase in a single bath was greater than that obtained by adding cellulase and laccase at the same time as in Example 4.

Table 10 Bleaching level, Standard deviation CIE*Lab Stonewashing 27.8/1.1/-13.2 0.3/0.1/0.1 Stonewashing+ bleaching, single bath 34.7/0.0/-12.2 0.5/0.1/0.1 [153] The results show that the amount of color modification obtained with sequential (i.e., two-step) addition of cellulase and laccase in a single bath is greater than that obtained by adding cellulase and laccase at the same time as in Example 4.

Example 8 - Color modification with laccase and pumice stones [154] This Example shows that effective stonewashing and color modification can be obtained using pumice stones and a laccase-mediator system in a single-bath process.

Enzyme [155] PRIMAGREEN EcoFade LT 100 laccase (Batch No. 7809136160, 6,292 GLacU/g).
Procedure [156] 12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.
[157] Following desizing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:

= 30 minutes, 30 C at 10:1 liquor ratio, 3 kg of pumice stone, with 3 %
PRIMAGREEN
EcoFade LT100 (Genencor). The blank/control was performed only with stones in water.
= 2 cold rinses for 5 minutes at 30:1 liquor ratio.
Evaluation of denim legs [158] Color modification on denim legs were evaluated after laccase treatment and after the stonewashing treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source, as before. The average of eight measurements taken on the outside of each leg were reported as the Bleaching level. The average of four measurements taken on the inside of each leg were reported as the Backstaining level.
[159] The results are summarized in Tables 11 and 12.

Table 11 Bleaching level, Standard deviation CIE*Lab Stonewashing 25.5/1.1/-11.4 0.3/0.1/0.2 Color modification 28.7/0.3/-12.0 0.6/0.1/0.1 Table 12 Backstaining level, Standard deviation CIE*Lab Stonewashing 50.6/-1.2/-5.5 0.4/0.1/0.3 Stonewashing+ color modification, 52.2/-1.2/-4.0 0.4/0.1/0.3 single bath [160] The results show that laccase treatment provides color modification even if pumice stones are present, and further shows reduction/removal of backstaining.

Example 9 - Stonewashing and color modification of sulphur dyed garments.
[161] The test garments were made of 100% cotton Twill fabric dyed with sulphur khaki brown dye. 21 garments weighing approximately 7 kg (total) were stonewashed in a 25 kg belly washer (36 rpm) under the following conditions:

= 45 minutes, 55 C at 18:1 liquor ratio, pH 4.5 at 1 g/l of INDIAGE 2XL
= 1 cold rinse for 3 minutes at 12:1 liquor ratio. No pumice stones were used.
= After washing the garments were dried for evaluation = 3 garments (approximately 1 kg, total) stonewashed as described above were treated with PRIMAGREEN EcoFade LT 100 under the following conditions:
= 15, 30 or 45 minutes, 40 C at 50:1 liquor ratio and 1, 2 or 3 g/1 of PRIMAGREEN
EcoFade LT 100. The blank/control was performed with the garment washed for 15, 30 or 45 min with only water.
= 1 cold rinse for 3 minutes.

= The denim was dried in an industrial dryer.
Evaluation of denim legs [162] Color modification and stonewashing of sulphur dyed garments were evaluated after laccase treatment and after the stonewashing treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source, as above. For each garment 10 measurements were taken and the results were averaged.
[163] The results are summarized in Tables 13 5 Table 13 Treatment CIE*Lab Before treatment 40.2/2.2/20.8 Whole complex cellulase 43.7/2.1/19.2 Blank, 15 min 44.9/1.3/18.3 Blank, 30 min 46.1/1.3/19.1 Blank, 45 min 46.6/1.3/18.5 PRIMAGREENR Ecofade (1 g/1) 15 min 45.1/2.9/16.5 PRIMAGREENR Ecofade (1 g/1) 30 min 45.5/3.3/16.8 PRIMAGREENR Ecofade (1 g/1) 45 min 44.7/3.2/16.4 PRIMAGREENR Ecofade (2 g/1) 15 min 44.7/3.3/15.9 PRIMAGREENR Ecofade (2 g/1) 30 min 45.3/3.5/15.9 PRIMAGREENR Ecofade (2 g/1) 45 min 45.0/3.5/15.6 PRIMAGREENR Ecofade (3 g/1) 15 min 44.4/3.4/15.5 PRIMAGREENR Ecofade (3 g/1) 30 min 44.6/3.6/15.7 PRIMAGREENR Ecofade (3 g/1) 45 min 45.0/3.6/15.4 [164] The results show that the a and the b values of the color space significantly change compared to the untreated fabric, as well as to the blank. The modification to the cast of the garments is visible by eye.

Example 10 - Color modification of sulphur dyed garments without stonewashing [165] 3 garments made of 100% cotton Twill fabric dyed with sulphur khaki brown dye and weighing approximately 1 kg (total) were treated in a 5 kg belly washer (36 rpm) under the following conditions:

= 15, 30 or 45 minutes, 40 C at 40:1 liquor ratio and 1, 2 or 3 g/1 of PRIMAGREEN
EcoFade LT 100. The blank/control was performed with the garment washed for 15, 30 or 45 min with just water.
= 1 cold rinses for 3 minutes = The denim was dried in an industrial dryer Evaluation of denim legs [166] Color modification and stonewashing on sulphur dyed garment were evaluated after laccase treatment and after the stonewashing treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. For each garment 10 measurements were taken and the results were averaged.
[167] The results are summarized in Table 14 Table 14 Treatment CIE*Lab Before treatment 40.2/2.2/20.8 Blank 15 min 41.1/2.0/19.7 Blank 30 min 41.9/2.1/20.4 Blank 45 min 42.4/2.1/20.29 PRIMAGREENR Ecofade (1 g/1) 15 min 41.1/3.7/17.6 PRIMAGREENR Ecofade (1 g/1) 30 min 41.5/4.2/18.6 PRIMAGREENR Ecofade (1 g/1) 45 min 41.6/4.0/18.1 PRIMAGREENR Ecofade (2 g/1) 15 min 40.4/3.9/17.0 PRIMAGREENR Ecofade (2 g/1) 30 min 41.2/4.2/17.3 PRIMAGREEN Ecofade (2 g/1) 45 min 41.6/4.3/17.3 PRIMAGREEN Ecofade (3 g/1) 15 min 40.5/4.0/16.6 PRIMAGREEN Ecofade (3 g/1) 30 min 41.0/4.2/17.1 PRIMAGREEN Ecofade (3 g/1) 45 min 40.6/4.3/17.0 [168] The results show that the a and the b values of the color space significantly change compared to the untreated fabric as well as to the blank. The modification to the cast of the garments is visible by eye.

Example 11 - Stonewashing and bleaching performance with cellulase and laccase in a single-bath process in the presence of surfactant and pumice stone Enzyme [169] PRIMAGREEN EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).
Procedure [170] 12 denim garments weighing 10 kg (total) and dyed with pure indigo were desized in a Tupesa front loading machine (36 rpm) under the following conditions:

= 10 minutes, 40 C at 10:1 liquor ratio, pH 7, and 0.5 g/1 of lubricant, 0.2 g/1 of dispersant (non ionic surfactant), and 0.2 g/1 of polyester blocker (non ionic hydrophilic co-polymer).
[171] Following desizing, the denim was de stonewashed under the following conditions:
= 30 minutes, 47 C at 5:1 liquor ratio, pH 6 with 7 kg of pumice stones 4 %
owg of INDIAGE Super GX cellulase (Genencor). 1 garment was taken out for evaluation = Following stonewashing, and without draining (dropping) the bath, the denim was bleached under the following conditions:
= 30 minutes, 47 C at 5:1 liquor ratio and 2% owg of RTU PRIMAGREEN EcoFade LT
100.

= 2 cold rinses for 2 minutes at 1:50 liquor ratio = The denim was dried in an industrial dryer Evaluation of denim legs [172] Color modification and stonewashing on denim were evaluated after laccase treatment and after cellulase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. For each leg 8 measurements were taken and the results were averaged.
[173] The results are summarized in Table 15.
Bleaching level Standard CIE*Lab deviation Stonewashing 27.6/0.6/-12.0 0.5/0.1/0.1 Stonewashing+ bleaching single bath in 32.3/-0.1/-12.8 0.7/0.1/0.1 presence of surfactant and pumice stone [174] The results show that color modification by laccase treatment occurs in the presence of pumice stones and in the presence of a surfactant.

[175] The aspects, embodiments, and examples described herein are for illustrative purposes only. Various modifications will be apparent to the skilled person, and are included within the spirit and purview of this application, and the scope of the appended claims.
All publications and patent documents cited herein are hereby incorporated by reference in their entirety.

Claims (27)

1. A textile processing method, comprising contacting a textile with a laccase enzyme and a mediator at a temperature less than 40°C, for a length of time and under conditions sufficient to cause a color modification of the textile.

2. The method of claim 2, wherein the color modification is selected from lightening of color, change of color, change in color cast, reduction of redeposition/backstaining, and bleaching.

3. The textile processing method of claim 1, wherein the temperature is from about 20°C
to less than 40°C.

4. The textile processing method of claim 1, wherein the temperature is from about 20°C
to about 30°C.

5. The textile processing method of claim 1, wherein the textile is indigo-dyed denim.

6. The textile processing method of claim 1, wherein the textile is sulfur-dyed denim.

7. The textile processing method of claim 1, wherein the denim is desized and/or stonewashed prior to or simultaneously with contacting the textile with the laccase enzyme and the mediator.

8. The textile processing method of claim 1, wherein the stonewashing and contacting the textile with the laccase enzyme and the mediator occur in the same bath.

9. The textile processing method of claim 1, further comprising contacting the textile with a cellulase enzyme, simultaneously or sequentially with contacting the textile with the laccase enzyme and the mediator.

10. The textile processing method of claim 9, wherein contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed sequentially, and wherein contacting the textile with the cellulase enzyme is performed prior to contacting the textile with the laccase enzyme and the mediator.

11. The textile processing method of claim 10, wherein contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed sequentially in the same bath without draining the bath between contacting the textile with a cellulase enzyme and contacting the textile with the laccase enzyme and the mediator.

12. The textile processing method of any of claims 9-11, wherein contacting the textile with the cellulase enzyme and contacting the textile with the laccase enzyme and the mediator are performed a temperature less than 40°C.

13. The method of any one of claims 1-12, wherein the laccase is a microbial laccase.

14. The method of any one of claims 1-12, wherein the laccase is from a Cerrena species.

15. The method of any one of claims 1-12, wherein the laccase is from Cerrena unicolor.

16. The method of any one of claims 1-12, wherein the laccase is laccase D
from C.
unicolor.

17. The method of any one of claims 1-12, wherein the laccase has an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ
ID NO: 20.

18. The method of any one of claims 1-12, wherein the laccase has an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ
ID NO: 20.

19. The method of any one of claims 1-12, wherein the laccase has an amino acid sequence that is at least 70% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

20. The method of any one of claims 1-12, wherein the laccase has an amino acid sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

21. The method of any one of claims 1-12, wherein the laccase has an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

22. The method of any one of claims 1-21, wherein the mediator is syringonitrile.

23. The method of any one of claims 1-21, wherein the temperature is from about 20° to about 35°C.

24. The method of any one of claims 1-21, wherein the temperature is from about 20°C
to about 23°C.

25. The method of any one of claims 1-21, wherein the temperature is the ambient temperature of tap water.

26. The method of any one of claims 1-25, wherein the laccase enzyme and the mediator are provided together in a ready-to-use composition.

27. The method of any one of claims 1-25, wherein the laccase enzyme and the mediator are provided in a solid form.