LACCASES AND METHODS OF USE THEREOF AT LOW TEMPERATURE

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

PRIORITY

The present application claims priority to U.S. Provisional Patent Application Ser. Nos. 61/140,724, filed on Dec. 24, 2008, 61/154,882, filed on Feb. 24, 2009, and 61/237,532, filed on Aug. 27, 2009, each of which is incorporated by reference in its entirety.

TECHNICAL FIELD

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

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.

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.

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.

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.

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.

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.

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

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° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., or 35° C. In some embodiments, the temperature is the ambient temperature of tap water.

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.

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.

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° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., or 35° C. In some embodiments, the temperature is the ambient temperature of tap water.

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.

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.

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.

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.

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.

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.

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 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.

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.

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

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

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

FIG. 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.

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

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

DETAILED DESCRIPTION

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

As used herein, the term “disinfecting” refers to the removal or killing of microbes, including fungi, bacteria, spores, and the like.

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.

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, 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 (—O—) 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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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.

As used herein, the term “desizing enzyme(s)” refers to an enzyme used to remove size. Exemplary enzymes are amylases, cellulases, and mannanases.

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.

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.

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.)

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.

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.”

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.

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.

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

μg microgram

mg milligram

kg kilogram

μL and μl microliter

mL and ml milliliter

mm millimeter

μm micrometer

M molar

mM millimolar

μM 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

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

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 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.

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

An expression vector containing a polynucleotide sequence encoding a laccase enzyme may be transformed into a suitable host cell. The host cell may be a fungal cell, such as a filamentous fungal cell, examples of which include but are not limited to species of Trichoderma [e.g., T. reesei (previously classified as T. longibrachiatum and currently also known as Hypocrea jecorina], T. viride, T. koningii, and T. harzianum), Aspergillus (e.g., A. niger, A. nidulans, A. oryzae, and A. awamori), Penicillium, Humicola (e.g., H. insolens and H. grisea), Fusarium (e.g., F. graminum and F. venenatum), Neurospora, Hypocrea, and Mucor. A host cell for expression of a laccase enzyme may also be from a species of Cerrena (e.g., C. unicolor). Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall using techniques known in the art.

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).

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.

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. Pat. No. 5,861,271). The expression vector may be, e.g., pTrex3g, as disclosed in U.S. Pat. No. 7,413,887. In some embodiments, laccases are expressed as described in U.S. Patent Publication Nos. 2008/0196173 or 2009/0221030.

The following laccase genes and laccases are described in U.S. Publication No. 2008/0196173:

A. Cerrena Laccase A1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 1):
ATGAGCTCAA AGCTACTTGC TCTTATCACT GTCGCTCTCG TCTTGCCACT 50
AGGCACCGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC 100
AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCGGTACT AGCTGGGGGC 150
ACATTCCCTG GAGCACTTAT TACCGGTCAG AAGGTATGGG AGATCAACTT 200
GGTTGAATAG AGAAATAAAA GTGACAACAA ATCCTTATAG GGAGACAGCT 250
TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG 300
ACATCCATTG TGAGTATAAT TTAGGTCCGC TCTTCTGGCT ATCCTTTCTA 350
ACTCTTACCG TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG 400
GGCCGATGGT CCTGCCTTCG TTACTCAATG CCCTATCGTC ACCGGAAATT 450
CCTTCCTGTA CGACTTTGAT GTTCCCGACC AACCTGGTAC TTTCTGGTAC 500
CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGTG GCCCGTTCGT 550
TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG 600
GTATGTGCAT CATCATAGAG ATATAATTCA TGCAGCTACT GACCGTGACT 650
GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC 700
TCGCAAGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTCA CTTATAGTGG 750
TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG 800
TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA 850
ACGTTAAACG CGGCAAACGG TATGTTATTG AACTCCCGAT TTCTCCATAC 900
ACAGTGAAAT GACTGTCTGG TCTAGTTATC GATTTCGTCT GGTCTCCATC 950
TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000
CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATTC 1050
AAATCTTCGC AGGCCAACGA TACTCGTTCG TCGTAAGTCT CTTTGCACGA 1100
TTACTGCTTC TTTGTCCATT CTCTGACCTG TTTAAACAGC TCCATGCCAA 1150
CCGTCCTGAA AACAACTATT GGATCAGGGC CAAACCTAAT ATCGGTACGG 1200
ATACTACCAC AGACAACGGC ATGAACTCTG CCATTCTGCG ATACAACGGC 1250
GCACCTGTTG CGGAACCGCA AACTGTTCAA TCTCCCAGTC TCACCCCTTT 1300
GCTCGAACAG AACCTTCGCC CTCTCGTGTA CACTCCTGTG GTATGTTTCA 1350
AAGCGTTGTA ATTTGATTGT GGTCATTCTA ACGTTACTGC GTTTGCATAG 1400
CCTGGAAACC CTACGCCTGG CGGCGCCGAT ATTGTCCATA CTCTTGACTT 1450
GAGTTTTGTG CGGAGTCAAC ATTCGTAAAG ATAAGAGTGT TTCTAATTTC 1500
TTCAATAATA GGATGCTGGT CGCTTCAGTA TCAACGGTGC CTCGTTCCTT 1550
GATCCTACCG TCCCCGTTCT CCTGCAAATT CTCAGCGGCA CGCAGAATGC 1600
ACAAGATCTA CTCCCTCCTG GAAGTGTGAT TCCTCTCGAA TTAGGCAAGG 1650
TCGTCGAATT AGTCATACCT GCAGGTGTCG TCGGTGGACC TCATCCGTTC 1700
CATCTCCATG GGGTACGTAA CCCGAACTTA TAACAGTCTT GGACTTACCC 1750
GCTGACAAGT GCATAGCATA ACTTCTGGGT CGTGCGAAGT GCCGGAACCG 1800
ACCAGTACAA CTTTAACGAT GCCATTCTCC GAGACGTCGT CAGTATAGGA 1850
GGAACCGGGG ATCAAGTCAC CATTCGTTTC GTGGTATGTT TCATTCTTGT 1900
GGATGTATGT GCTCTAGGAT ACTAACCGGC TTGCGCGTAT AGACCGATAA 1950
CCCCGGACCG TGGTTCCTCC ATTGCCATAT CGACTGGCAC TTGGAAGCGG 2000
GTCTCGCTAT CGTATTTGCA GAGGGAATTG AAAATACTGC TGCGTCTAAT 2050
TTAACCCCCC GTACGCGGTT TCCCTCACAT CCTGGAGCTA AGCAGCTTAC 2100
TAACATACAT TTGCAGAGGC TTGGGATGAG CTTTGCCCGA AGTATAACGC 2150
GCTCAGCGCA CAAAAGAAGG TTGCATCTAA GAAAGGCACT GCCATCTAAT 2200
TTTTGTAACA AACAAGGAGG GTCTCTTGTA CTTTTATTGG GATTTCTTTC 2250
TTGGGGTTTA TTGTTAAACT TGACTCTACT ATGTTTGGAA GACGAAAGGG 2300
GCTCGCGCAT TTATATACTA TCTCTCTTGG CATCACCTGC AGCTCAATCC 2350
TTCAACCACC TAA                   2363
Translated protein sequence (SEQ ID NO: 2):
MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50
TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100
AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150
KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG 200
PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250
SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDSGMNSAI 300
LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350
DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400
VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG 450
TGDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNLTPQ 500
AWDELCPKYN ALSAQKKLNP STT        523

B. Cerrena Laccase A2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 3):
ATGAGCTCAA AGCTACTTGC TCTTATTACT GTCGCTCTCG TCTTGCCACT 50
AGGCACTGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC 100
AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCTGTACT GGCTGGGGGC 150
ACATTCCCCG GAGCACTGAT TACCGGTCAG AAGGTATGGG AGATCGATTT 200
CGTTGAATAG AGAAATACAA CTGAAAACAA ATTCTTATAG GGAGACAGCT 250
TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG 300
ACATCCATTG TGAGTATAAT ATGGGTCCGC TCTTCTAGCT ATCCTTTCTA 350
ACTCTTACCC TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG 400
GGCCGATGGT CCTGCCTTCG TTACTCAATG TCCTATCGTC ACCGGAAATT 450
CCTTCCTGTA CGACTTTGAT GTCCCCGACC AACCTGGTAC TTTCTGGTAC 500
CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGGG GCCCGTTCGT 550
TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG 600
GTATGTGCAT CATCATAAAA ATATAATTCA TGCAGCTACT GACCGCGACT 650
GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC 700
TCGCACGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTGA CTTATAGTGG 750
TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG 800
TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA 850
ACGTTAAACG CGGCAAACGG TATGTCATTG AACTCCCGAT TTCTCCATTC 900
ACATTGAAAT GACTGTCTGG TCTAGTTATC GATTCCGTCT GGTCTCCATC 950
TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000
CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATCC 1050
AAATCTTCGC AGGCCAACGC TACTCGTTCG TCGTAAGTCT CTTTGAATGG 1100
TTGGTGCTTT TTCTGTCCAT TCTCTAACCT GTTTATACAG CTCCATGCCA 1150
ACCGTCCTGA AAACAACTAT TGGATCAGGG CCAAACCTAA TATCGGTACG 1200
GATACTACCA CAGACAACGG CATGAACTCT GCCATTCTGC GATACAACGG 1250
CGCACCTGTT GCGGAACCGC AAACTGTTCA ATCTCCCAGT CTCACCCCTT 1300
TGCTCGAACA GAACCTTCGC CCTCTCGTGT ACACTCCTGT GGTATGTTTC 1350
AAAGCGTTGT AATTTGATTG TGGTCATTCT AACGTTACTG CCTTTGCACA 1400
GCCTGGAAAT CCTACGCCTG GCGGGGCCGA TATTGTCCAT ACTCTTGACT 1450
TGAGTTTTGT GCGGAGTCAA CATTCGTAAA GATAAGAGTG TTTCTAATTT 1500
CTTCAATAAT AGGATGCTGG TCGCTTCAGT ATCAACGGTG CCTCGTTCCT 1550
TGATCCTACC GTCCCTGTTC TCCTGCAAAT TCTCAGCGGC ACGCAGAATG 1600
CACAAGATCT ACTCCCTCCT GGAAGTGTGA TTCCTCTCGA ATTAGGCAAG 1650
GTCGTCGAAT TAGTCATACC TGCAGGTGTT GTCGGTGGAC CTCATCCGTT 1700
CCATCTCCAT GGGGTACGTA ACCCGAACTT ATAACAGTCT TGGACTTACC 1750
CGCTGACAAG TGTATAGCAT AACTTCTGGG TCGTGCGAAG TGCCGGAACC 1800
GACCAGTACA ACTTTAACGA TGCCATTCTC CGAGACGTCG TCAGTATAGG 1850
AGGAACCGAG GATCAAGTCA CCATTCGATT CGTGGTATAT ACTTCATTCT 1900
TGTGGATGTA TGTGCTCTAG GATACTAACT GGCTTGCGCG TATAGACCGA 1950
TAACCCCGGA CCGTGGTTCC TCCATTGCCA TATCGACTGG CACTTGGAAG 2000
CGGGTCTCGC TATCGTATTT GCAGAGGGAA TTGAAAATAC TGCTGCGTCT 2050
AATCCAACCC CCCGTATGCG GTTTCCCACA CATTCTGAAT CTAAGCAGCT 2100
TACTAATATA CATTTGCAGA GGCTTGGGAT GAGCTTTGCC CGAAGTATAA 2150
CGCGCTCAAC GCACAAAAGA AGGTTGCATC TAAGAAAGGC ACTGCCATCT 2200
AATCCTTGTA ACAAACAAGG AGGGTCTCTT GTACTTTTAT TGGGATTTAT 2250
TTCTTGGGGT TTATTGTTCA ACTTGATTCT ACTATGTTTG GAAGTAGCGA 2300
TTACGAAAGG GGCTTGCGCA TTTATATACC ATCTTTCTTG GCACCACCTG 2350
CAGCTCAATC CTTCAACCAC CTAA       2374
Translated protein sequence (SEQ ID NO: 4):
MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50
TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100
AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150
KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG 200
PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250
SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDNGMNSAI 300
LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350
DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400
VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG 450
TEDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNPTPQ 500
AWDELCPKYN ALNAQKKLNP STT        523

C. Cerrena Laccase B1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 5):
ATGTCTCTTC TTCGTAGCTT GACCTCCCTC ATCGTACTAG TCATTGGTGC 50
ATTTGCTGCA ATCGGTCCAG TCACTGACCT ACATATAGTG AACCAGAATC 100
TCGACCCAGA TGGTTTCAAC CGCCCCACTG TACTCGCAGG TGGTACTTTC 150
CCCGGTCCTC TGATTCGTGG TAACAAGGTA CGCTTCATAA CCGCCCTCCG 200
TAGACGTAGG CTTCGGCTGA CATGACCATC ATCTGTAGGG AGATAACTTT 250
AAAATTAATG TGATTGACGA CTTGACAGAG CACAGTATGC TCAAGGCTAC 300
GTCCATCGTA AGTCCCTGAT TAACGTTTCA CCTGGTCATA TCGCTCAACG 350
TCTCGAAGCA CTGGCATGGG TTCTTCCAGA AGGGAACCAA CTGGGCCGAT 400
GGCCCCGCCT TTGTCACCCA ATGTCCTATC ACATCAGGAA ACGCCTTCCT 450
GTATGATTTC AACGTTCCGG ACCAAGCTGG TACTTTCTGG TACCACAGCC 500
ATCTCTCTAC ACAGTATTGT GACGGTCTTC GTGGTGCCTT TGTCGTCTAT 550
GATCCTAATG ATCCCAACAA GCAACTCTAT GATGTTGATA ACGGCAAGTT 600
CCTTGCATAT TTCATTTCTA TCATATCCTC ACCTGTATTG GCACAGAAAG 650
CACCGTGATT ACCTTGGCTG ATTGGTATCA TGCCCTTGCT CAGACTGTCA 700
CTGGTGTCGC GTGAGTGACA AATGGCCCTC AATTGTTCAC ATATTTTCCT 750
GATTATCATA TGATAGAGTA TCTGATGCAA CGTTGATCAA CGGATTGGGA 800
CGTTCGGCCA CCGGCCCCGC AAATGCCCCT CTGGCGGTCA TCAGTGTCGA 850
GCGGAATAAG AGGTCAGTTC CATAATTATG ATTATTTCCC GCGTTACTTC 900
CTAACAATTA TTTTTGTATC CCTCCACAGA TATCGTTTCC GATTGGTTTC 950
TATTTCTTGC GACCCTAACT TTATTTTCTC AATTGACCAC CACCCAATGA 1000
CCGTAATTGA GATGGACGGT GTTAATACCC AATCTATGAC CGTAGATTCG 1050
ATCCAAATAT TCGCAGGTCA ACGATATTCA TTTGTCGTAG GTTATTATAA 1100
ACTGCCCACC GATCATCTCT CACGTAACTG TTATAGATGC AAGCCAACCA 1150
ACCAGTTGGA AATTATTGGA TCCGCGCTAA ACCTAATGTT GGGAACACAA 1200
CTTTCCTTGG AGGCCTGAAC TCCGCTATAT TACGATATGT GGGAGCCCCT 1250
GACCAAGAAC CGACCACTGA CCAAACACCC AACTCTACAC CGCTCGTTGA 1300
GGCGAACCTA CGACCCCTCG TCTATACTCC TGTGGTATGT TGTTCTCGTT 1350
ACATATACCA AACCTAATAT GAAGACTGAA CGGATCTACT AGCCGGGACA 1400
GCCATTCCCT GGCGGTGCTG ATATCGTCAA GAACTTAGCT TTGGGTTTCG 1450
TACGTGTATT TCACTTCCCT TTTGGCAGTA ACTGAGGTGG AATGTATATA 1500
GAATGCCGGG CGTTTCACAA TCAATGGAGC GTCCCTCACA CCTCCTACAG 1550
TCCCTGTACT ACTCCAGATC CTCAGTGGTA CTCACAATGC ACAGGATCTT 1600
CTCCCAGCAG GAAGCGTGAT CGAACTTGAA CAGAATAAAG TTGTCGAAAT 1650
CGTTTTGCCC GCTGCGGGCG CCGTTGGCGG TCCTCATCCT TTTCACTTAC 1700
ATGGTGTAAG TATCAGACGT CCTCATGCCC ATATTGCTCC GAACCTTACA 1750
CACCTGATTT CAGCACAATT TCTGGGTGGT TCGTAGCGCC GGTCAAACCA 1800
CATACAATTT CAATGATGCT CCTATCCGTG ATGTTGTCAG TATTGGCGGT 1850
GCAAACGATC AAGTCACGAT CCGATTTGTG GTATGTATCT CGTGCCTTGC 1900
ATTCATTCCA CGAGTAATGA TCCTTACACT TCGGGTTCTC AGACCGATAA 1950
CCCTGGCCCA TGGTTCCTTC ACTGTCACAT TGACTGGCAT TTGGAGGCTG 2000
GGTTCGCTGT AGTCTTTGCG GAGGGAATCA ATGGTACTGC AGCTGCTAAT 2050
CCAGTCCCAG GTAAGACTCT CGCTGCTTTG CGTAATATCT ATGAATTTAA 2100
ATCATATCAA TTTGCAGCGG CTTGGAATCA ATTGTGCCCA TTGTATGATG 2150
CCTTGAGCCC AGGTGATACA TGA        2173
Translated protein sequence (SEQ ID NO: 6):
MSLLRSLTSL IVLVIGAFAA IGPVTDLHIV NQNLDPDGFN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300
VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350
FNAGRFTING ASLTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400
IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN 450
DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500
NQLCPLYDAL SPGDT                 515

D. Cerrena Laccase B2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 7):
CACCGCGATG TCTCTTCTTC GTAGCTTGAC CTCCCTCATC GTACTAGCCA 50
CTGGTGCATT TGCTGCAATC GGTCCAGTCA CCGACCTACA TATAGTGAAC 100
CAGAATCTCG CCCCAGATGG TTTAAACCGC CCCACTGTAC TCGCAGGTGG 150
TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGTACGC TTCATAACCG 200
CCCTCCGTAG ACGTAGGCTT CGGCTGACAT GACCATCATC TGTAGGGAGA 250
TAACTTTAAA ATTAATGTGA TTGACGACTT GACAGAACAC AGTATGCTCA 300
AGGCTACGTC CATTGTAAGT CCCTGATTAA CGTTTCACCT GGTCATATCG 350
CTCAACGTCT CGAAGCACTG GCATGGGTTC TTCCAGAAGG GAACCAACTG 400
GGCCGATGGC CCCGCCTTTG TCACCCAATG TCCTATCACA TCAGGAAACG 450
CCTTCTTGTA TGATTTCAAC GTTCCGGACC AAGCTGGTAC TTTCTGGTAC 500
CACAGCCATC TCTCYACACA GTATTGTGAC GGTCTTCGTG GTGCCTTTGT 550
CGTCTATGAT CCTAATGATC CCAACAAGCA ACTCTATGAT GTTGATAACG 600
GCAAGTCCCT TGCATATTTC AGTTCTATCA TATCCTCACC TGTATTGGCA 650
CAGAAAGCAC CGTGATTACC TTGGCTGATT GGTATCATGC CCTTGCTCAG 700
ACTGTCACTG GTGTCGCGTG AGTGACAAAT GGCCCTTAAT TGTTCACATA 750
TTTTCCTGAT TATCATATGA TAGAGTATCT GATGCAACGT TGATCAACGG 800
ATTGGGACGT TCGGCCACCG GCCCCGCAAA TGCCCCTCTG GCGGTCATCA 850
GTGTCGAGCG GAATAAGAGG TCAGTTCCAT AATTATGATT ATTTCCCGCG 900
TTACTTCCTA ACGATTATTT TTGTATCCCT CCACAGATAT CGTTTCCGAT 950
TGGTTTCTAT TTCTTGCGAC CCTAACTTTA TTTTCTCAAT TGACCACCAC 1000
CCAATGACCG TAATTGAGAT GGACGGTGTT AATACCCAAT CTATGACCGT 1050
AGATTCGATC CAAATATTCG CAGGTCAACG ATATTCATTT GTCGTAGGTT 1100
ATTATAAACT GCCCACCGAT CATCTCTCAC GTAACTGTTA TAGATGCAAG 1150
CCAACCAACC AGTTGGAAAT TATTGGATCC GYGCTAAACC TAATGTTGGG 1200
AACACAACTT TCCTTGGAGG CCTGAACTCC GCTATATTAC GATATGTGGG 1250
AGCCCCTGAC CAAGAACCGA CCACTGACCA AACACCCAAC TCTACACCGC 1300
TCGTCGAGGC GAACCTACGT CCCCTCGTCT ATACTCCTGT GGTATGTTGT 1350
TCTCGTTACA TATACCAAAC CTAATATGAG GACTGAACGG ATCTACTAGC 1400
CGGGACAGCC ATTCCCTGGC GGTGCTGATA TCGTCAAGAA CTTAGCTTTG 1450
GGTTTCGTAC GTGTATTTCA CTTCCCTTTT GGCAGTAACT GAGGTGGAAT 1500
GTATATAGAA TGCCGGGCGT TTCACAATCA ATGGAACATC CTTCACACCT 1550
CCTACAGTCC CTGTACTACT CCAGATCCTC AGTGGTACTC ACAATGCACA 1600
GGATCTTCTT CCAGCAGGAA GCGTGATCGA ACTTGAACAG AATAAAGTTG 1650
TCGAAATCGT TCTGCCCGCT GCGGGCGCCG TTGGCGGTCC TCATCCTTTC 1700
CACTTACATG GTGTAAGTAT CAGACGTCCT CATGCCTATA TTGCTCCGAA 1750
CCTTACACAC CTGATTTCAG CACAATTTCT GGGTGGTTCG TAGCGCCGGT 1800
CAAACCACAT ACAATTTCAA TGATGCTCCT ATCCGTGATG TTGTCAGTAT 1850
TGGCGGTGCA AACGATCAAG TCACGATCCG ATTTGTGGTA TGTATCTCGT 1900
GCCTTGCATT CATTCCACGA GTAATGATCC TTACACTTCG GGTTCTCAGA 1950
CCGATAACCC TGGCCCATGG TTCCTTCACT GTCACATTGA CTGGCATTTG 2000
GAGGCTGGGT TCGCTGTAGT CTTTGCGGAG GGAATCAATG GCACTGCAGC 2050
TGCTAATCCA GTCCCAGGTA AGACTCTCGC TGCTTTGCGT AATATCTATG 2100
AATTTAAAGC ATATCAATTT GCAGCGGCTT GGAATCAATT GTGCCCGTTG 2150
TATGATGCCT TGAGCCCAGG TGATACATGA TTACTCGTAG CTGTGCTTTC 2200
TTATACATAT TCTATGGGTA TATCGGAGTA GCTGTACTAT AGTATGTACT 2250
ATACTAGGTG GGATATGYTG ATGTTGATTT ATATAATTTT GTTTGAAGAG 2300
TGACTTTATC GACTTGGGAT TTAGCCGAGT ACATACTGAT CTCTCACTAC 2350
AGGCTTGTTT TGTCTTTGGG CGCTTACTCA ACAGTTGACT GTTTTTGCTA 2400
TTACGCATTG AACCGCATTC CGGTCYGACT CGTGTCCTCT ACTGTGACTT 2450
GTATTGGCAT TCTAGCACAT ATGTCTCTTA CCTATAGGAA CAATATGTCT 2500
CAACACTGTT CCAAAACCTG CGTAAACCAA ATATCGTCCA TCAGATCAGA 2550
TCATTAACAG TGCCGCACTA ACCTAATACA CTGGCARGGA CTGTGGAAAT 2600
CCCTATAAAT GACCTCTAGA CCGTGAGGTC ATTGCAAGGT CGCTCTCCTT 2650
GTCAAGATGA CCC                   2663
Translated protein sequence (SEQ ID NO: 8):
MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGLN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300
VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350
FNAGRFTING TSFTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400
IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN 450
DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500
NQLCPLYDAL SPGDT                 515

E. Cerrena Laccase B3 Gene (Partial) from ATCC20013 Strain

Polynucleotide sequence (SEQ ID NO: 9):
GTGGGGGCGG ATCCCTAACT GTTTCGAATC GGCACCGAAG TATGCAGGTG 50
TGACGGAGAT GAGGCGTTTT TTCATCTTCC ACTGCAGTAT AAAATGTCTC 100
AGGTAACGTC CAGCTTTTTG TACCAGAGCT ACCTCCAAAT ACCTTTACTC 150
GCAAAGGTTT CGCGATGTCT CTTCTTCGTA GCTTGACCTC CCTCATCGTA 200
CTAGCCACTG GTGCATTTGC TGCAATCGGT CCAGTCACTG ACCTACATAT 250
AGTGAACCAG AATCTCGCCC CAGATGGTTT CAACCGCCCC ACTGTACTCG 300
CAGGTGGTAC TTTCCCCGGT CCTCTGATTC GTGGTAACAA GGTACGCTTC 350
ATAACCGCCC TCCGTAGACG TAGGCTTCGG CTGACATGAC CATCATCTGT 400
AGGGAGATAA CTTTAAAATT AATGTGATTG ACGACTTGAC AGAACACAGT 450
ATGCTCAAGG CCACGTCCAT TGTAAGTCCC TGATTAACGT TTCACCTGGT 500
CATATCGCTC AACGTCTCGA AGCACTGGCA TGGGTTCTTC CAGAAGGGAA 550
CCAACTGGGC CGATGGCCCC GCCTTTGTCA CCCAATGTCC TATCACATCA 600
GGAAACTCCT TCCTGTATGA TTTCAACGTT CCGGACCAAG CTGGTACTTT 650
CTGGTACCAC AGCCATCTCT CTACACAGTA TTGTGACGGT CTTCGTGGTG 700
CCTTTGTCGT CTATGATCCT AATGATCCCA ACAAGCAACT CTATGATGTT 750
GATAACGGCA AGTCCCTTGC ATATTTCATT TCTATCATAT CCTCACCTGT 800
ATTGGCACAG AAAGCACCGT GATTACCTTG GCTGATTGGT ATCATGCCCT 850
TGCTCAGACT GTCACTGGTG TCGCGTGAGT GACAAATGGC CCTCAATTGT 900
TCACATATTT TCCTGATTAT CATATGATAG AGTATCTGAT GCAACGTTGA 950
TCAACGGATT GGGACGTTCG GCCACCGGCC CCGCAAATGC CCCTCTGGCG 1000
GTCATCAGTG TCGAGCGGAA TAAGAGGTCA GTTCCATAAT TATGATTATT 1050
TCCCGCGTTA CTTCCTAACA ATTATTCTTG TATCCCTCCA CAGATATCGC 1100
TTCCGATTGG TGTCTATTTC TTGCGACCCT AACTTTATTT TCTCAATTGA 1150
TCACCACCCA ATGACCGTAA TTGAGATGGA CGGTGTTAAT ACCCAATCTA 1200
TGACCGTAGA TTCGATCCAA ATATTCGCAG GTCAACGATA TTCATTTGTC 1250
GTAGGTTATT ATAAACTGCC CACCGATCAT CTCTCACGTA ACTGTTATAG 1300
ATGCAAGCCA ACCAACCRGT TGGAAATTAT TGGATCC 1337
Translated protein sequence (SEQ ID NO: 10):
MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGFN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN SFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GKTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWI   278

F. Cerrena Laccase C Gene (Partial) from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 11):
TGCAATCGGA CCGGTBGCTG ACCTTCACAT TACGGACGAT ACCATTGCCC 50
CCGATGGTTT CTCTCGTCCT GCTGTTCTCG CTGGCGGGGG TTTCCCTGGC 100
CCTCTCATCA CCGGAAACAA GGTAATGCCT AATGGTTGCG TCTTTGTTGG 150
TGCTCTCATT CATCCACGAC ATTTTGTACC AGGGCGACGC CTTTAAACTC 200
AATGTCATCG ATGAACTAAC GGACGCATCC ATGCTGAAGY CGACTTCCAT 250
CGTAAGTCTC GCTGTATTGC TCCTTGAGCC ATTTCATTGA CTATAACTAC 300
AACCAGCACT GGCATGGATT CTTCCAAAAG GGTACTAATT GGGCAGATGG 350
TCCCGCTTTT GTGAACCAAT GCCCCATCAC CACGGGAAAC TCCTTCTTGT 400
ACGACTTCCA GGTTCCTGAT CAAGCTGGTA AGCATGAGAT TACACTAGGA 450
AAGTTTAATT TAATAACTAT TCAATCAGGA ACCTACTGGT ATCATAGTCA 500
TTTGTCTACG CAATACTGTG ATGGTCTCAG AGGTGCATTC GTTGTCTACG 550
ACCCTTCAGA TCCTCACAAG GATCTCTACG ACGTCGACGA CGGTGAGCTT 600
TGCTTTTTTC ATTGGTATCC ATTATCGCTC ACGTGTCATT ACTGCGCCAC 650
AGAAAGTACC GTCATCACTT TGGCTGATTG GTATCATACT TTGGCTCGTC 700
AGATTGTTGG CGTTGCGTGA GTAGTCTTGT ACCGACTGAA ACATATTCCA 750
GTTGCTGACT TCCCCACAGC ATTTCTGATA CTACCTTGAT AAACGGTTTG 800
GGCCGCAATA CCAATGGTCC GGCTGATGCT GCTCTTGCTG TGATCAATGT 850
TGACGCTGGC AAACGGTGTG TCCAGATTAC TATACTCCCC ATGACGTCTC 900
AATGCTGATG TGTACTACTT CCAGGTACCG TTTCCGTCTT GTTTCCATAT 950
CCTGTGACCC CAATTGGGTA TTCTCGATTG ACAACCATGA CTTTACGGTC 1000
ATTGAAGTCG ATGGTGTTAA CAGTCAACCT CTCAACGTCG ATTCTGTTCA 1050
GATCTTCGCC GGACAACGTT ACTCGTTCGT 1080
Translated protein sequence (SEQ ID NO: 12):
AIGPVADLHI TDDTIAPDGF SRPAVLAGGG FPGPLITGNK GDAFKLNVID 50
ELTDASMLKX TSIHWHGFFQ KGTNWADGPA FVNQCPITTG NSFLYDFQVP 100
DQAGTYWYHS HLSTQYCDGL RGAFVVYDPS DPHKDLYDVD DESTVITLAD 150
WYHTLARQIV GVAISDTTLI NGLGRNTNGP ADAALAVINV DAGKRYRFRL 200
VSISCDPNWV FSIDNHDFTV IEVDGVNSQP LNVDSVQIFA GQRYSF 246

G. Cerrena Laccase D1 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 13):
GATTCTAATA GACCAGGCAT ACCAAGAGAT CTACAGGTTG ACAGACCATT 50
CTTCTAGGCG GCATTTATGC TGTAGCGTCA GAAATTATCT CTCCATTTGT 100
ATCCCACAGG TCCTGTAATA ACACGGAGAC AGTCCAAACT GGGATGCCTT 150
TTTTCTCAAC TATGGGCGCA CATAGTCTGG ACGATGGTAT ATAAGACGAT 200
GGTATGAGAC CCATGAAGTC AGAACACTTT TGCTCTCTGA CATTTCATGG 250
TTCACACTCT CGAGATGGGA TTGAACTCGG CTATTACATC GCTTGCTATC 300
TTAGCTCTGT CAGTCGGAAG CTATGCTGCA ATTGGGCCCG TGGCCGACAT 350
ACACATTGTC AACAAAGACC TTGCTCCAGA TGGCGTACAA CGTCCAACCG 400
TGCTTGCCGG AGGCACTTTT CCTGGGACGT TGATCACCGG TCAGAAAGTA 450
AGGGATATTA GTTTGCGTCA AAGAGCCAAC CAAAACTAAC CGTCCCGTAC 500
TATAGGGTGA CAACTTCCAG CTCAATGTCA TCGATGATCT TACCGACGAT 550
CGGATGTTGA CGCCAACTTC CATTGTGAGC CTATTATTGT ATGATTTATC 600
CGAATAGTTT CGCAGTCTGA TCATTGGATC TCTATCGCTA GCATTGGCAC 650
GGTTTCTTCC AGAAGGGAAC CGCTTGGGCC GACGGTCCCG CCTTCGTAAC 700
TCAGTGCCCT ATAATAGCAG ATAACTCTTT TCTGTATGAC TTCGACGTCC 750
CAGACCAAGC TGGTACTTTC TGGTATCATA GTCATCTATC CACTCAGTAC 800
TGTGACGGTT TACGTGGTGC CTTCGTTGTG TACGATCCTA ACGATCCTCA 850
CAAAGACCTA TACGATGTTG ATGACGGTGG GTTCCAAATA TTTGTTCTGC 900
AGACATTGTA TTGACGGTGT TCATTATAAT TTCAGAGAGC ACCGTGATTA 950
CCCTTGCGGA TTGGTACCAT GTTCTCGCCC AGACCGTTGT CGGCGCTGCG 1000
TGAGTAACAC ATACACGCGC TCCGGCACAC TGATACTAAT TTTTTTTTAT 1050
TGTAGCACTC CTGATTCTAC CTTGATCAAC GGGTTAGGCC GTTCACAGAC 1100
CGGACCCGCT GATGCTGAGC TGGCTGTTAT CAGCGTTGAA CATAACAAAC 1150
GGTATGTCAT CTCTACCCAG TATCTTCTCT CCTGCTCTAA TTCGCTGTTT 1200
CACCATAGAT ACCGTTTCCG TTTGGTTTCG ATTTCGTGCG ACCCCAACTT 1250
TACCTTCTCC GTTGATGGTC ATAATATGAC TGTCATCGAA GTCGATGGTG 1300
TCAACACACG ACCCCTGACC GTTGACTCTA TTCAAATCTT CGCCGGACAG 1350
AGGTATTCCT TTGTCGTAAG TTAATCGATA TATTCTCCTT ATTACCCCTG 1400
TGTAATTGAT GTCAATAGCT CAATGCTAAC CAACCCGAAG ACAATTACTG 1450
GATCCGTGCT ATGCCAAACA TCGGTAGAAA TACAACAACA CTGGACGGAA 1500
AGAATGCCGC TATCCTTCGA TACAAGAATG CTTCTGTAGA AGAGCCCAAG 1550
ACCGTTGGGG GCCCCGCTCA ATCCCCGTTG AATGAAGCGG ACCTGCGTCC 1600
ACTCGTACCT GCTCCTGTGG TATGTCTTGT CGCGCTGTTC CATCGCTATT 1650
TCATATTAAC GTTTTGTTTT TGTCAAGCCT GGAAACGCTG TTCCAGGTGG 1700
CGCAGACATC AATCACAGGC TTAACTTAAC TTTCGTACGT ACACCTGGTT 1750
GAAACATTAT ATTTCCAGTC TAACCTCTCT TGTAGAGTAA CGGCCTCTTC 1800
AGCATCAACA ACGCCTCCTT CACTAATCCT TCGGTCCCCG CCTTATTACA 1850
AATTCTGAGC GGTGCTCAGA ACGCTCAAGA TTTACTTCCA ACGGGTAGTT 1900
ACATTGGCCT TGAACTAGGC AAGGTTGTGG AGCTCGTTAT ACCTCCTCTG 1950
GCAGTTGGAG GACCGCACCC TTTCCATCTT CATGGCGTAA GCATACCACA 2000
CTCCCGCAGC CAGAATGACG CAAACTAATC ATGATATGCA GCACAATTTC 2050
TGGGTCGTCC GTAGTGCAGG TAGCGATGAG TATAACTTTG ACGATGCTAT 2100
CCTCAGGGAC GTCGTRAGCA TTGGAGCGGG GACTGATGAA GTCACAATCC 2150
GTTTCGTGGT ATGTCTCACC CCTCGCATTT TGAGACGCAA GAGCTGATAT 2200
ATTTTAACAT AGACCGACAA TCCGGGCCCG TGGTTCCTCC ATTGCCATAT 2250
TGATTGGCAT TTGGAGGCAG GCCTTGCCAT CGTCTTCGCT GAGGGCATCA 2300
ATCAGACCGC TGCAGCCAAC CCAACACCCC GTACGTGACA CTGAGGGTTT 2350
CTTTATAGTG CTGGATTACT GAATCGAGAT TTCTCCACAG AAGCATGGGA 2400
TGAGCTTTGC CCCAAATATA ACGGGTTGAG TGCGAGCCAG AAGGTCAAGC 2450
CTAAGAAAGG AACTGCTATT TAAACGTGGT CCTAGACTAC GGGCATATAA 2500
GTATTCGGGT AGCGCGTGTG AGCAATGTTC CGATACACGT AGATTCATCA 2550
CCGGACACGC TGGGACAATT TGTGTATAAT GGCTAGTAAC GTATCTGAGT 2600
TCTGGTGTGT AGTTCAAAGA GACAGCCCTT CCTGAGACAG CCCTTCCTGA 2650
GACAGCCCTT CCTGAGACGT GACCTCCGTA GTCTGCACAC GATACTYCTA 2700
AATACGTATG GCAAGATGAC AAAGAGGAGG ATGTGAGTTA CTACGAACAG 2750
AAATAGTGCC CGGCCTCGGA GAGATGTTCT TGAATATGGG ACTGGGACCA 2800
ACATCCGGA                        2809
Translated protein sequence (SEQ ID NO: 14):
MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50
TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100
AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150
NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200
PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250
PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300
LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350
NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400
VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450
GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500
AWDELCPKYN GLSASQKVKP KKGTAI     526

H. Cerrena Laccase D2 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 15):
GATCTGGACG ATGGTATATA AGACGATGGT ATGAGACCCA TGAAGTCTGA 50
ACACTTTTGC TCTCTGACAT TTCATGGTTC ATACTCTCGA GATGGGATTG 100
AACTCGGCTA TTACATCGCT TGCTATCTTA GCTCTGTCAG TCGGAAGCTA 150
TGCTGCAATT GGGCCCGTGG CCGACATACA CATTGTCAAC AAAGACCTTG 200
CTCCAGATGG TGTACAACGT CCAACCGTGC TCGCCGGAGG CACTTTTCCT 250
GGGACGTTGA TCACCGGTCA GAAAGTAAGG AATATTAGTT TGCGTCAAAG 300
AGCCAACCAA AATTAACCGT CCCGTCCCAT AGGGTGACAA CTTCCAGCTC 350
AATGTCATTG ATGATCTTAC CGACGATCGG ATGTTGACAC CAACTTCCAT 400
TGTGAGCCTA TTATTGTATG ATTTATCCGT ATAGTTTCTC AGTCTGATCA 450
TTGGCTCTCT ATCGCTAGCA TTGGCACGGT TTCTTCCAGA AGGGAACCGC 500
TTGGGCCGAC GGTCCCGCCT TCGTAACTCA GTGCCCTATA ATAGCAGATA 550
ACTCTTTTCT GTATGACTTC GACGTCCCCG ACCAAGCTGG TACTTTCTGG 600
TATCATAGTC ATCTATCCAC TCAGTACTGT GACGGTTTAC GTGGTGCCTT 650
CGTTGTGTAC GATCCTAACG ATCCTCACAA AGACCTATAC GATGTTGATG 700
ACGGTGGGTT CCAAATACTT GACCAAGAAA CATTATATTG ATAGTATCCA 750
CTCTGATTTT CAGAGAGCAC CGTGATTACC CTTGCGGATT GGTACCATGT 800
TCTCGCCCAG ACCGTTGTCG GCGCTGCGTG AGTAACACAT ACACGCGCTC 850
CGGCACACTG ATACTAATTT TTTATTGTAG CACTCCTGAT TCTACCTTGA 900
TCAACGGGTT AGGCCGTTCA CAGACCGGAC CCGCTGATGC TGAGCTGGCT 950
GTTATCAGCG TTGAACATAA CAAACGGTAT GTCATCTCTA CCCATTATCT 1000
TCTCTCCTGC TTTAATTCGC TGTTTCACCA TAGATACCGA TTCCGTTTGG 1050
TTTCGATTTC GTGCGACCCC AACTTTACCT TCTCCGTTGA TGGTCATAAT 1100
ATGACTGTCA TCGAAGTCGA CGGTGTCAAC ACACGACCCC TGACCGTTGA 1150
CTCTATTCAA ATCTTCGCCG GACAGAGGTA TTCCTTTGTC GTAAGTTAAT 1200
CGATATATTC TCCCTATTAC CCCTGTGTAA TTGATGTCAA CAGCTCAATG 1250
CTAACCAACC CGACGACAAT TACTGGATCC GTGCTATGCC AAACATCGGT 1300
AGAAATACAA CAACACTGGA CGGAAAGAAT GCCGCTATCC TTCGATACAA 1350
GAATGCTTCT GTAGAAGAGC CCAAGACCGT TGGGGGCCCC GCTCAATCCC 1400
CGTTGAATGA AGCGGACCTG CGTCCACTCG TACCTGCTCC TGTGGTATGT 1450
CTTGTCGTGC TGTTCCATCG CTATTTCATA TTAACGTTTT GTTTTTGTCA 1500
AGCCTGGAAA CGCTGTTCCA GGTGGCGCAG ACATCAATCA CAGGCTTAAC 1550
TTAACTTTCG TACGTACACC TGGTTGAAAC ATTATATTTC CAGTCTAACC 1600
TCTTGTAGAG TAACGGCCTT TTCAGCATCA ACAACGCCTC CTTCACTAAT 1650
CCTTCGGTCC CCGCCTTATT ACAAATTCTG AGCGGTGCTC AGAACGCTCA 1700
AGATTTACTT CCAACGGGTA GTTACATTGG CCTTGAACTA GGCAAGGTTG 1750
TGGAGCTCGT TATACCTCCT CTGGCAGTTG GAGGACCGCA CCCTTTCCAT 1800
CTTCATGGCG TAAGCATACC ACACTCCCGC AGCCAGAATG ACGCAAACTA 1850
ATCATGATAT GCAGCACAAT TTCTGGGTCG TCCGTAGTGC AGGTAGCGAT 1900
GAGTATAACT TTGACGATGC TATCCTCAGG GACGTCGTGA GCATTGGAGC 1950
GGGGACTGAT GAAGTCACAA TCCGTTTCGT GGTATGTCTC ACCCCTCGCA 2000
TTTTGAGACG CAAGAGCTGA TATATTTTAA CATAGACCGA CAATCCGGGC 2050
CCGTGGTTCC TCCATTGCCA TATTGATTGG CATTTGGAGG CAGGCCTTGC 2100
CATCGTCTTC GCTGAGGGCA TCAATCAGAC CGCTGCAGCC AACCCAACAC 2150
CCCGTACGTG ACACTGAGGG TTTCTTTATA GTGCTGGATT ACTGAATCGA 2200
GATTTCTCCA CAGAAGCATG GGATGAGCTT TGCCCCAAAT ATAACGGGTT 2250
GAGTGCGAGC CAGAAGGTCA AGCCTAAGAA AGGAACTGCT ATTTAAACG 2299
Translated protein sequence (SEQ ID NO: 16):
MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50
TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100
AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150
NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200
PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250
PLTVDSIQIF AGQRYSFVLN ANQPDDNYWI RAMPNIGRNT TTLDGKNAAI 300
LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350
NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400
VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450
GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500
AWDELCPKYN GLSASQKVKP KKGTAI     526

I. Cerrena Laccase E Gene (Partial) from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 17):
TGCAATCGGA CCGGTGGCCG ACCTCAAGAT CGTAAACCGA GACATTGCAC 50
CTGACGGTTT TATTCGTCCC GCCGTTCTCG CTGGAGGGTC GTTCCCTGGT 100
CCTCTCATTA CAGGGCAGAA AGTACGTTAC GCTATCTCGG TGCTTTGGCT 150
TAATTAAACT ATTTGACTTT GTGTTCTCTT AGGGGAACGA GTTCAAAATC 200
AATGTAGTCA ATCAACTGAC CGATGGTTCT ATGTTAAAAT CCACCTCAAT 250
CGTAAGCAGA ATGAGCCCTT TGCATCTCGT TTTATTGTTA ATGCGCCCAC 300
TATAGCATTG GCATGGATTC TTCCAGAAGG GAACAAACTG GGCAGACGGT 350
CCTGCGTTCG TGAACCAATG TCCAATCGCC ACGAACAATT CGTTCTTGTA 400
TCAGTTTACC TCACAGGAAC AGCCAGGTGA GTATGAGATG GAGTTCATCC 450
GAGCATGAAC TGATTTATTT GGAACCTAGG CACATTTTGG TACCATAGTC 500
ATCTTTCCAC ACAATACTGC GATGGTTTGC GAGGGCCACT CGTGGTGTAT 550
GACCCACAAG ACCCGCATGC TGTTCTCTAC GACGTCGACG ATGGTTCGTA 600
CTTCGCATAT CCACGCTCGC TTTCATACAA TGTAAACTTT GTTCCTCCAG 650
AAAGTACAAT CATCACGCTC GCGGATTGGT ATCATACCTT GGCTCGGCAA 700
GTGAAAGGCC CAGCGTAAGG CACTTTAGTG TTTCCTCATA GTCCAAGAAA 750
TTCTAACACG CCTTCTTCAT CAGGGTTCCT GGTACGACCT TGATCAACGG 800
GTTGGGGCGT CACAACAATG GTCCTCTAGA TGCTGAACTA GCGGTGATCA 850
GTGTTCAAGC CGGCAAACGG CAAGTTCAAT TCACACTTTT CACTCTGTAC 900
CTTCTTCCTG ACATTCTTTT CTTGTAGTTA CCGCTTCCGC CTGATTTCAA 950
TTTCATGCGA TCCCAACTAC GTATTCTCCA TTGATGGCCA TGATATGACT 1000
GTCATCGAAG TGGATAGTGT TAACAGTCAA CCTCTCAAGG TAGATTCTAT 1050
CCAAATATTT GCAGGTCAGA GATATTCGTT CGTGGTGAGT CAGATCAGGG 1100
CATATCCTTT TGTCGATACG TCATTGACCA TATAATGCTA CAAGCTGAAT 1150
GCCAACCAAC CAG                   1163
Translated protein sequence (SEQ ID NO: 18):
AIGPVADLKI VNRDIAPDGF IRPAVLAGGS FPGPLITGQK GNEFKINVVN 50
QLTDGSMLKS TSIHWHGFFQ KGTNWADGPA FVNQCPIATN NSFLYQFTSQ 100
EQPGTFWYHS HLSTQYCDGL RGPLVVYDPQ DPHAVLYDVD DESTIITLAD 150
WYHTLARQVK GPAVPGTTLI NGLGRHNNGP LDAELAVISV QAGKRQVQFT 200
LFTLYRFRLI SISCDPNYVF SIDGHDMTVI EVDSVNSQPL KVDSIQIFAG 250
QRYSFVLNAN QP                    262

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:

MGLNSAITSL AILALSVGSY AAIGPVADLH IVNKDLAPDG VQRPTVLAGG 50
TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100
AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150
NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200
PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250
PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300
LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350
NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400
VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450
GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500
AWDELCPKYN GLSASQKVKP KKGTAI     526

The mature processed form of this polypeptide is as follows (SEQ ID NO: 20):

AIGPVADLHIVNKDLAPDGVQRPTVLAGGTFPGILITGQKGDNFQLNVID
DLTDDRMLTPTSIHWHGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVP
DQAGTFWYHSHLSTQYCDGLRGAFVVYDPNDPHKDLYDVDDGGTVITLAD
WYHVLAQTVVGAATPDSTLINGLGRSQTGPADAELAVISVEHNKRYRFRL
VSISCDPNFTFSVDGHNMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVLNA
NQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSP
LNEADLRPLVPAPVPGNAVPGGADINHRLNLIFSNGLFSINNASFTNPSV
PALLQILSGAQNAQDLLPTGSYIGLELGKVVELVIPPLAVGGPHPFHLHG
HNFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEVTIRFVTDNPGPWFLHC
HIDWHLEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQKVKPK
KGTAI

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.

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

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.

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.

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

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

in which A is a group such as —R, -D, —CH═CH-D, —CH═CH—CH═CH-D, —CH═N-D, —N═N-D, or —N═CH-D, D is selected from the group consisting of —CO-E, —SO₂-E, —CN, —NXY, and —N⁺XYZ, E is —H, —OH, —R, —OR, or —NXY, and X, Y, and Z are independently selected from —H, —OH, —OR, and —R; where R is a 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 C_mH_2m+1; 1≦m≦5.

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 C_mH_2m+1; 1≦m≦5. In some embodiments, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as “syringonitrile” or “SN”).

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.

For applications such as textile processing, the mediator may be present in a concentration of about 0.005 to about 1.000 mmole per g denim, about 0.05 to about 500 mmole per g denim, about 0.1 to about 100 mmole per g denim, about 1 to about 50 μmole per g denim, or about 2 to about 20 μmole per g denim

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. Pat. No. 5,752,980. Other suitable mediators are described in, e.g., U.S. Patent Publication No. 2008/0189871.

Methods of Use

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.

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.

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.

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.

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.

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 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 bath between treatments. Such methods are referred to as “single-bath” methods.

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 enzymes include Humicola insolens, Fusarium oxysporum or Trichoderma reesei. Non-limiting examples of suitable cellulases are disclosed in U.S. Pat. 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.

In some embodiments, enzymatic “stonewashing” using a cellulase, bleaching using an 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.

Laccases can also be used in other aspects of textile manufacturing, generally including 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.

In further embodiments, the present systems and compositions may also be used in a method for modifying the color of wool. For example, European Patent No. EP 0 504 005 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 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.

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 7.0.

Ready to Use Compositions and Kits

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

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.

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

The following enzyme nomenclature is used in the Examples:

Trade name              Description
PRIMAGREEN ® EcoWhite 1 Mycobacterium smegmatis perhydrolase, S54V variant of
                        SEQ ID NO: 1
PRIMAGREEN ® 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. 11AG8
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

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 and an increase in absorbance at 420 nm. The color formation is proportional to laccase activity and is monitored against a laccase standard.

Mediator

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

Procedure

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 10:1 liquor ratio 50° C. with 0.5 g/l (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of a non-ionic surfactant [e.g., Rucogen BFA (Rudolf Chemie) or Ultravon RW (Huntsman)].
  • 2 cold rinses for 5 minutes at 30:1 liquor ratio.

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.
  • Stonewashing for 60 minutes at 10:1 liquor ratio 55° C. with 1 kg of pumice stone, pH 4.5 (1 g/l tri-sodium citrate dihydrate and 1 g/l citric acid monohydrate) and 1.2 g/l INDIAGE® 2XL cellulase (Genencor).
  • 2 cold rinses for 5 minutes at 30:1 liquor ratio.

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, with either (i) C. unicolor laccase D and syringonitrile at pH 6 (0.7 g/l monosodium phosphate and 0.17 g/l 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/l monosodium phosphate and 0.56 g/l 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

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:

$L^{*}=116{\left(\frac{Y}{Y_0}\right)}^{1/3}-16$ $\mathrm{when}\frac{Y}{Y_0}>0.008856$ $u^{*}=13L^{*}\left(u^{\prime }-u_0^{\prime }\right)$ $v^{*}=13L^{*}\left(v^{\prime }-v_0^{\prime }\right)$

• • where
  • Y: Tristimulus value Y (tristimulus value Y₁₀ can also be used.)
  • u′,v′: Chromaticity coordinates from the CIE 1976 UCS diagram
  • Y₀, u′₀, v′₀: Tristimulus value Y (or Y₁₀) and chromaticity coordinates u′, v′ of the perfect reflecting diffuser.

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 FIG. 1.

TABLE 1
Results using C. unicolor laccase and syringonitrile
C. unicolor
laccase	Syringonitrile		Bleaching
concentration,	concentration,	Temp.,	level,	Standard
g/l (U/ml)	g/l (mM)	° C.	CIE* Lab	deviation
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	30	36.2/−0.2/−12.8	0.5/0.1/0.1
(20.5)	(0.39)
0.3	0.07	30	36.1/−0.2/−13.0	0.5/0.1/0.1
(11.4)	(0.39)
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	34.0/0.3/−13.5	0.6/0.1/0.1
(5.7)	(0.39)	(no steam)

TABLE 2
Results using A. oryzae laccase from and methyl syringate
NOVOPRIME ®	NOVOPRIME ®		Bleaching
Base 268	F258 conc.,	Temp.,	level,	Standard
conc., g/l	g/l (mM)	° C.	CIE* Lab	deviation
0.47	0.07	40	36.2/−0.5/	0.6/0.1/0.2
	(0.33)		−11.2
0.27	0.07	40	36.5/−0.4/	0.6/0.1/0.2
	(0.33)		−11.4
0.15	0.07	40	35.7/−1.0/	0.5/0.1/0.2
	(0.33)		−11.9
0.15	0.07	30	33.9/0.1/	0.5/0.1/0.2
	(0.33)		−12.6

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

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

Color modification of denim legs was evaluated as described in Example 1. The results are shown in Table 3 and FIG. 2.

TABLE 3
Results using C. unicolor laccase and syringonitrile in different ratios
C. unicolor
laccase	Mediator		Bleaching
concentration,	concentration,	Temp.,	level,	Standard
g/l (U/ml)	g/l (mM)	° C.	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	40	37.1/−0.4/−12.7	0.6/0.1/0.1
(5.7)	(0.55)

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

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

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

Color modification of denim legs was evaluated as described in Example 1. The results are shown in Tables 5 and 6 and in FIGS. 3 and 4.

TABLE 5
Results using C. unicolor RTU composition
	RTU laccase,	Temp.,	Bleaching level,	Standard
	% owg*	° C.	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 ®	Temp.,	Bleaching level,	Standard
	II S, % owg	° C.	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

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.

12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.

Following desizing, the denim was stonewashed and bleached in a Unimac UF 50 washing machine under the following conditions:

• • 30 minutes, 30° C. at 10:1 liquor ratio, pH 6, (i) 0.4% owg INDIAGE® Super GX 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 FIG. 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

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.

12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.

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)

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.

The results are shown in Table 8 and FIG. 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,	Standard
	(° C.)	CIE* Lab	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

The results show that color modification by laccase treatment can be achieved following stonewashing.

Example 6

Laccase-Mediated Color Modification of Denim at 30° without Stonewashing

12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.

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.

The results are shown in Table 9 and FIG. 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.	Bleaching level,	Standard
	(° C.)	CIE* Lab	deviation
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

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

This Example shows that effective stonewashing and color modification can be obtained using laccase and cellulase in a single-bath process.

Enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).

Procedure

Starting material was desized denim weighing approximately 3 kg (ballast+2 legs for evaluation).

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 100
  • 2 cold rinses for 3 minutes
  • The denim was dried in an industrial dryer

Evaluation of Denim Legs

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.

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
	CIE*Lab	deviation
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

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

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

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 7809136160, 6,292 GLacU/g).

Procedure

12 denim legs weighing approximately 3 kg (total) were desized in a Unimac UF 50 washing machine as described in Example 1.

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

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.

The results are summarized in Tables 11 and 12.

	TABLE 11
		Bleaching level,	Standard
		CIE*Lab	deviation
	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
	CIE*Lab	deviation
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

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

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/l 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

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.

The results are summarized in Tables 13

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
PRIMAGREEN ® Ecofade (1 g/l) 15 min 45.1/2.9/16.5
PRIMAGREEN ® Ecofade (1 g/l) 30 min 45.5/3.3/16.8
PRIMAGREEN ® Ecofade (1 g/l) 45 min 44.7/3.2/16.4
PRIMAGREEN ® Ecofade (2 g/l) 15 min 44.7/3.3/15.9
PRIMAGREEN ® Ecofade (2 g/l) 30 min 45.3/3.5/15.9
PRIMAGREEN ® Ecofade (2 g/l) 45 min 45.0/3.5/15.6
PRIMAGREEN ® Ecofade (3 g/l) 15 min 44.4/3.4/15.5
PRIMAGREEN ® Ecofade (3 g/l) 30 min 44.6/3.6/15.7
PRIMAGREEN ® Ecofade (3 g/l) 45 min 45.0/3.6/15.4

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

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/l 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

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.

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
PRIMAGREEN ® Ecofade (1 g/l) 15 min 41.1/3.7/17.6
PRIMAGREEN ® Ecofade (1 g/l) 30 min 41.5/4.2/18.6
PRIMAGREEN ® Ecofade (1 g/l) 45 min 41.6/4.0/18.1
PRIMAGREEN ® Ecofade (2 g/l) 15 min 40.4/3.9/17.0
PRIMAGREEN ® Ecofade (2 g/l) 30 min 41.2/4.2/17.3
PRIMAGREEN ® Ecofade (2 g/l) 45 min 41.6/4.3/17.3
PRIMAGREEN ® Ecofade (3 g/l) 15 min 40.5/4.0/16.6
PRIMAGREEN ® Ecofade (3 g/l) 30 min 41.0/4.2/17.1
PRIMAGREEN ® Ecofade (3 g/l) 45 min 40.6/4.3/17.0

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

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).

Procedure

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/l of lubricant, 0.2 g/l of dispersant (non ionic surfactant), and 0.2 g/l of polyester blocker (non ionic hydrophilic co-polymer).

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

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.

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

The results show that color modification by laccase treatment occurs in the presence of pumice stones and in the presence of a surfactant.

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

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 claim 9, 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 claim 1, wherein the laccase is a microbial laccase.

14. The method of claim 1, wherein the laccase is from a Cerrena species.

15. The method of claim 1, wherein the laccase is from Cerrena unicolor.

16. The method of claim 1, wherein the laccase is laccase D from C. unicolor.

17. The method of claim 1, 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 claim 1, 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 claim 1, 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 claim 1, 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 claim 1, 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 claim 1, wherein the mediator is syringonitrile.

23. The method of claim 1, wherein the temperature is from about 20° to about 35° C.

24. The method of claim 1, wherein the temperature is from about 20° C. to about 23° C.

25. The method of claim 1, wherein the temperature is the ambient temperature of tap water.

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

27. The method of claim 1, wherein the laccase enzyme and the mediator are provided in a solid form.