JP2013241721A - Laccase and method of use thereof at low temperature - Google Patents

Abstract

PROBLEM TO BE SOLVED: To apply laccase enzymes to a color modification of denim fabrics.SOLUTION: Laccase enzymes and nucleic acid sequences encoding such laccase enzymes are described. The laccase enzymes can be employed in conjunction with mediators in improved methods for modifying the color of denim fabrics. Low-temperature and single-bath textile processing using the laccase enzymes are also described.

Description

Priority This application is based on US Provisional Patent Application No. 61 / 140,724 filed December 24, 2008, 61 / 154,882 filed February 24, 2009, and 2009. Claim priority to 61 / 237,532 filed on August 27th. Each document is incorporated in its entirety by reference.

The systems, compositions and methods of the present invention relate to laccase enzymes and nucleic acid sequences encoding such laccase enzymes. The laccase enzyme can be used with a transmitter in an improved way to modify the color of denim fabric.

Laccase is a copper-containing phenol oxidase that has been found to be an excellent oxidant in the presence of oxygen. Laccase is found in microorganisms, fungi and higher organisms. Laccase enzymes are pulp or paper bleaching, pulp waste liquor treatment, deinking, industrial color removal, bleaching in laundry detergents, oral care tooth whitening agents, and catalysts or accelerators for polymerization and oxidation reactions Used in a number of applications involving agents.

Laccase can be used in a variety of applications in a number of industries including the detergent industry, the pulp and paper industry, the textile industry and the food industry. In some applications, phenol oxidase is used as an aid to remove stains such as food stains from clothes during detergent cleaning. Most laccases are inert at neutral or alkaline pH, but exhibit pH optimum in the acidic pH region.

Laccases are genii Aspergillus, Neurospora, Podospora, Botrytis, Pleurotus, Fornes, Phlebia, Trametes, Polyporus , Stachybotris, Rhizoctonia, Bipolaris, Curvularia, Amerosporium, Lentinus, Myceliophtora, Coprinus, Thielavia, rena It has been found that it is made by a variety of fungi including Streptomyces and Melanocarpus species. However, there is still a need for laccases with different performance characteristics in various applications.

For many applications, the laccase's ability to oxidize can be improved by the use of a transmitter, also known as an accelerator. A system comprising a laccase and a transmitter is known in the art as a laccase transmitter system (LMS). The same compound can be used to activate or initiate the action of laccase.

There are several known transmitters for use in laccase transmitter systems. 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 a number of compounds that contain NH—OH or N—O groups that have been found to be useful as transmitters.

Functional groups and substituents greatly affect the ability of the transmitter. Even within the same class of compounds, substituents can change the laccase specificity for a substrate, thereby greatly increasing or decreasing the ability of the transmitter. In addition, the mediator is effective for certain applications, but may not be suitable for other applications. Another drawback with current mediators is their tendency to polymerize during use. Therefore, there is a need to find efficient mediators for specific applications. One such application is fiber bleaching, and it is also important that the mediator is not overly expensive or dangerous. Other uses of the laccase transmitter system are given below.

The method of using laccase at a low temperature is advantageous in terms of energy saving, for example, in a fiber processing method that can reduce the amount of energy input for heating the treatment tank. It would be desirable to develop methods for using laccase enzymes in applications such as enzymatic bleaching at low temperatures.

Enzymatic oxidation systems, compositions and methods involving laccase are described. In one aspect, a fiber processing method is provided comprising contacting the fiber with a laccase enzyme and optionally a transmitter at a temperature below 40 ° C. for a time and under conditions sufficient to cause color modification of the fiber. In some embodiments, the color modification is selected from color thinning, color change, tint change, redeposition / reverse dyeing reduction, and bleaching. In some embodiments, the temperature is greater than or equal to about 20 ° C and less than 40 ° C. In some embodiments, the temperature is from about 20 ° C 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. ° C, 34 ° C or 35 ° C. In some embodiments, the temperature is the ambient temperature of tap water.

In some embodiments, the fiber is indigo dyed denim. In some embodiments, the fibers are sulfur dyed denim. In some embodiments, the denim is densified and / or stonewashed prior to or simultaneously with the step of contacting the fiber with the laccase enzyme and the transmitter. In some embodiments, the stonewashing and contacting the fiber with the laccase enzyme and the transmitter are performed in the same vessel.

In some embodiments, the method further comprises contacting the fiber with the cellulase enzyme simultaneously or sequentially with contacting the fiber with the laccase enzyme and the transmitter. In some embodiments, prior to the step of contacting the fiber with the cellulase enzyme and the step of contacting the fiber with the laccase enzyme and the mediator, prior to contacting the fiber with the laccase enzyme and mediator. And the step of contacting the fiber with a cellulase enzyme. In some embodiments, contacting the fiber with a cellulase enzyme and contacting the fiber with a laccase enzyme and a mediator, contacting the fiber with a cellulase enzyme and contacting the fiber with a laccase enzyme and mediator. It is continuously performed in the same tank without draining the tank between the steps.

In some embodiments, the steps of contacting the fiber with a cellulase enzyme and contacting the fiber with a laccase enzyme and a transmitter are performed at a temperature of less than 40 ° C. In some embodiments, the temperature is greater than or equal to about 20 ° C and less than 40 ° C. In some embodiments, the temperature is from about 20 ° C 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. ° 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 provide a greater degree of fiber color thinning, color change, tint change, reduced redeposition / back dyeing, and / Or produce fibers with bleaching. In some embodiments, the cellulase enzyme acts additively with the laccase enzyme, resulting in a greater degree of fiber color thinning, color change compared to the same method without cellulase. To produce fibers having a color change, reduced redeposition / back dyeing, and / or bleaching.

In some embodiments, the laccase is a microbial laccase. In some embodiments, the laccase is derived from a Cerrena species. In some embodiments, the laccase is derived from Cerrena unicolor. In some embodiments, the laccase is laccase D derived from Serena unicolor.

In some embodiments, the laccase is 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 It 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: 20. In some embodiments, the laccase is 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 It 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: 20. In some embodiments, the laccase is 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 It has an amino acid sequence that is at least 90% or at least 95% identical to an amino acid sequence selected from the group consisting of 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, a laccase enzyme and a transmitter are provided together in a ready-to-use composition. In some embodiments, the laccase enzyme and the mediator are provided in solid form. In some embodiments, the laccase enzyme and the mediator are provided as granules. In certain embodiments, the transmitter is syringonitrile.

In another aspect, laccases, nucleic acid sequences encoding such laccases, and vectors and host cells for expressing laccases are provided. Laccases can be used at low temperatures in methods where a reduction in energy input is desired, such as fiber processing. In some embodiments, the laccase enzyme comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. Or consist essentially of the amino sequence or at least about 60 to any of SEQ ID NOs: 2, 4, 6, 8, 12, 14, 16, 18, 19 or 20. %, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical amino acid sequences It is what you have. In certain embodiments, the laccase is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% relative to SEQ ID NO: 19 or SEQ ID NO: 20, It has 98%, 99% or 99.5% identical amino acid sequence. In a further specific embodiment, the laccase has the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Such polypeptides preferably have laccase enzyme activity, and the activity can be measured, for example, using the assays described herein.

In another aspect, there is provided a composition comprising a laccase enzyme comprising, consisting essentially of, or consisting of any of the aforementioned amino acid sequences. In some embodiments, the composition further comprises a buffer system for maintaining the pH of the composition in solution from about 5.5 to about 6.5. In some embodiments, the composition further comprises a transmitter. This transmitter is, for example, acetosyringone, shilling aldehyde, shilling amide, methyl silling amide, 2-hydroxyethyl syring amide, syringic acid methyl, dimethyl silling amide, shrine acid, 4-hydroxy-3,5 -May be selected from dimethoxybenzonitrile (syringonitrile). In one embodiment, the transmitter is 4-hydroxy-3,5-dimethoxybenzonitrile. In some embodiments, the composition is in solid form. In some embodiments, the laccase enzyme and the mediator are provided together in the ready-to-use composition. In some embodiments, the laccase enzyme and the mediator are provided in solid form. In some embodiments, the laccase enzyme and transmitter are provided as granules. In certain embodiments, the transmitter is syringonitrile.

In some embodiments, the laccase enzyme has a pH of about 5 to about 7, a temperature of about 20 ° C. to about 30 ° C., a liquid ratio of about 5: 1 to about 10: 1, and about 15 minutes to about 60. Used in minutes time.

These and other aspects and embodiments of the systems, compositions and methods of the present invention will become apparent from the specification and the accompanying drawings.

FIG. 1 shows the effect of changing color in denim stonewashed with laccase enzyme at various temperatures, as described in Example 1.

FIG. 2 shows the effect of the ratio of laccase and transmitter on the color change of stonewashed denim, as described in Example 2.

FIG. 3 shows the effect of temperature on the color change of denim stonewashed with a “ready for use” laccase composition, as described in Example 3.

FIG. 4 shows the effect of temperature on the ability to change the color of the laccase enzyme on stonewashed denim, as described in Example 3.

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

Described are systems, compositions and methods of enzymatic oxidation involving laccase. These systems, compositions and methods are useful, for example, for low temperature processing of fibers to affect color modification. Such treatment uses chemical reagents that use less energy and are more environmentally friendly than conventional fiber treatment techniques. Various aspects and embodiments of these systems, compositions and methods are described.

Definitions Unless defined otherwise, 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, NY (1991) '' A number of common dictionaries of terms used herein are provided. The following terms are defined for further clarity.

As used herein, the term “enzyme” means a protein that catalyzes a chemical reaction. The catalytic function of an enzyme constitutes its “enzyme activity” or “activity”. Enzymes are generally classified according to the type of reaction to be catalyzed, for example, phenol oxidation, peptide bond hydrolysis, nucleotide linkage, and the like.

As used herein, the term “substrate” refers to a substance (eg, a compound) for which an enzyme exerts its catalytic ability to produce a product.

As used herein, “laccase” is the oxidation of phenol, polyphenol, and aniline by simultaneously reducing oxygen to water and removing one electron in a four-electron transfer process. It is a multiple copper-containing oxidase (EC 1.10.3.2) that catalyzes.

As used herein, “variant” proteins include related and derivative proteins that differ from the parent / reference protein by a few 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, the variants differ by about 1 to about 10 amino acid residues. In some embodiments, the variant protein is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% relative to the parent / reference protein. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% amino acid sequence identity.

As used herein, the term “similar sequence” refers to a polypeptide in a protein that provides a function, three-dimensional structure, and / or conserved residue similar to the sequence in the parent / reference protein. Means an array. For example, in structural regions that include an α helix or β sheet structure, substituted amino acid residues in similar sequences maintain the same structural characteristics. In some embodiments, a similar sequence provides a variant protein that exhibits similar or improved function as the parent protein from which the variant was derived.

As used herein, a “homologous protein” or “homolog” is a function similar to a reference protein (eg, a laccase enzyme from a different source) (eg, an enzyme Activity) and / or a protein having a structure (for example, a laccase enzyme). Homologues may be derived from evolutionarily related or unrelated species. In some embodiments, the homologue has a quaternary, tertiary and / or primary structure similar to the reference protein, thereby comparing part or fragment substitution with sequences derived from heterologous proteins, Potentially allows replacement of a portion or fragment in a reference protein by a similar portion or fragment from a homologue while reducing disruption of the structure and / or function of the reference protein.

As used herein, “wild type” proteins, “natural” proteins and “naturally occurring” proteins are those found in nature. The term “wild-type sequence” means an amino acid or nucleic acid sequence found in nature or naturally occurring. In some embodiments, the wild type sequence is the starting point for a protein engineering business (eg, production of a mutant protein).

As used herein, “signal sequence” means a sequence of amino acids attached to the N-terminal portion of a protein, facilitating secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved during the secretion process.

As used herein, the term “culture” refers to culturing a population of microbial cells under suitable conditions in a liquid, semi-solid or solid medium in order to express the polypeptide of interest. Means. As is known in the art, in some embodiments, culturing is performed in a tube or in a reactor.

As used herein, the term “derivative” refers to the addition of one or more amino acids to either or both the N-terminus and the C-terminus, at one or more different positions in the amino acid sequence. Substitution of one or more amino acid residues, deletion of one or more amino acid residues at one or both ends of the protein or at one or more positions in the amino acid sequence, and / or one in the amino acid sequence By the insertion of one or more amino acids at the above sites is meant a protein derived from the parent / reference protein. In many cases, protein derivatives are prepared by modifying a DNA base sequence encoding a natural protein, introducing the DNA base sequence into an appropriate host, and expressing the modified DNA base sequence to produce a derivative protein. Achieved by making.

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. This process includes both transcription and translation.

As used herein, the term “expression vector” refers to a DNA code operably linked to one or more suitable control sequences capable of expressing a coding sequence in a host. It means a DNA construct comprising a sequence (eg gene sequence). Such control sequences control promoters to achieve transcription, selective operator sequences to control such transcription, sequences encoding appropriate mRNA ribosome binding sites, and termination of transcription and translation Containing sequences to The vector may be a plasmid, a phage particle or simply a potential genomic insert. When introduced into a suitable host, the vector can replicate and function independently of the host genome or, in some instances, can integrate into the genome itself.

As used herein, the term “host cell” can be transfected, transformed, or expressed in a recombinant expression vector for polypeptide production. Means a cell or cell line that can be introduced in other ways. A host cell includes the progeny of a single host cell, which progeny are either parental (in form or in whole genomic DNA complement) due to natural, incidental, or intentional mutations. It may not always be the same as the cell. The host cell may be bacterial or fungal. Host cells include cells transfected or transformed in vivo with an expression vector.

As used herein, the term “introduction” in the context of inserting a nucleic acid sequence into a cell includes “transfection”, “transformation”, and “transduction” and includes eukaryotic or Integration of a nucleic acid sequence into a prokaryotic cell, wherein the nucleic acid sequence is integrated into the cell's genome (eg, chromosome, plasmid, plastid or mitochondrial DNA), converted to an independent replicon, or Means integration that is temporarily expressed.

As used herein, “cleansing composition” and “cleansing formulation” include dough, dish, contact lens, hair (shampoo), skin (soap and cream), teeth (mouth cleaning solution, toothpaste). ) As well as other solids and surfaces, and compositions that can be used to remove unwanted compounds from the member to be cleaned. This term is selected for a particular type of desired cleaning composition and product form (eg, liquid, gel, granule or spray composition) as long as the composition is compatible with the enzyme used in the composition. All substances / compounds to be used are included. The specific selection of cleaning composition material is facilitated by considering the surface, member or fabric to be cleaned and the desired form of the composition for the cleaning conditions in use.

The term further means any composition suitable for cleaning, bleaching, disinfecting and / or disinfecting objects and / or surfaces. The term includes, but is not limited to surfactant compositions (eg, liquid and / or solid laundry detergents and fine fabric detergents; glass, wood, ceramic and metal countertops, As well as hard surface cleaning formulations for windows and the like; carpet cleaners; oven cleaners; fabric deodorants; fabric softeners; and textile and pre-wash stains and dish detergents).

Indeed, the terms “cleaning composition” and “cleaning formulation” refer to a universal or heavy detergent in particulate or powder form (especially unless otherwise specified), in particular a cleansing detergent; universal in liquid, gel or paste form Detergents, especially the so-called heavy liquid (HDL) type; fine detergent liquid detergents; dishwashing detergents or light dishwashing detergents, especially high foaming types; various tablets, granules, liquids and washings for home and business use Detergents for dishwashers including auxiliary aid types; antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, car shampoos or carpet shampoos, liquid cleaners and disinfectants including bathroom cleaners; hair shampoos And hair rinses; shower gels and bubble baths and metal cleaners; and bleach additives and “stain-stick” Or a cleaning aid such as a pretreatment type.

As used herein, the terms “surfactant composition” and “detergent formulation” are used for mixtures intended for use in cleaning media for cleaning contaminated items. The In some embodiments, the term is used for laundry and / or garment laundry (eg, “laundry detergent”). In an alternative embodiment, the term refers to other detergents such as those used to clean dishes, knives, etc. (eg, “dishwashing detergent”). In addition to enzymes, “surfactant compositions” and “cleaning formulations” include surfactants, builders, bleaches, bleach accelerators, bluing and fluorescent dyes, anti-caking agents, masking agents, enzyme activity Including detergents, antioxidants, and detergents containing solubilizers.

As used herein, the phrase “detergent stability” means the stability of the enzyme in the surfactant composition, and optionally the stability of the associated substrate or transmitter. . In some embodiments, this stability is assessed during use of the detergent, while in other embodiments, the term refers to the stability of the surfactant composition during storage.

As used herein, the term “hard surface cleaning composition” refers to cleaning hard surfaces such as floors, walls, tiles, stainless steel tubes (eg, fermenters), bathtubs and kitchen fixtures, etc. Means a surfactant composition. Such compositions can be provided in any form including, but not limited to, solids, liquids, emulsions, and the like.

As used herein, the term “dish cleaning composition” refers to all of the compositions for cleaning dishes, including but not limited to granular and liquid forms. Means form.

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

As used herein, the term “dough cleaning composition” includes, but is not limited to, surfactants for cleaning dough, including granular, liquid and solid forms. It means the form of the composition.

As used herein, the terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are single stranded or multiple stranded (eg, single stranded, double stranded, triple helix, etc.). Any length, including deoxyribonucleotides, ribonucleotides, and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides (including modified nucleotides or bases or analogs thereof), whether or not And are used interchangeably to mean polymer forms of nucleotides of any three-dimensional structure. Since 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, including modifications that increase nuclease resistance (eg, deoxy, 2'-O-Me, phosphorothioate, etc.) so long as the polynucleotide retains the desired function under the conditions of use The body can be used. For detection or supplemental purposes, for example, radioactive or non-radioactive labels or labels such as anchors (eg biotin) can be incorporated. The term polynucleotide also encompasses peptide nucleic acids (PNA). A polynucleotide may be naturally occurring or non-naturally occurring. The sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester bonds can be replaced by alternative linking groups. For example, phosphates include P (O) S (“thioate”), P (S) S (“dithioate”), (O) NR ₂ (“amidate”), P (O) R, P (O ) OR ′, CO or CH ₂ (“form acetal”). Where R or R ′ are each independently H or optionally substituted with an ether (—O—) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Unsubstituted alkyl (having 1 to 20 carbon atoms). Not all bonds in a polynucleotide need be the same. The polynucleotide may be linear or circular, or may be composed of a combination of a linear part and a circular part.

As used herein, the terms “polypeptide”, “protein” and “peptide” refer to components comprising amino acids (ie, amino acid residues). Conventional one-letter or three-letter codes representing amino acid residues are used. The polypeptide may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The term is modified either naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations or modifications such as conjugation with a labeling moiety. Also included are amino acid polymers prepared. The definition also includes polypeptides, including, for example, analogs of one or more amino acids (including, for example, unnatural amino acids) and other modifications known in the art.

As used herein, the term “primer” is used regardless of whether it is naturally occurring or synthesized, eg, as a purified restriction fragment. It means an oligonucleotide that can function as a starting point for nucleic acid synthesis when incubated with complementary nucleic acids in the presence of nucleotides and polymerase at a suitable temperature and appropriate 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. The primer is preferably an oligodeoxyribonucleotide. The exact length of the primer will vary depending on many factors, including temperature, primer origin, and the method used.

As used herein, the terms “recovered”, “isolated”, “purified” and “isolated” are associated with the native state or as a result of heterologous expression. Means material (eg protein, nucleic acid or cells) removed from at least one component.

As used herein, the term “fiber” means fibers, yarns, fabrics, garments, and nonwoven fabric materials. The term includes natural and synthetic materials (eg, manufactured), as well as fibers made from a mixture of natural and synthetic materials. The term “fiber” means both untreated or treated fibers, yarns, woven or knitted fabrics, non-woven fabrics, and garments. In some embodiments, the fiber comprises cellulose.

As used herein, the phrase “fibers that require processing” means fibers that need to be desizing, ground, bleached and / or biopolished to produce the desired effect. To do.

As used herein, the phrase “fibers that require color modification” refers to fibers that need to be changed with respect to color. These fibers may have already been provided for other treatments, or may not have been provided yet. Similarly, these fibers may or may not require post-treatment.

As used herein, the term “fabric” is a manufactured assembly of fibers and / or yarns that has a relatively large surface area relative to thickness and is useful for the assembly. Means an assembly having sufficient cohesive strength to provide sufficient mechanical strength.

As used herein, the term “color modification” refers to the color saturation, saturation, strength associated with materials of fibers, yarns, fabrics, clothing or nonwoven fabrics, collectively referred to as fiber materials. , Brightness, and / or change in color tone. Without being bound by theory, it is proposed that color modification is due to the modification of the chromophoric groups associated with the fiber material, thereby changing its appearance. The chromophore may be one that naturally accompanies the material used to produce the fiber (eg, cotton white) or one that accompanies a special finish such as dyeing or printing It may be. Color modification includes not only chemical modification to the material to which the chromophore is attached, but also chemical modification to the chromophore. Examples of color modification include fading, bleaching, changing shades. A special color modification for indigo-dyed denim is to fade to a “vintage look”, which is less blue / purple shade and less dull than newly dyed denim. It has a gray appearance.

As used herein, the term “bleaching” refers to the process of treating fiber materials such as fibers, yarns, fabrics, garments or nonwoven fabric materials to produce a lighter color. To do. The term also encompasses, for example, the production of lighter or whiter fibers in the context of fiber treatment applications and the reduction of soil color, for example in the context of cleaning applications.

As used herein, the terms “size” and “sizing” are used to improve weaving performance by increasing the wear resistance and strength of yarns in the textile industry. Means a compound used in The glue is usually made from starch or starch compounds.

As used herein, the terms "desize" and "desizing" usually refer to fibers before applying a special finish, dye or bleach. Means the process of removing / removing glue (usually starch) from

As used herein, the term “desizing enzyme” means an enzyme used to remove glue. Typical enzymes are amylases, cellulases and mannanases.

As used herein, the term “% identity” refers to the identity of a nucleic acid sequence between a nucleic acid sequence encoding a laccase and other nucleic acid sequences as described herein. Or the level of amino acid sequence identity between a laccase enzyme and other amino acid sequences as described herein. The sequence comparison can be performed using a conventional sequence comparison program. Typical levels of nucleic acid and amino acid sequence identity include, but are not limited to, defined sequences (eg, coding sequences for laccases or laccases as described herein). Amino acid sequence) 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 at least 99 % Or more sequence identity.

A typical computer program that can be used to determine identity between two sequences is not limited to the following, but is available on the Internet at <www.ncbi.nlm.nih.gov/BLAST>. For example, BLASTN, BLASTX, and combinations of BLAST programs such as TBLASTX, BLASTP and TBLASTN. See also “Altschul, et al., 1990” and “Altschul, et al., 1997”.

A sequence search is usually performed using the BLASTN program when evaluating a given nucleic acid sequence by comparing it with a GenBank DNA base sequence and nucleic acid sequences in other public databases. The BLASTX program is preferred for searching nucleic acid sequences that are translated into all reading frames against GenBank protein sequences and other public database amino acid sequences. Both BLASTN and BLASTX are performed using the default parameters of 11.0 open gap penalty and 1.0 extended gap penalty and utilize the BLOSUM-62 matrix (see, eg, Altschul, et al., 1997). Please refer.)

Sequence comparisons of multiple sequences selected to determine “% identity” between two or more sequences include, for example, an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix. It can be implemented using the MacVector version 6.5 CLUSTAL-W program operated with the included default parameters.

As used herein, the terms “chemical mediator” and “transmitter” transport electrons between an enzyme exhibiting oxidase activity (eg, laccase) and a second substrate or electron donor. Are used interchangeably to mean compounds that function as redox mediators. 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 tank in which fiber material is present are used to completely or partially remove solvents and reagents present in the tank. Means emptying / empty. The step of draining the bath is usually performed during the process steps so that the solvents and reagents present in one process step do not interfere with later process steps. Dehydration may involve one or more rinsing steps to further remove such solvents and reagents.

As used herein, the terms “second substrate” and “electron donor” refer to dyes, pigments (eg, indigo) that can donate and receive electrons by enzymes that exhibit oxidase activity. ), Chromophores (eg, polyphenols, anthocyanins, or carotenoids) or other second substrates to be used interchangeably.

The following abbreviations / acronyms have the following meanings unless otherwise specified.
EC Enzyme Committee EDTA ethylenediaminetetraacetic acid kDa kilodalton MW molecular weight w / v weight / volume w / w weight / weight v / v volume / volume wt% weight percent ° C Celsius temperature H2O water dH20 or DI deionized water dIH2O deionized water , Milli-Q filter g or gm gram μg microgram mg milligram kg kilogram μL and μl microliter mL and ml milliliter mm millimeter μm micrometer M mol mM mmol μM micromol U unit sec and `` second min and 'min hr time 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. Unless expressly clear from the context, the singular articles “a”, “an” and “the” include plural referents. Unless otherwise specified, polypeptides are described in a standard orientation from N-terminus to C-terminus, and polynucleotides are described in a standard orientation from 5 'to 3'. It is understood that the particular methodologies, protocols and reagents described are not intended to be limiting as equivalent methods and materials may be used in the practice or testing of the compositions and methods of the present invention. Must be. The description has been divided into chapters to assist the reader, but the chapter headings should not be construed as limiting, and descriptions in one chapter can be applied to other chapters. All publications cited in this specification are expressly incorporated by reference.

Laccase and laccase-related enzymatic enzymatic oxidation systems, compositions and methods include one or more of laccases or laccase-related enzymes, collectively referred to herein as “laccases” or “laccase enzymes”. Such laccases are any of those included in EC 1.10.3.2 by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Includes laccase enzyme. Laccase enzymes derived from microorganisms and plants are known in the art. Microbial laccase enzymes may be derived from bacteria or fungi (including filamentous fungi and yeasts). Suitable examples include Aspergillus, Neurospora (eg, N. crassa), Podospora, Botrytis, Collybia, Cerrena (eg, Unicolor), Stachybotrys, Panus (eg P. Rudis), Thielavia, Fomes, Lentinus, Pleurotus, Trametes (eg T. et al.). T. villosa and T. versicolor), Rhizoctonia (for example R. solani), Coprinus (for example C. plicatilis, C. C. cinereus), Psatyrella, Miseriotra (for example, M. thermonhila), Cytalidium, Phlebia (for example, P. radita ( O92 / 01046), or Coriolus (eg, C. hirsutus (JP2238885)), Spongipellis, Polyporus, Ceriporiopsis subvermispora, Ganoderma tsunodae, Ganoderma tsunodae Also included are laccases derived from or derivable from Trichoderma species.

Laccase can be produced by culturing host cells transformed with a recombinant DNA vector comprising a nucleotide sequence encoding laccase. The DNA vector may further include a nucleotide sequence that allows expression of the laccase in the medium and allows the laccase to be selectively recovered from the culture medium.

An expression vector containing a polynucleotide sequence encoding a laccase enzyme can 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, Trichoderma [e.g. Liesei (formerly classified as T. longibrachiatum, also now known as Hypocrea jecorina), T. Viride, T. Koningii and T. Harzianum], Aspergillus (eg, A. niger, A. nidulans, A. oryzae and A. awamori) Penicillium, Humicola (eg, H. insolens and H. grisea), Fusarium (eg, F. Graminum and F. Benenatum). (F. venenatum), Neurospora, Button Bamboo (Hypocrea), and Mucor species. The host cell for expression of the laccase enzyme may be derived from a type of Cerrena (eg C. unicolor). Fungal cells can be transformed, using techniques known in the art, by processes including protoplast formation and protoplast transformation, followed by cell wall regeneration.

Alternatively, the host organism is a Bacillus [e.g. Subtilis, B. Rikeniformis, B.M. Rentas, B.H. (Naugeobacillus) stearothermophilus and Brevis], Pseudomonas streptomyces (for example, S. coelicolor, S. lividans), or one derived from a kind of bacteria such as E. coli. Transformation of bacterial cells can be performed according to conventional methods, for example, as described in “Maniatis, T. et al.,“ Molecular Cloning: A Laboratory Manual, ”Cold Spring Harbor, 1982”. . Screening of appropriate DNA sequences and construction of vectors can also be performed by standard procedures (see above).

The medium used to culture the transformed host cells can be any conventional medium suitable for culturing host cells. In some embodiments, the expressed enzyme is secreted into the medium and can be recovered from the medium according to known procedures. Laccase can be recovered from media such as described in, for example, US Patent Publication No. 2008/0196173. In some embodiments, the enzyme is expressed intracellularly and is recovered after disruption of the cell membrane.

In certain embodiments, the expression host may be a Trichoderma reesei having a laccase coding region under the control of a CBH1 promoter and terminator (see, eg, US Pat. No. 5,861,271). The expression vector may be pTrex3g, for example as disclosed in US Pat. No. 7,413,887. In some embodiments, the laccase is expressed as described in US Patent Publication Nos. 2008/0196173 or 2009/0221030.

The following laccase genes and laccases are described in US Publication No. 2008/0196173.

A. Cerrena laccase A1 gene polynucleotide sequence derived from strain CBS 115.075 (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 polynucleotide sequence (SEQ ID NO: 3) derived from strain CBS154.29:
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 polynucleotide sequence derived from strain CBS 115.075 (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 polynucleotide sequence derived from strain CBS154.29 (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 derived from ATCC 20013 strain (partial)
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 (part) derived 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 polynucleotide sequence from CBS154.29 strain (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 polynucleotide sequence (SEQ ID NO: 15) derived from strain CBS 115.075:
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 from CBS154.29 strain (partial)
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) can 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 processed mature form of this polypeptide is as follows (SEQ ID NO: 20).
AIGPVADLHIVNKDLAPDGVQRPTVLAGGTFPGTLITGQKGDNFQLNVIDDLTDDRMLTPTSIHWHGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVPDQAGTFWYHSHLSTQYCDGLRGAFVVYDPNDPHKDLYDVDDGGTVITLADWYHVLAQTVVGAATPDSTLINGLGRSQTGPADAELAVISVEHNKRYRFRLVSISCDPNFTFSVDGHNMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVLNANQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSPLNEADLRPLVPAPVPGNAVPGGADINHRLNLTFSNGLFSINNASFTNPSVPALLQILSGAQNAQDLLPTGSYIGLELGKVVELVIPPLAVGGPHPFHLHGHNFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEVTIRFVTDNPGPWFLHCHIDWHLEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQKVKPKKGTAI

In some embodiments, a laccase enzyme suitable for use in the compositions and methods of the invention is a mature polypeptide that lacks a signal sequence that can be used to direct secretion of a full-length polypeptide from a cell. It is. Suitable mature polypeptides are 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: 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% with respect to the amino acid sequence selected from the group consisting of number 20 , At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or more amino acid sequence identity. It is preferred that 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 compositions and methods of the invention are those that have been cleaved relative to the full-length or mature parent / reference sequence. Such cleaved peptides can be generated by proteolysis of full-length or mature polypeptide sequences, or by designing a polynucleotide to encode a cleaved peptide. Exemplary polypeptides are 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 Compared with an amino acid sequence selected from the group consisting of: those cleaved at the amino and / or carboxyl terminus. The cleavage portion may be a small number (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues, or Or the entire structural domain or functional domain. Suitable cleaved peptides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least relative to one or more corresponding portions of the reference amino acid sequence 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 at least 99%, or more amino acid sequence identity It may have sex. Preferably, such polypeptides have enzymatic laccase activity, as determined using the assays and procedures described herein.

In some embodiments, the enzymatic oxidation systems, compositions and methods further comprise one or more chemical mediator agents that enhance the activity of the laccase enzyme. Transmitters (also called enhancers or accelerators) include enzymes that exhibit oxidase activity, dyes, pigments (eg, indigo), chromophores (eg, in colored soils, such as polyphenols, anthocyanins or carotenoids), Alternatively, it is a chemical substance that acts as a redox transmitter for effectively transporting electrons to and from another second substrate or electron donor.

In some embodiments, the chemical messenger is a phenolic compound, such as methyl syringate, or related compounds, as described, for example, in PCT applications WO 95/01426 and WO 96/12845. The transmitter may be, for example, an N-hydroxy compound such as N-hydroxybenzotriazole, violuric acid, N-hydroxyacetanilide, an N-oxime compound or an N-oxide compound. The transmitter may also be a phenoxazine / phenothiazine compound such as, for example, phenothiazine-10-propionate. The mediator can also be 2,2′-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid. Other chemical mediators are, for example, compounds disclosed in PCT application WO 95/01426 (this compound is Are known in the art, such as acetosyringone, methyl syringate, ethyl syringate, propyl syringate, butyl syringate, hexyl syringate Or it may be octyl syringate.

In some embodiments, the transmitter is, for example, 4- (N-methylcarboxamido) -2,6-dimethoxyphenol, 4- [N- (2-hydroxyethyl) carboxamido] -2,6-dimethoxy. Phenol), or 4-cyano-2,6-dimethoxyphenol, 4-carboxyamide-2,6-dimethoxyphenol, such as 4- (N, N-dimethylcarboxyamide) -2,6-dimethoxyphenol, Or these N substituted derivatives.

（ここで、Ａは、−Ｒ、−Ｄ、−ＣＨ＝ＣＨ−Ｄ、−ＣＨ＝ＣＨ−ＣＨ＝ＣＨ−Ｄ、−ＣＨ＝Ｎ−Ｄ、−Ｎ＝Ｎ−Ｄ、又は、−Ｎ＝ＣＨ−Ｄなどの基であり、Ｄは、−ＣＯ−Ｅ、−ＳＯ_２−Ｅ、−ＣＮ、−ＮＸＹ、及び、−Ｎ^＋ＸＹＺからなる群より選択され、Ｅは、−Ｈ、−ＯＨ、−Ｒ、−ＯＲ、又は、−ＮＸＹであり、Ｘ、Ｙ及びＺは、−Ｈ、−ＯＨ、−ＯＲ及び−Ｒから独立して選択され；Ｒは、Ｃ_１−Ｃ_１６アルキル、好ましくはＣ_１−Ｃ_８アルキルであり、該アルキルは、飽和又は不飽和のいずれであってもよく、分岐又は非分岐のいずれであってもよく、また、選択的にカルボキシ基、スルフォ基又はアミノ基で置換されていてもよく；Ｂ及びＣは、Ｃ_ｍＨ_２ｍ＋１（１≦ｍ≦５）から独立して選択される）によって表される。 In some embodiments, the transmitter substance has the formula:

(Where A is -R, -D, -CH = CH-D, -CH = CH-CH = CH-D, -CH = ND, -N = ND, or -N = A group such as CH—D, wherein D is selected from the group consisting of —CO—E, —SO ₂ —E, —CN, —NXY, and —N ⁺ XYZ, where E is —H, —OH; , -R, -OR, or a -NXY, X, Y and Z are, -H, -OH, is independently selected from -OR and -R; R _is, C 1 _{-C 16} alkyl, preferably Is C ₁ -C ₈ alkyl, which may be either saturated or unsaturated, branched or unbranched, and optionally carboxy, sulfo or amino It may be substituted with a group; B and _C, the C _{m H 2m +} 1 are selected (1 ≦ m ≦ 5) independently from) It is.

In some embodiments, A in the above formula is —CN or —CO—E, wherein E is —H, —OH, —R, —OR, or —NXY, and X and Y is -H, -OH, is independently selected from -OR and -R, R _is C 1 _{-C 16} alkyl, preferably _C 1 -C ₈ alkyl, the alkyl is a saturated or unsaturated It may be any of saturated, branched or unbranched, and may be optionally substituted with a carboxy group, a sulfo group or an amino group; B and C are C _m H It is independently selected from _{2m + 1} (1 ≦ m ≦ 5). In some embodiments, the transmitter is 4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as “syringonitrile” or “SN”).

Note that in the above formula, A may be located in the meta relative to the hydroxyl group, instead of in the para position as shown.

For applications such as fiber processing, the transfer material is about 0.005 μmol to about 1000 μmol per gram denim, about 0.05 μmol to about 500 μmol per gram denim, and about 0.1 μm per gram denim. It may be present in a concentration from about 1 to about 100 μmol, from about 1 μmol to about 50 μmol per gram of denim, or from about 2 μmol to about 20 μmol per gram of denim.

The transmitter can be prepared by methods known to those skilled in the art as disclosed in PCT applications WO 97/11217 and WO 96/12845 and US Pat. No. 5,752,980. Other suitable mediators are described, for example, in US Patent Publication No. 2008/0189871.

Methods of Use The systems and compositions of the present invention can be used in applications where enzymatic laccase activity is useful or desirable. Among these, the use / method is color modification of the substrate, which may be related to fibers. In some embodiments, such methods include incubating the laccase enzyme with a suitable substrate at a low temperature, such as, 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 ° C and about 35 ° C. In some embodiments, the temperature is about 20 ° C, about 25 ° C, about 30 ° C, or about 35 ° C. In some embodiments, the temperature is an ambient temperature of tap water, such as, for example, from about 20 ° C to about 23 ° C. The temperature may be maintained within a narrow range or may be varied without significantly affecting the performance of the system and composition.

This method contemplates the use of one or more laccases described herein. In some embodiments, the laccase is C.I. Derived from Cerrena species such as unicolor. In some embodiments, the laccase is a C.I. The amino acid sequence of any of the unicolor laccase enzymes, or C.I. At least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 for any of the unicolor laccase enzymes %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity and includes amino acid sequences having laccase enzyme activity Or consist essentially of their amino acid sequence or consist essentially of their amino acid sequence.

In some embodiments, for example, the systems and methods are used in fiber processing methods, such as, for example, methods for changing the color of textiles, including fibers, yarns, fabrics, or finished garments. Is done. Generally, the method includes at least one (ie, one or more) selected from, for example, color change, color change, decolorization, bleaching, fading, and / or reduction of redeposition / backside dyeing. Contacting the fiber with the laccase and the transmitter for a time and under conditions sufficient to produce a measurable effect. In some embodiments, this method is used to give a “vintage look” to a dyed denim product. In the case of indigo-dyed denim, the vintage appearance has a weaker blue / purple hue than freshly dyed denim and a gray appearance that is less constrained than freshly dyed denim. In the case of sulfur dyed denim, the vintage appearance is faded in color without the brown shade that can be caused by hypochlorite treatment. Thus, although the color modification embodiment obtained with laccase can be characterized as a “bleaching” action, this term does not fully represent the color modification possible with the use of laccase.

The fibers provided for color modification may be cellulose fibers or a mixture of cellulose fibers and synthetic fibers. In some embodiments, the fibers are denim dyed with indigo and / or sulfur based dyes. In certain embodiments, the fibers are dyed with indigo and the laccase enzyme and transmitter are used to oxidize indigo to isatin. Denim can be selectively desizing and / or stonewashed prior to color modification by the laccase enzyme.

In general, assuming the same degree of wear in the fiber treatment method, the strength of the denim is further reduced at higher temperatures compared to lower temperatures. The method of the present invention has the advantage of reducing damage to the fiber during processing compared to the conventional method because it can be performed at a lower temperature compared to the conventional method. Furthermore, laccase enzymes generally do not reduce their strength by reaction with cellulosic fabric fibers during processing. Accordingly, in some embodiments, the method of the present invention does not affect the physical strength of the denim compared to the conventional method, and the decrease in physical strength is small compared to the conventional method. When the denim is composed of stretched denim (eg, elastane, spandex), the method of the present invention does not affect the stretch performance of the fabric as compared to the conventional method, and reduces the decrease in stretch performance.

In some embodiments, laccase is used in fiber processing methods in combination with at least one other enzyme. When such treatments are performed simultaneously, the enzyme treatment can be performed at a low temperature as described herein. If the treatment is continuous, laccase can be used at low temperatures as described herein, and optionally other enzymes can be used at low temperatures. In some embodiments, the laccase is used simultaneously or sequentially in combination with the cellulase enzyme. In one embodiment, the fibers are contacted simultaneously with laccase and cellulase. In another embodiment, the fiber is continuously contacted with laccase and cellulase. In one embodiment, the fiber is first contacted with cellulase to achieve a “stone wash” and then contacted with a laccase to affect color modification. In another embodiment, the fiber is first contacted with laccase and then with cellulase. When cellulase treatment and laccase treatment are continuous, the two treatment steps can be performed in the same vessel and without draining the vessel between the two treatments. Such a method is referred to as a “single tank” method.

Suitable cellulases include, for example, Humicola, Thermomyces, Bacillus, Trichoderma, Fusarium, Myceliophthora, Phanerochaete, Phanerochaete, Irpex, Irpex, It may be derived from microorganisms known to be able to produce cellulolytic enzymes, such as Scytalidium, Schizophyllum, Penicillium, Aspergillus, or Geotricum species . Known species capable of producing cellulolytic enzymes include Humicola insolens, Fusarium oxysporum, or Trichoderma reesei. Non-limiting examples of suitable cellulases are disclosed in US Pat. No. 4,435,307; European Patent Application No. 0495257; PCT Patent Application No. WO 91/17244; and European Patent Application No. EP-A2-271004. All of which are incorporated herein by reference.

In some embodiments, enzymatic “stone wash” using cellulase, bleaching using allyl esterase, and color modification using laccase are combined to provide a comprehensive enzyme fiber processing system. May be. Such a system allows the fiber processing equipment to produce fibers with various finishing processes without the need to use conventional fiber processing chemicals.

Laccases can also be used in other aspects of fiber manufacturing, which generally include processing aspects, processing, finishing, polishing, or fiber production. In addition to modifying the color of the dyed denim, in the decolorization of dyed waste (including indigo dyed waste), in fabric dyeing, soil in fiber bleaching, in fiber denaturation; improved Laccases can also be used in the realization of fiber or fabric properties and the like.

In a further embodiment, the systems and compositions of the present invention can also be used in methods of changing the color of wool. For example, European Patent No. EP0504005 discloses that laccase can be used to stain wool. Laccase can also be used in the leather industry. For example, laccase can be used in animal skin treatment including, but not limited to, skin hair removal, lime treatment, fermentation, and / or leather.

The systems and compositions of the present invention can also be used in methods of modifying the color of pulp or paper products. Such methods include contacting a pulp or paper product in need of color modification with a laccase as described herein for a time and under conditions sufficient for color modification to occur. . In certain embodiments, the color modification is bleaching.

The systems and compositions of the present invention can also be used in methods for modifying the color of hair. Laccase has been reported to be useful for hair dyes (see, for example, WO 95/33836, WO 95/33837). Such a method comprises contacting the hair having the color to be modified with laccase for a time and under conditions suitable for changing the color of the hair.

The systems and compositions of the present invention can also be used in the field of wastewater treatment. For example, laccase can be used in the decolorization of colored compounds, in detoxification of phenolic components, for antimicrobial activity (eg in water recycling), in bioremediation and the like.

The systems and compositions of the present invention include depolymerization of high molecular weight aggregates, deinking of waste paper, polymerization of aromatic compounds, radical mediated polymerization and crosslinking reactions (eg, paints, coatings, biomaterials), It can also be used in dye activation and organic compound linking.

The systems and compositions of the present invention can also be used as cleaning compositions or components thereof, or as detergents for use in cleaning methods. For example, laccases can be used in the washing, processing or care of laundry, such as clothing or fabrics, in the cleaning of domestic hard surfaces; in the care of dishes including dishwasher applications; and the soap solids and liquids; It can also be used as a stick and liquid for synthetic surfactants. The enzymes shown herein may be useful, for example, in soil removal / decolorization and / or odor removal and / or purification. Laccase transmitters can be used as cleansing agents and antibiotics (eg, wood protection, detergents) separately from or together with the laccase enzyme.

Laccase can be used in other embodiments of the personal care field. For example, laccase is used in the preparation of human body products such as perfumes and products for human skin care, hair care, oral care, body cleaning and deodorization and / or antiperspirants. can do. Laccases are for example in hair dyeing and / or decolorization, nail dyeing and / or bleaching; skin dyeing and / or bleaching; surface modification (eg as a coupling reagent); as an antibiotic; in odor removal; It may be useful for whitening, etc. Laccase can be used in the field of contact lens cleaning. Laccases can be used, for example, in the cleaning, storage, sterilization and / or preservation of contact lenses.

Laccase can be used in the field of biomaterials. For example, laccase as a biocatalyst for various organic reactions; and / or in connection with biopolymers; in connection with packaging; in connection with adhesives; surface modification (activation and cupping) In the production of primary alcohols; in connection with biosensors and / or organic synthesis; Laccase can oxidize a variety of colored compounds with various chemical structures using oxygen as an electron acceptor.

The systems and compositions of the present invention provide for removal of lignin from lignocellulose-containing materials (eg, pulp delignification), bleaching of lignocellulose-containing materials (ie, enzymatic deinking of recycled paper), and / or paper. Alternatively, it can be used for the treatment of wastewater generated from the production of cellulose. Such a process may use a laccase enzyme in combination with the addition or metering of a non-aromatic redox agent in addition to a phenol aromatic redox compound and / or a non-phenol aromatic redox compound. The phenolic and non-phenolic units of lignin were directly oxidized by the action of these phenol aromatics and / or non-phenolic aromatic compounds, or lignin was produced by the oxidizing action of these compounds Oxidized by other phenols and / or non-phenols.

Laccase can be used in other embodiments involving pulp and paper. For example, laccase is used for the production of paper and fluff pulp from raw materials such as wood, bamboo and grain rice straw; for the production of paper and board for printing and writing, packing, hygiene applications and other technical applications; Recycling of acetate fibers for the purpose of making paper and board; used in the treatment of waste generated and processed by pulp or paper factories and other equipment specially dedicated to the production of paper, pulp or fluff be able to. Laccases are, for example, in wood processing; in pulp bleaching; in wood fiber denaturation; in bioadhesives (lignin activators) for MDF production; to improve paper properties; in ink removal; in paper dyeing; In adhesives (eg, lignin-based adhesives for slab or fiberboard) and the like;

Laccase can be used in the field of feed. For example, laccase can be used alone or as part of a feed additive to enhance the nutritional value of all types of animal feed such as chickens, cows, pigs, fish and pets; and / or It can be used as a processing aid to process plant materials and food industry by-products to produce materials / products suitable as feed ingredients.

Laccase can be used in the field of starch processing. For example, laccase can be used in processing a substrate comprising starch and / or cereal into glucose (dextrose) syrup, fructose syrup or other syrup, alcohol (drink or fuel) or sugar. Such starch processing may include processing steps such as substrate liquefaction, saccharification, isomerization and debranching.

Laccase can be used in the field of food. Laccase can be used, for example, in its preparation, processing or as an active ingredient in foods such as yellow fat, tea-based beverages, cooking products, bread and frozen foods for human consumption. it can. Laccase can be used, for example, as a bread improving agent, as a deoxidizing agent, etc. in food preservation. Laccase can be used to reduce or eliminate the microbial burden of various foods (eg, meat) or feed.

Any of the methods or uses for laccase described herein can be performed at low temperatures, eg, temperatures below about 40 ° C., eg, below about 40 ° C., below about 37 ° C., below about 35 ° C., about 32 ° C. It can be carried out at less than 0 ° C, less than about 30 ° C, less than about 27 ° C, less than about 25 ° C, less than about 22 ° C. A typical temperature range is about 20 ° C. or more and less than about 40 ° C. Typical 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 degreeC. In some embodiments, the temperature is room temperature or ambient temperature of tap water, for example, from about 20 ° C to about 23 ° C.

Any of the methods or uses for laccase described herein use any of the laccase enzymes described herein, e.g., laccase from Cerrena unicolor. Can be implemented. In some embodiments, the laccase is from about 0.005 mg / liter to about 5000 mg / liter, from about 0.05 mg / liter to about 500 mg / liter, from about 0.1 mg / liter to about 100 mg / liter, or from about 0 Used at a concentration of 5 mg / liter to about 10 mg / liter. In some denim processing embodiments, the laccase is about 0.005 mg to about 5000 mg per kg denim, about 0.05 mg to about 500 mg per kg denim, about 0.1 mg to about 100 mg per kg denim, or per kg denim Used at a concentration of about 0.5 mg to about 10 mg. In some embodiments, the laccase is from about 5 to about 7, from about 5.5 to about 6.5,
Used at a pH of about 5 to about 6, or about 6. Typical pH is approximately 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 noted above, the compositions of the present invention comprise one or more laccases and optionally further comprise one or more transmitter substances. In some embodiments, the composition comprises 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, comprising a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from SEQ ID NO: 20, or a variant or fragment thereof. In certain embodiments, the composition comprises a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 19 and 20, or variants or fragments thereof. including. Preferably, such a polypeptide has an enzymatic laccase activity. Its activity can be measured using the assays and procedures described herein.

Providing such a composition in the form of a “ready to use” (RTU) composition comprising, consisting essentially of, or consisting essentially of a laccase enzyme and a transmitter, comprising a laccase enzyme and a transmitter You can also. In some embodiments, the transmitter is from acetosyringone, syringaldehyde, syringamide, methyl syringamide, 2-hydroxyethyl syringamide, methyl syringate, syringonitrile, dimethyl syringamide, and syringic acid. Selected. In one embodiment, the transmitter is syringonitrile (4-hydroxy-3,5-dimethoxybenzonitrile). The RTU composition may further comprise one or more compounds for providing pH buffering when the composition is in solution. For example, in some embodiments, the composition comprises monosodium phosphate and adipic acid as a buffer system. The RTU composition may be in solid granular form for ease of storage and transport. The composition is then diluted with water, for example, to provide an aqueous solution for use as described. The RTU composition may include any number of additional reagents such as dispersants, surfactants, blocking agents, polymers, and preservatives.

The following examples are provided to illustrate the systems, compositions and methods and should not be construed as limiting in any way. Other aspects and embodiments will be apparent to those skilled in the art upon reference to the specification.

実施例１−ストーンウォッシュされたデニムのラッカーゼによる色変性に対する温度の影響 EXAMPLES The following enzyme nomenclature is used in the examples.

Example 1-Effect of Temperature on Color Modification by Stonewashed Denim Laccase

Enzymes In this test, granular laccase D enzyme (38,000 U / g) derived from Serena Unicolor was used. One laccase unit is subject to analytical conditions based on the ability of the laccase enzyme to oxidize ABTS (2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonate)) to its corresponding stable cation radical, ABTS ^+. Defined as the amount of laccase activity that oxidizes 1 nmol of ABTS substrate. The accumulation of radicals makes ABTS dark green and increases the absorbance at 420 nm. Color production is proportional to laccase activity and is monitored against laccase criteria. Transmitter

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

Twelve denim legs weighing approximately 3 kg (total) were de-glueed in a Unimac UF50 washer under the following conditions: 0.5 g / l (15 g) OPTISIZE® 160 amylase (15 g) (Genencor) and 0.5 g / l non-ionic surfactant [eg Rucogen BFA (Rudolf Chemie) or Ultravon RW (Huntsman )] For 15 minutes at 50 ° C. at a liquid ratio of 10: 1 and two cold water rinses for 5 minutes at a liquid ratio of 30: 1.

After desizing, the denim was stonewashed in a Unimac UF50 washer under the following conditions. 5 minutes cold water rinse in a 10: 1 liquid ratio 1 kg pumice, pH 4.5 (1 g / l sodium tricitrate dihydrate and 1 g / l citric acid monohydrate), and Stone wash for 60 minutes at 55 ° C. in a 10: 1 liquid ratio with 2 g / l INDIAGE® 2XL cellulase (Genencor) • 2 cold water rinses for 5 minutes in a 30: 1 liquid ratio

After stone wash, laccase treatment was performed in a Unimac UF50 washer according to the following process. (I) pH 6 C.I. Unicolor laccase D and syringonitrile (0.7 g / l monosodium phosphate and 0.17 g / l adipic acid) and a temperature of 40 ° C., 30 ° C. or 23 ° C. (ii) pH 4.8 NOVOPRIME® Base268 and NOVOPRIME® F258 (0.29 g / l monosodium phosphate and 0.56 g / l adipic acid) and a temperature of 40 ° C. or 30 ° C., 30: 1 Two minutes of cold water rinse for 5 minutes at a liquid ratio of denim leg evaluation

ここで、Ｙ：三刺激値Ｙ（三刺激値Ｙ_１０を使用することもできる）ｕ’、ｖ’：ＣＩＥ１９７６ＵＣＳダイアグラムから得た色度座標Ｙ_０、ｕ’_０、ｖ’_０：完全反射拡散器の三刺激値Ｙ（又はＹ_１０）及び色度座標ｕ’、ｖ’ The amount of color modification reported as “bleaching” of the denim leg after laccase treatment with a Minolta Chromameter CR 310 in the CIE test color space with a D65 light source was evaluated. The CIE color space system, also known as the CIELV color space, was adopted by the International Commission on Illumination (CIE) in 1976 and has the values L ^* , u ^* and v ^* calculated as follows: Including.

Here, Y: tristimulus values Y (it is also possible to use the tristimulus values _{Y 10) u ', v'} : CIE1976UCS chromaticity coordinates _Y 0 obtained from the _{diagram, u '0, v' 0} : completely diffuse reflection Tristimulus value Y (or Y ₁₀ ) and chromaticity coordinates u ′, v ′

表２

Eight measurements were obtained for each denim leg and the results for 12 legs (96 measurements total) were averaged. The results are shown in Tables 1 and 2 and FIG.
Table 1

Table 2

This result shows that C.I. in affecting the color change of stonewashed denim. It demonstrates the effectiveness of unicolor laccase and syringonitrile. Example 2 Effect of Laccase: Transmitter Ratio for Color Modification of Stone Washed Denim Procedure

As described in Example 1, twelve denim legs weighing about 3 kg (total) were desizing and stonewashed. After stone wash, laccase treatment was performed in a Unimac UF50 washer according to the following process.・ C. Unicolor laccase D and syringonitrile, pH 6 (0.7 g / l monosodium phosphate and 0.17 g / l adipic acid) at 40 ° C. for 30 minutes in a 10: 1 solution ratio, 30: 1 Two cold water rinses over 5 minutes at a liquid ratio of denim leg evaluation

Denim leg color modification was evaluated as described in Example 1. The results are shown in Table 3 and FIG.
Table 3

This result indicates that color modification can be altered by manipulating the ratio of laccase enzyme to transmitter. Example 3-Effect of temperature on the color modification performance of a composition comprising laccase and a transmitter on stonewashed denim

手順 To examine the color modification performance with laccase at low temperatures, “Ready to Use” (RTU) compositions were prepared as shown in Table 4. Monosodium phosphate and adipic acid provide a buffering function of about pH 6 in applications for use as described below.
Table 4

procedure

As described in Example 1, twelve denim legs weighing about 3 kg (total) were desizing and stonewashed. After stone wash, laccase treatment was performed in a Unimac UF50 washer according to the following process. The RTU laccase composition at the concentrations and temperatures described in Tables 5 and 6 at 30 ° C. for 30 minutes at 30 ° C. or without inflow steam (ie, temperatures between 21 ° C. and 22 ° C.) Or use DENILITE (R) IIS (Novozymes)-Two cold water rinses for 5 minutes at 30: 1 liquid ratio Evaluation of denim legs

表６

Denim leg color modification was evaluated as described in Example 1. The results are shown in Tables 5 and 6 and FIGS.
Table 5

Table 6

These results are shown in C.I. Unicolor laccase RTU compositions have been shown to provide better color modification at low temperatures compared to conventional commercial laccase compositions. Example 4 One Step Stone Wash and Color Modification at 30 ° C

Twelve denim legs weighing about 3 kg (total) as described in Example 1 were desizing in a Unimac UF50 washer.

After desizing, the denim was stone washed and bleached in a Unimac UF50 washer under the following conditions: • 0.4% by weight of INDIAGE® Super GX Cellulase (Genencor) + Example 3 (ie 3% by weight RTU laccase composition, or (ii) INDIAGE® Super GX cellulase only (ie “Stonewash”), as described in “Stonewash + one step of bleaching”) Only) • 2 cold water rinses for 5 minutes at 30: 1 liquid ratio. No pumice was used. These results are shown in Table 7 and FIG.
Table 7

These results indicate that color modification can be achieved using laccase and cellulase simultaneously. Example 5-Two Step Stone Wash and Color Modification at 30 ° C

As described in Example 1, twelve denim legs weighing about 3 kg (total) were desizing in a Unimac UF50 washer.

After desizing, the denim was stonewashed in a Unimac UF50 washer under the following conditions. -0.4% by weight of INDIAGE® Super GX cellulase (Genencor) at 30 ° C., 30 ° C., pH 5.5 for 30 minutes at pH 5.5

After the stone wash, the denim was bleached in a Unimac UF50 washer under the following conditions. • RTU laccase composition as described in Example 3, 30% at 30 ° C., pH 6 at 30 ° C., pH 6 for 30 minutes, 5 minutes at 30: 1 solution ratio Rinse twice with cold water. No pumice was used.

These results are shown in Table 8 and FIG. The results from the two steps of stone wash and color modification were compared with the results of only stone wash described in Example 4.
Table 8

These results show that color modification by laccase treatment can be achieved after stone wash. Example 6 Color Modification by Denim Laccase at 30 ° C. without Stone Wash

As described in Example 1, twelve denim legs weighing about 3 kg (total) were desizing in a Unimac UF50 washer.

After desizing, the denim was bleached in a Unimac UF50 washer under the following conditions. • RTU laccase composition as described in Example 3 at 30 ° C., 30 ° C., pH 6, at 3% by weight with respect to the product for 30 minutes • 2 for 5 minutes at 30: 1 solution ratio Rinse with cold water. No pumice was used.

These results are shown in Table 9 and FIG. As described in Example 4, the color modification results were compared to the stonewash only results.
Table 9

These results show that the degree of color modification caused by laccase treatment without stone wash is higher than that of stone wash alone. Example 7-Stone wash and color modification with cellulase and laccase in a single tank process without pumice

This example shows that effective stone wash and color modification can be obtained using laccase and cellulase in a single bath process. enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 780931616, 6,292 GLacU / g). procedure

The starting material was desizing denim (ballast + 2 legs for evaluation) weighing about 3 kg.

This denim was stonewashed in a Renzacci LX 22 washer under the following conditions. 40 minutes, 50 ° C., 10: 1 liquid ratio, pH 6.5, 0.4% by weight of INDIAGE® Neutra L cellulase (batch No. 40105358001, activity 5197 NPCNU / g) (Genencor) . -After stone wash, one leg was removed and dried for evaluation. After the stone wash, the second denim leg was subjected to color modification under the following conditions without draining the tank (ie draining). • PRIMAGREEN® EcoFade LT 100 ready for use (RTU) 40 minutes at 40 ° C., 10: 1 liquid ratio, and 1% weight to product. • 2 cold water rinses for 3 minutes.・ Denim was dried with an industrial dryer. Denim leg evaluation

After laccase treatment and after cellulase treatment, color denaturation and stone wash on denim legs were evaluated using a Minolta Chromameter CR 310 in a CIE test color space equipped with a D65 light source. Six measurements were taken for each leg and the results were averaged.

These results are summarized in Table 10. The degree of color modification obtained by continuous (ie, two-step) addition of cellulase and laccase in a single tank was obtained by adding cellulase and laccase simultaneously as in Example 4. Greater than the degree. Table 10

These results show that the degree of color modification obtained by the continuous (ie, two-step) addition of cellulase and laccase in a single bath is that cellulase and laccase are added simultaneously as in Example 4. Is greater than the degree obtained. Example 8-Color modification with laccase and pumice

This example shows that a pumice and laccase transmitter system can be used in a single tank process to obtain effective stone wash and color modification. enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch 7809136160, 6,292 GLacU / g). procedure

As described in Example 1, twelve denim legs weighing about 3 kg (total) were de-sanded in a Unimac UF50 washer.

After desizing, the denim was stonewashed in a Unimac UF50 washer under the following conditions. -30 minutes, 30 degreeC, 10: 1 liquid ratio, 3 kg pumice, 3% PRIMAGREEN (registered trademark) EcoFade LT100 (Genencor) was used. Blank / control was performed using only stones in water. Two cold water rinses for 5 minutes at a 30: 1 liquid ratio.

As before, color modification to denim legs was evaluated using a Minolta Chromameter CR 310 in a CIE test color space with a D65 light source after laccase treatment and after stone wash treatment. The average of 8 measurements taken outside each leg was reported as the bleaching level. The average of the 4 measurements taken inside each leg was reported as the back staining level.

表１２

These results are summarized in Tables 11 and 12.
Table 11

Table 12

These results indicate that, even in the presence of pumice, laccase treatment provides color modification and further demonstrates the reduction / removal of backside staining. Example 9-Stone wash and color modification of sulfur dyed clothing

The test garment was made of a 100% cotton twill fabric dyed with sulfur kirky brown dye. Under the following conditions, 21 garments weighing approximately 7 kg (total) were stonewashed in a 25 kg belly washer (36 rpm).
• INDIAGE® 2XL at 45 ° C., 55 ° C., 18: 1, pH 4.5, 1 g / l • 1 cold water rinse for 3 minutes at 12: 1 solution ratio. No pumice was used. • After washing, the clothes were dried for evaluation.
-Three clothes (total of about 1 kg) stonewashed as described above were treated with PRIMAGREEN (registered trademark) EcoFade LT 100 under the following conditions. 15 min, 30 min or 45 min, 50: 1 liquid ratio, 40 ° C. and 1 g / l, 2 g / l or 3 g / l PRIMAGREEN® EcoFade LT 100. Blanks / controls were performed using clothes washed with water alone for 15 minutes, 30 minutes or 45 minutes.
-One cold water rinse for 3 minutes. -Denim was dried with an industrial dryer.

After laccase treatment and after stone wash treatment, color modification and stone wash of sulfur-stained clothes were evaluated using a Minolta Chromameter CR 310 in a CIE test space equipped with a D65 light source as described above. Ten measurements were taken for each garment and the results averaged.

These results are summarized in Table 13.
Table 13

These results show that the a and b values of the color space vary significantly compared to untreated and blank fabrics. Modification to the color of the clothes is confirmed visually. Example 10-Color modification of sulfur-dyed garments without stone wash

Three garments made of 100% cotton twill fabric weighing about 1 kg (total) dyed with sulfur carky brown dye were treated (36 rpm) in a 5 kg Billy washer under the following conditions.
15 min, 30 min or 45 min, 40: 1 solution ratio, 40 ° C., and 1 g / l, 2 g / l or 3 g / l PRIMAGREEN® EcoFade LT 100. Blanks / controls were performed using clothes washed with water alone for 15 minutes, 30 minutes or 45 minutes.
-One cold water rinse for 3 minutes.・ Denim was dried with an industrial dryer.

After laccase treatment and after stone wash treatment, color modification and stone wash on sulfur-stained clothing was evaluated using a Minolta Chromameter CR 310 in a CIE test color space with a D65 light source. For each garment, 10 measurements were taken and the results were averaged.

These results are summarized in Table 14.
Table 14

These results show that the a and b values of the color space vary significantly compared to untreated and blank fabrics. Modification to the color of the clothes is confirmed visually. Example 11-Performance of Stone Wash and Bleaching with Cellulase and Laccase in Single Tank Process in the Presence of Surfactant and Pumice Enzyme

PRIMAGREEN® EcoFade LT 100 Laccase (Batch No. 780931616, 6,292GLacU / g) Procedure

Twelve denim garments weighing 10 kg (total) dyed with pure indigo were desizing in a Tupesa front loading machine (36 rpm) under the following conditions. 10 minutes, 40 ° C., 10: 1 liquid ratio, pH 7, and 0.5 g / l lubricant, 0.2 g / l dispersant (nonionic surfactant), and 0.2 g / l l Polyester blocker (nonionic hydrophilic copolymer).

After desizing, the denim was stonewashed under the following conditions. 30 minutes, 47 ° C., 5: 1 liquid ratio, pH 6, 7 kg of pumice, 4% by weight of INDIAGE® Super GX cellulase (Genencor) is used. One piece of clothing was removed for evaluation. -After the stone wash, the denim was bleached under the following conditions without draining (dripping) the tank. • 30 minutes, 5: 1 liquid ratio, 47 ° C. and 2% by weight of ready to use (RTU) PRIMAGREEN® EcoFade LT100 • 2 times over 2 minutes at a liquid ratio of 1:50 Rinse with cold water. -Denim was dried with an industrial dryer. Denim leg evaluation

After laccase treatment and after cellulase treatment, color denaturation and stone wash for denim were evaluated using a Minolta Chromameter CR 310 in a CIE test color space equipped with a D65 light source. Eight measurements were taken for each leg and the results averaged.

These results are summarized in Table 15.
Table 15

These results indicate that color modification is caused by laccase treatment in the presence of pumice and surfactant.

The aspects, embodiments and examples described herein are for illustrative purposes only. Various modifications will be apparent to those skilled in the art and are encompassed within the spirit and scope of this application and the scope of the appended claims. All publications and patent literature cited herein are hereby incorporated by reference in their entirety.

Claims (26)

Contacting the fiber with a laccase enzyme and a transmitter at a temperature less than 40 ° C. for a time and under conditions sufficient to cause color modification of the fiber, wherein the laccase comprises SEQ ID NO: 19 or SEQ ID NO: 20 A fiber treatment method characterized by having an amino acid sequence that is at least 90% identical. The method of claim 1, wherein the color modification is selected from color thinning, color change, tint change, redeposition / reverse dyeing reduction, and bleaching. The fiber treatment method according to claim 1, wherein the temperature is 20 ° C or higher and lower than 40 ° C. The fiber treatment method according to claim 1, wherein the temperature is 20 ° C to 30 ° C. The fiber processing method according to claim 1, wherein the fiber is indigo-dyed denim. The fiber processing method according to claim 1, wherein the fiber is sulfur-dyed denim. The fiber processing method according to claim 1, wherein the denim is desizing and / or stonewashed before or simultaneously with the step of contacting the fiber with a laccase enzyme and a transmitter. The fiber processing method according to claim 1, wherein the step of stonewashing and the step of contacting the fiber with a laccase enzyme and a transmitter are performed in the same tank. The fiber processing method according to claim 1, further comprising the step of contacting the fiber with a cellulase enzyme simultaneously or sequentially with the step of contacting the fiber with a laccase enzyme and a transmitter. The step of contacting the fiber with the cellulase enzyme and the step of contacting the fiber with the laccase enzyme and the transmitter are performed sequentially and before the step of contacting the fiber with the laccase enzyme and the transmitter The fiber treatment method according to claim 9, wherein the step of bringing a cellulase into contact with a cellulase enzyme is performed. The step of contacting the fiber with the cellulase enzyme without draining the bath between the step of contacting the fiber with the cellulase enzyme and the step of contacting the fiber with the laccase enzyme and the transmitter; and the fiber with the laccase enzyme and transfer The fiber treatment method according to claim 10, wherein the step of bringing into contact with a substance is continuously performed in the same tank. 12. The method according to any one of claims 9 to 11, wherein the step of contacting the fiber with a cellulase enzyme and the step of contacting the fiber with a laccase enzyme and a transmitter are performed at a temperature of less than 40C. The fiber processing method as described. The method according to any one of claims 1 to 12, wherein the laccase is a microbial laccase. 13. A method according to any one of the preceding claims, wherein the laccase is derived from a Cerrena species. 13. The method according to any one of claims 1 to 12, characterized in that the laccase is derived from C. unicolor. The method according to any one of claims 1 to 12, characterized in that the laccase is laccase D derived from C. unicolor. The laccase is a 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. 13. The method according to any one of claims 1 to 12, wherein the method has an amino acid sequence that is at least 70% identical to a more selected amino acid sequence. The laccase is a 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 method according to any one of claims 1 to 12, wherein the method has an amino acid sequence that is at least 80% identical to the amino acid sequence selected. 13. The method according to any one of claims 1 to 12, wherein the laccase has an amino acid sequence that is at least 70% identical to SEQ ID NO: 19 or SEQ ID NO: 20. 13. The method according to any one of claims 1 to 12, wherein the laccase has an amino acid sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO: 20. 21. A method according to any one of the preceding claims, wherein the transmitter substance is syringonitrile. The method according to any one of claims 1 to 20, wherein the temperature is 20 to 35 ° C. 21. A method according to any one of the preceding claims, wherein the temperature is between 20C and 23C. The method according to any one of claims 1 to 20, wherein the temperature is an outside temperature of tap water. 25. A method according to any one of claims 1 to 24, wherein the laccase enzyme and the transmitter are provided together in a ready-to-use composition. 25. A method according to any one of claims 1 to 24, wherein the laccase enzyme and the transmitter are provided in solid form.

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