KR20110117064A - Lacasees and their use at low temperatures - Google Patents

Abstract

Lacase enzymes and nucleic acid sequences encoding said laccase enzymes are described. Lacase enzymes can be used with mediators in an improved method for modifying the color of denim fabrics. Low temperature and single-bath fabric processing using laccase enzymes is also described.

Description

Lacase and its use at low temperatures {LACCASES AND METHODS OF USE THEREOF AT LOW TEMPERATURE}

preference

The present invention discloses US Provisional Serial No. 61 / 140,724, filed Dec. 24, 2008, 61 / 154,882, filed Feb. 24, 2009, and No. 61 /, filed Aug. 27, 2009. Priority is given to 237,532, each of which is incorporated by reference in its entirety.

Technical field

The present systems, compositions and methods relate to laccase enzymes and nucleic acid sequences encoding such laccase enzymes. Lacase enzymes can be used in conjunction with mediators in improved methods for modifying the color of denim fabrics.

Lacasees are copper-containing phenol oxidases known as good oxygenating agents in the presence of oxygen. Lacases are found in microorganisms, fungi and higher organisms. Lacase enzymes are catalysts for pulp and paper bleaching, pulp wastewater treatment, printing ink de-inking, industrial stain removal, bleaching in laundry detergents, oral care tooth whitening agents, and polymerization and oxidation reactions, or Used as an accelerator.

Lacases can be used for a wide range of applications in numerous industries, including the detergent industry, paper and pulp industry, the textile industry and the food industry. In one application, phenol oxidase is used as an aid in removing stains such as food stains from clothing during detergent washes. Most laccases exhibit a pH optimum in the acidic pH range, while they are inert at neutral or alkaline pH.

Lacasees include Aspergillus, Neurospora, Podospora, Botrytis, Pleurotus, Fornes, Plelebia, Tramethes, Polyporus, Stachybotrys, Rhizoctonia, Bipolaris, Curvularia, Amerosporium, Lentinus To a wide range of fungi, including varieties of the genus Lentinus, Myceliophtora, Coprinus, Thielavia, Serrena, Streptomyces and Melanocarpus It is known to be produced by. However, there remains a need for laccases with different performance profiles in various applications.

In many applications, the oxidative efficacy of laccases can be enhanced through the use of mediators, also known as enhancers. Systems comprising laccases and mediators are known in the art as laccase-mediated systems (LMS). The same compound can also be used to activate or initiate the action of laccases.

Several mediators for use in laccase-mediated systems are known. It is 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-benzotriazine-4 (3H) -one), and VIO (violuric acid). In addition, there are several compounds that contain NH-OH or N-O groups found to be useful as mediators.

Functional groups and substituents have a great impact on the mediator efficacy. Even within the same class of compounds, substituents can significantly increase or decrease mediator efficacy by altering laccase specificity for the substrate. In addition, a mediator may be effective for a particular application unless suitable for another application. Another obstacle to current mediators is their tendency to polymerize during use. Therefore, it is necessary to find effective mediators for certain applications. One such application is the bleaching of fabrics, and it is also important that the mediators are not excessively expensive or harmful. Other applications of the laccase-mediating system are given below.

The method of using laccases at low temperatures can provide advantages in terms of energy savings in textile processing, for example, where the energy input for heating the processing bath can be reduced. It may be desirable to develop a method of using laccase enzymes at low temperatures for applications such as enzyme bleaching.

Summary of the Invention

Enzymatic oxidation systems, compositions, and methods are described that include laccases. In one aspect, a fabric processing method is provided comprising contacting a fabric with a laccase enzyme, and optionally at a temperature below 40 ° C., under conditions sufficient for causing color deformation of the fabric and for a time. In some embodiments, the color variations are selected from color brightening, color changing, color cast changing, redepositing / backstaining and bleaching. In some embodiments, the temperature is about 20 ° C to less than 40 ° C. In some embodiments, the temperature is about 20 ° C to about 35 ° C. In some embodiments, the temperature is about 20 ° C to about 30 ° C. In some embodiments, the temperature is about 20 ° C to about 23 ° C. In some embodiments, the temperature is 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 Or 35 ° C. In some embodiments, the temperature is the ambient temperature of the tap water.

In some embodiments, the fabric is indigo-dyed denim. In some embodiments, it is a fabric yellow-dyed denim. In some embodiments, the denim is desize and / or stonewashed prior to or concurrently with contacting the fabric with the laccase enzyme and the mediator. In some embodiments, the stonewash and contact of the fabric with the laccase enzyme and mediator are in the same bath.

In some embodiments, the method further comprises contacting the fabric with the cellulase enzyme simultaneously or sequentially with contacting the fabric with the laccase enzyme and the mediator. In some embodiments, contacting the fabric with the cellulase enzyme and contacting the fabric with the laccase enzyme and the mediator are performed sequentially, and contacting the fabric with the cellulase enzyme is performed prior to contacting the fabric with the laccase enzyme and the mediator. do. In some embodiments, contacting the fabric with the cellulase enzyme and contacting the fabric with the laccase enzyme and the mediator comprise draining the bath between contacting the fabric with the cellulase enzyme and contacting the fabric with the laccase enzyme and the mediator. They are performed sequentially in the same bath.

In some embodiments, contacting the fabric with the cellulase enzyme and contacting the fabric with the laccase enzyme and the mediator are performed at a temperature below 40 ° C. In some embodiments, the temperature is about 20 ° C to less than 40 ° C. In some embodiments, the temperature is about 20 ° C to about 35 ° C. In some embodiments, the temperature is about 20 ° C to about 30 ° C. In some embodiments, the temperature is about 20 ° C to about 23 ° C. In some embodiments, the temperature is 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 Or 35 ° C. In some embodiments, the temperature is the ambient temperature of the tap water.

In some embodiments, the cellulase enzyme synergizes with the laccase enzyme to produce a fabric having a high degree of fabric color lightening, color change, color cast change, redeposition / recoloration reduction and / or bleaching. In some embodiments, the cellulase enzyme additionally acts with the laccase enzyme, resulting in a greater degree of fabric color lightening, color change, color cast change, redeposition / recoloration reduction and / or bleaching as compared to the same method without cellulase. Produces a fabric with

In some embodiments, the laccase is a microbial laccase. In some embodiments, the laccase is from the Serrena species. In some embodiments, the laccase is from Serrena unicolor. In some embodiments, the laccase is Lacasease D from C. unicolor.

In some embodiments, the laccases comprise 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, At least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the laccases comprise 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, At least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the laccases comprise 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, At least 90%, or at least 95% identical amino acid sequence to the amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

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

In some embodiments, the laccase enzyme and mediator are provided together in a ready-to-use composition. In some embodiments, the laccase enzyme and mediator are provided in solid form. In some embodiments, the laccase enzyme and mediator are provided in granules. In certain embodiments, the mediator is cyringonitrile.

In another aspect, provided are laccases, nucleic acid sequences encoding the laccases, and vectors and host cells for expression of laccases. Lacases can be used at low temperatures in ways in which a reduction in energy input, such as textile processing, is desired. In some embodiments, the laccase enzyme is an amino acid sequence represented by any of the following SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, or 20, or SEQ ID NO: 2 At least about 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94% of any of 4, 6, 8, 12, 14, 16, 18, 19, or 20 , 95%, 96%, 97%, 98%, 99% or 99.5% identical amino acid sequences, consisting of, or consisting essentially of. In certain embodiments, the laccases comprise SEQ ID NO: 19 or SEQ ID NO: 20 and at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 99.5% identical amino acid sequence. In even more specific embodiments, the laccase has the amino acid sequence SEQ ID NO: 19 or SEQ ID NO: 20. Preferably, such polypeptides have a laccase enzyme activity that can be measured using, for example, the assays described herein.

In another aspect, a composition is provided comprising a laccase enzyme comprising, consisting essentially of, or consisting of any of the amino acid sequences described above. 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 mediator. The mediators are, for example, acetosyringone, cyringalaldehyde, cyringimide, methyl cyringimide, 2-hydroxyehil syringimide, methyl syringe, dimethylsyringamide, shrine acid and 4-hydroxy- 3,5-dimethoxybenzonitrile (cyringonitrile). In one embodiment, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile. In some embodiments, the composition is in solid form. In some embodiments, the laccase enzyme and mediator are provided together in a ready-to-use composition. In some embodiments, the laccase enzyme and mediator are in solid form. In some embodiments, the laccase enzyme and mediator are provided in granules. In certain embodiments, the mediator is cyringonitrile.

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

These and other aspects and embodiments of the present systems, compositions, and methods will be apparent from the description and the accompanying drawings.

1 shows the effect of modifying the color of the stonewashed denim with the laccase enzyme at different temperatures, as described in Example 1. FIG.
2 shows the effect of the ratio of laccase and mediators on modifying the color of the stonewashed denim, as described in Example 2. FIG.
FIG. 3 shows the effect of temperature on modifying the color of the stonewashed denim with a "ready to use" laccase composition, as described in Example 3. FIG.
4 shows the effect of temperature on the color-modifying performance of the laccase enzyme on stonewashed denim, as shown in Example 3. FIG.
5 shows the effect of cellulase treatment in combination with laccase-mediated color modification, as described in Examples 4-6.

details

Enzymatic oxidation systems, compositions, and methods are described that include laccases. The systems, compositions and methods are useful for low temperature processing of fabrics, for example, to influence color deformation. Such processing uses less energy than conventional fabric processing techniques and involves more environmentally friendly chemical reagents. Various aspects and embodiments of systems, compositions, and methods are described.

Justice

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provides the general dictionary meanings of many terms used herein. The following terms are defined for further clarity.

As used herein, the term "enzyme" refers to a protein that promotes chemical reactions. The catalytic function of an enzyme constitutes its "enzyme activity" or "activity". Enzymes are usually classified according to the type of reaction catalyzed by the enzyme, for example, oxidation of phenols, hydrolysis of peptide bonds, incorporation of nucleotides, and the like.

As used herein, the term “substrate” means a substrate (eg a chemical compound) from which an enzyme performs its catalytic activity to produce a product.

As used herein, "lacase" refers to a multi-copper containing oxidase (EC) that catalyzes the oxidation of phenols, polyphenols and anilines by single-electron extraction with incidental reduction of oxygen to water in a 4-electron transition process. 1.10.3.2).

As used herein, “variant” proteins include related and derivative proteins that differ from the parent / reference protein by minor 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%, 85%, 90 relative to the parent / reference protein. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% amino acid identity.

As used herein, the term “similar sequence” refers to a polypeptide sequence in a protein that provides similar function, tertiary structure, and / or conserved residues relative to the sequence in the parent / reference protein. For example, at structural sites that include alpha helix or beta sheet structures, the replacement amino acid residues in the analogous sequence retain the same structural features. In some embodiments, the analogous sequence results in a variant protein that exhibits similar or improved function with respect to the parent protein from which the variant is derived.

As used herein, a "homologous protein" or "homolog" has a function (eg enzymatic activity) and / or structure similar to a reference protein (eg a laccase enzyme from different sources). Protein (eg a laccase enzyme). Homologs may be from evolutionarily related or unrelated species. In some embodiments, homologues have a quaternary, tertiary and / or primary structure similar to that of the reference protein, such that the structure and / or structure of the reference protein is compared to the replacement of segments or fragments with sequences from non-homologous proteins. Or similar segments or segments from homologues with reduced cleavage of function, potentially allowing the segment or segment in the reference protein to be replaced.

As used herein, "wild type", "natural", and "naturally occurring" proteins are those found in nature. The term “wild type sequence” means an amino acid or nucleic acid found or naturally occurring in nature. In some embodiments, the wild type sequence is the starting point for protein engineering projects, such as the generation of variant proteins.

As used herein, “signal sequence” means an amino acid sequence that binds to the N-terminal portion of a protein and promotes the secretion of protein mature forms 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” means growing a microbial cell population under suitable conditions in liquid, semisolid, or solid medium to express the polypeptide of interest. In some embodiments, the culturing is carried out in a vessel or reactor as known in the art.

As used herein, the term "derivative" means one or more amino acid additions to either or both of the N- and C-terminal end (s), one or more amino acids at one or a large number of different sites in the amino acid sequence. In the parent / reference protein by residue substitution, one or both of the terminal ends of the protein, or one or more amino acid residue deletions in one or more sites in an amino acid sequence, and / or one or more amino acid insertions in one or more sites in an amino acid sequence Derived protein means. Preparation of protein derivatives is often done by modifying the DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host, and expressing the modified DNA sequence to form the derivative protein.

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

As used herein, the term “expression vector” includes a DNA coding sequence (eg, a gene sequence) operably linked to one or more suitable control sequence (s) that can affect the expression of a coding sequence in a host. Means a DNA construct. Such control sequences include promoters that affect transcription, any operator sequence that controls such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the completion of transcription and translation. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into a suitable host, the vector can be replicated and function independently of the host genome, or in some cases fused to the genome itself.

As used herein, the term “host cell” refers to a cell or cell line into which a recombinant expression vector for polypeptide production can be transfected, transformed, or otherwise introduced for expression of a polypeptide. Host cells include the progeny of a single host cell, which may not necessarily be identical to the parent cell due to natural, accidental, or intentional mutations (in form or in total genomic DNA complement). The host cell can be bacterial or fungal. Host cells include cells transfected or transformed in vivo with an expression vector.

As used herein, the term “introduced” in the context of introduction of a nucleic acid sequence into a cell includes “transfection”, “transformation” and “transduction”, wherein the nucleic acid sequence is in eukaryotic or prokaryotic cells. By fusion, the nucleic acid sequence is fused into the genome of the cell (eg chromosome, plasmid, plastid or mitochondrial DNA), converted into autonomic replicon, or transiently expressed.

The terms "cleaning composition" and "cleaning formulation" as used herein refer to fabrics, utensils, contact lenses, hair (shampoo), skin (soaps and creams), teeth (mouthwashes, toothpastes) and other solids and surfaces. By a composition that can be used to remove unwanted compounds from the item to be cleaned. The term refers to any substance / compound selected for the particular type and product form (eg liquid, gel, granule or spray composition) of the desired cleaning composition so long as the composition is compatible with the enzyme (s) used in the composition. Include. Specific selection of cleaning composition materials is readily made by considering the desired form of the composition for the surface, item or fabric to be cleaned, and cleaning conditions during use.

The term also refers to any composition suitable for cleaning, bleaching, sterilizing and / or sterilizing a subject and / or surface. The term includes, but is not limited to, detergent compositions (eg, liquid and / or solid laundry detergents and fine fabric detergents; hard surface cleaning, such as for glass, wood, ceramic and metal counter tops and windows). Formulations; carpet cleaners, oven cleaners, fabric cleaners, fabric softeners, and dish and detergent pre-spotter as well as dish detergents.

Indeed, the terms “cleaning composition” and “cleaning formulation” (unless otherwise indicated) refer to multipurpose or heavy-duty cleaning agents, in particular cleaning detergents in the form of granules or powders; Liquid, gel or paste-type multipurpose cleaners, in particular so-called heavy liquid (HDL) forms; Liquid pure-fabric detergents; Manual dishwashing agents or light duty dishwashing agents, in particular high foaming type; Mechanical dishwashing agents, including various types of tablets, granules, liquids and rinse aids for home and institutional use; Liquid cleaning and disinfecting agents, including antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, vehicle or carpet shampoos, bathroom cleaners; Hair shampoos and hair rinses; Shower gels and foam baths and metal cleaners; As well as bleaching aids and cleaning aids such as "stain-stick" or pretreatment types.

As used herein, “detergent composition” and “detergent formulation” are used with reference to mixtures intended for use in wash media for cleaning contaminated subjects. In some embodiments, the term is used with reference to washing fabrics and / or garments (eg “laundry detergents”). In alternative embodiments, the term refers to other detergents, such as those used for utensils, cutlery, and the like (eg, “dishwashing detergents”). In addition to enzyme (s), "detergent compositions" and "detergent formulations" include surfactants, builders, bleaches, bleach activators, blue agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants And detergents containing solubilizers.

As used herein, the phrase “detergent stability” refers to the stability of enzymes, and optionally associated substrates or mediators, in detergent compositions. In some embodiments stability is assessed during detergent use, while in other embodiments the term means stability of the detergent composition during storage.

As used herein, the term “hard surface cleaning composition” refers to detergent compositions for cleaning hard surfaces such as floors, walls, tiles, stainless steel containers (eg fermentation tanks), bathtubs and kitchen facilities. it means. Such compositions may be provided in any form, including but not limited to solids, liquids, emulsions and the like.

As used herein, the term “dishwashing composition” means any form of composition for cleaning dishware, including but not limited to granular and liquid forms.

As used herein, the term “sterilization” means the removal or killing of microorganisms including fungi, bacteria, spores and the like.

As used herein, the term “fabric cleaning composition” means a form of detergent composition for cleaning textiles, including but not limited to granules, liquids and bar forms.

As used herein, the terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably to deoxyribonucleotide, ribonucleotide and / or analogue or modification of deoxyribonucleotide or ribonucleotide. Any length and any three-dimensional structure, which is a single- or multi-strand (eg, single-stranded, double-stranded, triple-helix, etc.) comprising a modified form (including modified nucleotides or bases or analogs thereof) Refers to the polymer form of nucleotides. Because the genetic code is degraded, more than one codon can be used to encode a particular amino acid. Any form of modified so long as the polynucleotide maintains the desired function under conditions of use, including modifications that increase nuclease resistance (eg, deoxy, 2′-O-Me, phosphorothioate, etc.). Nucleotides or nucleotide analogues can be used. For example, radioactive or non-radioactive labels or anchors such as biotin may also be incorporated for the purpose of detection or capture. The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides may be naturally occurring or non-naturally occurring. Nucleotide sequences can be blocked by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. For example, phosphate can be P (O) S ("thioate"), P (S) S ("dithioate"), (O) NR ₂ ("amidate"), P (O) R, P (O) OR ′, CO or CH ₂ (“formacetal”), each of which R or R ′ is independently H, or optionally a substitution comprising an ether (—O—) linkage or Unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aradyl. All linkages in the polynucleotide need not be identical. The polynucleotides may be linear or cyclic or may comprise a combination of linear or cyclic moieties.

As used herein, the terms “polypeptide”, “protein” and “peptide” mean a composition consisting of amino acids (ie, amino acid residues). Conventional one or three letter codes for amino acids are used. Polypeptides can be linear or branched, can include modified amino acids, and can be blocked by non-amino acids. The term also refers to amino acid polymers modified naturally or by intervention: for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with labeled components. It includes. Also included within the definition are polypeptides that include, for example, one or more analogues of amino acids (eg, unnatural amino acids, etc.) as well as other modifications known in the art.

As used herein, the term “primer” is to be incubated with complementary nucleic acids in the presence of nucleotides and polymerases at suitable temperatures and pHs, either naturally occurring (eg, as purified restriction fragments) or synthetically produced. Oligonucleotide, which can serve as a starting point for nucleic acid synthesis. The primer is preferably single stranded but may alternatively be double stranded. In the case of double strands, the primer is first treated to separate its strands before use to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. The exact length of the primer will depend on many factors, including temperature, primer source and the use of the method.

As used herein, the terms "recovered", "isolated", "purified" and "isolated" mean a substance that is removed from one or more components that are associated or naturally associated as a result of heterologous expression ( For example proteins, nucleic acids or cells).

As used herein, the term "fabric (s)" refers to fibers, yarns, fabrics, clothing, and non-woven materials. The term includes fabrics made from natural and synthetic blends as well as natural and synthetic (eg, produced) materials. The term "fabric" refers to raw and processed fibers, twisted yarns, woven fabrics or knits, nonwovens and garments. In some embodiments, the fabric contains cellulose.

As used herein, the phrase “fabric (s) in need of processing” means a fabric that needs to be invoked, wiped, bleached and / or biopolish to produce the desired effect.

As used herein, the phrase "fabric (s) requiring color modification" means a fabric that needs to be changed for its color. Such fabrics may or may not have already undergone other treatments. Similarly, such fabrics may or may not require subsequent treatment.

As used herein, the term "fabric" means a fabricated assembly of fibers and / or yarns that has sufficient adhesion to obtain a substantial surface area, and a collection of useful mechanical strength for its thickness.

As used herein, the term "color variation" refers to the saturation, purity, intensity, luminescence, and / or hue of a color associated with a fiber, yarn, fabric, clothing, or nonwoven material collectively represented as a fabric material. Means change. Without wishing to be bound by theory, it is proposed that the color deformation results from the change in its visual appearance due to the deformation of the chromophores associated with the fabric material. Chromophores can be naturally associated with the materials used to make the fabric (eg white of cotton) or can be associated with specific finishes such as dyeing or printing. Color modifications include chemical modifications to the chromophore to which the chromophore is attached as well as chemical modifications to the chromophore. Examples of color variations include color fading, bleaching, and color tone changes. Certain color variations for indigo-dyed denim are faded to a "vintage look" with a less intense blue / purple shade and more relaxed gray appearance than newly dyed denim.

As used herein, the term “bleaching” refers to the process of treating textile materials such as fibers, yarns, fabrics, clothing or nonwoven materials such that a pale color is produced. The term includes, for example, making the fabric brighter and / or whiter in the context of textile processing applications, as well as lightening the color of the stain, for example in the context of cleaning applications.

As used herein, the terms “size” and “sizing” refer to compounds used in the textile industry to improve weaving performance by increasing corrosion resistance and yarn strength. Pools are usually made from starch or starch compounds.

As used herein, the terms "deburst" and "determine" generally mean the process of excluding / removing grass (generally starch) from a fabric prior to a particular finish, dye or bleaching application.

As used herein, the term “calling enzyme (s)” means an enzyme used to remove grass. Exemplary enzymes are amylases, cellulases and mannanases.

As used herein, the term “% identity” refers to the level of nucleic acid sequence identity between a nucleic acid sequence that encodes a laccase as described herein and another nucleic acid sequence, or another amino acid that is a laccase enzyme as described herein. Amino acid sequence identity level between sequences. Alignment can be performed using conventional sequence alignment programs. Exemplary levels of nucleic acid and amino acid sequence identity include, but are not limited to, at least 60%, at least 65%, at least 70% of a coding sequence for a given sequence, eg, a laccase as described herein, or at least an amino acid sequence of a laccase. , 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% sequence identity.

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

Sequence searches are typically performed using the BLASTN program in evaluating a given nucleic acid sequence against nucleic acid sequences in GenBank DNA sequences and other public databases. The BLASTX program is preferred for searching for translated nucleic acid sequences within all reading frames for GenBank protein sequences and amino acid sequences in other public databases. Both BLASTN and BLASTX are implemented using default parameters of open gap penalty 11.0, and extended gap penalty 1.0, using the BLOSUM-62 matrix (see, eg, Altschul, et al., 1997).

Alignment of selected sequences to determine the "% identity" between two or more sequences can be performed with default parameters including, for example, open gap penalty 10.0, extended gap penalty 0.1, and BLOSUM 30 similarity matrix. Can be performed using the CLUSTAL-W program.

As used herein, the terms “chemical mediator” and “mediator” are used interchangeably to shuttle electrons between an enzyme that exhibits oxidase activity (eg, a laccase) and a secondary substrate or electron donor. It means a chemical compound that functions as a redox mediator to make. Such chemical mediators are also known in the art as "enhancers" and "accelerators."

As used herein, the term "drainage" or "dropping" for a bath in which the textile material is present means to completely or partially release / eliminate solvents and reagents present in the bath. Draining the bath is typically performed between process steps so that solvents and reagents present in one processing step do not interfere with subsequent processing steps. Drainage may involve one or more rinsing steps to further remove these solvents and reagents.

As used herein, the terms “secondary substrate” and “electron donor” are used interchangeably to form dyes, pigments (eg indigo), chromophores (eg polyphenols, anthocyanins or carotenoids), or By other secondary substrates the electrons can be reciprocated by enzymes that exhibit oxidase activity.

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

EC Enzyme Panel

EDTA ethylenediaminetetraacetic acid

kDa kilodalton

MW molecular weight

w / v weight / volume

w / w weight / weight

v / v volume / volume

wt% wt%

℃ Celsius

H ₂ O water

dH ₂ O or DI deionized water

dIH ₂ O Deionized Water, Milli-Q Filtrate

g or gm gram

Μg microgram

mg milligram

kg kg

μL and μl microliters

mL and ml milliliters

mm millimeter

Μm micrometer

M Mall

mM millimoles

μM micromolar

U unit

sec and "seconds

min and 'min

hr hour

eq. Equivalent

N normal

RTU ready to use

U unit

owg about product weight

CIE International Commission on Illumination

The numerical range includes the numbers defining the range. Singular expressions include plural expressions unless the context clearly dictates otherwise. Unless otherwise specified, polypeptides are indicated in the standard N-terminus to C-terminal direction and polynucleotides are indicated in the standard 5 'to 3' direction. It is understood that the specific methodologies, protocols, and reagents described are not intended to be limited to the equivalent methods and materials that can be used in the practice or testing of the compositions and methods. To assist the reader, the description is divided into sections, but section headings should not be considered limiting, and the description in one section may apply to another. All publications cited herein are expressly incorporated by reference.

Lacase  And Lacase  Related Enzymes

Enzymatic oxidation systems, compositions, and methods include one or more laccases or laccase-associated enzymes, which are collectively referred to herein as "lacases" or "lacase enzymes." Such laccases include any laccase enzymes covered by EC 1.10.3.2 according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Lacase enzymes of microbial and plant origin are known in the art. Microbial laccase enzymes can be derived from bacteria or fungi (including fungi and yeast). Suitable examples include Aspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis, Collybia. , Serrena (e.g. C. unicolor), Stachybotrys, Panus (e.g. P. rudis), Tierra Viala, Pomese, Lentinus, Pleurotus, Trametes (e.g., T. villosa and T. Versico) T. versicolor), Rhizoctonia (e.g., R. solani), Coprinus (e.g., C. plicatilis) And C. cinereus), Satyrella, Miceliophthora (eg, M. thermonhila), Chitalidium, Phlebia (e.g. P. radita) (WO 92/01046)), or Coriolus (e.g., C. hirsutus (JP 2238885)), Spongipellis, Polyporus, Seri And racases derived from or derivable from strains of Ceriporiopsis subvermispora, Ganoderma tsunodae, and Trichoderma.

Lacases can be prepared by culturing a host cell transformed with a recombinant DNA vector comprising a nucleotide sequence encoding the laccase. The DNA vector may further comprise a nucleotide sequence that allows expression of the laccase in the culture medium and optionally enables the recovery of the laccase from the culture.

Expression vectors comprising 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 [eg, T. reesei (this time T. longibrachiatum). And now also known as Hypocrea jecorina), T. viride, T. koningii and T. harzianum], As Pergillus (e.g., A. niger, A. nidulans, A. oryzae and A. awamori), penicill Penicillium, Humicola (e.g. H. insolens and H. grisea), Fusarium (e.g. F. Grami) F. graminum and F. venenatum, Neurospora, Hypocrea and Mucor. Host cells for the expression of laccase enzymes can also be from the type of serena (eg C. unicolor). Fungal cells can be transformed by processes including protoplast formation and transformation of the protoplasts followed by cell wall regeneration (using techniques known in the art).

Alternatively, the host organism may be a bacterium such as Bacillus [eg, B. subtilis, B. licheniformis, B. lentus (B. lentus), B. (now Geobacillus) Stearothermophilus and B. brevis], Pseudomonas, Streptomyces (e.g. S S. coelicolor, S. lividans, or 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 and vector construction of appropriate DNA sequences can also be performed by standard procedures (see above).

The medium used to culture the transformed host cell may be any conventional medium suitable for growing host cells. In some embodiments, the expressed enzyme is secreted into the culture medium and can be recovered therefrom by well known procedures. For example, laccases can be recovered from the culture medium as described in US Patent Publication No. 2008/0196173. In some embodiments, the enzyme is expressed intracellularly and recovered after cell membrane division.

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

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

A. Serena Lacase A1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 1):

Translated protein sequence (SEQ ID NO: 2):

B. Serena Lacase A2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 3):

Translated protein sequence (SEQ ID NO: 4):

C. Serena Lacase B1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 5):

Translated protein sequence (SEQ ID NO: 6):

D. Serena Lacase B2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 7):

Translated protein sequence (SEQ ID NO: 8):

E. serena laccase B3 gene from the ATCC20013 strain (part)

Polynucleotide sequence (SEQ ID NO: 9):

Translated protein sequence (SEQ ID NO: 10):

F. Serena Lacase C Gene from CBS154.29 Strain (Partial)

Polynucleotide sequence (SEQ ID NO: 11):

Translated protein sequence (SEQ ID NO: 12):

G. Serena Lacase D1 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 13):

Translated protein sequence (SEQ ID NO: 14):

Serena Lacase D2 Gene from H. CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 15):

Translated protein sequence (SEQ ID NO: 16):

I. Serena Lacase E Gene from CBS154.29 Strain (Partial)

Polynucleotide sequence (SEQ ID NO: 17):

Translated protein sequence (SEQ ID NO: 18):

Lacasease D enzymes having the following amino acid sequence (SEQ ID NO: 19; signal sequence is italic) can be used in the methods described herein:

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

In some embodiments, the laccase enzymes suitable for use in the compositions and methods are mature polypeptides lacking signal sequences that can be used for direct secretion of full length polypeptides from cells. Suitable mature polypeptides include 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, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, for an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, 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%. . Preferably, the polypeptide has enzymatic laccase activity as measured using the assays and procedures described herein.

In some embodiments, laccase enzymes suitable for use in the compositions and methods are truncated to the full length or to the mature parent / reference sequence. Such truncated polypeptides can be produced by proteolytic digestion of full length or mature polypeptide sequences, or by polynucleotide engineering to encode a truncated polypeptide. Exemplary polypeptides include 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 are cleaved at the amino and / or carboxyl-terminus for an amino acid sequence selected from the group. Cleavage can be a minority (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), or of the entire structural or functional domain. Suitable truncated polypeptides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92 relative to a corresponding portion of one or more of the above referenced amino acid sequences. At least%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Preferably such polypeptides have enzymatic laccase activity as measured using the assays and procedures described herein.

Intermediary

In some embodiments, enzymatic oxidation systems, compositions, and methods further comprise one or more chemical mediating agents that enhance the activity of the laccase enzyme. Mediators (also referred to as enhancers or accelerators) are enzymes and dyes that exhibit oxidase activity, pigments (e.g. indigo), chromophores (e.g. in colored stains, e.g. polyphenols, anthocyanins or carotenoids). ), Or other secondary substrate or electron donor, is a chemical that acts as a redox mediator for effectively reciprocating electrons.

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

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

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

[In the meal,

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

In some embodiments, A in the aforementioned formula is -CN or -CO-E, wherein E can be -H, -OH, -R, -OR or -NXY, and X and Y are -H, Independently selected from —OH, —OR and —R, R is C ₁ -C ₁₆ alkyl, preferably C ₁ -C ₈ alkyl, which may be saturated or unsaturated, branched or unbranched, and Optionally substituted with sulfo or amino groups; B and C are independently selected from C _m H _{2m +1} ; 1≤m≤5. In some embodiments, the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as "cyringonitrile" or "SN").

Note that in the above-mentioned formula, A may be positioned meta to the hydroxyl group instead of being placed in the para position as shown.

For applications such as textile processing, the mediator may be from about 0.005 to about 1,000 μmol per gram of denim, from about 0.05 to about 500 μmol per gram of denim, from about 0.1 to about 100 μmol per gram of denim and about 1 per gram of denim. To about 50 μmol, or about 2 to about 20 μmol per gram of denim.

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

How to use

The present systems and compositions can be used in applications where enzymatic laccase activity is useful or desirable. Among these applications / methods are color variations of the substrate that may be associated with the fabric. In some embodiments, such methods include incubating the laccase enzyme with a suitable substrate at low temperature, for example about 40 ° C. or less. In some embodiments, the temperature is about 20 ° C to about 40 ° C. In some embodiments, the temperature is about 20 ° C to about 35 ° C. In some embodiments, the temperature is about 20 ° C, 25 ° C, 30 ° C or 35 ° C. In some embodiments, the temperature is at an ambient temperature of the tap water, eg, about 20 ° C to about 23 ° C. The temperature can be varied or maintained within a narrow range without significantly affecting the performance of the system and the composition.

The method envisions the use of one or more laccases described herein. In some embodiments, the laccase is from a serena species, such as C. unicolor. In some embodiments, the laccase comprises at least about 35%, 40%, 45% of the amino acid sequence of any of the C. unicholor laccase enzymes described herein, or any of the C. unicolor laccase enzymes described herein , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 It comprises, consists of, or consists essentially of an amino acid sequence having%, 99% or 99.5% identity and having a laccase enzyme activity.

In some embodiments, the systems and methods are used in fabric processing methods, such as methods for modifying the color of textile products (including, for example, fibers, yarns, cloths or finished garments). In general, the method is capable of measuring at least one (eg one or more) of the fabric, for example selected from color change, color cast change, color brightening, bleaching, color fading and / or redeposition / recoloring reduction. Contacting the laccase and the mediator under conditions and for a time sufficient to effect. In some embodiments, the method is used to impart a "vintage look" to dyed denim articles. In the case of indigo-dyed denim, the vintage look has a less intense blue / purple tone and a more relaxed gray appearance than the newly dyed denim. In the case of sulfur-dyed denim, the vintage look fades without the brownish color that can result from hypochlorite treatment. Thus, while the aspect of color modification obtained using a laccase may be characterized as a "bleaching" effect, the term does not describe all of the color variations that can be used with laccase.

Fabrics provided for color modification may be cellulose fabrics or a combination of cellulose and synthetic fibers. In some embodiments, the fabric is denim dyed with indigo and / or sulfur-based dyes. In certain embodiments, the fabric is dyed with indigo and the indigo is oxidized to isatin using laccase enzymes and mediators. Denim may be optionally called and / or stonewashed prior to color transformation with the laccase enzyme.

In general, given the same amount of wear in the fabric processing method, the denim strength is reduced to a greater extent at high temperatures compared to low temperatures. Since the present method can be carried out at a lower temperature than the conventional method, this has the advantage of reducing damage to the fabric during processing as compared to the conventional method. In addition, laccase enzymes generally do not react with cellulose fabric fibers such that their strength is reduced during processing. Thus, in some embodiments, the method does not affect the physical strength of the denim or reduces the physical strength loss compared to conventional methods. If the denim is stretch denim, for example comprising elastin or spandex, the method does not affect the stretch performance of the fabric or reduces the loss of stretch performance compared to conventional methods.

In some embodiments, laccases are used in textile processing methods in combination with one or more other enzymes. If such processing is continuous, enzymatic treatment may be performed at low temperatures as described herein. If processing is sequential, the laccase can be used at low temperatures as described herein and other enzyme (s) can optionally also be used at low temperatures. In some embodiments, laccases are used in combination with cellulase enzymes simultaneously or sequentially. In one embodiment, the fabric is in contact with the laccase and cellulase simultaneously. In another embodiment, the fabric is sequentially contacted with laccase and cellulase. In one embodiment, the fabric is first contacted with cellulase to result in a "stonewash," and then contacted with laccase to affect color deformation. In another embodiment, the fabric is first contacted with laccase and then contacted with cellulase. If cellulase and laccase treatments are sequential, both processing steps are performed in the same bath without draining the bath between treatments. This method is referred to as the "single-bath" method.

Suitable cellulases are, for example, Humicola, Thermomyces, Bacillus, Trichoderma, Fusarium, Myceliophthora, Paneroch Celluloses such as Phanerochaete, Irpex, Scytalidium, Schizophyllum, Penicillium, Aspergillus or Geotricum It can be derived from microorganisms known to be able to produce degradable enzymes. Kinds known to be able to produce cellulase degrading enzymes include Humicola insolens, Fusarium oxysporum or Trichoderma reesei. Non-limiting examples of suitable cellulases include US Pat. No. 4,435,307, all of which is incorporated herein by reference; European Patent Application No. 0 495 257; PCT Patent Application No. WO 91/17244; And European Patent Application No. EP-A2-271 004.

In some embodiments, an enzymatic “stonewash” using cellulase, bleaching using aryl esterases, and color modifications using laccases may be combined to provide a comprehensive enzymatic fabric processing system. This system allows fabric processors to produce fabrics with various finishes without the need to use conventional fabric processing chemicals.

Lacasees can also be used in other aspects of fabric making, including generally in aspects such as processing, processing, finishing, gloss, fiber making, and the like. In addition to the color variations of the dyed denim, laccases can be used for bleaching dyeing wastes (including indigo-dyeing wastes), fabric dyeing, fabric bleaching operations, fiber modifications; It may be used to achieve enhanced fiber or fabric properties, and the like.

In further embodiments, the present systems and compositions can also be used in methods of color modification of wool. For example, European patent number EP 0 504 005 discloses that laccases can be used for wool dyeing. Lacases can also be used in the leather industry. For example, laccases can be used for processing animal skins including, but not limited to, hair loss, liming, bating and / or tanning of the leather.

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

The present systems and compositions can also be used in hair color modification methods. Lacases are reported to be useful for hair dyeing (see for example WO 95/33836 and WO 95/33837). Such methods include contacting the hair having the color to be modified with laccase under conditions and for a time sufficient to change the color of the hair.

The present systems and compositions can also be used in the wastewater treatment field. For example, laccases may include decolorization of colored compounds; Detoxification of the phenolic component; For antimicrobial activity (eg in water recycle); Bio-remediation; Or the like.

The systems and compositions can also be used in depolymerization of high molecular weight aggregates, waste ink removal, polymerization of aromatic compounds, radical-mediated polymerization and crosslinking reactions (e.g. paints, coatings, biomaterials), dye activation and organic compound combinations. have.

The present systems and compositions can also be used in cleaning compositions or components thereof, or detergents for use in cleaning methods. For example, laccases can be used to clean, process or care for laundry items such as clothing or fabrics; Cleaning of household hard surfaces; Tableware management including mechanical dishwashing applications; And soap bars and liquid and / or synthetic surfactant bars and liquids. The enzymes shown herein may be useful, for example, in stain removal / bleaching, and / or odor removal, and / or sanitation. Lacase mediators may be used as sanitizers and antimicrobial agents (eg tree protectants, detergents), independently or in combination with laccase enzymes.

Lacases can be used in other aspects of the personal care field. For example, laccases can be used in the manufacture of personal products for humans, such as perfumes, and skin care, hair care, oral hygiene, personal cleansing and deodorant and / or antiperspirant products for humans. Lacases include, for example, hair dyeing and / or bleaching, nail dyeing and / or bleaching; Skin pigmentation and / or bleaching; Surface modification (for example as a coupling reagent); As an antimicrobial agent; Odor removal; Teeth whitening; Or the like. Lacases can be used in the field of contact lens cleaning. For example, laccases can be used to clean, store, sterilize and / or preserve contact lenses.

Lacases can be used in the field of biomaterials. For example, laccases are biocatalysts for various organic reactions; And / or in relation to biopolymers; In relation to packaging; In relation to adhesives; In surface modification (active and binder); In the preparation of primary alcohols; In connection with biosensors and / or organic synthesis; Or the like. Lacases can use oxygen as the electron acceptor to oxidize various colored compounds having different chemical structures.

The present systems and compositions may also be used for the removal of lignin from lignocellulosic-containing materials (eg delignification of pulp), bleaching of lignocellulosic-containing materials (ie enzymatic ink removal of recycled paper) and / or paper or It can be used for the treatment of wastewater resulting from the production of cellulose. Such methods are prepared by non-aromatic redox agents + phenol and / or non-phenol aromatic redox compounds, lignin which is directly oxidized by the action of such phenols and / or non-phenolic aromatic compounds, or by the oxidative action of such compounds. Lacase enzymes can be used with metering or addition of lignin in phenolic and non-phenolic units oxidized by other phenolic and / or non-phenolic compounds.

Lacases can be used in other aspects related to pulp and paper. For example, laccases can be used to prepare paper pulp and fluff pulp from raw materials such as wood, bamboo and cereal rice straw; Manufacturing paper and boards for printing and writing, packaging, hygiene and other technical purposes; Use of cellulose fiber regeneration for paper and board manufacturing purposes; And wastes processed and produced in pulp or paper mills and other facilities specialized in the manufacture of paper, pulp or fluff. Lacases are for example wood processing; Pulp bleaching; Wood fiber deformation; Bio-glue for preparing MDF; For enhancing paper properties; Ink removal; Paper dyeing; Adhesives (eg lignin based adhesives for particle- or fiber boards); Or the like.

Lacases can be used in the field of feed. For example, laccases can be used as part of feed supplements aimed at increasing the nutritional value of any kind of animal feed, such as chickens, cattle, pigs, fish and pets, or as feed additives alone; And / or as processing aids for plant material processing and the food industry, with products aimed at producing materials / products suitable as feed raw materials.

Lacases can be used in the field of starch processing. For example, laccases can be used to process substrates containing starch and / or grains into glucose (dextrose) syrups, fructose syrups or any other syrups, alcohols (edible or fueled) or sugars. . Such starch processing may include liquefaction, saccharification, isomerization and debranching of the substrate.

Lacases can be used in the food sector. For example, laccase can be used as an active ingredient in food manufacturing, processing, or foods such as yellow fat, tea based beverages, culinary products, confectionery and frozen foods for human consumption. For example, laccases can be used as bread improvers, in food preservatives, as oxygen scavengers, and the like. Lacasees can be used to reduce or eliminate the microbial load of various foods (eg meat) or feed.

Any method or use for a laccase described herein may be, for example, a temperature of less than about 40 ° C., such as less than about 40 ° C., less than about 37 ° C., less than about 35 ° C., less than about 32 ° C., less than about 30 ° C. , Less than about 27 ° C., less than about 25 ° C., and less than about 22 ° C. Exemplary temperature ranges range from about 20 ° C to less than about 40 ° C. Exemplary temperatures are 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 ℃. In some embodiments, the temperature is at ambient temperature or at room temperature, for example from about 20 ° C to about 23 ° C.

Any method or use for the laccases described herein can be performed using any of the laccase enzymes described herein, eg, laccases from Serrena unicolor. In some embodiments, the laccase is used at a concentration of about 0.005 to about 5000 mg / l, about 0.05 to about 500 mg / l, about 0.1 to about 100 mg / l, or about 0.5 to about 10 mg / l. In some denim processing embodiments, the laccase is about 0.005 to about 5000 mg / kg (denim), about 0.05 to about 500 mg / kg (denim), about 0.1 to about 100 mg / kg (denim), or about 0.5 to It is used at a concentration of about 10 mg / kg (denim). In some embodiments, the laccase is used at a pH of about 5 to about 7, about 5.5 to about 6.5, about 5 to about 6, or about 6. Exemplary pH values are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

Immediately Available  Composition and Kit

As described above, the composition comprises one or more laccases, and optionally one or more mediators. 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 A polypeptide comprising, consisting of or consisting essentially of an amino acid sequence selected from ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, 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 SEQ ID NOs: 19 and 20, or a variant or fragment thereof. Preferably, the polypeptide has enzymatic laccase activity that can be measured using the assays and procedures described herein.

Such compositions may also be provided in the form of “right-to-use” (RTU) compositions comprising, consisting essentially of, or consisting of laccase enzymes and mediators. In some embodiments, the mediator may include acetosyringone, cyringaldehyde, cyringimide, methyl cyringimide, 2-hydroxyethyl syringimide, methyl syringegate, cyringonitrile, dimethylsyringimide, and syringic acid. Is selected. In one embodiment, the mediator is cyringonitrile (4-hydroxy-3,5-dimethoxybenzonitrile). The RTU composition may further contain one or more compounds to provide a pH buffer when the composition is in solution. For example, in some embodiments, the composition contains monosodium phosphate and adipic acid as a buffer system. The RTU composition may be in solid, granular form for ease of storage and transport. The composition is then diluted with water to provide an aqueous solution, for example as described. The RTU composition may also include any number of additional reagents such as dispersants, surfactants, blockers, polymers, preservatives, and the like.

The following examples are provided to illustrate the systems, compositions, and methods, which should not be construed as limiting in any way. Other aspects and embodiments will be apparent to those skilled in the art in view of the detailed description.

Example

The following enzyme nomenclature was used in the examples.

Example  One - Stonewashed Denim Lacase Effect of Temperature on Media Color Deformation

enzyme

Granular laccase D enzyme (38,000 U / g) from Serena Uricolor was used for this experiment. One unit of laccase is based on the ability of the laccase enzyme to oxidize ABTS (2,2'-azinobis (3-ethylbenzthiazoline6-sulfonate)) to its corresponding stable cationic radical, ABTS ⁺ . Is defined as the amount of laccase activity that oxidizes 1 nmol of ABTS substrate per second under the conditions of the assay. The accumulation of radicals causes the ABTS to turn dark green and cause an increase in absorbance at 420 nm. Color formation is proportional to laccase activity and monitored for laccase standards.

Intermediary

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

step

Twelve denim legs weighed at approximately 3 kg (total) were called on a Unimac UF 50 washing machine under the following conditions:

160 아밀라아제 (Genencor) 및 0.5 g/ℓ (15 g) 의 비이온성 계면활성제 [예를 들어, Rucogen BFA (Rudolf Chemie) 또는 Ultravon RW (Huntsman)] 로, 10:1 액비 50℃ 에서 15 분 동안 호발시킴. And 0.5 g / ℓ (15 g) OPTISIZE of

Called with 160 amylase (Genencor) and 0.5 g / l (15 g) of nonionic surfactant [eg Rucogen BFA (Rudolf Chemie) or Ultravon RW (Huntsman)] for 10 minutes at 50 ° C Sikkim.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

After the firing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:

And 10: 1 liquid ratio in the cooled rinse for 5 minutes.

2XL 셀룰라아제 (Genencor) 로, 10:1 액비 55℃ 에서 60 분 동안 스톤워시함. And 1 kg of pumice (pumice stone), pH 4.5 ( 1 g / ℓ tri-sodium citrate dihydrate and 1 g / ℓ of citric acid monohydrate) and 1.2 g / ℓ INDIAGE

Stone wash with 2XL cellulase (Genencor) at 55 ° C. for 10 min ratio.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

After stonewash, the laccase treatment was carried out in a Unimac UF 50 washing machine according to the following procedure:

Base 268 및 NOVOPRIME

F258 (pH 4.8) (0.29 g/ℓ 제1인산염 및 0.56 g/ℓ 의 아디프산) 로, 40℃ 또는 30℃ 의 온도에서, 10:1 액비에서 30 분. And (i) C. Wu Nicole L'lacquer kinase D and ache and nitriles (pH 6) (0.7 g / ℓ first phosphate and 0.17 g / ℓ as adipic acid) with, 40 ℃, at 30 ℃ or temperature of 23 ℃ Or (ii) NOVOPRIME

Base 268 and NOVOPRIME

30 min at 10: 1 liquid ratio at a temperature of 40 ° C. or 30 ° C. with F258 (pH 4.8) (0.29 g / l monophosphate and 0.56 g / l adipic acid).

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

Denim  Pants rating

The amount of color deformation of the denim, represented by "bleaching", was evaluated with a Minolta colorimeter CR 310 in a CIE Lab color space with a D 65 light source after lacase treatment. The CIE color space (also known as CIELUV color space) was adapted by the 1976, International Commission on Illumination (CIE), which calculated the values L *, u * and v * calculated as follows: Contains:

In the above formula,

Y: tristimulus Y (tristimulus Y ₁₀ may also be used)

u ', v': chromaticity coordinates from the CIE 1976 UCS plot

Y ₀ , U ' ₀ , V' ₀ : Tristimulus values Y (or Y ₁₀ ) and chromaticity coordinates u ', v' of the fully diffuse reflecting surface

For denim pants, 8 measurements were taken and the results from 12 pants (96 measurements in total) were averaged. The results are shown in Tables 1 and 2, and FIG. 1.

C. Unikollor Lacase  And Cyringonitrile  Results used C. Woori Color Laca Agase concentration, g / l (U / ml) Cyringonitrile  Concentration, g / ℓ ( mM ) Temperature, ℃ Bleaching level,
CIE * Lab Standard Deviation 0.54
(20.5) 0.07
(0.39) 40 38.3 / -1.2 / -12.0 0.5 / 0.1 / 0.1 0.3
(11.4) 0.07
(0.39) 40 37.6 / -0.5 / -12.3 0.6 / 0.1 / 0.1 0.15
(5.7) 0.07
(0.39) 40 36.4 / -0.2 / -12.8 0.5 / 0.1 / 0.1 0.54
(20.5) 0.07
(0.39) 30 36.2 / -0.2 / -12.8 0.5 / 0.1 / 0.1 0.3
(11.4) 0.07
(0.39) 30 36.1 / -0.2 / -13.0 0.5 / 0.1 / 0.1 0.15
(5.7) 0.07
(0.39) 30 35.3 / 0.0 / -13.3 0.5 / 0.1 / 0.1 0.15
(5.7) 0.07
(0.39) 23
(No steam) 34.0 / 0.3 / -13.5 0.6 / 0.1 / 0.1

A. Orizae  And methyl The syringe gate  Results used

Base 268 농도, g/ℓ NOVOPRIME

Base 268 concentration, g / l NOVOPRIME

F258 concentration, g / l ( mM ) Temperature, ℃ Bleaching level,
CIE * Lab Standard Deviation 0.47 0.07
(0.33) 40 36.2 / -0.5 / -11.2 0.6 / 0.1 / 0.2 0.27 0.07
(0.33) 40 36.5 / -0.4 / -11.4 0.6 / 0.1 / 0.2 0.15 0.07
(0.33) 40 35.7 / -1.0 / -11.9 0.5 / 0.1 / 0.2 0.15 0.07
(0.33) 30 33.9 / 0.1 / -12.6 0.5 / 0.1 / 0.2

The results show the effectiveness of C. unicolor laccases and cyringonitrile in influencing the color change of stonewashed denim.

Example  2 - Stonewashed Denim  For color variations Lacase : Intermediary  Influence of Rate

step

Twelve denim pants weighed at approximately 3 kg (total) were called and stonewashed as described in Example 1. After Stonewash, lacase treatment was performed on a Unimac UF 50 washing machine according to the following procedure:

And C. Wu Nicole L'lacquer kinase D and ache and a nitrile, in 40 ℃ 10: 1 liquid ratio, for 30 minutes at pH 6 (0.7 g / ℓ first phosphate and 0.17 g / ℓ as adipic acid).

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

Denim  Pants rating

Color variations of the denim pants were evaluated as described in Example 1. The results are shown in Table 3 and FIG. 2.

At different rates C. Unikollor Lacase  And Cyringonitrile  Results used C. Unikollor la Casein concentration, g / l (U / ml) Intermediary  Concentration, g / ℓ ( mM ) Temperature, ℃ Bleaching level,
CIE * Lab Standard Deviation 0.15
(5.7) 0.07
(0.39) 40 36.4 / -0.2 / -12.8 0.5 / 0.1 / 0.1 0.15
(5.7) 0.08
(0.44) 40 37.0 / -0.4 / -12.7 0.5 / 0.1 / 0.1 0.15
(5.7) 0.1
(0.55) 40 37.1 / -0.4 / -12.7 0.6 / 0.1 / 0.1

The results indicate that the ratio of laccase enzyme to mediator can be manipulated to alter color modification.

Example  3 - Stonewashed In denim Lacase  And Intermediary  Effect of temperature on color deformation performance of containing composition

In order to investigate the laccase-mediated color modification performance at low temperatures, a "ready to use" (RTU) composition was prepared as shown in Table 4. Monophosphate and adipic acid provide a buffering function at about pH 6 for use applications as described below.

 Immediately Available  Formulation ingredient % w / w Monophosphate (anhydrous) 70 Adipic acid 7 C. Unikol Lacase D Granules (38,000 U / g) 15 Cyringonitrile 8

step

Twelve denim pants weighed at approximately 3 kg (total) were called and stonewashed as described in Example 1. After Stonewash, lacase treatment was performed on a Unimac UF 50 washing machine according to the following procedure:

II S (Novozymes) 로, 유입 스팀이 없이 (즉, 21-22℃ 의 온도) 또는 30℃ 에서 10:1 액비에서 30 분. And to the concentration and temperature as described in Tables 5 and 6, the above-described kinase RTU lacquer composition or DENILITE

With II S (Novozymes), 30 minutes without inlet steam (ie, a temperature of 21-22 ° C.) or at 10: 1 liquid ratio at 30 ° C.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

Denim  Pants rating

Color variations of the denim pants were evaluated as described in Example 1. The results are shown in Tables 5 and 6 and FIGS. 3 and 4.

C. Unikollor RTU  Results of using the composition RTU  Lacase,
% owg * Temperature, ℃ Bleaching level,
CIE * Lab Standard Deviation One 30 35.1 / -0.7 / -13.5 0.6 / 0.1 / 0.2 3 30 38.5 / -1.3 / -12.6 0.7 / 0.1 / 0.2 One 21-22 33.3 / -0.4 / -13.6 0.6 / 0.1 / 0.1 3 21-22 37.2 / -1.013.3 0.7 / 0.1 / 0.1 * "owg" = for product weight

A. Orizae Lacase RTU  Results of using the composition

II S,
% owg DENILITE

II S,
% owg Temperature, ℃ Bleaching level,
CIE * Lab Standard Deviation 3 30 36.1 / -1.3 / -10.9 0.6 / 0.1 / 0.2 3 21-22 33.8 / -0.8 / -12.1 0.5 / 0.1 / 0.2

The results indicate that the C. unicalor laccase RTU composition provides good color modification at low temperatures compared to conventional commercial laccase compositions.

Example  4 - At 30 ° C  Stage 1 Stone Wash  And color variations

Twelve denim trousers weighed at approximately 3 kg (total) were called on a Unimac UF 50 washing machine as described in Example 1.

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

Super GX 셀룰라아제 (Genencor) + 실시예 3 에서 기재된 바와 같은 3% owg RTU 라카아제 조성물 (즉, "스톤워시 + 표백 1-단계") 또는 (ii) INDIAGE

Super GX 셀룰라아제 단독 (즉, "스톤워시 단독"), 10:1 액비, pH 6 에서 30℃, 30 분. And (i) 0.4% owg INDIAGE

Super GX Cellulase (Genencor) + 3% owg RTU Lacase Composition as described in Example 3 (ie "Stonewash + Bleaching 1-Step") or (ii) INDIAGE

Super GX Cellulase alone (ie “Stonewash only”), 10: 1 liquid ratio, 30 ° C., pH 6, 30 min.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes. Pumice was not used.

The results are shown in Table 7 and FIG. 5.

Stage 1 Stone Wash  And the result of color variations Temperature (℃) Bleaching level, CIE * Lab Standard Deviation Stone Wash  Exclusive 30 25.4 / 1.3 / -12 0.3 / 0.1 / 0.3 Stone Wash + Bleaching, step 1 30 27.3 / 0.6 / -12.2 0.5 / 0.2 / 0.2

The results indicate that color modifications can be achieved using both laccase and cellulase simultaneously.

Example  5 - At 30 ° C  2-step Stone Wash  And color variations

Twelve denim trousers weighed at approximately 3 kg (total) were called on a Unimac UF 50 washing machine as described in Example 1.

After the firing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:

Super GX 셀룰라아제 (Genencor), 10:1 액비, pH 5.5 에서 30℃, 30 분. And 0.4% owg INDIAGE

Super GX Cellulase (Genencor), 10: 1 liquid ratio, 30 ° C., pH 5.5, 30 min.

After stonewash, the denim was bleached in a Unimac UF 50 washing machine under the following conditions:

3% as described in Example 3 and owg RTU azepin lacquer composition, 10: 1 liquid ratio, 30 ℃ at pH 6, 30 min.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes. Pumice was not used.

The results are shown in Table 8 and FIG. 5. The two-step stonewash and color deformation results were compared with the results for the stonewash alone described in Example 4.

2-step Stone Wash  And color transformation results Temperature
(° C) Bleaching level,
CIE * Lab Standard Deviation Stone Wash  Exclusive 30 25.4 / 1.3 / -12 0.3 / 0.1 / 0.3 Stone Wash + Bleaching, step 2 30 31.9 / -0.3 / -12.9 0.6 / 0.2 / 0.1

The results indicate that color modification by laccase treatment can be achieved after stone wash.

Example  6 - Stone Wash  without At 30 ° C Denim Lacase -Media color variations

Twelve denim trousers weighed at approximately 3 kg (total) were called on a Unimac UF 50 washing machine as described in Example 1.

After firing, the denim was bleached in a Unimac UF 50 washing machine under the following conditions:

3% as described in Example 3 and owg RTU azepin lacquer composition, 10: 1 liquid ratio, 30 ℃ at pH 6, 30 min.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes. Pumice was not used.

The results are shown in Table 9 and FIG. 5. The color deformation results were compared with the results for the Stonewash alone described in Example 4.

Stone Wash  The result of missing color variations Temperature (℃) Bleaching level,
CIE * Lab Standard Deviation Stone Wash  Exclusive 30 25.4 / 1.3 / -12 0.3 / 0.1 / 0.3 Bleached, No- Stone Wash
30 26.9 / 0.7 / 12.1 0.5 / 0.1 / 0.2

The results indicate that the amount of color modification produced by the lacasease treatment without stonewash is higher than that with stonewash alone.

Example  7 - Pumice  Without single- Bath  Cellulase in the process and Lacazero  Stone wash and color variations

This example demonstrates that effective stone washes and color modifications can be obtained using laccases and cellulases in a single-bath process.

enzyme

EcoFade LT 100 라카아제 (배치 번호 780913616, 6,292 GLacU/g).PRIMAGREEN

EcoFade LT 100 Lacase (Batch No. 780913616, 6,292 GLacU / g).

step

Starting material was called denim weighed approximately 3 kg (ballast + 2 pants (evaluation)).

The denim was stonewashed in a Renzacci LX 22 washing machine under the following conditions:

Neutra L 셀룰라아제 (배치 번호 40105358001 활성 5197 NPCNU/g) (Genencor), 10:1 액비, pH 6.5 에서 50℃, 40 분. ㆍ 0.4% owg INDIAGE

Neutra L Cellulase (Batch No. 40105358001 Activity 5197 NPCNU / g) (Genencor), 10: 1 liquid ratio, 50 ° C., pH 6.5 at pH 6.5, 40 min.

And-rescue dry for evaluation Remove the 1 after the stone wash pants.

And after the stone wash, without drainage of the bath (i.e., loading), the two color-rescue transformation under the conditions of the second to denim:

EcoFade LT 100, 10:1 액비에서 40℃, 40 분. ㆍ 1% owg RTU PRIMAGREEN

EcoFade LT 100, 40 ° C., 10 min at 10: 1 liquid ratio.

And two-thirds times during cooling rinsing.

And the denim was dried in industrial dryers.

Denim  Pants rating

After the laccase treatment and after the cellulase treatment, color variations and stonewashes for the denim pants were evaluated with a Minolta colorimeter CR 310 in a CIE Lab color space with a D 65 light source. Six measurements were taken for each pant and the results averaged.

The results are summarized in Table 10 below. The amount of color modification obtained by sequential (ie, two-step) addition of cellulases and laccases in a single bath was greater than that obtained by adding cellulases and laccases at the same time as in Example 4.

Bleaching level, CIE * Lab Standard Deviation Stone Wash 27.8 / 1.1 / -13.2 0.3 / 0.1 / 0.1 Stone Wash + Bleaching, single Bath 34.7 / 0.0 / -12.2 0.5 / 0.1 / 0.1

The results indicate that the amount of color modification obtained by the sequential (ie two-step) addition of cellulase and laccase in a single bath is greater than that obtained by adding cellulase and laccase at the same time as in Example 4.

Example  8 - Lacase  And Pumice  Color variations

This example shows that effective stone washes and color variations can be obtained using pumice and laccase-mediated systems in a single-bath process.

enzyme

EcoFade LT 100 라카아제 (배치 번호 7809136160, 6,292 GLacU/g).PRIMAGREEN

EcoFade LT 100 Lacase (Batch No. 7809136160, 6,292 GLacU / g).

step

Twelve denim trousers weighed at approximately 3 kg (total) were called on a Unimac UF 50 washing machine as described in Example 1.

After the firing, the denim was stonewashed in a Unimac UF 50 washing machine under the following conditions:

EcoFade LT100 (Genencor) 로, 3 kg 의 부석, 10:1 액비에서 30℃, 30 분. 물 중 부석만을 사용하여 블랭크/대조군을 수행하였음. And 3% PRIMAGREEN

With EcoFade LT100 (Genencor), 3 kg pumice, 30 ° C., 30 min at 10: 1 liquor ratio. Blank / control was performed using only pumice in water.

And 30: 1 liquid ratio in the rinse twice cooling for 5 minutes.

Denim  Pants rating

After the laccase treatment and after the stonewash treatment, the color deformation for the denim pants was evaluated with a Minolta colorimeter CR 310 in a CIE Lab color space with a D 65 light source. The average of eight measurements taken outside each pants was reported as the level of bleaching. The average of four measurements taken inside each pant was reported as the restaining level.

The results are summarized in Tables 11 and 12 below.

Bleaching level, CIE * Lab Standard Deviation Stone Wash 25.5 / 1.1 / -11.4 0.3 / 0.1 / 0.2 Color variations 28.7 / 0.3 / -12.0 0.6 / 0.1 / 0.1

Restaining level, CIE * Lab Standard Deviation Stone Wash 50.6 / -1.2 / -5.5 0.4 / 0.1 / 0.3 Stone Wash + Color variations, single Bath 52.2 / -1.2 / -4.0 0.4 / 0.1 / 0.3

The results show that the laccase treatment provides color modification even in the presence of pumice and also shows a reduction / elimination of restaining.

Example  9-of yellow-dyed clothing Stone Wash  And color variations

Test garments were made of 100% cotton Twill fabric dyed with sulfur khaki brown dye. 21 garments weighed at approximately 7 kg (total) were stonewashed at 25 kg belly washer (36 rpm) under the following conditions:

2XL 에서, 18:1 액비, pH 4.5 에서 55℃, 45 분. And 1 g / ℓ of INDIAGE

In 2XL, 18: 1 liquid ratio, pH 4.5 at 55 ° C., 45 min.

Once cooled in the rinsing liquid ratio 1: 12 and 3 minutes for. Pumice was not used.

After washing and drying clothes hayeoteum for evaluation.

EcoFade LT 100 으로 처리하였음: And PRIMAGREEN under the following conditions for the three clothes (total of approximately 1 kg) Stone Wash as described above

Treated with EcoFade LT 100:

EcoFade LT 100, 50:1 액비에서 40℃, 15, 30 또는 45 분. 물만으로 15, 30 또는 45 분 동안 세척한 의류로 블랭크/대조군을 수행하였음. And 1, 2 or 3 g / ℓ PRIMAGREEN

EcoFade LT 100, 40 ° C., 15, 30 or 45 minutes at 50: 1 liquid ratio. Blank / control was performed with clothing washed for 15, 30 or 45 minutes with water only.

And a third time for cooling the rinse.

The denim-rescue and dried in industrial dryers.

Denim  Pants rating

After the laccase treatment and after the stonewash treatment, the color deformation and stonewash of the yellow dyed garments were evaluated with Minolta colorimeter CR 310 in the CIE Lab color space with D 65 light source as above. Ten measurements were taken for each garment and the results averaged.

The results are summarized in Table 13 below.

process CIE * Lab Before processing 40.2 / 2.2 / 20.8 Whole complex cellulase 43.7 / 2.1 / 19.2 Blank, 15 minutes 44.9 / 1.3 / 18.3 Blank, 30 minutes 46.1 / 1.3 / 19.1 Blank, 45 minutes 46.6 / 1.3 / 18.5

Ecofade (1 g/ℓ) 15 분PRIMAGREEN

Ecofade (1 g / ℓ) 15 minutes 45.1 / 2.9 / 16.5 PRIMAGREEN

Ecofade (1 g / ℓ) 30 minutes 45.5 / 3.3 / 16.8 PRIMAGREEN

Ecofade (1 g / ℓ) 45 minutes 44.7 / 3.2 / 16.4 PRIMAGREEN

Ecofade (2 g / ℓ) 15 minutes 44.7 / 3.3 / 15.9 PRIMAGREEN

Ecofade (2 g / ℓ) 30 minutes 45.3 / 3.5 / 15.9 PRIMAGREEN

Ecofade (2 g / ℓ) 45 minutes 45.0 / 3.5 / 15.6 PRIMAGREEN

Ecofade (3 g / ℓ) 15 minutes 44.4 / 3.4 / 15.5 PRIMAGREEN

Ecofade (3 g / ℓ) 30 minutes 44.6 / 3.6 / 15.7 PRIMAGREEN

Ecofade (3 g / ℓ) 45 minutes 45.0 / 3.6 / 15.4

The results indicate that the a and b values of the color space changed significantly as compared to the blank as well as the untreated fabric. Deformation to the cast of the garment is visible to the naked eye.

Example  10 - Stone Wash  Color variation of yellow-dyed clothing without

Three garments made of 100% cotton Twill fabric dyed with sulfur khaki brown dye and weighed at approximately 1 kg (total) were treated at 5 kg belly washer (36 rpm) under the following conditions:

EcoFade LT 100, 40:1 액비에서 40℃, 15, 30 또는 45 분. 물 만으로 15, 30 또는 45 분 동안 세척한 의류로 블랭크/대조군을 수행하였음. And 1, 2 or 3 g / ℓ PRIMAGREEN

EcoFade LT 100, 40 ° C., 15, 30 or 45 minutes at 40: 1 liquid ratio. Blank / control was performed with clothing washed for 15, 30 or 45 minutes with water only.

And a third time for cooling the rinse.

The denim-rescue and dried in industrial dryers.

Denim  Pants rating

After laccase treatment and stonewash treatment, color variations and stonewashes for sulfur dyed garments were evaluated with Minolta colorimeter CR 310 in CIE Lab color space with D 65 light source. Ten measurements were taken for each garment and the results averaged.

The results are summarized in Table 14 below.

process CIE * Lab Before processing 40.2 / 2.2 / 20.8 Blank 15 minutes 41.1 / 2.0 / 19.7 Blank 30 minutes 41.9 / 2.1 / 20.4 Blank 45 minutes 42.4 / 2.1 / 20.29

Ecofade (1 g/ℓ) 15 분PRIMAGREEN

Ecofade (1 g / ℓ) 15 minutes 41.1 / 3.7 / 17.6 PRIMAGREEN

Ecofade (1 g / ℓ) 30 minutes 41.5 / 4.2 / 18.6 PRIMAGREEN

Ecofade (1 g / ℓ) 45 minutes 41.6 / 4.0 / 18.1 PRIMAGREEN

Ecofade (2 g / ℓ) 15 minutes 40.4 / 3.9 / 17.0 PRIMAGREEN

Ecofade (2 g / ℓ) 30 minutes 41.2 / 4.2 / 17.3 PRIMAGREEN

Ecofade (2 g / ℓ) 45 minutes 41.6 / 4.3 / 17.3 PRIMAGREEN

Ecofade (3 g / ℓ) 15 minutes 40.5 / 4.0 / 16.6 PRIMAGREEN

Ecofade (3 g / ℓ) 30 minutes 41.0 / 4.2 / 17.1 PRIMAGREEN

Ecofade (3 g / ℓ) 45 minutes 40.6 / 4.3 / 17.0

The results indicate that the a and b values of the color space were significantly changed compared to the blank as well as the untreated fabric. Deformation to the cast of the garment is visible to the naked eye.

Example  11-surfactants and Pumice  Single-present Bath  Cellulase in the process and Lacazero Stone Wash  And bleaching performance

enzyme

EcoFade LT 100 라카아제 (배치 번호 780913616, 6,292 GLacU/g).PRIMAGREEN

EcoFade LT 100 Lacase (Batch No. 780913616, 6,292 GLacU / g).

step

Twelve denim garments weighed to 10 kg (total) and dyed with pure indigo were called on a Tupesa front loading machine (36 rpm) under the following conditions:

And 0.5 g / ℓ lubricant, 0.2 g / ℓ dispersing agents (nonionic surfactant) of and 0.2 g / ℓ of polyester blockers (non-ionic hydrophilic copolymer), 10: 1 liquid ratio, 40 ℃ at pH 7, 10 bun .

After firing, the denim was stonewashed under the following conditions:

Super GX 셀룰라아제 (Genencor) 로, 5:1 액비, pH 6 에서 47℃, 30 분. 1 개의 의류를 평가를 위해 빼내었음. And 7 kg of pumice, 4% owg of INDIAGE

With Super GX Cellulase (Genencor), 5: 1 liquid ratio, 47 ° C. at pH 6, 30 min. One garment was taken out for evaluation.

And after the stone wash, without drainage (dropping) the bath, under the conditions to hayeoteum bleaching denim:

EcoFade LT 100, 5:1 액비에서 47℃, 30 분. ㆍ 2% owg RTU PRIMAGREEN

EcoFade LT 100, 47 ° C. in a 5: 1 liquor, 30 min.

In liquid ratio 1:50 and 2 minutes 2 times for rinsing cooling.

The denim-rescue and dried in industrial dryers.

Denim  Pants rating

After laccase treatment and after cellulase treatment, color variations and stonewashes for denim were evaluated with Minolta colorimeter CR 310 in the CIE Lab color space with D 65 light source. Eight measurements were taken for each pants and the results averaged.

The results are summarized in Table 15 below.

Bleaching level CIE * Lab Standard Deviation Stone Wash 27.6 / 0.6 / -12.0 0.5 / 0.1 / 0.1 Surfactants and Pumice  In existence,
Stone Wash + Bleaching single Bath 32.3 / -0.1 / -12.8 0.7 / 0.1 / 0.1

The results indicate that color modification by laccase treatment occurs in the presence of pumice and in the presence of surfactants.

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 included within the spirit and scope of the present application and the scope of the appended claims. All publications and patent documents cited herein are incorporated by reference in their entirety.

                         SEQUENCE LISTING <110> Danisco US Inc.        Ashton, Wayne        Krouwer, Andreas J.        McAuliffe, Joseph C.        Pericu, Piera        Wang, Huaming   <120> Laccases and Methods of Use Thereof at Low Temperature <130> 31342WO-2 <140> PCT / US2009 / 069229 <141> 2009-12-22 <150> US 61 / 237,532 <151> 2009-08-27 <150> US 61 / 154,882 <151> 2009-02-24 <150> US 61 / 140,724 <151> 2008-12-24 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 2363 <212> DNA <213> Cerrena unicolor <400> 1 atgagctcaa agctacttgc tcttatcact gtcgctctcg tcttgccact aggcaccgac 60 gccggcatcg gtcctgttac cgacttgcgc atcaccaacc aggatatcgc tccagatggc 120 ttcacccgac cagcggtact agctgggggc acattccctg gagcacttat taccggtcag 180 aaggtatggg agatcaactt ggttgaatag agaaataaaa gtgacaacaa atccttatag 240 ggagacagct tccaaatcaa tgtcatcgac gagcttaccg atgccagcat gttgacccag 300 acatccattg tgagtataat ttaggtccgc tcttctggct atcctttcta actcttaccg 360 tctagcattg gcacggcttc tttcagaagg gatctgcgtg ggccgatggt cctgccttcg 420 ttactcaatg ccctatcgtc accggaaatt ccttcctgta cgactttgat gttcccgacc 480 aacctggtac tttctggtac catagtcact tgtctactca atattgcgat ggtcttcgtg 540 gcccgttcgt tgtatacgat ccaaaggatc ctaataaacg gttgtacgac attgacaatg 600 gtatgtgcat catcatagag atataattca tgcagctact gaccgtgact gatgctgcca 660 gatcatacgg ttattaccct ggcagactgg taccacgttc tcgcaagaac tgttgtcgga 720 gtcgcgtaag tacagtctca cttatagtgg tcttcttact cattttgaca taggacaccc 780 gacgcaacct tgatcaacgg tttgggccgt tctccagacg ggccagcaga tgctgagttg 840 gctgtcatca acgttaaacg cggcaaacgg tatgttattg aactcccgat ttctccatac 900 acagtgaaat gactgtctgg tctagttatc gatttcgtct ggtctccatc tcatgtgacc 960 ctaattacat cttttctatc gacaaccatt ctatgactgt catcgaagtc gatggtgtca 1020 acacccaatc cctgaccgtc gattctattc aaatcttcgc aggccaacga tactcgttcg 1080 tcgtaagtct ctttgcacga ttactgcttc tttgtccatt ctctgacctg tttaaacagc 1140 tccatgccaa ccgtcctgaa aacaactatt ggatcagggc caaacctaat atcggtacgg 1200 atactaccac agacaacggc atgaactctg ccattctgcg atacaacggc gcacctgttg 1260 cggaaccgca aactgttcaa tctcccagtc tcaccccttt gctcgaacag aaccttcgcc 1320 ctctcgtgta cactcctgtg gtatgtttca aagcgttgta atttgattgt ggtcattcta 1380 acgttactgc gtttgcatag cctggaaacc ctacgcctgg cggcgccgat attgtccata 1440 ctcttgactt gagttttgtg cggagtcaac attcgtaaag ataagagtgt ttctaatttc 1500 ttcaataata ggatgctggt cgcttcagta tcaacggtgc ctcgttcctt gatcctaccg 1560 tccccgttct cctgcaaatt ctcagcggca cgcagaatgc acaagatcta ctccctcctg 1620 gaagtgtgat tcctctcgaa ttaggcaagg tcgtcgaatt agtcatacct gcaggtgtcg 1680 tcggtggacc tcatccgttc catctccatg gggtacgtaa cccgaactta taacagtctt 1740 ggacttaccc gctgacaagt gcatagcata acttctgggt cgtgcgaagt gccggaaccg 1800 accagtacaa ctttaacgat gccattctcc gagacgtcgt cagtatagga ggaaccgggg 1860 atcaagtcac cattcgtttc gtggtatgtt tcattcttgt ggatgtatgt gctctaggat 1920 actaaccggc ttgcgcgtat agaccgataa ccccggaccg tggttcctcc attgccatat 1980 cgactggcac ttggaagcgg gtctcgctat cgtatttgca gagggaattg aaaatactgc 2040 tgcgtctaat ttaacccccc gtacgcggtt tccctcacat cctggagcta agcagcttac 2100 taacatacat ttgcagaggc ttgggatgag ctttgcccga agtataacgc gctcagcgca 2160 caaaagaagg ttgcatctaa gaaaggcact gccatctaat ttttgtaaca aacaaggagg 2220 gtctcttgta cttttattgg gatttctttc ttggggttta ttgttaaact tgactctact 2280 atgtttggaa gacgaaaggg gctcgcgcat ttatatacta tctctcttgg catcacctgc 2340 agctcaatcc ttcaaccacc taa 2363 <210> 2 <211> 523 <212> PRT <213> Cerrena unicolor <400> 2 Met Ser Ser Lys Leu Leu Ala Leu Ile Thr Val Ala Leu Val Leu Pro 1 5 10 15 Leu Gly Thr Asp Ala Gly Ile Gly Pro Val Thr Asp Leu Arg Ile Thr             20 25 30 Asn Gln Asp Ile Ala Pro Asp Gly Phe Thr Arg Pro Ala Val Leu Ala         35 40 45 Gly Gly Thr Phe Pro Gly Ala Leu Ile Thr Gly Gln Lys Gly Asp Ser     50 55 60 Phe Gln Ile Asn Val Ile Asp Glu Leu Thr Asp Ala Ser Met Leu Thr 65 70 75 80 Gln Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Ser Ala Trp                 85 90 95 Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Val Thr Gly Asn             100 105 110 Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Pro Gly Thr Phe Trp         115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro     130 135 140 Phe Val Val Tyr Asp Pro Lys Asp Pro Asn Lys Arg Leu Tyr Asp Ile 145 150 155 160 Asp Asn Asp His Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu                 165 170 175 Ala Arg Thr Val Val Gly Val Ala Thr Pro Asp Ala Thr Leu Ile Asn             180 185 190 Gly Leu Gly Arg Ser Pro Asp Gly Pro Ala Asp Ala Glu Leu Ala Val         195 200 205 Ile Asn Val Lys Arg Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile     210 215 220 Ser Cys Asp Pro Asn Tyr Ile Phe Ser Ile Asp Asn His Ser Met Thr 225 230 235 240 Val Ile Glu Val Asp Gly Val Asn Thr Gln Ser Leu Thr Val Asp Ser                 245 250 255 Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu His Ala Asn             260 265 270 Arg Pro Glu Asn Asn Tyr Trp Ile Arg Ala Lys Pro Asn Ile Gly Thr         275 280 285 Asp Thr Thr Thr Asp Ser Gly Met Asn Ser Ala Ile Leu Arg Tyr Asn     290 295 300 Gly Ala Pro Val Ala Glu Pro Gln Thr Val Gln Ser Pro Ser Leu Thr 305 310 315 320 Pro Leu Leu Glu Gln Asn Leu Arg Pro Leu Val Tyr Thr Pro Val Pro                 325 330 335 Gly Asn Pro Thr Pro Gly Gly Ala Asp Ile Val His Thr Leu Asp Leu             340 345 350 Ser Phe Asp Ala Gly Arg Phe Ser Ile Asn Gly Ala Ser Phe Leu Asp         355 360 365 Pro Thr Val Pro Val Leu Leu Gln Ile Leu Ser Gly Thr Gln Asn Ala     370 375 380 Gln Asp Leu Leu Pro Pro Gly Ser Val Ile Pro Leu Glu Leu Gly Lys 385 390 395 400 Val Val Glu Leu Val Ile Pro Ala Gly Val Val Gly Gly Pro His Pro                 405 410 415 Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Thr             420 425 430 Asp Gln Tyr Asn Phe Asn Asp Ala Ile Leu Arg Asp Val Val Ser Ile         435 440 445 Gly Gly Thr Gly Asp Gln Val Thr Ile Arg Phe Val Thr Asp Asn Pro     450 455 460 Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly 465 470 475 480 Leu Ala Ile Val Phe Ala Glu Gly Ile Glu Asn Thr Ala Ala Ser Asn                 485 490 495 Leu Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Ala Leu             500 505 510 Ser Ala Gln Lys Lys Leu Asn Pro Ser Thr Thr         515 520 <210> 3 <211> 2374 <212> DNA <213> Cerrena unicolor <400> 3 atgagctcaa agctacttgc tcttattact gtcgctctcg tcttgccact aggcactgac 60 gccggcatcg gtcctgttac cgacttgcgc atcaccaacc aggatatcgc tccagatggc 120 ttcacccgac cagctgtact ggctgggggc acattccccg gagcactgat taccggtcag 180 aaggtatggg agatcgattt cgttgaatag agaaatacaa ctgaaaacaa attcttatag 240 ggagacagct tccaaatcaa tgtcatcgac gagcttaccg atgccagcat gttgacccag 300 acatccattg tgagtataat atgggtccgc tcttctagct atcctttcta actcttaccc 360 tctagcattg gcacggcttc tttcagaagg gatctgcgtg ggccgatggt cctgccttcg 420 ttactcaatg tcctatcgtc accggaaatt ccttcctgta cgactttgat gtccccgacc 480 aacctggtac tttctggtac catagtcact tgtctactca atattgcgat ggtcttcggg 540 gcccgttcgt tgtatacgat ccaaaggatc ctaataaacg gttgtacgac attgacaatg 600 gtatgtgcat catcataaaa atataattca tgcagctact gaccgcgact gatgctgcca 660 gatcatacgg ttattaccct ggcagactgg taccacgttc tcgcacgaac tgttgtcgga 720 gtcgcgtaag tacagtctga cttatagtgg tcttcttact cattttgaca taggacaccc 780 gacgcaacct tgatcaacgg tttgggccgt tctccagacg ggccagcaga tgctgagttg 840 gctgtcatca acgttaaacg cggcaaacgg tatgtcattg aactcccgat ttctccattc 900 acattgaaat gactgtctgg tctagttatc gattccgtct ggtctccatc tcatgtgacc 960 ctaattacat cttttctatc gacaaccatt ctatgactgt catcgaagtc gatggtgtca 1020 acacccaatc cctgaccgtc gattctatcc aaatcttcgc aggccaacgc tactcgttcg 1080 tcgtaagtct ctttgaatgg ttggtgcttt ttctgtccat tctctaacct gtttatacag 1140 ctccatgcca accgtcctga aaacaactat tggatcaggg ccaaacctaa tatcggtacg 1200 gatactacca cagacaacgg catgaactct gccattctgc gatacaacgg cgcacctgtt 1260 gcggaaccgc aaactgttca atctcccagt ctcacccctt tgctcgaaca gaaccttcgc 1320 cctctcgtgt acactcctgt ggtatgtttc aaagcgttgt aatttgattg tggtcattct 1380 aacgttactg cctttgcaca gcctggaaat cctacgcctg gcggggccga tattgtccat 1440 actcttgact tgagttttgt gcggagtcaa cattcgtaaa gataagagtg tttctaattt 1500 cttcaataat aggatgctgg tcgcttcagt atcaacggtg cctcgttcct tgatcctacc 1560 gtccctgttc tcctgcaaat tctcagcggc acgcagaatg cacaagatct actccctcct 1620 ggaagtgtga ttcctctcga attaggcaag gtcgtcgaat tagtcatacc tgcaggtgtt 1680 gtcggtggac ctcatccgtt ccatctccat ggggtacgta acccgaactt ataacagtct 1740 tggacttacc cgctgacaag tgtatagcat aacttctggg tcgtgcgaag tgccggaacc 1800 gaccagtaca actttaacga tgccattctc cgagacgtcg tcagtatagg aggaaccgag 1860 gatcaagtca ccattcgatt cgtggtatat acttcattct tgtggatgta tgtgctctag 1920 gatactaact ggcttgcgcg tatagaccga taaccccgga ccgtggttcc tccattgcca 1980 tatcgactgg cacttggaag cgggtctcgc tatcgtattt gcagagggaa ttgaaaatac 2040 tgctgcgtct aatccaaccc cccgtatgcg gtttcccaca cattctgaat ctaagcagct 2100 tactaatata catttgcaga ggcttgggat gagctttgcc cgaagtataa cgcgctcaac 2160 gcacaaaaga aggttgcatc taagaaaggc actgccatct aatccttgta acaaacaagg 2220 agggtctctt gtacttttat tgggatttat ttcttggggt ttattgttca acttgattct 2280 actatgtttg gaagtagcga ttacgaaagg ggcttgcgca tttatatacc atctttcttg 2340 gcaccacctg cagctcaatc cttcaaccac ctaa 2374 <210> 4 <211> 523 <212> PRT <213> Cerrena unicolor <400> 4 Met Ser Ser Lys Leu Leu Ala Leu Ile Thr Val Ala Leu Val Leu Pro 1 5 10 15 Leu Gly Thr Asp Ala Gly Ile Gly Pro Val Thr Asp Leu Arg Ile Thr             20 25 30 Asn Gln Asp Ile Ala Pro Asp Gly Phe Thr Arg Pro Ala Val Leu Ala         35 40 45 Gly Gly Thr Phe Pro Gly Ala Leu Ile Thr Gly Gln Lys Gly Asp Ser     50 55 60 Phe Gln Ile Asn Val Ile Asp Glu Leu Thr Asp Ala Ser Met Leu Thr 65 70 75 80 Gln Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Ser Ala Trp                 85 90 95 Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Val Thr Gly Asn             100 105 110 Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Pro Gly Thr Phe Trp         115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro     130 135 140 Phe Val Val Tyr Asp Pro Lys Asp Pro Asn Lys Arg Leu Tyr Asp Ile 145 150 155 160 Asp Asn Asp His Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu                 165 170 175 Ala Arg Thr Val Val Gly Val Ala Thr Pro Asp Ala Thr Leu Ile Asn             180 185 190 Gly Leu Gly Arg Ser Pro Asp Gly Pro Ala Asp Ala Glu Leu Ala Val         195 200 205 Ile Asn Val Lys Arg Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile     210 215 220 Ser Cys Asp Pro Asn Tyr Ile Phe Ser Ile Asp Asn His Ser Met Thr 225 230 235 240 Val Ile Glu Val Asp Gly Val Asn Thr Gln Ser Leu Thr Val Asp Ser                 245 250 255 Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu His Ala Asn             260 265 270 Arg Pro Glu Asn Asn Tyr Trp Ile Arg Ala Lys Pro Asn Ile Gly Thr         275 280 285 Asp Thr Thr Thr Asp Asn Gly Met Asn Ser Ala Ile Leu Arg Tyr Asn     290 295 300 Gly Ala Pro Val Ala Glu Pro Gln Thr Val Gln Ser Pro Ser Leu Thr 305 310 315 320 Pro Leu Leu Glu Gln Asn Leu Arg Pro Leu Val Tyr Thr Pro Val Pro                 325 330 335 Gly Asn Pro Thr Pro Gly Gly Ala Asp Ile Val His Thr Leu Asp Leu             340 345 350 Ser Phe Asp Ala Gly Arg Phe Ser Ile Asn Gly Ala Ser Phe Leu Asp         355 360 365 Pro Thr Val Pro Val Leu Leu Gln Ile Leu Ser Gly Thr Gln Asn Ala     370 375 380 Gln Asp Leu Leu Pro Pro Gly Ser Val Ile Pro Leu Glu Leu Gly Lys 385 390 395 400 Val Val Glu Leu Val Ile Pro Ala Gly Val Val Gly Gly Pro His Pro                 405 410 415 Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Thr             420 425 430 Asp Gln Tyr Asn Phe Asn Asp Ala Ile Leu Arg Asp Val Val Ser Ile         435 440 445 Gly Gly Thr Glu Asp Gln Val Thr Ile Arg Phe Val Thr Asp Asn Pro     450 455 460 Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly 465 470 475 480 Leu Ala Ile Val Phe Ala Glu Gly Ile Glu Asn Thr Ala Ala Ser Asn                 485 490 495 Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Ala Leu             500 505 510 Asn Ala Gln Lys Lys Leu Asn Pro Ser Thr Thr         515 520 <210> 5 <211> 2173 <212> DNA <213> Cerrena unicolor <400> 5 atgtctcttc ttcgtagctt gacctccctc atcgtactag tcattggtgc atttgctgca 60 atcggtccag tcactgacct acatatagtg aaccagaatc tcgacccaga tggtttcaac 120 cgccccactg tactcgcagg tggtactttc cccggtcctc tgattcgtgg taacaaggta 180 cgcttcataa ccgccctccg tagacgtagg cttcggctga catgaccatc atctgtaggg 240 agataacttt aaaattaatg tgattgacga cttgacagag cacagtatgc tcaaggctac 300 gtccatcgta agtccctgat taacgtttca cctggtcata tcgctcaacg tctcgaagca 360 ctggcatggg ttcttccaga agggaaccaa ctgggccgat ggccccgcct ttgtcaccca 420 atgtcctatc acatcaggaa acgccttcct gtatgatttc aacgttccgg accaagctgg 480 tactttctgg taccacagcc atctctctac acagtattgt gacggtcttc gtggtgcctt 540 tgtcgtctat gatcctaatg atcccaacaa gcaactctat gatgttgata acggcaagtt 600 ccttgcatat ttcatttcta tcatatcctc acctgtattg gcacagaaag caccgtgatt 660 accttggctg attggtatca tgcccttgct cagactgtca ctggtgtcgc gtgagtgaca 720 aatggccctc aattgttcac atattttcct gattatcata tgatagagta tctgatgcaa 780 cgttgatcaa cggattggga cgttcggcca ccggccccgc aaatgcccct ctggcggtca 840 tcagtgtcga gcggaataag aggtcagttc cataattatg attatttccc gcgttacttc 900 ctaacaatta tttttgtatc cctccacaga tatcgtttcc gattggtttc tatttcttgc 960 gaccctaact ttattttctc aattgaccac cacccaatga ccgtaattga gatggacggt 1020 gttaataccc aatctatgac cgtagattcg atccaaatat tcgcaggtca acgatattca 1080 tttgtcgtag gttattataa actgcccacc gatcatctct cacgtaactg ttatagatgc 1140 aagccaacca accagttgga aattattgga tccgcgctaa acctaatgtt gggaacacaa 1200 ctttccttgg aggcctgaac tccgctatat tacgatatgt gggagcccct gaccaagaac 1260 cgaccactga ccaaacaccc aactctacac cgctcgttga ggcgaaccta cgacccctcg 1320 tctatactcc tgtggtatgt tgttctcgtt acatatacca aacctaatat gaagactgaa 1380 cggatctact agccgggaca gccattccct ggcggtgctg atatcgtcaa gaacttagct 1440 ttgggtttcg tacgtgtatt tcacttccct tttggcagta actgaggtgg aatgtatata 1500 gaatgccggg cgtttcacaa tcaatggagc gtccctcaca cctcctacag tccctgtact 1560 actccagatc ctcagtggta ctcacaatgc acaggatctt ctcccagcag gaagcgtgat 1620 cgaacttgaa cagaataaag ttgtcgaaat cgttttgccc gctgcgggcg ccgttggcgg 1680 tcctcatcct tttcacttac atggtgtaag tatcagacgt cctcatgccc atattgctcc 1740 gaaccttaca cacctgattt cagcacaatt tctgggtggt tcgtagcgcc ggtcaaacca 1800 catacaattt caatgatgct cctatccgtg atgttgtcag tattggcggt gcaaacgatc 1860 aagtcacgat ccgatttgtg gtatgtatct cgtgccttgc attcattcca cgagtaatga 1920 tccttacact tcgggttctc agaccgataa ccctggccca tggttccttc actgtcacat 1980 tgactggcat ttggaggctg ggttcgctgt agtctttgcg gagggaatca atggtactgc 2040 agctgctaat ccagtcccag gtaagactct cgctgctttg cgtaatatct atgaatttaa 2100 atcatatcaa tttgcagcgg cttggaatca attgtgccca ttgtatgatg ccttgagccc 2160 aggtgataca tga 2173 <210> 6 <211> 515 <212> PRT <213> Cerrena unicolor <400> 6 Met Ser Leu Leu Arg Ser Leu Thr Ser Leu Ile Val Leu Val Ile Gly 1 5 10 15 Ala Phe Ala Ala Ile Gly Pro Val Thr Asp Leu His Ile Val Asn Gln             20 25 30 Asn Leu Asp Pro Asp Gly Phe Asn Arg Pro Thr Val Leu Ala Gly Gly         35 40 45 Thr Phe Pro Gly Pro Leu Ile Arg Gly Asn Lys Gly Asp Asn Phe Lys     50 55 60 Ile Asn Val Ile Asp Asp Leu Thr Glu His Ser Met Leu Lys Ala Thr 65 70 75 80 Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp                 85 90 95 Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Thr Ser Gly Asn Ala Phe             100 105 110 Leu Tyr Asp Phe Asn Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His         115 120 125 Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala Phe Val     130 135 140 Val Tyr Asp Pro Asn Asp Pro Asn Lys Gln Leu Tyr Asp Val Asp Asn 145 150 155 160 Gly Asn Thr Val Ile Thr Leu Ala Asp Trp Tyr His Ala Leu Ala Gln                 165 170 175 Thr Val Thr Gly Val Ala Val Ser Asp Ala Thr Leu Ile Asn Gly Leu             180 185 190 Gly Arg Ser Ala Thr Gly Pro Ala Asn Ala Pro Leu Ala Val Ile Ser         195 200 205 Val Glu Arg Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys     210 215 220 Asp Pro Asn Phe Ile Phe Ser Ile Asp His His Pro Met Thr Val Ile 225 230 235 240 Glu Met Asp Gly Val Asn Thr Gln Ser Met Thr Val Asp Ser Ile Gln                 245 250 255 Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Met Gln Ala Asn Gln Pro             260 265 270 Val Gly Asn Tyr Trp Ile Arg Ala Lys Pro Asn Val Gly Asn Thr Thr         275 280 285 Phe Leu Gly Gly Leu Asn Ser Ala Ile Leu Arg Tyr Val Gly Ala Pro     290 295 300 Asp Gln Glu Pro Thr Thr Asp Gln Thr Pro Asn Ser Thr Pro Leu Val 305 310 315 320 Glu Ala Asn Leu Arg Pro Leu Val Tyr Thr Pro Val Pro Gly Gln Pro                 325 330 335 Phe Pro Gly Gly Ala Asp Ile Val Lys Asn Leu Ala Leu Gly Phe Asn             340 345 350 Ala Gly Arg Phe Thr Ile Asn Gly Ala Ser Leu Thr Pro Pro Thr Val         355 360 365 Pro Val Leu Leu Gln Ile Leu Ser Gly Thr His Asn Ala Gln Asp Leu     370 375 380 Leu Pro Ala Gly Ser Val Ile Glu Leu Glu Gln Asn Lys Val Val Glu 385 390 395 400 Ile Val Leu Pro Ala Ala Gly Ala Val Gly Gly Pro His Pro Phe His                 405 410 415 Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Gln Thr Thr             420 425 430 Tyr Asn Phe Asn Asp Ala Pro Ile Arg Asp Val Val Ser Ile Gly Gly         435 440 445 Ala Asn Asp Gln Val Thr Ile Arg Phe Val Thr Asp Asn Pro Gly Pro     450 455 460 Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly Phe Ala 465 470 475 480 Val Val Phe Ala Glu Gly Ile Asn Gly Thr Ala Ala Ala Asn Pro Val                 485 490 495 Pro Ala Ala Trp Asn Gln Leu Cys Pro Leu Tyr Asp Ala Leu Ser Pro             500 505 510 Gly asp thr         515 <210> 7 <211> 2663 <212> DNA <213> Cerrena unicolor <400> 7 caccgcgatg tctcttcttc gtagcttgac ctccctcatc gtactagcca ctggtgcatt 60 tgctgcaatc ggtccagtca ccgacctaca tatagtgaac cagaatctcg ccccagatgg 120 tttaaaccgc cccactgtac tcgcaggtgg tactttcccc ggtcctctga ttcgtggtaa 180 caaggtacgc ttcataaccg ccctccgtag acgtaggctt cggctgacat gaccatcatc 240 tgtagggaga taactttaaa attaatgtga ttgacgactt gacagaacac agtatgctca 300 aggctacgtc cattgtaagt ccctgattaa cgtttcacct ggtcatatcg ctcaacgtct 360 cgaagcactg gcatgggttc ttccagaagg gaaccaactg ggccgatggc cccgcctttg 420 tcacccaatg tcctatcaca tcaggaaacg ccttcttgta tgatttcaac gttccggacc 480 aagctggtac tttctggtac cacagccatc tctcyacaca gtattgtgac ggtcttcgtg 540 gtgcctttgt cgtctatgat cctaatgatc ccaacaagca actctatgat gttgataacg 600 gcaagtccct tgcatatttc agttctatca tatcctcacc tgtattggca cagaaagcac 660 cgtgattacc ttggctgatt ggtatcatgc ccttgctcag actgtcactg gtgtcgcgtg 720 agtgacaaat ggcccttaat tgttcacata ttttcctgat tatcatatga tagagtatct 780 gatgcaacgt tgatcaacgg attgggacgt tcggccaccg gccccgcaaa tgcccctctg 840 gcggtcatca gtgtcgagcg gaataagagg tcagttccat aattatgatt atttcccgcg 900 ttacttccta acgattattt ttgtatccct ccacagatat cgtttccgat tggtttctat 960 ttcttgcgac cctaacttta ttttctcaat tgaccaccac ccaatgaccg taattgagat 1020 ggacggtgtt aatacccaat ctatgaccgt agattcgatc caaatattcg caggtcaacg 1080 atattcattt gtcgtaggtt attataaact gcccaccgat catctctcac gtaactgtta 1140 tagatgcaag ccaaccaacc agttggaaat tattggatcc gygctaaacc taatgttggg 1200 aacacaactt tccttggagg cctgaactcc gctatattac gatatgtggg agcccctgac 1260 caagaaccga ccactgacca aacacccaac tctacaccgc tcgtcgaggc gaacctacgt 1320 cccctcgtct atactcctgt ggtatgttgt tctcgttaca tataccaaac ctaatatgag 1380 gactgaacgg atctactagc cgggacagcc attccctggc ggtgctgata tcgtcaagaa 1440 cttagctttg ggtttcgtac gtgtatttca cttccctttt ggcagtaact gaggtggaat 1500 gtatatagaa tgccgggcgt ttcacaatca atggaacatc cttcacacct cctacagtcc 1560 ctgtactact ccagatcctc agtggtactc acaatgcaca ggatcttctt ccagcaggaa 1620 gcgtgatcga acttgaacag aataaagttg tcgaaatcgt tctgcccgct gcgggcgccg 1680 ttggcggtcc tcatcctttc cacttacatg gtgtaagtat cagacgtcct catgcctata 1740 ttgctccgaa ccttacacac ctgatttcag cacaatttct gggtggttcg tagcgccggt 1800 caaaccacat acaatttcaa tgatgctcct atccgtgatg ttgtcagtat tggcggtgca 1860 aacgatcaag tcacgatccg atttgtggta tgtatctcgt gccttgcatt cattccacga 1920 gtaatgatcc ttacacttcg ggttctcaga ccgataaccc tggcccatgg ttccttcact 1980 gtcacattga ctggcatttg gaggctgggt tcgctgtagt ctttgcggag ggaatcaatg 2040 gcactgcagc tgctaatcca gtcccaggta agactctcgc tgctttgcgt aatatctatg 2100 aatttaaagc atatcaattt gcagcggctt ggaatcaatt gtgcccgttg tatgatgcct 2160 tgagcccagg tgatacatga ttactcgtag ctgtgctttc ttatacatat tctatgggta 2220 tatcggagta gctgtactat agtatgtact atactaggtg ggatatgytg atgttgattt 2280 atataatttt gtttgaagag tgactttatc gacttgggat ttagccgagt acatactgat 2340 ctctcactac aggcttgttt tgtctttggg cgcttactca acagttgact gtttttgcta 2400 ttacgcattg aaccgcattc cggtcygact cgtgtcctct actgtgactt gtattggcat 2460 tctagcacat atgtctctta cctataggaa caatatgtct caacactgtt ccaaaacctg 2520 cgtaaaccaa atatcgtcca tcagatcaga tcattaacag tgccgcacta acctaataca 2580 ctggcargga ctgtggaaat ccctataaat gacctctaga ccgtgaggtc attgcaaggt 2640 cgctctcctt gtcaagatga ccc 2663 <210> 8 <211> 515 <212> PRT <213> Cerrena unicolor <400> 8 Met Ser Leu Leu Arg Ser Leu Thr Ser Leu Ile Val Leu Ala Thr Gly 1 5 10 15 Ala Phe Ala Ala Ile Gly Pro Val Thr Asp Leu His Ile Val Asn Gln             20 25 30 Asn Leu Ala Pro Asp Gly Leu Asn Arg Pro Thr Val Leu Ala Gly Gly         35 40 45 Thr Phe Pro Gly Pro Leu Ile Arg Gly Asn Lys Gly Asp Asn Phe Lys     50 55 60 Ile Asn Val Ile Asp Asp Leu Thr Glu His Ser Met Leu Lys Ala Thr 65 70 75 80 Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp                 85 90 95 Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Thr Ser Gly Asn Ala Phe             100 105 110 Leu Tyr Asp Phe Asn Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His         115 120 125 Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala Phe Val     130 135 140 Val Tyr Asp Pro Asn Asp Pro Asn Lys Gln Leu Tyr Asp Val Asp Asn 145 150 155 160 Gly Asn Thr Val Ile Thr Leu Ala Asp Trp Tyr His Ala Leu Ala Gln                 165 170 175 Thr Val Thr Gly Val Ala Val Ser Asp Ala Thr Leu Ile Asn Gly Leu             180 185 190 Gly Arg Ser Ala Thr Gly Pro Ala Asn Ala Pro Leu Ala Val Ile Ser         195 200 205 Val Glu Arg Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys     210 215 220 Asp Pro Asn Phe Ile Phe Ser Ile Asp His His Pro Met Thr Val Ile 225 230 235 240 Glu Met Asp Gly Val Asn Thr Gln Ser Met Thr Val Asp Ser Ile Gln                 245 250 255 Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Met Gln Ala Asn Gln Pro             260 265 270 Val Gly Asn Tyr Trp Ile Arg Ala Lys Pro Asn Val Gly Asn Thr Thr         275 280 285 Phe Leu Gly Gly Leu Asn Ser Ala Ile Leu Arg Tyr Val Gly Ala Pro     290 295 300 Asp Gln Glu Pro Thr Thr Asp Gln Thr Pro Asn Ser Thr Pro Leu Val 305 310 315 320 Glu Ala Asn Leu Arg Pro Leu Val Tyr Thr Pro Val Pro Gly Gln Pro                 325 330 335 Phe Pro Gly Gly Ala Asp Ile Val Lys Asn Leu Ala Leu Gly Phe Asn             340 345 350 Ala Gly Arg Phe Thr Ile Asn Gly Thr Ser Phe Thr Pro Pro Thr Val         355 360 365 Pro Val Leu Leu Gln Ile Leu Ser Gly Thr His Asn Ala Gln Asp Leu     370 375 380 Leu Pro Ala Gly Ser Val Ile Glu Leu Glu Gln Asn Lys Val Val Glu 385 390 395 400 Ile Val Leu Pro Ala Ala Gly Ala Val Gly Gly Pro His Pro Phe His                 405 410 415 Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Gln Thr Thr             420 425 430 Tyr Asn Phe Asn Asp Ala Pro Ile Arg Asp Val Val Ser Ile Gly Gly         435 440 445 Ala Asn Asp Gln Val Thr Ile Arg Phe Val Thr Asp Asn Pro Gly Pro     450 455 460 Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly Phe Ala 465 470 475 480 Val Val Phe Ala Glu Gly Ile Asn Gly Thr Ala Ala Ala Asn Pro Val                 485 490 495 Pro Ala Ala Trp Asn Gln Leu Cys Pro Leu Tyr Asp Ala Leu Ser Pro             500 505 510 Gly asp thr         515 <210> 9 <211> 1337 <212> DNA <213> Cerrena unicolor <400> 9 gtgggggcgg atccctaact gtttcgaatc ggcaccgaag tatgcaggtg tgacggagat 60 gaggcgtttt ttcatcttcc actgcagtat aaaatgtctc aggtaacgtc cagctttttg 120 taccagagct acctccaaat acctttactc gcaaaggttt cgcgatgtct cttcttcgta 180 gcttgacctc cctcatcgta ctagccactg gtgcatttgc tgcaatcggt ccagtcactg 240 acctacatat agtgaaccag aatctcgccc cagatggttt caaccgcccc actgtactcg 300 caggtggtac tttccccggt cctctgattc gtggtaacaa ggtacgcttc ataaccgccc 360 tccgtagacg taggcttcgg ctgacatgac catcatctgt agggagataa ctttaaaatt 420 aatgtgattg acgacttgac agaacacagt atgctcaagg ccacgtccat tgtaagtccc 480 tgattaacgt ttcacctggt catatcgctc aacgtctcga agcactggca tgggttcttc 540 cagaagggaa ccaactgggc cgatggcccc gcctttgtca cccaatgtcc tatcacatca 600 ggaaactcct tcctgtatga tttcaacgtt ccggaccaag ctggtacttt ctggtaccac 660 agccatctct ctacacagta ttgtgacggt cttcgtggtg cctttgtcgt ctatgatcct 720 aatgatccca acaagcaact ctatgatgtt gataacggca agtcccttgc atatttcatt 780 tctatcatat cctcacctgt attggcacag aaagcaccgt gattaccttg gctgattggt 840 atcatgccct tgctcagact gtcactggtg tcgcgtgagt gacaaatggc cctcaattgt 900 tcacatattt tcctgattat catatgatag agtatctgat gcaacgttga tcaacggatt 960 gggacgttcg gccaccggcc ccgcaaatgc ccctctggcg gtcatcagtg tcgagcggaa 1020 taagaggtca gttccataat tatgattatt tcccgcgtta cttcctaaca attattcttg 1080 tatccctcca cagatatcgc ttccgattgg tgtctatttc ttgcgaccct aactttattt 1140 tctcaattga tcaccaccca atgaccgtaa ttgagatgga cggtgttaat acccaatcta 1200 tgaccgtaga ttcgatccaa atattcgcag gtcaacgata ttcatttgtc gtaggttatt 1260 ataaactgcc caccgatcat ctctcacgta actgttatag atgcaagcca accaaccrgt 1320 tggaaattat tggatcc 1337 <210> 10 <211> 278 <212> PRT <213> Cerrena unicolor <400> 10 Met Ser Leu Leu Arg Ser Leu Thr Ser Leu Ile Val Leu Ala Thr Gly 1 5 10 15 Ala Phe Ala Ala Ile Gly Pro Val Thr Asp Leu His Ile Val Asn Gln             20 25 30 Asn Leu Ala Pro Asp Gly Phe Asn Arg Pro Thr Val Leu Ala Gly Gly         35 40 45 Thr Phe Pro Gly Pro Leu Ile Arg Gly Asn Lys Gly Asp Asn Phe Lys     50 55 60 Ile Asn Val Ile Asp Asp Leu Thr Glu His Ser Met Leu Lys Ala Thr 65 70 75 80 Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp                 85 90 95 Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Thr Ser Gly Asn Ser Phe             100 105 110 Leu Tyr Asp Phe Asn Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His         115 120 125 Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala Phe Val     130 135 140 Val Tyr Asp Pro Asn Asp Pro Asn Lys Gln Leu Tyr Asp Val Asp Asn 145 150 155 160 Gly Lys Thr Val Ile Thr Leu Ala Asp Trp Tyr His Ala Leu Ala Gln                 165 170 175 Thr Val Thr Gly Val Ala Val Ser Asp Ala Thr Leu Ile Asn Gly Leu             180 185 190 Gly Arg Ser Ala Thr Gly Pro Ala Asn Ala Pro Leu Ala Val Ile Ser         195 200 205 Val Glu Arg Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys     210 215 220 Asp Pro Asn Phe Ile Phe Ser Ile Asp His His Pro Met Thr Val Ile 225 230 235 240 Glu Met Asp Gly Val Asn Thr Gln Ser Met Thr Val Asp Ser Ile Gln                 245 250 255 Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Met Gln Ala Asn Gln Pro             260 265 270 Val Gly Asn Tyr Trp Ile         275 <210> 11 <211> 1080 <212> DNA <213> Cerrena unicolor <400> 11 tgcaatcgga ccggtbgctg accttcacat tacggacgat accattgccc ccgatggttt 60 ctctcgtcct gctgttctcg ctggcggggg tttccctggc cctctcatca ccggaaacaa 120 ggtaatgcct aatggttgcg tctttgttgg tgctctcatt catccacgac attttgtacc 180 agggcgacgc ctttaaactc aatgtcatcg atgaactaac ggacgcatcc atgctgaagy 240 cgacttccat cgtaagtctc gctgtattgc tccttgagcc atttcattga ctataactac 300 aaccagcact ggcatggatt cttccaaaag ggtactaatt gggcagatgg tcccgctttt 360 gtgaaccaat gccccatcac cacgggaaac tccttcttgt acgacttcca ggttcctgat 420 caagctggta agcatgagat tacactagga aagtttaatt taataactat tcaatcagga 480 acctactggt atcatagtca tttgtctacg caatactgtg atggtctcag aggtgcattc 540 gttgtctacg acccttcaga tcctcacaag gatctctacg acgtcgacga cggtgagctt 600 tgcttttttc attggtatcc attatcgctc acgtgtcatt actgcgccac agaaagtacc 660 gtcatcactt tggctgattg gtatcatact ttggctcgtc agattgttgg cgttgcgtga 720 gtagtcttgt accgactgaa acatattcca gttgctgact tccccacagc atttctgata 780 ctaccttgat aaacggtttg ggccgcaata ccaatggtcc ggctgatgct gctcttgctg 840 tgatcaatgt tgacgctggc aaacggtgtg tccagattac tatactcccc atgacgtctc 900 aatgctgatg tgtactactt ccaggtaccg tttccgtctt gtttccatat cctgtgaccc 960 caattgggta ttctcgattg acaaccatga ctttacggtc attgaagtcg atggtgttaa 1020 cagtcaacct ctcaacgtcg attctgttca gatcttcgcc ggacaacgtt actcgttcgt 1080 <210> 12 <211> 246 <212> PRT <213> Cerrena unicolor <220> <221> misc_feature (222) (60) .. (60) <223> Xaa can be any naturally occurring amino acid <400> 12 Ala Ile Gly Pro Val Ala Asp Leu His Ile Thr Asp Asp Thr Ile Ala 1 5 10 15 Pro Asp Gly Phe Ser Arg Pro Ala Val Leu Ala Gly Gly Gly Phe Pro             20 25 30 Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Ala Phe Lys Leu Asn Val         35 40 45 Ile Asp Glu Leu Thr Asp Ala Ser Met Leu Lys Xaa Thr Ser Ile His     50 55 60 Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala 65 70 75 80 Phe Val Asn Gln Cys Pro Ile Thr Thr Gly Asn Ser Phe Leu Tyr Asp                 85 90 95 Phe Gln Val Pro Asp Gln Ala Gly Thr Tyr Trp Tyr His Ser His Leu             100 105 110 Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala Phe Val Val Tyr Asp         115 120 125 Pro Ser Asp Pro His Lys Asp Leu Tyr Asp Val Asp Asp Glu Ser Thr     130 135 140 Val Ile Thr Leu Ala Asp Trp Tyr His Thr Leu Ala Arg Gln Ile Val 145 150 155 160 Gly Val Ala Ile Ser Asp Thr Thr Leu Ile Asn Gly Leu Gly Arg Asn                 165 170 175 Thr Asn Gly Pro Ala Asp Ala Ala Leu Ala Val Ile Asn Val Asp Ala             180 185 190 Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys Asp Pro Asn         195 200 205 Trp Val Phe Ser Ile Asp Asn His Asp Phe Thr Val Ile Glu Val Asp     210 215 220 Gly Val Asn Ser Gln Pro Leu Asn Val Asp Ser Val Gln Ile Phe Ala 225 230 235 240 Gly Gln Arg Tyr Ser Phe                 245 <210> 13 <211> 2809 <212> DNA <213> Cerrena unicolor <400> 13 gattctaata gaccaggcat accaagagat ctacaggttg acagaccatt cttctaggcg 60 gcatttatgc tgtagcgtca gaaattatct ctccatttgt atcccacagg tcctgtaata 120 acacggagac agtccaaact gggatgcctt ttttctcaac tatgggcgca catagtctgg 180 acgatggtat ataagacgat ggtatgagac ccatgaagtc agaacacttt tgctctctga 240 catttcatgg ttcacactct cgagatggga ttgaactcgg ctattacatc gcttgctatc 300 ttagctctgt cagtcggaag ctatgctgca attgggcccg tggccgacat acacattgtc 360 aacaaagacc ttgctccaga tggcgtacaa cgtccaaccg tgcttgccgg aggcactttt 420 cctgggacgt tgatcaccgg tcagaaagta agggatatta gtttgcgtca aagagccaac 480 caaaactaac cgtcccgtac tatagggtga caacttccag ctcaatgtca tcgatgatct 540 taccgacgat cggatgttga cgccaacttc cattgtgagc ctattattgt atgatttatc 600 cgaatagttt cgcagtctga tcattggatc tctatcgcta gcattggcac ggtttcttcc 660 agaagggaac cgcttgggcc gacggtcccg ccttcgtaac tcagtgccct ataatagcag 720 ataactcttt tctgtatgac ttcgacgtcc cagaccaagc tggtactttc tggtatcata 780 gtcatctatc cactcagtac tgtgacggtt tacgtggtgc cttcgttgtg tacgatccta 840 acgatcctca caaagaccta tacgatgttg atgacggtgg gttccaaata tttgttctgc 900 agacattgta ttgacggtgt tcattataat ttcagagagc accgtgatta cccttgcgga 960 ttggtaccat gttctcgccc agaccgttgt cggcgctgcg tgagtaacac atacacgcgc 1020 tccggcacac tgatactaat ttttttttat tgtagcactc ctgattctac cttgatcaac 1080 gggttaggcc gttcacagac cggacccgct gatgctgagc tggctgttat cagcgttgaa 1140 cataacaaac ggtatgtcat ctctacccag tatcttctct cctgctctaa ttcgctgttt 1200 caccatagat accgtttccg tttggtttcg atttcgtgcg accccaactt taccttctcc 1260 gttgatggtc ataatatgac tgtcatcgaa gtcgatggtg tcaacacacg acccctgacc 1320 gttgactcta ttcaaatctt cgccggacag aggtattcct ttgtcgtaag ttaatcgata 1380 tattctcctt attacccctg tgtaattgat gtcaatagct caatgctaac caacccgaag 1440 acaattactg gatccgtgct atgccaaaca tcggtagaaa tacaacaaca ctggacggaa 1500 agaatgccgc tatccttcga tacaagaatg cttctgtaga agagcccaag accgttgggg 1560 gccccgctca atccccgttg aatgaagcgg acctgcgtcc actcgtacct gctcctgtgg 1620 tatgtcttgt cgcgctgttc catcgctatt tcatattaac gttttgtttt tgtcaagcct 1680 ggaaacgctg ttccaggtgg cgcagacatc aatcacaggc ttaacttaac tttcgtacgt 1740 acacctggtt gaaacattat atttccagtc taacctctct tgtagagtaa cggcctcttc 1800 agcatcaaca acgcctcctt cactaatcct tcggtccccg ccttattaca aattctgagc 1860 ggtgctcaga acgctcaaga tttacttcca acgggtagtt acattggcct tgaactaggc 1920 aaggttgtgg agctcgttat acctcctctg gcagttggag gaccgcaccc tttccatctt 1980 catggcgtaa gcataccaca ctcccgcagc cagaatgacg caaactaatc atgatatgca 2040 gcacaatttc tgggtcgtcc gtagtgcagg tagcgatgag tataactttg acgatgctat 2100 cctcagggac gtcgtragca ttggagcggg gactgatgaa gtcacaatcc gtttcgtggt 2160 atgtctcacc cctcgcattt tgagacgcaa gagctgatat attttaacat agaccgacaa 2220 tccgggcccg tggttcctcc attgccatat tgattggcat ttggaggcag gccttgccat 2280 cgtcttcgct gagggcatca atcagaccgc tgcagccaac ccaacacccc gtacgtgaca 2340 ctgagggttt ctttatagtg ctggattact gaatcgagat ttctccacag aagcatggga 2400 tgagctttgc cccaaatata acgggttgag tgcgagccag aaggtcaagc ctaagaaagg 2460 aactgctatt taaacgtggt cctagactac gggcatataa gtattcgggt agcgcgtgtg 2520 agcaatgttc cgatacacgt agattcatca ccggacacgc tgggacaatt tgtgtataat 2580 ggctagtaac gtatctgagt tctggtgtgt agttcaaaga gacagccctt cctgagacag 2640 cccttcctga gacagccctt cctgagacgt gacctccgta gtctgcacac gatactycta 2700 aatacgtatg gcaagatgac aaagaggagg atgtgagtta ctacgaacag aaatagtgcc 2760 cggcctcgga gagatgttct tgaatatggg actgggacca acatccgga 2809 <210> 14 <211> 526 <212> PRT <213> Cerrena unicolor <400> 14 Met Gly Leu Asn Ser Ala Ile Thr Ser Leu Ala Ile Leu Ala Leu Ser 1 5 10 15 Val Gly Ser Tyr Ala Ala Ile Gly Pro Val Ala Asp Ile His Ile Val             20 25 30 Asn Lys Asp Leu Ala Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala         35 40 45 Gly Gly Thr Phe Pro Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn     50 55 60 Phe Gln Leu Asn Val Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr 65 70 75 80 Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp                 85 90 95 Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn             100 105 110 Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp         115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala     130 135 140 Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val 145 150 155 160 Asp Asp Gly Gly Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu                 165 170 175 Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn             180 185 190 Gly Leu Gly Arg Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val         195 200 205 Ile Ser Val Glu His Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile     210 215 220 Ser Cys Asp Pro Asn Phe Thr Phe Ser Val Asp Gly His Asn Met Thr 225 230 235 240 Val Ile Glu Val Asp Gly Val Asn Thr Arg Pro Leu Thr Val Asp Ser                 245 250 255 Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn             260 265 270 Gln Pro Glu Asp Asn Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg         275 280 285 Asn Thr Thr Thr Leu Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys     290 295 300 Asn Ala Ser Val Glu Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser 305 310 315 320 Pro Leu Asn Glu Ala Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro                 325 330 335 Gly Asn Ala Val Pro Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu             340 345 350 Thr Phe Ser Asn Gly Leu Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn         355 360 365 Pro Ser Val Pro Ala Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala     370 375 380 Gln Asp Leu Leu Pro Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys 385 390 395 400 Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro                 405 410 415 Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Ser             420 425 430 Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile         435 440 445 Gly Ala Gly Thr Asp Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro     450 455 460 Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly 465 470 475 480 Leu Ala Ile Val Phe Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn                 485 490 495 Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly Leu             500 505 510 Ser Ala Ser Gln Lys Val Lys Pro Lys Lys Gly Thr Ala Ile         515 520 525 <210> 15 <211> 2299 <212> DNA <213> Cerrena unicolor <400> 15 gatctggacg atggtatata agacgatggt atgagaccca tgaagtctga acacttttgc 60 tctctgacat ttcatggttc atactctcga gatgggattg aactcggcta ttacatcgct 120 tgctatctta gctctgtcag tcggaagcta tgctgcaatt gggcccgtgg ccgacataca 180 cattgtcaac aaagaccttg ctccagatgg tgtacaacgt ccaaccgtgc tcgccggagg 240 cacttttcct gggacgttga tcaccggtca gaaagtaagg aatattagtt tgcgtcaaag 300 agccaaccaa aattaaccgt cccgtcccat agggtgacaa cttccagctc aatgtcattg 360 atgatcttac cgacgatcgg atgttgacac caacttccat tgtgagccta ttattgtatg 420 atttatccgt atagtttctc agtctgatca ttggctctct atcgctagca ttggcacggt 480 ttcttccaga agggaaccgc ttgggccgac ggtcccgcct tcgtaactca gtgccctata 540 atagcagata actcttttct gtatgacttc gacgtccccg accaagctgg tactttctgg 600 tatcatagtc atctatccac tcagtactgt gacggtttac gtggtgcctt cgttgtgtac 660 gatcctaacg atcctcacaa agacctatac gatgttgatg acggtgggtt ccaaatactt 720 gaccaagaaa cattatattg atagtatcca ctctgatttt cagagagcac cgtgattacc 780 cttgcggatt ggtaccatgt tctcgcccag accgttgtcg gcgctgcgtg agtaacacat 840 acacgcgctc cggcacactg atactaattt tttattgtag cactcctgat tctaccttga 900 tcaacgggtt aggccgttca cagaccggac ccgctgatgc tgagctggct gttatcagcg 960 ttgaacataa caaacggtat gtcatctcta cccattatct tctctcctgc tttaattcgc 1020 tgtttcacca tagataccga ttccgtttgg tttcgatttc gtgcgacccc aactttacct 1080 tctccgttga tggtcataat atgactgtca tcgaagtcga cggtgtcaac acacgacccc 1140 tgaccgttga ctctattcaa atcttcgccg gacagaggta ttcctttgtc gtaagttaat 1200 cgatatattc tccctattac ccctgtgtaa ttgatgtcaa cagctcaatg ctaaccaacc 1260 cgacgacaat tactggatcc gtgctatgcc aaacatcggt agaaatacaa caacactgga 1320 cggaaagaat gccgctatcc ttcgatacaa gaatgcttct gtagaagagc ccaagaccgt 1380 tgggggcccc gctcaatccc cgttgaatga agcggacctg cgtccactcg tacctgctcc 1440 tgtggtatgt cttgtcgtgc tgttccatcg ctatttcata ttaacgtttt gtttttgtca 1500 agcctggaaa cgctgttcca ggtggcgcag acatcaatca caggcttaac ttaactttcg 1560 tacgtacacc tggttgaaac attatatttc cagtctaacc tcttgtagag taacggcctt 1620 ttcagcatca acaacgcctc cttcactaat ccttcggtcc ccgccttatt acaaattctg 1680 agcggtgctc agaacgctca agatttactt ccaacgggta gttacattgg ccttgaacta 1740 ggcaaggttg tggagctcgt tatacctcct ctggcagttg gaggaccgca ccctttccat 1800 cttcatggcg taagcatacc acactcccgc agccagaatg acgcaaacta atcatgatat 1860 gcagcacaat ttctgggtcg tccgtagtgc aggtagcgat gagtataact ttgacgatgc 1920 tatcctcagg gacgtcgtga gcattggagc ggggactgat gaagtcacaa tccgtttcgt 1980 ggtatgtctc acccctcgca ttttgagacg caagagctga tatattttaa catagaccga 2040 caatccgggc ccgtggttcc tccattgcca tattgattgg catttggagg caggccttgc 2100 catcgtcttc gctgagggca tcaatcagac cgctgcagcc aacccaacac cccgtacgtg 2160 acactgaggg tttctttata gtgctggatt actgaatcga gatttctcca cagaagcatg 2220 ggatgagctt tgccccaaat ataacgggtt gagtgcgagc cagaaggtca agcctaagaa 2280 aggaactgct atttaaacg 2299 <210> 16 <211> 526 <212> PRT <213> Cerrena unicolor <400> 16 Met Gly Leu Asn Ser Ala Ile Thr Ser Leu Ala Ile Leu Ala Leu Ser 1 5 10 15 Val Gly Ser Tyr Ala Ala Ile Gly Pro Val Ala Asp Ile His Ile Val             20 25 30 Asn Lys Asp Leu Ala Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala         35 40 45 Gly Gly Thr Phe Pro Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn     50 55 60 Phe Gln Leu Asn Val Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr 65 70 75 80 Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp                 85 90 95 Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn             100 105 110 Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp         115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala     130 135 140 Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val 145 150 155 160 Asp Asp Gly Gly Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu                 165 170 175 Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn             180 185 190 Gly Leu Gly Arg Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val         195 200 205 Ile Ser Val Glu His Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile     210 215 220 Ser Cys Asp Pro Asn Phe Thr Phe Ser Val Asp Gly His Asn Met Thr 225 230 235 240 Val Ile Glu Val Asp Gly Val Asn Thr Arg Pro Leu Thr Val Asp Ser                 245 250 255 Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn             260 265 270 Gln Pro Asp Asp Asn Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg         275 280 285 Asn Thr Thr Thr Leu Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys     290 295 300 Asn Ala Ser Val Glu Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser 305 310 315 320 Pro Leu Asn Glu Ala Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro                 325 330 335 Gly Asn Ala Val Pro Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu             340 345 350 Thr Phe Ser Asn Gly Leu Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn         355 360 365 Pro Ser Val Pro Ala Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala     370 375 380 Gln Asp Leu Leu Pro Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys 385 390 395 400 Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro                 405 410 415 Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Ser             420 425 430 Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile         435 440 445 Gly Ala Gly Thr Asp Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro     450 455 460 Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly 465 470 475 480 Leu Ala Ile Val Phe Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn                 485 490 495 Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly Leu             500 505 510 Ser Ala Ser Gln Lys Val Lys Pro Lys Lys Gly Thr Ala Ile         515 520 525 <210> 17 <211> 1163 <212> DNA <213> Cerrena unicolor <400> 17 tgcaatcgga ccggtggccg acctcaagat cgtaaaccga gacattgcac ctgacggttt 60 tattcgtccc gccgttctcg ctggagggtc gttccctggt cctctcatta cagggcagaa 120 agtacgttac gctatctcgg tgctttggct taattaaact atttgacttt gtgttctctt 180 aggggaacga gttcaaaatc aatgtagtca atcaactgac cgatggttct atgttaaaat 240 ccacctcaat cgtaagcaga atgagccctt tgcatctcgt tttattgtta atgcgcccac 300 tatagcattg gcatggattc ttccagaagg gaacaaactg ggcagacggt cctgcgttcg 360 tgaaccaatg tccaatcgcc acgaacaatt cgttcttgta tcagtttacc tcacaggaac 420 agccaggtga gtatgagatg gagttcatcc gagcatgaac tgatttattt ggaacctagg 480 cacattttgg taccatagtc atctttccac acaatactgc gatggtttgc gagggccact 540 cgtggtgtat gacccacaag acccgcatgc tgttctctac gacgtcgacg atggttcgta 600 cttcgcatat ccacgctcgc tttcatacaa tgtaaacttt gttcctccag aaagtacaat 660 catcacgctc gcggattggt atcatacctt ggctcggcaa gtgaaaggcc cagcgtaagg 720 cactttagtg tttcctcata gtccaagaaa ttctaacacg ccttcttcat cagggttcct 780 ggtacgacct tgatcaacgg gttggggcgt cacaacaatg gtcctctaga tgctgaacta 840 gcggtgatca gtgttcaagc cggcaaacgg caagttcaat tcacactttt cactctgtac 900 cttcttcctg acattctttt cttgtagtta ccgcttccgc ctgatttcaa tttcatgcga 960 tcccaactac gtattctcca ttgatggcca tgatatgact gtcatcgaag tggatagtgt 1020 taacagtcaa cctctcaagg tagattctat ccaaatattt gcaggtcaga gatattcgtt 1080 cgtggtgagt cagatcaggg catatccttt tgtcgatacg tcattgacca tataatgcta 1140 caagctgaat gccaaccaac cag 1163 <210> 18 <211> 262 <212> PRT <213> Cerrena unicolor <400> 18 Ala Ile Gly Pro Val Ala Asp Leu Lys Ile Val Asn Arg Asp Ile Ala 1 5 10 15 Pro Asp Gly Phe Ile Arg Pro Ala Val Leu Ala Gly Gly Ser Phe Pro             20 25 30 Gly Pro Leu Ile Thr Gly Gln Lys Gly Asn Glu Phe Lys Ile Asn Val         35 40 45 Val Asn Gln Leu Thr Asp Gly Ser Met Leu Lys Ser Thr Ser Ile His     50 55 60 Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala 65 70 75 80 Phe Val Asn Gln Cys Pro Ile Ala Thr Asn Asn Ser Phe Leu Tyr Gln                 85 90 95 Phe Thr Ser Gln Glu Gln Pro Gly Thr Phe Trp Tyr His Ser His Leu             100 105 110 Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Leu Val Val Tyr Asp         115 120 125 Pro Gln Asp Pro His Ala Val Leu Tyr Asp Val Asp Asp Glu Ser Thr     130 135 140 Ile Ile Thr Leu Ala Asp Trp Tyr His Thr Leu Ala Arg Gln Val Lys 145 150 155 160 Gly Pro Ala Val Pro Gly Thr Thr Leu Ile Asn Gly Leu Gly Arg His                 165 170 175 Asn Asn Gly Pro Leu Asp Ala Glu Leu Ala Val Ile Ser Val Gln Ala             180 185 190 Gly Lys Arg Gln Val Gln Phe Thr Leu Phe Thr Leu Tyr Arg Phe Arg         195 200 205 Leu Ile Ser Ile Ser Cys Asp Pro Asn Tyr Val Phe Ser Ile Asp Gly     210 215 220 His Asp Met Thr Val Ile Glu Val Asp Ser Val Asn Ser Gln Pro Leu 225 230 235 240 Lys Val Asp Ser Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val                 245 250 255 Leu Asn Ala Asn Gln Pro             260 <210> 19 <211> 526 <212> PRT <213> Artificial sequence <220> <223> synthetic laccase D enzyme <400> 19 Met Gly Leu Asn Ser Ala Ile Thr Ser Leu Ala Ile Leu Ala Leu Ser 1 5 10 15 Val Gly Ser Tyr Ala Ala Ile Gly Pro Val Ala Asp Leu His Ile Val             20 25 30 Asn Lys Asp Leu Ala Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala         35 40 45 Gly Gly Thr Phe Pro Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn     50 55 60 Phe Gln Leu Asn Val Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr 65 70 75 80 Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp                 85 90 95 Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn             100 105 110 Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp         115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala     130 135 140 Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val 145 150 155 160 Asp Asp Gly Gly Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu                 165 170 175 Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn             180 185 190 Gly Leu Gly Arg Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val         195 200 205 Ile Ser Val Glu His Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile     210 215 220 Ser Cys Asp Pro Asn Phe Thr Phe Ser Val Asp Gly His Asn Met Thr 225 230 235 240 Val Ile Glu Val Asp Gly Val Asn Thr Arg Pro Leu Thr Val Asp Ser                 245 250 255 Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn             260 265 270 Gln Pro Glu Asp Asn Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg         275 280 285 Asn Thr Thr Thr Leu Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys     290 295 300 Asn Ala Ser Val Glu Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser 305 310 315 320 Pro Leu Asn Glu Ala Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro                 325 330 335 Gly Asn Ala Val Pro Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu             340 345 350 Thr Phe Ser Asn Gly Leu Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn         355 360 365 Pro Ser Val Pro Ala Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala     370 375 380 Gln Asp Leu Leu Pro Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys 385 390 395 400 Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro                 405 410 415 Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Ser             420 425 430 Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile         435 440 445 Gly Ala Gly Thr Asp Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro     450 455 460 Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly 465 470 475 480 Leu Ala Ile Val Phe Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn                 485 490 495 Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly Leu             500 505 510 Ser Ala Ser Gln Lys Val Lys Pro Lys Lys Gly Thr Ala Ile         515 520 525 <210> 20 <211> 505 <212> PRT <213> Artificial sequence <220> <223> synthetic mature processed laccase D enzyme <400> 20 Ala Ile Gly Pro Val Ala Asp Leu His Ile Val Asn Lys Asp Leu Ala 1 5 10 15 Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala Gly Gly Thr Phe Pro             20 25 30 Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn Phe Gln Leu Asn Val         35 40 45 Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr Pro Thr Ser Ile His     50 55 60 Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp Ala Asp Gly Pro Ala 65 70 75 80 Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn Ser Phe Leu Tyr Asp                 85 90 95 Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu             100 105 110 Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala Phe Val Val Tyr Asp         115 120 125 Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val Asp Asp Gly Gly Thr     130 135 140 Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu Ala Gln Thr Val Val 145 150 155 160 Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg Ser                 165 170 175 Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val Ile Ser Val Glu His             180 185 190 Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys Asp Pro Asn         195 200 205 Phe Thr Phe Ser Val Asp Gly His Asn Met Thr Val Ile Glu Val Asp     210 215 220 Gly Val Asn Thr Arg Pro Leu Thr Val Asp Ser Ile Gln Ile Phe Ala 225 230 235 240 Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn Gln Pro Glu Asp Asn                 245 250 255 Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg Asn Thr Thr Thr Leu             260 265 270 Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys Asn Ala Ser Val Glu         275 280 285 Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser Pro Leu Asn Glu Ala     290 295 300 Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro Gly Asn Ala Val Pro 305 310 315 320 Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu Thr Phe Ser Asn Gly                 325 330 335 Leu Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn Pro Ser Val Pro Ala             340 345 350 Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala Gln Asp Leu Leu Pro         355 360 365 Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys Val Val Glu Leu Val     370 375 380 Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro Phe His Leu His Gly 385 390 395 400 His Asn Phe Trp Val Val Arg Ser Ala Gly Ser Asp Glu Tyr Asn Phe                 405 410 415 Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile Gly Ala Gly Thr Asp             420 425 430 Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro Gly Pro Trp Phe Leu         435 440 445 His Cys His Ile Asp Trp His Leu Glu Ala Gly Leu Ala Ile Val Phe     450 455 460 Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn Pro Thr Pro Gln Ala 465 470 475 480 Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly Leu Ser Ala Ser Gln Lys                 485 490 495 Val Lys Pro Lys Lys Gly Thr Ala Ile             500 505

Claims (27)

A fabric processing method comprising contacting a fabric with a laccase enzyme and a mediator at temperatures below 40 ° C. under conditions and for a time sufficient to cause color deformation of the fabric. The method of claim 2, wherein the color deformation is selected from color lightening, color change, color cast change, redeposition / backstaining and bleaching. The method of claim 1 wherein the temperature is from about 20 ° C. to less than 40 ° C. 7. The method of claim 1 wherein the temperature is from about 20 ° C. to about 30 ° C. 7. The method of claim 1 wherein the fabric is indigo-dyed denim. The method of claim 1 wherein the fabric is a sulfur-dyed denim. The method of claim 1 wherein the denim is desize and / or stonewashed prior to or concurrently with contacting the fabric with a laccase enzyme and a mediator. The method of claim 1 wherein the stonewash and contact of the fabric with the laccase enzyme and mediator occur in the same bath. The method of claim 1, further comprising contacting the fabric with the cellulase enzyme simultaneously or sequentially with contacting the fabric with the laccase enzyme and the mediator. 10. The method according to claim 9, wherein the contact of the fabric with the cellulase enzyme and the contact of the fabric with the laccase enzyme and the mediator are performed sequentially, and the contact of the fabric with the cellulase enzyme is carried out with the fabric with the laccase enzyme and the mediator. Fabric processing method performed before contact. The method of claim 10, wherein the contact of the fabric with the cellulase enzyme and the contact of the fabric with the laccase enzyme and the mediator are performed between the contact of the fabric with the cellulase enzyme and the contact of the fabric with the laccase enzyme and the mediator. Fabric processing method performed sequentially in the same bath without draining. The fabric processing method according to any one of claims 9 to 11, wherein the contact of the fabric with the cellulase enzyme and the contact of the fabric with the laccase enzyme and the mediator are performed at a temperature of less than 40 ° C. The method according to any one of claims 1 to 12, wherein the laccase is a microbial laccase. 13. The method of any one of claims 1 to 12, wherein the laccase is from the Serrena species. 13. The method of any one of claims 1 to 12, wherein the laccase is from Serrena unicolor.  The method according to any one of claims 1 to 12, wherein the laccase is Lacasease D from C. unicolor. The method according to any one of claims 1 to 12, wherein 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 a method having an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 . The method according to any one of claims 1 to 12, wherein 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 a method having 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 . 13. The method of 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 of 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. 13. The method of any one of claims 1 to 12, wherein the laccase has at least 90% identical amino acid sequence to SEQ ID NO: 19 or SEQ ID NO: 20. 22. The method of any one of claims 1 to 21, wherein the mediator is cyringonitrile. 22. The method of any one of claims 1 to 21, wherein the temperature is from about 20 ° C to about 35 ° C. 22. The method of any one of claims 1 to 21, wherein the temperature is from about 20 ° C to about 23 ° C. The method of claim 1, wherein the temperature is the ambient temperature of the tap water. 26. The method of any one of claims 1 to 25, wherein the laccase enzyme and the mediator are provided together in a ready-to-use composition. 26. The method of any one of claims 1 to 25, wherein the laccase enzyme and the mediator are provided in solid form.

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