CA2763882A1 - Enzymatic textile colour modification - Google Patents

Abstract

A method for adjusting the colour tone of dyed cellulosic textile fibre material comprising contacting said textile material with an enzymatic textile treatment composition comprising (i) a perhydrolase enzyme, (ii) an ester substrate for said perhydrolase enzyme, and (iii) a hydrogen peroxide source, for a length of time and under conditions suitable to permit measurable brightening of the textile material.

Description

Enzymatic Textile Colour Modification The present application claims priority to U.S. Provisional Application Serial No. 61/223,348, filed on July 7, 2009, U.S. Provisional Application Serial No. 61/322,743, filed on April 9, 2010, European Patent Application No. 09162047.6, filed on June 5, 2009 and European Patent Application No. 09163751.2, filed on June 25, 2009, which are hereby incorporated by reference in their entirety.

The present invention relates to methods for the enzymatic colour modification of dyed cellulosic textile fibre material, in particular denim dyed with indigo or sulphide dyes.

Some textile materials are washed after dyeing with the objective of adjusting the colour tone or shade on the dyed textile, also known as washdown effect. For instance, blue jeans made from indigo-dyed denim can be washed in the presence of pumice stones and enzymatic desizing agents, followed by an on tone-washdown process to obtain a desired worn appearance. Conventional washdown processes comprise treatment of the coloured denim with sodium hypochlorite, which is undesirable in view of the appearance of fibre damages and because of ecological reasons.
Washdown with hydrogen peroxide is an alternative solution. The adjusting effect obtainable with hydrogen peroxide, however, is rather limited. Furthermore, the required high pH is ecologically undesirable.

An enzymatic washdown process which does not show the above indicated disadvantages would be desirable.
There is a need for an effective enzymatic washdown process for coloured cotton textiles which provides the desired wash-out effect under mild conditions and minimizes the adverse environmental impact, when compared to conventional textile colour modification processes.
The present invention accordingly relates to a method for adjusting the colour tone of dyed cellulosic textile fibre material comprising contacting said textile material with an enzymatic textile treatment composition comprising (i) a perhydrolase enzyme, (ii) an ester substrate for said perhydrolase enzyme, and (iii) a hydrogen peroxide source.

The enzymatic treatment step of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J.
Gait, ed., 1984);
Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1994); PCR:
The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transfer and Expression: A
Laboratory Manual (Kriegler, 1990).

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 to which this invention pertains.

Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, New York (1991) provide one of skill in the art with a general dictionary of many of the biotechnology related terms used in this invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Numeric ranges provided herein are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The following terms and phrases are defined for clarity:

As used here, the term "adjusting" means the process of treating a textile material for a sufficient length of time and under appropriate pH and temperature conditions to produce a lighter colour in said textile material by removal, modification or masking of colour-causing compounds in the textile material. Thus, "adjusting" refers to the treatment of a textile material to effect a brightening of the textile material.
In some embodiments, brightening is defined as a percentage of colour removal from the textile material. The amount of colour removal can be determined by comparing the colour level can be determined by comparing the colour level following treatment with an enzymatic textile treatment composition according to the invention (i.e. residual colour level) to the colour level of the starting textile material (i.e. original colour level) using known spectrophotometric or visual inspection methods.

As used here, the term "original colour level" refers to the colour level of a dyed textile material prior to contact with an enzymatic textile treatment composition according to the invention.
Original colour level may be measured using known spectrophotometric or visual inspection methods.

As used here, the term "residual colour level" refers to the colour level of a dyed textile material after contact with an enzymatic textile treatment composition according to the invention.
Residual colour level may be measured using known spectrophotometric or visual inspection methods.

As used here, the term "cellulosic textile fibre material" refers to a material that comprises natural cellulosic fibres such as cotton, linen and hemp, semi-synthetic cellulosic fibres such as viscose and lyocell as well as blends of cellulosic fibres and synthetic fibres such as elastane.
Suitable cellulosic textile fibre material substrates that can be treated with the method according to the invention are yarns, wovens, knits and garments.

As used here, a "perhydrolase" refers to an enzyme that is capable of catalyzing a perhydrolysis reaction that results in the production of a sufficiently high amount of peracid suitable for use in an enzymatic textile adjusting composition according to the method described herein. Generally, a perhydrolase enzyme used in the methods described herein exhibits a high perhydrolysis to hydrolysis ratio. In some embodiments, the perhydrolase comprises, consists of, or consists essentially of the Mycobacterium smegmatis perhydrolase amino acid sequence set forth in SEQ ID NO: 1, or a variant or homolog thereof. In some embodiments, the perhydrolase enzyme comprises acyl transferase activity and catalyzes an aqueous acyl transfer reaction.

As used here, a "peracid" is an organic acid of the formula RC(=O)OOH, wherein R is an aliphatic, aromatic or araliphatic radical.

As used here, an "ester substrate" is a perhydrolase substrate that contains an ester linkage.
Esters comprising aliphatic and/or aromatic carboxylic acids and alcohols may be utilized as substrates with perhydrolase enzymes. In some embodiments, the ester source is selected from the esters of one or more of the following acids: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. In some embodiments, the ester source is an acetate ester. In some embodiments, the ester source is selected from one or more of propylene glycol diacetate, ethylene glycol diacetate, glycerol triacetate, ethyl acetate, and glycerol tributyrate.

As used here, the terms "perhydrolyzation," "perhydrolyze" and "perhydrolysis"
refer to a reaction wherein a peracid is generated from an ester substrate and a hydrogen peroxide source. The perhydrolyzation reaction is catalyzed with a perhydrolase, e.g., acyl transferase or aryl esterase, enzyme. In some embodiments, a peracid is produced by perhydrolysis of an ester substrate of the formula RC(=O)OR*, where R and R* are the same or different organic moieties, in the presence of hydrogen peroxide (H202). In one embodiment, -OR*
is -OH. In one embodiment, -OR* is replaced by -NH2. In some embodiments, a peracid is produced by perhydrolysis of a carboxylic acid or amide substrate.

As used here, the term "peracid," as used herein, refers to a molecule derived from, or capable of being derived from, a carboxylic acid ester which has been reacted with hydrogen peroxide to form a highly reactive product that is able to transfer one of its oxygen atoms. It is this ability to transfer oxygen atoms that enables a peracid, for example, peracetic acid, to function as a brightening agent.

As used here, the phrase "perhydrolysis to hydrolysis ratio" refers to the ratio of the amount of enzymatically produced peracid to the amount of enzymatically produced acid by a perhydrolase enzyme from an ester substrate under defined conditions and within a defined time. In some embodiments, the assays provided in WO 05/056782 are used to determine the amounts of peracid and acid produced by the enzyme.

As used herein, "effective amount of perhydrolase enzyme" refers to the quantity of perhydrolase enzyme necessary to achieve the enzymatic activity required in the processes or methods described herein. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the pH used, the temperature used and the like, as well as the results desired (e.g., level of brightening).

As used herein, the term "transferase" refers to an enzyme that catalyzes the transfer of a functional group from one substrate to another substrate. For example, an acyl transferase may transfer an acyl group from an ester substrate to a hydrogen peroxide substrate to form a peracid.

As used herein, the term "acyl" refers to an organic group with the general formula RCO-, derived from an organic acid by removal of the -OH group. Typically, acyl group names end with the suffix "-oyl," e.g., ethanoyl chloride, CH3CO-CI, is the acyl chloride formed from ethanoic acid, CH3CO-OH.

As used herein, the term "acylation" refers to a chemical transformation in which one of the substituents of a molecule is substituted by an acyl group, or the process of introduction of an acyl group into a molecule.

As used herein, an "oxidizing chemical" is a chemical capable of brightening a textile. The oxidizing chemical is present at an amount, pH and temperature suitable for brightening. The term includes, but is not limited to hydrogen peroxide and peracids.

As used herein, the terms "purified" and "isolated" refer to the removal of contaminants from a sample and/or to a material (e.g., a protein, nucleic acid, cell, etc.), such that the sample or material is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the polynucleotides applied applied within the context of the present invention encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O--Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring.
The terms "polynucleotide" and "nucleic acid" and "oligonucleotide" are used herein interchangeably. Polynucleotides of the invention may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S
("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (C,-C2o) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions. Suitable polynucleotides are described in WO 2005/056782.

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

As used herein, the terms "analogous sequence," "homologous protein," "wild-type or native proteins," "wild-type sequence," "native sequence," "naturally-occuring sequence," "wild-type gene," "related proteins," "derivative proteins," "variant proteins" and the like, are familiar to those skilled in the art and are described in more detail in WO 2005/056782 on pages 12, 13 and 50 to 52, which are herein incorporated by reference. In some embodiments, homologous proteins are engineered to produce enzymes with desired activity(ies).

Several methods are known in the art that are suitable for generating variants of the enzymes described herein, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

The degree of homology between sequences may be determined using any suitable method known in the art. For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. Another example of a useful algorithm is the BLAST
algorithm. Useful methods and programs are referred to in WO 2005/056782 on pages 59 and 60, which are herein incorporated by reference.

The terms "substantially similar" and "substantially identical" as generally used in the context of a polynucleotide or polypeptide sequence compared to a reference (i.e., wild-type) sequence as well as methods to determine sequence identity are described in more detail in WO
2005/056782 on pages 61 and 62, which are herein incorporated by reference.

As used herein, the term "surfactant" refers to a substance that reduces surface tension of a liquid.

As used herein, the term "emulsifier" refers to a substance that promotes the suspension of one liquid in another.

As used herein, the term "sequestering agent" refers to a substance capable of reacting with metallic ions by forming a water-soluble complex in which the metal is held in a non-ionizable form.

As used herein, the term "catalase" refers to an enzyme (i.e., a polypeptide having catalytic activity) that catalyzes the decomposition of hydrogen peroxide to water and oxygen.

As used herein, the term the term "batch process" or "batchwise process" or "discontinuous process" or "exhaust process" refers to processing of textiles as lots or batches in which the whole of each batch is subjected to a process or one stage of a process at a time.

As used herein, the term "liquor ratio" refers to the ratio of the weight of liquor (liquid) employed in a textile treatment process to the weight of the textile treated.

The enzymatic colour tone adjusting composition used in accordance with the enzymatic textile adjusting method of the present invention contain a perhydrolase enzyme, an ester substrate for the perhydrolase enzyme suitable for production of a peracid upon catalytic reaction of the perhydrolase enzyme on the substrate in the presence of a hydrogen peroxide source and/or hydrogen peroxide. The enzymatic colour tone adjusting composition may, optionally, further contain a surfactant and/or an emulsifier, a peroxide stabilizer, a fluorescence whitening agent, an enzymatic desizing agent, a biopolishing agent, a combination product, a sequestering agent or a bufferwhich maintains a pH of about 6 to about 8 during a textile colour tone adjusting process.

In the following the components applied in accordance with the method of the present invention along with information on the quantities of these components are described in more detail. Parts (ppm) are parts by weight, unless noted otherwise.

Perhydrolase Enzyme One or more perhydrolase enzymes may be used in the compositions according to the methods for enzymatic textile colour tone adjusting as described herein.

In some embodiments, the perhydrolase enzyme is naturally-occurring (i.e., a perhydrolase enzyme encoded by a genome of a cell). In some embodiments, the perhydrolase enzyme comprises, consists of, or consists essentially of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of a naturally-occurring perhydrolase enzyme.

In some embodiments, the perhydrolase enzyme is a naturally-occurring M.
smegmatis perhydrolase enzyme. In some embodiments, the perhydrolase enzyme comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID
NO:1 or a variant or homologue thereof. In some embodiments, the perhydrolase enzyme comprises, consists of, or consists essentially of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence set forth in SEQ
ID NO:1.
The amino acid sequence of M. smegmatis perhydrolase is shown below:
MAKRI LCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVI EEGLSARTTN I D
DPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGG
VGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVI
STDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO:1).

The corresponding polynucleotide sequence encoding M. smegmatis perhydrolase is:
5'-ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCC
CCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACCGGTG
TGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGACTGAGCGCGC
GCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGGCGCGAGCTACCTGC
CGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTGATCATCATGCTGGGCACCAA
CGACACCAAGGCCTACTTCCGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGT
GCTCGTCACGCAGGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCCGGCACC
CAAGGTGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCA
GTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGC
GCTCGCGTCGTTCATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGAC
GGCGTCGACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTC
GCGGAACAGGTGCGGAGCCTGCTGTAA-3' (SEQ ID NO:2).

In some embodiments, the perhydrolase enzyme comprises one or more substitutions at one or more amino acid positions equivalent to position(s) in the M. smegmatis perhydrolase amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the perhydrolase enzyme comprises any one or any combination of substitutions of amino acids selected from M1, K3, R4,15, L6, C7, D10, S11, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35, G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47, V48, 149, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, 160, D61, D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, R101, R102, P104, L105, D106, 1107, A108, L109, G110, M111, S112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P146, P148, W149, F150, 1153, F154, 1194, and F196.
In some embodiments, the perhydrolase enzyme comprises one or more of the following substitutions at one or more amino acid positions equivalent to position(s) in the M. smegmatis perhydrolase amino acid sequence set forth in SEQ ID NO:1: L1 2C, Q, or G;
T25S, G, or P;
L53H, Q, G, or S; S54V, L A, P, T, or R; A55G or T; R67T, Q, N, G, E, L, or F;
K97R; V125S, G, R, A, or P; F154Y; F196G.

In some embodiments, the perhydrolase enzyme is the S54V variant of SEQ ID NO:
1, which is shown, below as SEQ ID NO: 3, wherein the S54V substitution is underlined.

The amino acid sequence of M. smegmatis perhydrolase is shown below:
MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLVARTTNI D
DPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGG
VGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVI
STDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO: 3).

In some embodiments, the perhydrolase enzyme comprises a combination of amino acid substitutions at amino acid positions equivalent to amino acid positions in the M. smegmatis perhydrolase amino acid sequence set forth in SEQ ID NO:1: L121 S54V; L12M
S54T; L12T
S54V; L12Q T25S S54V; L53H S54V; S54P V125R; S54V V125G; S54V F196G; S54V K97R
V125G; or A55G R67T K97R V1 25G.

In some embodiments, the perhydrolase enzyme comprises a perhydrolysis to hydrolysis ratio of at least 1. In some embodiments, the perhydrolase enzyme comprises a perhydrolysis to hydrolysis ratio greater than 1.

In some embodiments, the perhydrolase enzyme is provided in the enzymatic textile colour tone adjusting composition used according to the textile colour tone adjusting method of the present invention at a concentration of about 0.5 to about 2.5 ppm, about 1.5 to about 2.0 ppm, for example, about 1.7 ppm, based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

Ester Substrate The enzymatic colour tone adjusting compositions used in accordance with the method described herein include an ester which serves as a substrate for the perhydrolase enzyme for production of a peracid in the presence of hydrogen peroxide. In some embodiments, the ester substrate is an ester of an aliphatic and/or aromatic carboxylic acid. In some embodiments, the ester substrate is an ester of one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid.
In some embodiments, glycerol triacetate, glycerol tributyrate, and other esters serve as acyl donors for peracid formation. In some embodiments, the ester substrate is selected from propylene glycol diacetate, ethylene glycol diacetate, glycerol triacetate, ethyl acetate, and glycerol tributyrate.
In some embodiments, the ester substrate is propylene glycol diacetate, ethylene glycol diacetate, or ethyl acetate. In one embodiment, the ester substrate is propylene glycol diacetate.

In some embodiments, the ester substrate, for example, propylene glycol diacetate, is provided at a concentration of about 2000 to about 4000 ppm, about 2500 to about 3500 ppm, about 2800 ppm to about 3200 ppm, or about 3000 ppm, based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

Hydrogen Peroxide Source The enzymatic colour tone adjusting compositions used in accordance with the method described herein include a hydrogen peroxide source. Hydrogen peroxide can be either added directly in batch, or generated continuously "in situ" by chemical, electro-chemical, and/or enzymatic means.

In some embodiments, the hydrogen peroxide source is hydrogen peroxide. In some embodiments, the hydrogen peroxide source is a solid compound that generates hydrogen peroxide spontaneously upon addition to water. Such compounds include adducts of hydrogen peroxide with various inorganic or organic compounds, of which the most widely employed is sodium carbonate perhydrate, also referred to as sodium percarbonate.
Inorganic perhydrate salts are one embodiment of hydrogen peroxide source.
Examples of inorganic perhydrate salts include perborate, percarbonate, perphosphate, persulfate and persilicate salts. The inorganic perhydrate salts are normally the alkali metal salts.

Other hydrogen peroxide adducts useful in the compositions used in accordance with the method described herein include adducts of hydrogen peroxide with zeolites, or urea hydrogen peroxide.

The hydrogen peroxide source compounds may be included as the crystalline and/or substantially pure solid without additional protection. For certain perhydrate salts however, the preferred executions of such granular compositions utilize a coated form of the material which provides better storage stability for the perhydrate salt in the granular product. Suitable coatings comprise inorganic salts such as alkali metal silicate, carbonate or borate salts or mixtures thereof, or organic materials such as waxes, oils, or fatty soaps.

In some embodiments, the hydrogen peroxide source is an enzymatic hydrogen peroxide generation system. In one embodiment, the enzymatic hydrogen peroxide generation system comprises an oxidase and its substrate. Suitable oxidase enzymes include, but are not limited to: glucose oxidase, sorbitol oxidase, hexose oxidase, choline oxidase, alcohol oxidase, glycerol oxidase, cholesterol oxidase, pyranose oxidase, carboxyalcohol oxidase, L-amino acid oxidase, glycine oxidase, pyruvate oxidase, glutamate oxidase, sarcosine oxidase, lysine oxidase, lactate oxidase, vanillyl oxidase, glycolate oxidase, galactose oxidase, uricase, oxalate oxidase, and xanthine oxidase.

The following equation provides an example of a coupled system for enzymatic production of hydrogen peroxide.
Glucose oxidase Glucose + H2O ---------------------------------------------------gluconic acid + H202 Perhydrolase H202 + ester substrate ----------------------------------------4 alcohol +
peracid It is not intended that the present invention be limited to any specific enzyme, as any enzyme that generates H202 with a suitable substrate may be used in the present invention. For example, lactate oxidases from Lactobacillus species which are known to create H202 from lactic acid and oxygen may be used. One advantage of the enzymatic generation of acid (e.g., gluconic acid in the above example) is that this reduces the pH of a basic solution to the pH
range in which a peracid is most effective in colour tone adjusting (i.e., at or below the pKa).
Other enzymes (e.g., alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, etc.) that can generate hydrogen peroxide also may be used with ester substrates in combination with the perhydrolase enzymes of the present invention to generate peracids.

In some embodiments, the hydrogen peroxide generating oxidase is a carbohydrate oxidase.
Hydrogen peroxide may also be generated electrochemically, for example using a fuel cell fed oxygen and hydrogen gas.

In some embodiments, the hydrogen peroxide source is hydrogen peroxide provided at a concentration of about 1000 to about 3200 ppm, about 1500 to about 2800 ppm, about 2000 ppm to about 2200 ppm, or about 2100 ppm, based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

Surfactants and Emulsifier The enzymatic textile colour tone adjusting compositions used in accordance with the present method may contain one or more, i.e., at least one surfactant and/or at least one emulsifier.
Surfactants suitable for use in practicing the present invention include, without limitation, nonionic (see, e.g., U.S. Pat. No. 4,565,647, which is herein incorporated by reference);
anionic; cationic; and zwitterionic surfactants (see, e.g., U.S. Pat. No.
3,929,678 which is herein incorporated by reference). Anionic surfactants include, without limitation, linear alkylbenzenesulfonate, a-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, and soap. Non-ionic surfactants include, without limitation, fatty alcohol ethoxylate, isotridecanol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, and N-acyl N-alkyl derivatives of glucosamine ("glucamides").

In some embodiments, the surfactant and/or emulsifier comprises a non-ionic surfactant. In one embodiment, the non-ionic surfactant is a fatty alcohol ethoxylate. In one embodiment, the non-ionic surfactant is isotridecanol ethoxylate. In one embodiment, the non-ionic surfactant is a fatty alcohol ethoxylate and isotridecanol ethoxylate.

In one embodiment, the composition used in accordance with the present method comprises a surfactant and an emulsifier.

A surfactant may be present at a concentration of about 300 ppm to about 4800 ppm, about 600 ppm to about 3600 ppm, or about 300 ppm to about 1200 ppm, based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

In some embodiments, the enzymatic colour tone adjusting composition contains isotridecanol ethoxylate at a concentration of about 300 ppm to about 3600 ppm, about 600 ppm to about 3000 ppm, or about 900 ppm to about 2400 ppm, based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

Peroxide Stabilizer The enzymatic colour tone adjusting compositions used in accordance with the method described herein may contain a peroxide stabilizer. Examples of peroxide stabilizers include, but are not limited to, sodium silicate, sodium carbonate, acrylic polymers, magnesium salts, and phosphonic acid. In one embodiment, the peroxide stabilizer is phosphonic acid.

A peroxide stabilizer may be present in the enzymatic textile colour tone adjusting composition at a concentration of about 60 ppm to about 600 ppm, about 60 ppm to about 1200 ppm, or about 120 ppm to about 960 ppm based on the total weight of the aqueous composition (bath) applied for treatment of the textile material.

Combination Product Appropriately, at least one surfactant and/or emulsifier, at least one peroxide stabilizer and at least one sequestering agent are applied as a combination product containing each of at least one surfactant and/or emulsifier, at least one peroxide stabilizer and at least one sequestering agent. The said combination product is designated bleaching processor and is commercially available, for example, CLARITE LTC, CLARITE WIN or CLARITE ONE (products of Huntsman).
A surfactant may be present at a concentration of about 5% to about 40%, about 20% to about 30%, or about 5% to about 10%, based on the total weight of the bleaching processor.

A peroxide stabilizer may be present in the combination product at a concentration of about 1 %
to about 5%, about 1 % to about 10%, or about 2% to about 8%, based on the total weight of the bleaching processor.

A sequestering agent may be present in the bleaching processor at a concentration of about 1 % to about 15%, about 5% to about 10%, or about 3% to about 10%, based on the total weight of the bleaching processor.

In some embodiments, the bleaching processor contains isotridecanol ethoxylate at a concentration of about 5% to about 30%, about 10% to about 25%, or about 15%
to about 20%, based on the total weight of the bleaching processor.

The bleaching processor is suitably provided as an aqueous composition comprising the above indicated components.

Buffer The enzymatic colour tone adjusting composition may contain a buffer that is capable of maintaining the pH of the composition at a pH of about 6 to about 8. The buffer may be, for example, a phosphate buffer, pH 7, or a sodium carbonate or potassium carbonate, pH 7.

Enzymatic Textile Colour Tone Adjusting Method The method according to the invention is particularly suitable for the treatment of denim dyed with vat dyes, reactive dyes, direct dyes and sulphur dyes, most preferably for indigo-dyed denim and/or sulfur-dyed denim. The method provides textile material with a soft handle and very good crease recovery properties compared to textiles treated using conventional methods. The methods described herein are particularly carried out as a discontinuous process but can also be carried out as a semi-continuous process like pad-batch or pad-roll.

Appropriately, the method of the invention utilizes a liquor ratio of about 2:1 to about 50:1, about 5:1 to about 20:1, for example, about 20:1 or 10:1.
Textiles are contacted with the enzymatic colour tone adjusting composition at a temperature of about 55 C to about 75 C, about 60 C to about 70 C, for a processing time of about 20 to about 60 minutes at a pH of about 6 to about 8. In one embodiment, the treatment temperature is about 65 C and the processing time is about 50 minutes. In some embodiments, the temperature of the enzymatic colour tone adjusting composition is raised by about 2 C per minute from a starting temperature of about 20 C to about 50 C, for example, about 20 C to about 40 C, until the processing temperature for colour tone adjusting is reached.
One or more rinsing steps are performed after treatment of the textile material with the enzymatic colour tone adjusting composition, to remove the colour tone adjusting composition.
Appropriately, the textile is rinsed with an aqueous composition (water or a composition containing water). In some embodiments, the rinsing temperature is about 40 C
to about 60 C, for example, about 50 C.

In some embodiments, the aqueous rinsing composition contains a catalase enzyme to catalyze the decomposition of hydrogen peroxide to water and oxygen. In one embodiment, the textile is rinsed twice with a catalase containing aqueous composition for about 10 minutes for each rinse. In one embodiment residual hydrogen peroxide is removed by rinsing twice with an aqueous composition containing catalase at about 50 C. In some embodiments, catalase may be added directly to the liquor containing the perhydrolase enzyme without first dropping the bath.

As the enzymes that hydrolyze polyester substrates suitable for use in the present invention may be used, for example, pectinases, cutinases or lipases. These enzymes, their application as well as assays to determine enzyme activity are described and referred to in more detail in WO 2007/136469 on pages 21 and 22, which are herein incorporated by reference.

Further suitable enzymes that may be used additionally in the method according to the invention are amylases (desizing agents) and cellulases (biopolishing agents).

The following examples are intended to illustrate, but not limit, the invention. Temperatures are in degrees Celsius, parts are parts by weight and the percentage data are percentages by weight, unless noted otherwise. Parts by weight bear the same relation to parts by volume as the kilogram to the litre.

Experimental Part A comparison between the method of the present invention and conventional oxidation bleach with hydrogen peroxide is performed according to the procedures given below by treating the fabric in exhaust using a Mathis AG Labomat.

Examples 1 and 2 and Comparative Example 3 Enzymatic colour tone adjusting (Examples 1 and 2) carried out as a one-bath exhaustion method:
Indigo dyed denim swatches (length: 11 cm, width: 9.5 cm), washed at 65 C/5 min and cold in overflow/5 min, are treated in a bath containing the combination product, the buffer, propylene glycol diacetate, hydrogen peroxide and the perhydrolase enzyme in the amounts given in Table 1 using a liquor ratio of 10:1. The temperature is raised from ambient temperature to a target temperature of 65 C at a rate of 2 C per minute. The bath is then held at 65 C for 5 0 minutes and after cooling and draining the swatches are rinsed twice for 10 minutes each at 50 C and then dried at 70 C. 0.5 g/I of a 25% solution of Catalase T100 (available from Genencor) is included in each rinse.
Alkali colour tone adjusting (Comparative Example 3) carried out as a one-bath exhaustion method:

The procedure described above is repeated, but perhydrolase enzyme and phosphate buffer are replaced with sodium hydroxide.

Table 1: Examples 1, 2 and Comparative Example 3 Example # 1 2 3 (comparison) Deionised water [g/I] 975.0 975.0 987.5 INVALON EC 1) [g/I] 1.0 CLARITE GS 3) [g/I] 0.5 CLARITE WIN 2) [g/I] 3.0 CLARITE ONE 4) [g/I] 3.0 Phosphate buffer solution [ml/1] 12.0 12.0 Propylene glycol diacetate [ml/1] 3.0 3.0 Hydrogen peroxide 35% [ml/1] 6.0 6.0 6.0 Perhydrolase enzyme 5) [g/I1 1.0 1.0 NaOH (30 %) [g/I] 5.0 pH 10.2 10.5 11.0 Aspect (initially) Colourless, dull Colourless, dull Colourless, dull pH after treatment 7.2 7.2 11.5 Aspect (after treatment) Dark green, Dark green, Dark green, clear clear clear INVALON EC: dispersing agent (commercial product supplied by Huntsman) 2) CLARITE WIN: bleaching processor (commercial product supplied by Huntsman) 3) CLARITE GS: bleaching processor (commercial product supplied by Huntsman) 4) CLARITE ONE: bleaching processor (commercial product supplied by Huntsman) 5) Primagreen EcoWhite (1x) (commercial product supplied by Genencor) Example 4 Enzymatic on-tone washdown carried out as a one-bath exhaustion method:
Indigo dyed denim swatches (length: 11 cm, width: 9.5 cm), desized at 10:1 liquor ratio, 60 C/10 min, with 0.5 g/I CLARITE WIN, 0.5 g/I Albfluid C and 1.5 g/I ULTRAVON
RW
(non-ionic surfactant, commercial product supplied by Huntsman) are treated in the same bath with 1.5 g/I of INVAZYME LTE (perhydrolase enzyme) at 10:1 liquor ratio, 60 C/10 min.
Following desizing, the denim is stonewashed in a rotary washing machine with 1 kg of pumice stones (60 C/40 min). Subsequently, 1.0 g/I CLARITE LTC (combination product, supplied by Huntsman), 2.5 g/I of soda ash, 3.0 g/I of propylene glycol diacetate, 3.0 g/I of hydrogen peroxide and 1.0 g/I of INVAZYME LTE (perhydrolase enzyme) are added to the liquor. The bath is then held at 65 C for 50 minutes and after cooling and draining the swatches are rinsed twice for 15 minutes each at 50 C with a bath containing 0.5 g/I INVAZYME
CAT (stabilized liquid catalase enzyme to remove residual peroxide) and afterwards for 15 min with a bath containing 1.0 g/I of a softening agent (Turpex CAN new) at room temperature, and then dried.
The denim swatches thus obtained have a soft handle and very good crease recovery properties.

Example 5 Effect of Perhydrolase Concentration on Dye Discoloration on Indigo-dyed Denim Materials Perhydrolase (PrimaGreen EcoWhite 1 (321 U/g), available from Genencor Division, Danisco US, Inc.), is used in this experiment. H202 analysis grade (30 wt %) and propylene glycol diacetate >99.7% (PDGA) were purchased from Sigma Aldrich.

Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized in a Unimac UF 50 washing machine under the following conditions:
= Desizing for 15 minutes at 10:1 liquor ratio 50 C with 0.5 g/I (15 g) of Optisize 160 amylase (Genencor) and 0.5 g/I (15 g) of a non-ionic surfactant (Ultravon RW
(Huntsman)).
= 2 cold rinse steps for 5 minutes at 30:1 liquor ratio.
Following desizing, the denim is stonewashed in a Unimac UF 50 rotary washing machine according to the following program:
= Cold rinsing for 5 minutes at 10:1 liquor ratio = Stonewashing for 60 minutes at 10:1 liquor ratio 55 C with 1 kg of pumice stone, pH 6.5 - 7 (1 g/I of disodium phosphate 2H20 + 0.53 g/I of citric acid H2O) and 0.025 g/I of MEX-500 neutral cellulase (Meiji).
= 2 cold rinse steps of 5 min each The denim is dried in a household dryer and then used to make swatches (7 x 7 cm).
After stonewashing, the experiments are performed in a Launder-O-meter (Rapid Laboratory Dyeing Machine type H12) according to the following process:
= 450 ml stainless steel reaction vessels are filled with 100 ml of pH 8 phosphate buffer (8.9 g/I of disodium phosphate 2H20 + 0.4 g/I of monosodium phosphate anhydrous).

= To each vessel five (7 x 7 cm) stonewashed denim swatches of 10 g weight are added.
= 6 ml/I of H202 solution (30% wt) and 2m1/I of PDGA (>99.7%) is added.
= Perhydrolase is added at concentrations of 0. 01, 0. 05, 0. 3, 1.0, 3.0, or 10 ml/l.
= The reaction vessels are closed and loaded into the launder-O-meter, which is pre-heated to 60 C.
= Incubation is performed for 60 minutes, after which the swatches are rinsed by overflow, spun dry in an AEG IPX4 centrifuge, and dried with an Elna Press Electronic iron at program cotton and evaluated.

Evaluation of denim swatches The denim swatches are evaluated after perhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. Measurements are done before and after perhydrolase treatment and the results from five swatches are averaged. The total color difference (TCD) is calculated using the formula: TCD = J (AL)2 + (Aa)2 + (Ab)2.
The results are shown in Table 2.

Table 2 Perhydrolase TCD L/ a/ b concentration (ml/1) Buffer 0.44 0.41/0.13/0.10 0.01 0.56 0.40/0.32/-0.23 0.05 1.46 1.10/0.31/-0.90 0.3 1.97 1.50/0.34/-1.23 1 2.11 1.37/0.51/-1.52 3 2.05 1.41/0.41/-1.43 1.49 1.19/0.42/-0.80 Example 6 Effect of H202 and PDGA Concentrations on Dye Discoloration Performance of Perhydrolase on Indigo-dyed Denim Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized and stonewashed as described in Example 5. After stonewashing, the experiments are performed in a Launder-O-meter (Rapid Laboratory Dyeing Machine type H12) according to the following process:
= 450 ml stainless steel reaction vessels are filled with 100 ml of pH 8 phosphate buffer (8.9 g/I of disodium phosphate 2H20 + 0.4 g/I of monosodium phosphate anhydrous).
= To each vessel five (7 x 7 cm) stonewashed denim swatches of 10 g weight are added.
= H202 solution (30% wt) and PDGA (>99.7%) are added according to the experimental design as shown in Table 3 =
Table 3 [H202] (MI/1) [PGDA] (ml/1) 7.55 3.8 15 7.5 0.1 7.5 7.55 3.8 0.1 0.1 15 0.1 7.55 3.8 6.0 3.0 0 3.0 6.0 0 15 3.8 7.55 7.5 = 1.0 mI/I of perhydrolase is added (PrimaGreen EcoWhite 1 (321 U/g)).
= The reaction vessels are closed and loaded into the Launder-O-Meter which was pre-heated to 60 C
= Incubation is performed for 60 minutes, after which the swatches are rinsed by overflow, spun dry in an AEG IPX4 centrifuge, and dried with an Elna Press Electronic iron at program cotton, and evaluated.

Evaluation of denim swatches The denim swatches are evaluated after perhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. Measurements are done before and after perhydrolase treatment and the results from five swatches are averaged. The total color difference (TCD) is calculated using the formula: TCD = J (AL)2 + (4a)2 + (4b)2.
The results are shown in Table 4.

Table 4 [H202] (ml/1) [PGDA] (ml/1) TCD L/ a/ b 7.55 3.8 2.33 1.03/0.36/-1.24 15 7.5 2.48 1.11/0.40/-1.37 0.1 7.5 1.09 0.57/0.02/0.00 7.55 3.8 2.31 1.04/0.45/-1.17 0.1 0.1 0.76 0.07/-0.04/-0.06 15 0.1 1.48 0.66/0.12/-0.49 6.0 3.0 2.55 1.50/0.24/-1.17 0 3.0 0.62 0.15/-0.06/0.22 6.0 0 0.80 -0.22/0.10/-0.15 15 3.8 2.17 0.62/0.43/-1.28 7.55 7.5 2.37 1.17/0.35/-1.19 Example 7 Effect of Time on Dye Discoloration Performance of Perhydrolase on Indigo-dyed Denim Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized and stonewashed as described in Example 5. After stonewashing, the experiments are performed in a Launder-O-meter (Rapid Laboratory Dyeing Machine type H12) according to the following process.
= 450 ml stainless steel reaction vessels are filled with 100 ml of pH 8 phosphate buffer (8.9 g/I Disodium phosphate 2H20 + 0.4 g/I Monosodium phosphate anhydrous) = To each vessel five (7 x 7 cm) stonewashed denim swatches of 10 g weight are added.
= 6 mI/I of H202 solution (30% wt) and 0.2 mI/I of PDGA (>99.7%) are added.
= 1.0 g/I of perhydrolase is added (PrimaGreen EcoWhite 1 (321 U/g)).
= The reaction vessels are closed and loaded into the Launder-O-Meter, which was pre-heated to 60 C.

Incubation is performed for 10, 20, 30, 40, 50, or 60 minutes, after which the swatches are rinsed by overflow, spun dry in an AEG IPX4 centrifuge, dried with an Elna Press Electronic iron at program cotton, evaluated.
Evaluation of denim swatches The denim swatches are evaluated after perhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. Measurements are done before and after perhydrolase treatment and the results from five swatches are averaged. The total color difference (TCD) is calculated using the formula: TCD = J (AL)2 + (Aa)2 + (Ab)2.
[01] The results are shown in Table 5.
Table 5 time TCD L/ a/ b buffer 1.09 1.05/0.27/0.05 1.48 0.97/0.30/-1.08 2.17 1.51/0.45/-1.49 2.05 1.28/0.53/-1.51 2.24 1.57/0.44/-1.55 2.45 1.80/0.49/-1.59 2.62 1.99/0.46/-1.64 Example 8 Effect of Temperature on Dye Discoloration Performance of Perhydrolase on Indigo-dyed Denim Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized and stonewashed as described in Example 5. After stonewashing, the experiments are performed in a Launder-O-meter (Rapid Laboratory Dyeing Machine type H12) according to the following process.
= 450 ml stainless steel reaction vessels are filled with 100 ml of pH 8 phosphate buffer (8.9 g/I of disodium phosphate 2H20 + 0.4 g/I of monosodium phosphate anhydrous).
= To each vessel five (7 x 7 cm) stonewashed denim swatches of 10 g weight are added.
= 6 ml/I of H202 solution (30% wt) and 2m1/I of PDGA (>99.7%) is added.

= 1.0 mI/l of perhydrolase is added (PrimaGreen EcoWhite 1 (321 U/g)).

= The reaction vessels are closed and loaded into the Launder-O-Meter, which was pre-heated to 30, 40, 50 or 60 C.
= Incubation is performed for 60 minutes, the swatches rinsed by overflow, spun dry in an AEG IPX4 centrifuge, dried with an Elna Press Electronic iron at program cotton, evaluated.

Evaluation of denim swatches The denim swatches are evaluated after perhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. Measurements are done before and after perhydrolase treatment and the results from five swatches are averaged. The total color difference (TCD) is calculated using the formula: TCD = J (AL)2 + (Aa)2 + (Ab)2.
The results are shown in Table 6.
Table 6 Temperature C TCD L/ a/ b 30 (only buffer) 0.93 0.91/0.07/0.16 30 1.36 1.20/0.28/-0.57 40 (only buffer) 0.78 0.77/0.11/-0.02 40 1.55 1.26/0.28/-0.86 50 (only buffer) 1.07 1.06/0.11/-0.02 50 2.02 1.63/0.32/-1.14 60 (only buffer) 0.9 0.86/0.24/-0.15 60 2.21 1.67/0.44/-1.38 Example 9 Color Adjustment Performance with a Cellulase + Perhydrolase Sequential Process on Indigo-dyed Denim in a Front Loading Washing Machine Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized in a Unimac UF 50 washing machine under the following conditions:
= Desizing for 15 minutes at 10:1 liquor ratio 50 C with 0.5 g/I (15 g) of Optisize 160 amylase (Genencor) and 0.5 g/I (15 g) of a non-ionic surfactant (Ultravon RW
(Huntsman)) = 2 cold rinses for 5 minutes at 30:1 liquor ratio Following desizing, the denim is stonewashed in a Unimac UF 50 rotary washing machine according to the following procedure:
= Cold rinse for 5 minutes at 10:1 liquor ratio = Stonewashing for 60 minutes at 10:1 liquor ratio 55 C with 1 kg of pumice stone, pH 4.8 (1 g/I of trisodium citrate 2 H2O + 0.87g/l of citric acid H2O) 1.17g/l of Indiage 2XL
cellulase (Genencor) = 2 cold rinse steps of 5 min each = 4 legs taken out as a control After stonewashing, treatment with perhydrolase is performed in a Unimac UF 50 washing machine according to the following process:
= 60 minutes at 10:1 liquor ratio, with 1 g/I perhydrolase (PrimaGreen EcoWhite 1 (321 U/g)), 6 g/I of H202 solution (30%wt) and 3g/I of PDGA(>99.7%) at pH 7 (1 g/I
of disodium phosphate 2 H2O and 0.17 g/I of citric acid) and temperature of 60 C.
The pH
was kept at 7 by adding 4 M of sodium hydroxide solution = 2 cold rinses for 5 minutes at 30:1 liquor ratio = The denim is dried in a household dryer.
Evaluation of denim legs Colour tone adjusting of denim legs is evaluated after treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. For each denim leg, 8 measurements are taken and the results of the 12 legs (96 measurements) were averaged.
The results are shown in Table 7.

Table 7 trials L/a/b Perhydrolase treatment 36.3/-0.29/-15.17 Example 10 Color Adjustment Performance of Cellulase + Laccase + Perhydrolase Sequential Process on Indigo-dyed Denim in a Front Loading Washing Machine Procedure Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, is desized in a Unimac UF 50 washing machine under the following conditions:

= Desizing for 15 minutes at 10:1 liquor ratio 50 C with 0.5 g/I (15 g) of Optisize 160 amylase (Genencor) and 0.5 g/I (15 g) of a non-ionic surfactant (Ultravon RW
(Huntsman).
= 2 cold rinses for 5 minutes at 30:1 liquor ratio Following desizing, the denim is stonewashed in a Unimac UF 50 rotary washing machine according to the following procedure:
= Cold rinse for 5 minutes at 10:1 liquor ratio = Stonewashing for 60 minutes at 10:1 liquor ratio 55 C with 1 kg of pumice stone, pH 4.8 (1 g/I of trisodium citrate 2 H2O + 0.87g/l of citric acid H2O) and 1.17g/l of Indiage 2XL
(Genencor) = 2 cold rinse steps of 5 min each After stonewashing, laccase treatment is performed in a Unimac UF 50 washing machine according to the following process:
= 30 minutes at 10:1 liquor ratio, with 3 g/I of ready to use PrimaGreen EcoFade LT 100 (Genencor) laccase and laccase mediator at pH 6 and temperature of 30 C
= 2 cold rinses for 5 minutes at 30:1 liquor ratio = The denim is dried in a household dryer.
After brightening with laccase, treatment with perhydrolase is performed in a Unimac UF 50 washing machine according to the following process:
= 60 minutes at 10:1 liquor ratio, with 1 g/I of perhydrolase (PrimaGreen EcoWhite 1 (321 U/g)), 6 g/I of H202 solution (30%wt) and 3g/I of PDGA(>99.7%) at pH 8 (8.9 g/I
disodium phosphate 2H20 + 0.4 g/I monosodium phosphate anhydrous) and temperature of 60 C
= 2 cold rinses for 5 minutes at 30:1 liquor ratio The denim is dried in a household dryer Evaluation of denim legs Brightening of denim legs is evaluated after laccase treatment and after perhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source. For each denim leg, 8 measurements are taken and the results of the 12 legs (96 measurements) are averaged.
The results are shown in Table 8.

Table 8 Trials L/a/b Laccase 40.5/-1.5/-12.1 Laccase + Perhydrolase 44.4/-1.3/-15.2 Example 11 Effect of Perhydrolase Treatment on a Dyed Fabric Materials Perhydrolase (S54V variant of M smegmatis, containing 1.5 mg/g active protein) is used in this Example. Hydrogen peroxide (analytical grade; 30% w/w) and propylene glycol diacetate (>99.7%) are purchased from Sigma Aldrich. Standard dyed fabrics are obtained from the Center for Test Materials, Vlaardingen, The Netherlands. The fabrics are dyed with one of the dyes (or combinations of dyes) listed in Table 15, which are identified by their Color Index (CI) number according to the Society of Dyers and Colourists (UK) and by the American Association of Textile Chemists and Colorists (USA).

Procedure 2 each swatches of dyed fabric (see Table 9) of approximately 12.5 cm x 12.5 cm are treated in a Launder-O-Meter (Rapid Laboratory Dyeing Machine type H 12) according to the following procedure:
= 3 each 450-ml stainless steel reaction vessels are filled with 100 ml of phosphate buffer (pH 8; 8.9 g/I of disodium phosphate-2H20 + 1.06 g/L of monosodium phosphate anhydrous).
= 2 each standard fabric swatches are added to each vessel.
= 6.0 ml/L H202 solution (30% wt/wt) and 3.0 ml/L of PGDA (>99.7%) are added to 2 each reaction vessels ("perhydrolase" and "blank"). Buffer only is added to the third reaction vessel.
= 1.0 ml/L perhydrolase is added to the "perhydrolase" reaction vessel.
= The reactions vessels are closed and placed in the Launder-O-Meter pre-heated to 60 C.
Incubation is carried out for 30 minutes. Following incubation, the swatches are rinsed by overflow, spin dried in an AEG IPX4 centrifuge, and dried in a Novotronic T
494 C household type dryer.

The resulting experimental samples are designated "perhydrolase" (perhydrolase + H202 +
PGDA), "blank" (H202 + PGDA), and "buffer only."

Table 9. Dyes applied to the different fabric swatches Cl Sulphur Black 1 Cl Sulphur Black 1 Cl Diazo component 13 (Fast Scarlet R) and Cl Coupler 2 (Naphthol AS) Cl Direct Black 22 Cl Direct Black 22 Cl Direct Black 22 Cl Reactive Black 5 Cl Reactive Black 5 Cl Reactive Orange 16 Cl Reactive Blue 71 Cl Reactive Blue 19 Mixture of Cl Reactive Orange 107, Reactive Red 198, and Reactive Black 5 Mixture of Cl Reactive Orange 107, Reactive Red 198, and Reactive Blue 220 Evaluation of standard fabric swatches The effect of perhydrolase treatment on the standard fabric swatches is evaluated using a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65 light source.
Measurements are performed before and after perhydrolase (or control) treatment. Three measurements are performed on each swatch (6 measurements total for each experimental condition) and the results averaged. Total color difference (TCD) is calculated using the formula: TCD = 4 (AL)2 + (Aa)2 + (Ab)2. The A values are the difference between the fabric before treatment and the fabric after treatment. The TCD* values are the difference between the values obtained for the blank values and for the perhydrolase treatment.
The results are shown, below, in the following Tables, which indicate the dyes applied to the fabrics.

Table 10. Cl Sulphur Black 1 Swatch L/a/b TCD TCD*
Fabric before treatment 24.5/0.5/-3.0 --Only buffer 24.1/0.4/-3.2 0.5 Blank 25.8/0.4/-3.5 1.4 Perhydrolase 41.4/0.5/-2.8 16.7 15.3 Table 11. Cl Sulphur Black 1 + CR resin Swatch L/a/b TCD TCD*
Fabric before treatment 24.4/0.8/-0.9 --Only buffer 23.4/0.7/-1.1 1.1 Blank 24.1/0.7/-1.7 0.8 Perhydrolase 34.7/0.2/-3.0 10.4 10.6 Table 12. Mixture of Cl Diazo component 13 and Cl Coupler 2 Swatch L/a/b TCD TCD*
Fabric before treatment 37.8/58.9/28.0 --Only buffer 37.1/59.5/28.6 1.1 Blank 38.3/60.4/30.4 2.9 Perhydrolase 39.0/60.8/32.3 4.8 2.0 Table 13. Cl Direct Black 22 Swatch L/a/b TCD TCD*
Fabric before treatment 23.2/-0.4/-1.0 --Only buffer 22.7/-0.6/-1.5 0.7 Blank 23.2/-0.6/-1.6 0.7 Perhydrolase 26.7/-0.3/-8.6 8.3 7.8 Table 14. Cl Direct Black 22 + cationic finish Swatch L/a/b TCD TCD*
Fabric before treatment 23.6/-0.1/-1.2 --Only buffer 22.8/0.1/-1.3 0.9 Blank 22.8/-0.2/-1.4 0.9 Perhydrolase 25.8/0.7/-5.5 4.9 5.1 Table 15. Cl Direct Black 22 + CR resin Swatch L/a/b TCD TCD*
Fabric before treatment 23.2/-0.2/-1.0 --Only buffer 22.7/-0.2/-1.2 0.5 Blank 22.9/-0.4/-1.4 0.7 Perhydrolase 24.1/-0.3/-2.9 2.1 1.9 Table 16. Cl Reactive Black 5 (light) Swatch L/a/b TCD TCD*
Fabric before treatment 44.1/-0.2/-17.7 --Only buffer 43.7/-0.1/-17.8 0.4 Blank 44.1/0.1/-17.8 0.3 Perhydrolase 47.1/-0.4/-15.0 4.0 4.1 Table 17. Cl Reactive Black 5 (dark) Swatch L/a/b TCD TCD*
Fabric before treatment 26.1/1.9/-12.8 --Only buffer 25.5/2.0/-12.7 0.5 Blank 25.5/2.0/-12.7 0.6 Perhydrolase 27.4/1.2/-12.0 1.6 2.1 Table 18. Cl Reactive Orange 16 Swatch L/a/b TCD TCD*
Fabric before treatment 60.0/50.5/35.8 --Only buffer 59.5/50.4/35.3 0.7 Blank 59.8/50.6/35.3 0.6 Perhydrolase 61.1/49.5/34.6 1.9 1.8 Table 19. Cl Reactive Blue 71 Swatch L/a/b TCD TCD*
Fabric before treatment 64.1/-21.3/-35.3 --Only buffer 63.3/-21.3/-36.0 1.0 Blank 63.4/-21.2/-35.7 0.8 Perhydrolase 64.0/-22.3/-33.6 2.0 2.4 Table 20. Cl Reactive Blue 19 Swatch L/a/b TCD TCD*
Fabric before treatment 41.4/11.3/-42.8 --Only buffer 40.6/11.7/-43.4 1.1 Blank 40.9/11.8/-43.6 1.1 Perhydrolase 41.4/10.9/-41.9 1.0 1.9 Table 21. Mixture of Cl Reactive Orange 107, Reactive Red 198, and Reactive Black 5 Swatch L/a/b TCD TCD*
Fabric before treatment 32.3/4.7/3.2 --Only buffer 31.6/4.6/3.1 0.7 Blank 31.6/4.9/3.4 0.7 Perhydrolase 33.7/5.2/5.3 2.7 2.9 Table 22. Mixture of Cl Reactive Orange 107, Reactive Red 198, and Reactive Blue 220 Swatch L/a/b TCD TCD*
Fabric before treatment 41.2/14.0/7.6 --Only buffer 39.7/14.0/8.2 1.6 Blank 40.3/14.0/8.0 1.0 Perhydrolase 41.8/12.9/6.2 1.9 2.6 Perhydrolase-mediated decolorization is observed for all the dyed swatches tested. These results demonstrate that enzymatic decolorization is effective for use with a wide range of dyes.

Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Claims (15)

1. A method for adjusting the colour tone of dyed cellulosic textile fibre material comprising contacting said textile material with an enzymatic textile treatment composition comprising (i) a perhydrolase enzyme, (ii) an ester substrate for said perhydrolase enzyme, and (iii) a hydrogen peroxide source.

2. A method according to claim 1, wherein said perhydrolase enzyme comprises the amino acid sequence set forth in SEQ ID NO:1 or a variant or homolog thereof.

3. A method according to either claim 1 or 2, wherein said perhydrolase enzyme is the S54V variant of SEQ ID NO:1.

4. A method according to any one of claims 1 to 3, wherein said perhydrolase enzyme comprises a perhydrolysis to hydrolysis ratio greater than 1.

5. A method according to any one of claims 1 to 4, wherein said ester substrate is selected from propylene glycol diacetate, ethylene glycol diacetate, glycerol triacetate, ethyl acetate, and glycerol tributyrate.

6. A method according to any one of claims 1 to 5, wherein said hydrogen peroxide source is hydrogen peroxide.

7. A method according to any one of claims 1 to 6, wherein said enzymatic textile treatment composition additionally comprises (iv) a surfactant and/or emulsifier.

8. A method according to any one of claims 1 to 7, wherein said enzymatic textile treatment composition additionally comprises (v) a fluorescence whitening agent.

9. A method according to any one of claims 1 to 8, wherein said enzymatic textile treatment composition additionally comprises (vi) an enzymatic desizing agent.

10. A method according to any one of claims 1 to 9, wherein said enzymatic textile treatment composition additionally comprises (vii) a biopolishing agent.

11. A method according to any one of claims 1 to 10, wherein said enzymatic textile treatment composition additionally comprises (viii) a combination product.

12. A method according to any one of claims 1 to 11, further comprising hydrolyzing said hydrogen peroxide with a catalase enzyme after said bleached textile is produced.

13. A method according to any one of claims 1 to 12, wherein said method is performed in a process selected from a batch process, an exhaust process, and a discontinuous process.

14. A method according to any one of claims 1 to 13, wherein the textile material is contacted with the enzymatic textile treatment composition at a temperature of 60°C to 75°C, for a processing time of 30 to 60 minutes.

15. A method according to any one of claims 1 to 14, wherein said dyed cellulosic textile fibre material is indigo-dyed denim.