HK1179313A - Treatment of keratinous fibers with an enzyme having perhydrolase activity - Google Patents

Description

Treatment of keratinous fibers with enzymes having perhydrolase activity

Technical Field

The compositions and methods of the present invention relate to the treatment of keratinous fibers and fabrics containing such fibers under aqueous conditions using enzymatically generated peracids. The treatment reduces felting and increases dye uptake.

Background

When textiles made of keratinous fibers, such as wool, are subjected to mechanical manipulation in the wet state, they have a tendency (sometimes significant) to shrink. Such shrinkage is referred to as "felting". Felting is generally undesirable and irreversible and renders the fabric or garment unusable. The felting tendency can be mitigated by chemically modifying the surface of the textile, which changes the frictional properties of the textile by reducing or masking the scale structure.

Chlorination is the oldest, most widely known method for reducing felting and increasing dye uptake. However, chlorine-containing compounds and their decomposition products are harmful to the environment and textile workers. Similar environmental and safety issues are associated with resin finishing, as the resin is typically crosslinked with epichlorohydrin.

The use of peracids to modify the surface of wool fibers has been described (e.g., U.S. patent No.3,634,020); however, such processes are carried out in anhydrous organic solutions. Environmental and safety issues associated with the use and storage of concentrated peracid solutions and organic solvents have hampered the commercial viability of such processes.

There is a need for safer, more environmentally friendly, and more effective compositions and methods for reducing felting and increasing the dye uptake of wool and other fabrics made from keratinous fibers.

Disclosure of Invention

Compositions and methods for chemically modifying keratinous fibers, e.g., to reduce felting, increase the dye uptake, and/or reduce the needle-prick feel of textiles comprising such fibers, are described.

In some aspects, a method for reducing felting of wool or another textile made from keratinous fibers is described, the method comprising: contacting the textile with an aqueous composition comprising an enzyme having perhydrolase activity, an ester substrate for the enzyme, and a hydrogen peroxide source, wherein the enzyme generates peracids in situ in the aqueous medium, and wherein the peracids modify the textile, thereby reducing the tendency of the textile to felt. In some embodiments, the enzyme generates peracids in situ prior to contacting the textile with the aqueous composition. In some embodiments, the enzyme generates peracids in situ after the textile is contacted with the aqueous composition. In some embodiments, contacting the textile with the aqueous composition and the enzyme generating the peracid in situ occur simultaneously.

In a particular aspect, there is provided a method for reducing felting of wool or other textiles formed from keratinous fibers, the method comprising: contacting the textile with an aqueous composition comprising an enzyme having perhydrolase activity, an ester substrate for the enzyme, and a hydrogen peroxide source for a time sufficient to generate a peracid, wherein the peracid modifies the textile, thereby reducing the tendency of the textile to felt.

In another particular aspect, there is provided a method for reducing felting of wool or other textiles formed from keratinous fibers, the method comprising: (i) providing an aqueous composition comprising: an enzyme having perhydrolase activity, an ester substrate for the enzyme and a hydrogen peroxide source, (ii) generating a peracid in situ in an aqueous composition, and (iii) contacting the textile comprising keratinous fibres with an aqueous composition comprising a peracid, thereby modifying the textile so as to reduce the tendency of the textile to felt.

In yet another specific aspect, a method for reducing felting of wool or other textiles formed from keratinous fibers is provided, the method comprising: (i) providing an aqueous composition comprising: an enzyme having perhydrolase activity, an ester substrate for the enzyme and a hydrogen peroxide source, (ii) generating a peracid in situ in an aqueous composition, and (iii) contacting keratinous fibers with an aqueous composition comprising a peracid, thereby modifying the fibers so as to reduce the tendency of textiles comprising said fibers to felt.

In yet another aspect, in a process for making a textile comprising keratinous fibers, there is provided a process for modifying the fibers to reduce felting of the textile, comprising: (i) providing an aqueous composition comprising: an enzyme having perhydrolase activity, an ester substrate for the enzyme and a hydrogen peroxide source, (ii) generating a peracid in situ in an aqueous composition, and (iii) contacting the keratinous fibers with an aqueous composition containing a peracid to modify the fibers to reduce the tendency of textiles comprising said fibers to felt.

In some embodiments of any of the above methods, the keratinous fibers are obtained from sheep. In some embodiments, the textile is wool.

In some embodiments of any of the above methods, the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence at least 70% identical to the amino acid sequence set forth in seq id No. 1 or seq id No. 2. In some embodiments, the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence at least 80% identical to the amino acid sequence set forth in seq id No. 1 or seq id No. 2. In some embodiments, the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence at least 90% identical to the amino acid sequence set forth in seq id No. 1 or seq id No. 2.

In some embodiments of any of the above methods, the enzyme having perhydrolase activity is derived from Mycobacterium smegmatis (Mycobacterium smegmatis) perhydrolase. In some embodiments, the enzyme having perhydrolase activity is a variant of mycobacterium smegmatis perhydrolase having the substitution S54V.

In some embodiments of any of the above methods, the enzyme having perhydrolase activity is not an enzyme that would be recognized by the skilled artisan as a protease, a peroxidase, or a haloperoxidase.

In some embodiments of any of the above methods, the ester substrate is Propylene Glycol Diacetate (PGDA), triacetin, ethyl acetate, tributyrin, and/or Ethylene Glycol Diacetate (EGDA). In some embodiments, the source of hydrogen peroxide is hydrogen peroxide, perborate, or percarbonate.

Some embodiments of any of the above methods further comprise the step of treating the keratinous fibers or textile comprising the keratinous fibers with sodium sulfite to further reduce felting of the textile. Some embodiments of these methods further comprise the step of treating the keratinous fibers or textile comprising the keratinous fibers with transglutaminase to improve textile strength.

In another aspect, there is provided a kit for performing any of the above methods, the kit comprising: an enzyme having perhydrolase activity, an ester substrate for the enzyme, a hydrogen peroxide source, and instructions for use.

In another aspect, there is provided a textile prepared by any of the above methods.

These and other aspects and embodiments of the compositions and methods of the present invention will be apparent from the specification, including the drawings.

Drawings

FIG. 1 is a scanning electron micrograph (magnification ≈ 2,000 ×) showing the morphology of wool fibers treated with buffer.

FIG. 2 shows the use of buffer + PGDA + H₂O₂Scanning electron micrographs of the morphology of the treated wool fibers (magnification ≈ 2,000 ×).

FIG. 3 shows the use of buffer + PGDA + H₂O₂+ aryl esterase treated wool fibers scanning electron micrographs (magnification ≈ 2,000 ×).

FIG. 4 shows the results of the analysis using (1) buffer, (2) buffer + PGDA + H₂O₂And (3) buffer + PGDA + H₂O₂+ FTIR/ATR spectra of aryl esterase treated wool fibers.

Figure 5 shows the results of dyeing differently treated wool fibers with identifying textile fiber dye a. The left fiber was not dyed (control). The right side fibers were dyed (dyed) using the identification fabric fiber dye a.

FIG. 6 shows the results obtained with buffer (left), buffer + PGDA + H₂O₂(intermediate) and buffer + PGDA + H₂O₂+ amount of shrinkage of the aryl esterase (right) treated wool jersey swatches.

Figure 7 shows the position of a reference mark (i.e. cotton/polyester thread; shown in cross) placed on a wool knit sample before treatment.

FIG. 8 shows the use of PGDA + H₂O₂(left) and PGDA + H₂O₂+ dimensions of the arylesterase (right) treated wool interlock swatches, and washed as described.

FIGS. 9 and 10 are graphs showing results from buffer + PGDA + H₂O₂(FIG. 9) and buffer + PGDA + H₂O₂+ aryl esterase (fig. 10) scanning electron micrograph of wool fiber morphology of treated sample (magnification ≈ 2,000 ×).

FIG. 11 shows FTIR/ATR spectra of control wool fibers and those wool fibers treated with enzymatically generated peracid.

FIG. 12 shows FTIR/ATR spectra of wool jersey treated with (1) non-enzyme, (2) aryl esterase, (3) sodium sulfite, (4) non-enzyme followed by sodium sulfite, and (4) aryl esterase followed by sodium sulfite.

Detailed Description

Definition of

Before describing in detail the compositions and methods of the present invention, the following terms are defined for clarity. Undefined terms should be accorded their ordinary meanings as used in the related art.

As used herein, "keratinous fibers" are fibers comprising keratin, a family of fibrous structural proteins that occur naturally in animal cells. Keratinous fibers are found primarily in wool (see below), hair, fur, nails, and other animal tissues. As used herein, keratinous fibers include not only naturally occurring fibers, but also synthetic fibers.

As used herein, "wool" refers to textiles made from keratinous fibers of animals in the ovine subfamily, primarily sheep. However, wool also encompasses cashmere and mohair from goats, alpaca from animals in the camelidae familyAlpaca and camel hair, and angora hair from rabbits. The keratinous fibers used to form wool are similar to but different from hair or fur.

As used herein, "felting" refers to shrinkage of a textile comprising keratinous fibers in one or more dimensions. Felting is easily caused, for example, by rubbing such textiles in a wet state, where the scales on the surface of the keratinous fibres interact, pulling the fibres together and causing the textiles to shrink.

As used herein, "shrinkage" refers to a reduction in the surface area of a fabric as measured along one or more dimensions. Shrinkage may be associated with increased density and changes in texture.

As used herein, the term "textile" refers to fibers, yarns, fabrics, garments, and nonwovens. The textile may be a natural textile, a synthetic (e.g., manufactured) textile, or a blend thereof. The term "textile" refers to both raw and processed fibers, yarns, woven or knitted fabrics, nonwovens, and garments.

As used herein, the term "fabric" refers to an assemblage of manufactured fibers and/or yarns having a substantial amount of surface area associated with the thickness thereof, as well as sufficient cohesion for imparting useful mechanical strength to the assemblage.

As used herein, the term "dye" refers to a colored substance (i.e., chromophore) that has affinity for the substrate (such as a textile) to which it is applied.

As used herein, the term "dyeing" refers to the application of chromophores to a substrate (such as a textile), particularly by soaking in a dyeing solution.

As used herein, an "aqueous medium" is a solution or mixed solution/suspension in which the solvent is predominantly water. The aqueous medium is substantially free of inorganic solvents.

As used herein, a "perhydrolase" is an enzyme capable of catalyzing the perhydrolysis reaction which results in the generation of peracid. Preferably, the perhydrolase exhibits high hydrogenolysis peroxide: the hydrolysis ratio. Perhydrolases may have acyltransferase and/or arylesterase activity and may be so named.

As used herein, the term "perhydrolysis" refers to a reaction in which an ester substrate and hydrogen peroxide are used to produce a peracid.

As used herein, "effective amount of perhydrolase" refers to the amount of perhydrolase necessary to modify keratinous fibers to reduce felting of fabrics made from the keratinous fibers.

As used herein, the term "peracid" refers to a molecule having the general formula RC (═ O) OOH, which is a highly reactive product formed by the reaction of a carboxylic acid ester with hydrogen peroxide. Such peracid products are capable of transferring one of their oxygen atoms to another molecule (such as keratinous fibers).

As used herein, the term "ester substrate" refers to a perhydrolase substrate that comprises at least one ester bond. Esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are useful as substrates for the use of perhydrolases.

As used herein, the term "acyl" refers to an organic group having the general formula RCO-, which can be derived from an organic acid by removing the-OH group. The name of acyl groups generally has the suffix "oxy", e.g., formyloxy Chloride (CH)₃CO-Cl) which is prepared from formic acid (CH)₃CO-OH) to form acid chlorides.

As used herein, the term "acylation" refers to a chemical transformation in which one substituent of a molecule is replaced with an acyl group, or a process in which an acyl group is introduced into a molecule.

As used herein, the term "hydrogen peroxide source" refers to a molecule capable of generating hydrogen peroxide (e.g., in situ). Sources of hydrogen peroxide include hydrogen peroxide itself, as well as molecules that spontaneously or enzymatically generate hydrogen peroxide as a reaction product.

As used herein, the phrase "hydrogenolysis: the hydrolysis ratio "refers to the ratio of enzymatically produced peracid to enzymatically produced acid (e.g., in moles), wherein the peracid and the acid are generated from an ester substrate by a perhydrolase enzyme under defined conditions and for a defined time. The amounts of peracid and acid produced by the enzyme can be determined using the assay provided in WO 05/056782.

As used herein, the term "hydrogen peroxide-producing oxidase" refers to a catalytic system involving molecular oxygen (O)₂) As electron acceptorsAn enzyme of the oxidation/reduction reaction of (1). In such reactions, oxygen is reduced to water (H)₂O) or hydrogen peroxide (H)₂O₂). An oxidase as used herein is an oxidase that acts on its substrate to produce hydrogen peroxide (as opposed to water). Examples of hydrogen peroxide-generating oxidases and substrates therefor suitable for use herein are glucose oxidase and glucose. Other oxidases that may be used to produce hydrogen peroxide include alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, and the like. The hydrogen peroxide-generating oxidase may be a carbohydrate oxidase.

As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of any length comprising amino acid residues joined by peptide bonds. The conventional single letter code or three letter code for amino acid residues is used herein. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acids that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as binding to a labeling component). Also included in the definition are, for example, polypeptides comprising 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, functionally and/or structurally similar proteins are considered "related proteins". Such proteins may be derived from organisms of different genera and/or species, or even organisms of different classes (e.g., bacteria and fungi). Related proteins also encompass homologues determined by basic sequence analysis, determined by tertiary structure analysis, or determined by immunological cross-reactivity.

As used herein, the term "derived polypeptide/protein" refers to a protein that is derived from another protein by adding one or more amino acids to the N-terminus and/or C-terminus, replacing one or more amino acids at one or more different sites in the amino acid sequence, and/or deleting one or more amino acids at one or both ends of the protein or at one or more sites in the amino acid sequence, and/or inserting one or more amino acids at one or more sites in the amino acid sequence. The preparation of protein derivatives may be accomplished by modifying the DNA sequence encoding the native protein, transferring the DNA sequence to a suitable host, and expressing the modified DNA sequence to form the derived protein.

Related (and derived) proteins include "variant proteins". Variant proteins differ from a reference/parent protein, such as a wild-type protein, by substitution, deletion and/or insertion of a small number of amino acid residues. The number of different amino acid residues may be one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more amino acid residues. A variant protein shares at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99% or more amino acid sequence identity with a reference protein. Variant proteins may also differ from a reference protein in selected motifs, domains, epitopes, conserved regions, etc.

As used herein, the term "similar sequence" refers to a sequence within a protein that provides similar function, tertiary structure, and/or conserved residues as the protein of interest (i.e., typically the original protein of interest). For example, in epitope regions comprising alpha-helical or beta-sheet structures, the substituted amino acids in similar sequences preferably retain the same specific structure. The term also refers to nucleotide sequences as well as amino acid sequences. In some embodiments, similar sequences are developed such that the substitution of amino acids results in variant enzymes exhibiting similar or improved function. In some embodiments, the tertiary structure of the amino acid and/or conserved residues in the protein of interest are located at or near the segment or fragment of interest. Thus, where the segment or fragment of interest comprises, for example, an alpha-helix or beta-sheet structure, the replacement amino acid preferably retains that particular structure.

As used herein, the term "homologous protein" refers to a protein having similar activity and/or structure as a reference protein. This does not mean that the respective homologues are necessarily evolutionarily related and therefore the term is intended to cover the same, similar or corresponding enzymes (i.e. in terms of structure and function) obtained from different organisms. In some embodiments, it is highly desirable to identify homologues having a similar quaternary, tertiary and/or primary structure to the reference protein. In some embodiments, the homologous protein elicits a similar immune response as the reference protein. In some embodiments, homologous proteins are engineered to produce enzymes with desired activities.

The degree of homology between the sequences may be determined using any suitable method known in the art (see, for example, Smith and Waterman (1981) adv. Appl. Math.2:482 (Smith and Waterman, 1981, advances in applied mathematics, Vol. 2, page 482), Needlemanand Wunsch (1970) J.Mol.biol.,48:443 (Needleman and Wunsch, 1970, J. mol. biol., Vol. 48, page 443), Pearson and Lipman (1988) Proc. Natl.Acad. Sci. USA 85:2444 (Pearson and Lipman, 1988, J. Natl. Acad. Sci. USA 85, page 2444), programs such as, for example, the Wisconsin Genetics Package (Wisconsin Genetics Package, Sodsware, Wisconsin. Patch., Van. W., Dev. Korea, Vol. No. 85, page 2444), programs, such as the Wisconsin Genetics Genencon. Genet al., Vol. FAS., P. No. 395, Masui. No. 12, Masui. Fa., Masui. upright, Masui. No. 12, Masui. Fa., Masui., Fa., Masson, Masui. No. 4, Masui., Masson, Massach., Masui., Massa, Masui., pages No. 4, Mas.

For example, PILEUP is a program that can be used to determine the degree of sequence homology. PILEUP generates multiple sequence alignments from a set of related sequences using progressive pair-wise alignments. It is also possible to draw a tree graph showing the clustering relationships (used to generate the alignments). PILEUP uses the simplified progressive alignment method of Feng and Doolittle (1987) J. mol. Evol.35: 351-. The method is similar to that described by Higgins and Sharp (1989) CABIOS5:151-153 (Higgins and Sharp, 1989, application of computer in bioscience, Vol. 5, page 151-153)). Available PILEUP parameters include: a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Another example of an algorithm that can be used is the BLAST algorithm described by Altschul et al (Altschul et al, (1990) J. mol. biol.215: 403-. A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al (1996) meth. enzymol.266:460-480 (Altschul et al, 1996, methods in enzymology, 266: 460-480)). The parameters "W", "T" and "X" determine the sensitivity and speed of the alignment. BLAST program uses default values: the word length (W)11, BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915 (Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. 89, Vol. 89, page 10915)) were compared 50 times for (B) and for (E)10, M '5, N' -4, for both strands.

As used herein, the phrases "substantially similar" and "substantially identical," in the context of at least two nucleic acids or polypeptides, generally mean that the polynucleotide or polypeptide comprises a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or even at least about 99% identical or greater identity, as compared to a reference (i.e., wild-type) sequence. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL, using standard parameters. (see, e.g., Altschul et al, (1990) J.mol.biol.215: 403-) -410 (Altschul et al, 1990, journal of molecular biology, 215: 403-); Henikoff et al (1989) Proc.Natl.Acad.Sci.USA 89:10915 (Henikoff et al, 1989, journal of the national academy of sciences USA, 89: 10915); Karin et al, (1993) Proc.Natl.Acad.Sci USA 90:5873 (Karin et al, 1993, journal of the national academy of sciences USA, 90: 5873), and Higgins et al (1988) Gene73:237 (237) (Higgins et al, 1988, 237: 237); 237) (1988, journal of the national academy of sciences), 237-)). Software for performing BLAST analysis is publicly available through the national center for Biotechnology Information. In addition, the database can be searched using FASTA (Pearson et al, (1988) Proc. Natl. Acad Sci. USA 85: 2444-. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, one polypeptide is substantially identical to a second polypeptide, e.g., where the two polypeptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., in the range of medium to high stringency).

As used herein, "wild-type" and "native" proteins are those proteins that occur in nature. The terms "wild-type sequence" and "wild-type gene" are used interchangeably herein to refer to a sequence that is native or naturally occurring within a host cell. In some embodiments, a wild-type sequence refers to a sequence of interest that is the starting point for a protein engineering project. Genes encoding naturally occurring proteins can be obtained according to general methods known to those skilled in the art. The method generally comprises: synthesizing a labeled probe having a putative sequence of a region encoding a protein of interest, preparing a genomic library from an organism expressing the protein, and screening the library by hybridization to the probe to obtain the gene of interest. The forward hybridizing clones were then mapped and sequenced.

As used herein, "packaging" refers to a container capable of providing the perhydrolase enzyme, the substrate for the perhydrolase enzyme, and/or the hydrogen peroxide source in a form that is easy to handle and transport. Exemplary packaging includes boxes, drums, cans, drums, bags, or even tanker trucks.

As used herein, the term "contacting" means incubating in the presence of an aqueous solution (typically in an aqueous solution).

As used herein, the term "modifying keratinous fibers" refers to any chemical change to the keratinous fibers that results in a reduction in felting of the fabric comprising the modified fibers. Chemical modifications include, but are not limited to, oxidation, formation of cysteic acid, and formation of Bunte salts.

As used herein, the singular articles "a," "an," and "the" encompass plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety.

The following abbreviations/acronyms have the following meanings, unless otherwise indicated:

overview

The compositions and methods of the present invention relate to chemically modifying keratinous fibers to reduce the amount of felting of fabrics comprising such fibers. The compositions and methods are characterized by: the use of enzymatically generated peracids in an aqueous medium to treat keratinous fibers eliminates many of the problems associated with conventional processes.

In one aspect, the compositions and methods involve contacting keratinous fibers or fabrics made from keratinous fibers with an aqueous composition comprising an enzyme having perhydrolase activity, an ester substrate for the enzyme, and a hydrogen peroxide source for a time sufficient to generate a peracid. While the in situ generated peracids are produced, they modify the keratinous fibers in an aqueous environment, thereby reducing the tendency of fabrics made from keratinous fibers to felt.

In another aspect, an aqueous composition comprising an enzyme having perhydrolase activity, an ester substrate for the enzyme, and a hydrogen peroxide source is provided, such that peracid is generated in situ in the aqueous composition. The keratinous fibres or fabric made from the keratinous fibres are then contacted under aqueous conditions with a peracid generated in situ, wherein the peracid modifies the fibres and reduces the tendency of the fabric made from the fibres to felt.

Various features and preferred embodiments of the compositions and methods are described below.

Perhydrolase

The compositions and methods of the present invention for reducing felting of fabrics made from keratinous fibers are characterized by the use of one or more perhydrolases. Generally, perhydrolases are capable of generating peracids from a suitable ester substrate in the presence of hydrogen peroxide.

In some embodiments, the perhydrolase enzyme is a naturally-occurring enzyme, or a perhydrolase enzyme comprising, consisting of, or consisting essentially of the amino acid sequence of seq id no: the amino acid sequence is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 99.5% identical to the amino acid sequence of a naturally-occurring perhydrolase. The perhydrolase may be obtained from a microbial source, such as a bacterium or a fungus.

In some embodiments, the perhydrolase enzyme is a naturally-occurring mycobacterium smegmatis perhydrolase or a variant thereof. The enzymes, their enzymatic properties, their structures and their various variants and homologues are described in detail in international patent application publications WO 05/056782A and WO 08/063400a and in U.S. patent application publications US2008145353 and US2007167344, all of which are incorporated herein by reference.

In some embodiments, the perhydrolase enzyme comprises, consists of, or consists essentially of the amino acid sequence set forth in sequence identification No. 1, or a variant or homolog thereof. In some embodiments, the perhydrolase enzyme comprises, consists of, or consists essentially of the amino acid sequence of seq id no: the amino acid sequence has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 99.5% identity to the amino acid sequence set forth in sequence identification No. 1.

The amino acid sequence of mycobacterium smegmatis perhydrolase is shown below (seq id No. 1):

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

in some embodiments, the perhydrolase comprises one or more substitutions at one or more amino acid positions equivalent to one or more positions in the mycobacterium smegmatis perhydrolase amino acid sequence set forth in sequence identification No. 1. In some embodiments, the perhydrolase comprises any one or any combination of amino acid substitutions selected from the group consisting of: m1, K3, R4, I5, L6, C7, D10, S11, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, a 22, P22, T22, E22, R22, F22, a 22, P22, D22, V22, R22, W22, T22, G22, L22, Q22, D22, L22, G22, a 22, F22, E22, V22, I22, E22, G22, L22, S22, a 22, R22, T22, N22, I22, D149, P22, L22, N22, P119, P22, P72, P22, P122, P22, N22, P122, P22, N22, P72, P22, N22, P122, N22, P22.

In some embodiments, the perhydrolase enzyme comprises one or more of the following substitutions at one or more amino acid positions equivalent to one or more positions in the mycobacterium smegmatis perhydrolase amino acid sequence set forth in sequence identification No. 1: L12C, 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 comprises a combination of the following amino acid substitutions at a plurality of amino acid positions equivalent to the plurality of amino acid positions in the mycobacterium smegmatis perhydrolase amino acid sequence set forth in sequence identification No. 1: L12I S54V; L12M S54T; L12T S54V; L12Q T25SS 54V; L53H S54V; S54P V125R; S54V V125G; S54V F196G; s54VK97R V125G; or a55G R67T K97R V125G.

In particular embodiments, the perhydrolase is the S54V variant of M.smegmatis perhydrolase, which is shown below (SEQ ID NO: 2; S54V substitutions are underlined):

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLVARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

in some embodiments, the perhydrolase enzyme comprises the S54V substitution, yet further has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% identity to the amino acid sequence set forth in sequence identification No. 1 or 2.

In some embodiments, the perhydrolase is subjected to hydrogenolysis by hydrogen peroxide: the hydrolysis ratio is at least 1. In some embodiments, the perhydrolase hydrogenolysis: the hydrolysis ratio is greater than 1.

The amount of perhydrolase is generally not a critical factor, and is selected to generate sufficient amounts of peracid to modify the keratinous fibers in the manner described.

Ester substrates

Another feature of the compositions and methods of the present invention is the use of ester substrates of perhydrolases to produce peracids in the presence of hydrogen peroxide. Suitable substrates may be monovalent (i.e., contain a single carboxylate moiety) or multivalent (i.e., contain more than one carboxylate moiety). The amount of substrate required to generate the peracid can be adjusted depending on the number of carboxylate moieties in the substrate molecule.

In some embodiments, the ester substrate is an ester of an aliphatic and/or aromatic carboxylic acid with an alcohol. The ester substrate may be a monovalent, divalent, or polyvalent ester, or mixtures thereof. For example, the ester substrate can be an ester of a carboxylic acid and a monohydric alcohol (monovalent esters such as ethyl acetate, propyl acetate), an ester of two carboxylic acids and a diol [ such as Propylene Glycol Diacetate (PGDA), Ethylene Glycol Diacetate (EGDA), or mixtures such as 2-acetoxy-1-propionate, where propylene glycol has an acetate on the second alcohol group and a propyl ester on the first alcohol group ], or an ester of three carboxylic acids and a triol (e.g., glycerol triacetate or a mixture of acetate/propionate linked to glycerol or another polyvalent alcohol, etc.). In some embodiments, the ester substrate is an ester of a nitroalcohol (e.g., 2-nitro-1-propanol). In some embodiments, the ester substrate is a polymeric ester, e.g., a partially acylated (acetylated, propionylated, etc.) polycarboxylic alcohol, acetylated starch, and the like. 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, pelargonic acid, capric acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. In some embodiments, triacetin, tributyrin, and other esters act as acyl donors for peracid formation. 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.

The amount of ester substrate is generally not a critical factor and is selected to generate a sufficient amount of peracid to modify the keratinous fibers in the manner described.

Hydrogen peroxide source

Another feature of the compositions and methods of the present invention is the use of a source of hydrogen peroxide. Generally, hydrogen peroxide can be provided directly, or can be generated continuously by chemical, electrochemical, and/or enzymatic means.

In some embodiments, the source of hydrogen peroxide is hydrogen peroxide itself. In some embodiments, the source of hydrogen peroxide is a compound that generates hydrogen peroxide immediately upon addition to water. The compound may be a solid compound. Such compounds include adducts of hydrogen peroxide with various inorganic or organic compounds, of which sodium carbonate hydroperoxide, also known as sodium percarbonate, is most widely used.

In some embodiments, the hydrogen peroxide source is an inorganic perhydrate salt. Examples of inorganic perhydrate salts are perborate, percarbonate, perphosphate, persulfate and persilicate salts. The inorganic perhydrate salts are normally the alkali metal salts.

Additional sources of hydrogen peroxide include adducts of hydrogen peroxide with zeolites, or urea peroxide.

The hydrogen peroxide source may be in crystalline form and/or in the form of a substantially pure solid without additional protection. For certain perhydrate salts, the preferred form is a granular composition comprising a coating which provides better storage stability of the perhydrate salt in the granular product. Suitable coatings include inorganic salts such as alkali metal silicates, carbonates or borates or mixtures thereof, or organic materials such as waxes, oils or fatty soaps.

In some embodiments, the hydrogen peroxide source is a system for enzymatically generating hydrogen peroxide. In one embodiment, the system for enzymatically generating hydrogen peroxide comprises an oxidase and a substrate therefor. Suitable oxidases include (but are not limited to): glucose oxidase, sorbitol oxidase, hexose oxidase, choline oxidase, alcohol oxidase, glycerol oxidase, cholesterol oxidase, pyranose oxidase, carboxyl alcohol 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 formula provides an example of a coupling system for the enzymatic generation of hydrogen peroxide.

H₂O2_{Is/are as follows}Production is not necessarily limited to any particular enzyme, as any enzyme capable of producing H by acting on a suitable substrate may be used₂O₂The enzyme of (1). For example, lactate oxidase derived from Lactobacillus species, which is known to produce H from lactic acid and oxygen, can be used₂O₂. One advantage of such reactions is the enzymatic generation of an acid (e.g., gluconic acid in the above example) that lowers the pH of the aqueous alkaline solution to a pH range (i.e., at or below pKa) where peracid is most effective for bleaching. Such pH reduction is also directly caused by the generation of peracids. Other enzymes capable of producing hydrogen peroxide (e.g., alcohol oxidase, ethylene glycol oxidation) may also be used in conjunction with the perhydrolase enzyme in conjunction with the ester substrateEnzymes, glycerol oxidase, amino acid oxidase, etc.) to produce peracids.

In the case where hydrogen peroxide is generated electrochemically, it may be prepared, for example, using a fuel cell supplied with oxygen and hydrogen.

The amount of hydrogen peroxide is generally not a critical factor and is selected to generate a sufficient amount of peracid to modify the keratinous fibers in the manner described. After treating keratinous fibers, it may be desirable in some cases to eliminate residual hydrogen peroxide using a hydrogen peroxide decomposing enzyme.

Fibers and fabrics

Keratinous fibers suitable for treatment as described include not only naturally occurring fibers, but also synthetic fibers, fibers comprising polypeptides classified as keratins, a family of fibrous structural proteins found primarily in animal cells. Keratin is rich in cysteine, and disulfide bonds between cysteine residues are important for the overall structural integrity of keratinous fibers. Natural products containing keratinous fibers include, but are not limited to, wool, hair, fur, nails, horns, hooves, feathers, and skin. In some cases, keratin constitutes a large fraction of the dry weight of these materials. Although keratinous fibers are generally available from natural sources, the use of keratinous fibers made from extracted keratin or even recombinant keratin is not intended to be excluded from this specification as a matter of course.

The term "wool" is sometimes used only to refer to keratinous fibers derived from sheep's specialized skin cells (known as hair follicles). However, the term "wool" is also used to refer more generally to keratinous fibers derived from animals in the ovine subfamily (including sheep, goats, camels, and rabbits). Wool from goats is also known as cashmere and mohair; wool from camels is also known as alpaca and alpaca, and wool from rabbits is also known as angora. Although sheep wool is exemplified herein, the compositions and methods of the present invention may be used to treat various types of wool in general.

The keratinous fibers may be treated prior to their incorporation into the textile, and/or after their incorporation into the yarn, fabric, textile, or garment. Such treatment may be carried out in an aqueous medium in a batch mode or a continuous mode. Fabrics comprising keratinous fibers may be blended with other fibers, including other natural or synthetic fibers. Exemplary fibers include, but are not limited to, cotton fibers, flax fibers, polyester fibers, polyamide fibers, hemp fibers, and the like.

Dye material

One feature of the compositions and methods of the present invention is the ability to increase the dye uptake of keratinous fibers or fabrics made therefrom. Examples of suitable dyes include, but are not limited to, azo dyes, monoazo dyes, disazo dyes, nitro dyes, xanthene dyes, quinoline dyes, anthraquinone dyes, triarylmethane dyes, p-azoaniline dyes, azine oxazine dyes, stilbene dyes, aniline dyes, and phthalocyanine dyes, or mixtures thereof. The dye may be an azo dye (e.g., reactive black 5 (4-amino-5-hydroxy-3, 6-bis ((4- ((2- (sulfoxy) ethyl) sulfonyl) phenyl) azo) -2, 7-naphthalenedisulfonic acid tetrasodium salt), reactive violet 5, methyl yellow, congo red, etc.), an anthraquinone dye (e.g., reactive blue), an indigo dye (indigo carmine), a triarylmethane/p-azoaniline dye (e.g., crystal violet, malachite green), or a sulfur-based dye. The dye may be a reactive dye, a direct dye, a disperse dye or a pigment dye. The dye may be a component of the ink.

Aqueous medium

It is a feature of the compositions and methods of the present invention that keratinous fibers or fabrics made from keratinous fibers are treated in an aqueous medium with an enzymatically generated peracid. The aqueous medium is a solution or mixed solution/suspension wherein the primary solvent is water. This feature readily distinguishes the compositions and methods of the present invention from conventional methods of treating keratinous fibers with peracids, the latter being carried out in substantially inorganic solvents (see, e.g., U.S. patent No.3,634,020). Although small amounts of such solvents are generally acceptable for use in the compositions and methods of the present invention, they are not required. Accordingly, the aqueous medium may be completely free of organic solvent, or may contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.7%, less than 0.5%, less than 0.2%, or even less than 0.1% organic solvent.

The aqueous medium may comprise any number of buffers, surfactants, salts, lubricants, binders, builders, polymers, chelating agents, complexing agents, anti-fouling agents, dyes, and the like. The aqueous medium may also comprise additional enzymes, substrates for such enzymes, cofactors, activators, and the like. However, in some embodiments, the aqueous medium specifically does not comprise a substantial amount of protease and/or peroxidase. Suitable aqueous media for treating textiles generally comprise buffers, salts and optionally surfactants.

Sodium sulfite treatment

In some embodiments of the compositions and methods of the present invention, the keratinous fibers or textiles comprising such fibers are additionally treated with sodium sulfite. It is known that treatment with sodium sulfite (or other sulfite salts) causes the formation of Bunte salts with oxidized cysteine residues. Conventional methods of forming Bunte salts rely on treatment of keratinous fibers with powerful oxidants (such as peracetic acid, permanganate, persulfate, hydrogen peroxide, and the like) followed by treatment with sodium sulfite.

In some embodiments of the compositions and methods of the present invention, the keratinous fibers or fabrics comprising keratinous fibers are first treated with an enzymatically generated peracid in an aqueous medium, followed by treatment with sodium sulfite. As exemplified herein, treatment with enzymatically generated peracid followed by treatment with sodium sulfite in an aqueous medium results in even less felting than treatment with enzymatically generated peracid alone.

Transglutaminase treatment

In some embodiments of the compositions and methods of the present invention, transglutaminase is used to crosslink enzymatically generated peracid-treated or enzymatically generated peracid and sodium sulfite-treated keratinous fibers, or textiles comprising such fibers. Such treatments are described in detail, for example, in cortex et al (2004) Enzymeand microbiological Technology 34:64-72 (cortex et al, 2004, enzyme and microbiological Technology, volume 34, pages 64-72). Transglutaminase treatment can increase the strength of wool or other textiles made from keratinous fibers, or restore the strength lost during treatment with enzymatically generated peracids.

Composition and reagent set

The compositions and methods of the present invention may be embodied as a kit (i.e., a kit) for treating keratinous fibers or fabrics comprising such fibers. Such kits may include a perhydrolase enzyme, a substrate for the perhydrolase enzyme, and a hydrogen peroxide source in amounts and ratios suitable to generate sufficient peracid to modify keratinous fibers.

The perhydrolase enzyme, the substrate for the perhydrolase enzyme, and the hydrogen peroxide source may be provided as separate components, which are combined prior to addition to the aqueous medium to generate the peracid for use in modifying the keratinous fibers. Alternatively, the perhydrolase enzyme, the substrate for the perhydrolase enzyme, and the hydrogen peroxide source may be provided in a single container, or in a single formulation for addition to the aqueous medium, as described in detail in international patent application publications WO 07/133263 and WO 08/140988, and U.S. patent application publication US 20090311395. All of which are incorporated by reference.

The kit may also include a sulfite salt (such as sodium sulfite) for further modifying the enzymatically generated peracid-treated keratinous fibers. The kit may further comprise catalase to eliminate residual hydrogen peroxide.

The kit desirably takes a form convenient for use by an end user, such as in a textile manufacturing or processing plant. However, home-use suits are also conceivable, which will enable consumers to treat woolen garments at home. Any such kit may include, in addition to the perhydrolase enzyme, the substrate for the perhydrolase enzyme, and hydrogen peroxide, suitable instructions for enzymatically generating peracid, and for treating keratinous fibers or fabrics comprising such fibers. The instructions may be provided in the form of printed matter or in the form of electronic media, such as a CD, DVD, or in the form of a website address where such instructions are available.

These and other aspects and embodiments of the compositions and methods of the invention will be apparent to the skilled person in light of the present specification. The following examples are intended to further illustrate, but not limit, the compositions and methods.

Examples of the invention

Example 1: morphological alteration of enzymatically treated wool fibers

50mg of 100% wool fibers (i.e., "top grade wool fibers") were placed in a glass tube and incubated at 65 ℃ with slow stirring for one hour in a reaction volume of 2 ml. The incubation conditions were as follows:

1) buffer (100 mM sodium phosphate buffer, pH 7),

2) buffer + 5. mu.l/ml PGDA + 5. mu.l/ml H₂O₂(50% w/w), or

3) Buffer + 5. mu.l/ml PGDA + 5. mu.l/ml H₂O₂(50% w/w) +0.8ppm perhydrolase.

The specific perhydrolase used was the S54V variant of mycobacterium smegmatis perhydrolase (also known as "arylesterase"). After treatment, the wool fibers were rinsed three times with DI water and then air dried. Morphology of the treated wool fibers was evaluated using Phenom scanning electron microscopy of FEI.

Buffer + PGDA + H was used₂O₂Treatment of wool fibers (figure 2) did not significantly change the morphology of the wool scales compared to treatment with buffer alone (figure 1). However, the addition of perhydrolase resulted in substantial removal of wool scales (fig. 3).

The chemical changes in the wool fibers caused by the different treatments were monitored by fourier transform infrared spectroscopy, using attenuated total reflectance mode (FTIR/ATR). Solid wool fibers were mounted directly on a MIRacle ATR fitting attached to a tensiometer 27 (tensiometer 27, Brucker optical instruments) to obtain spectra after different treatments. As shown in fig. 4, at about 1040cm in the spectrum^-1And 1170cm^-1The large increase in (b) indicates an increase in the content of cysteic acid (oxidation product of cysteine) after treatment with perhydrolase (fig. 4).

These results indicate that treatment with perhydrolase reduces the surface scaling of wool fibers, probably due to the presence of oxidized cysteine residues in the wool protein.

Example 2: dyeing affinity modification of enzymatically treated wool fibers

50mg of treated wool fibers (see example 1) were stained with identifying fabric fiber stain A (combination of picric acid, G150 chlorazol blue and 3BS crocus scarlet) for 5 minutes at room temperature. A significant increase in dye affinity was observed in wool fibers treated with the hydrolytic enzyme using identifying fabric fiber stain a (fig. 5). In the presence of buffer or buffer + PGDA + H₂O₂The treated wool fibers were dyed a medium orange color with buffer or buffer + PGDA + H₂O₂+ perhydrolase treated wool fibres were dyed deep red. These results demonstrate that treatment of wool fibers with perhydrolases increases dye uptake.

Example 3: knitted wool fabric treated in a Launder-ometer (laundry-O-meter) Reduction of shrinkage of articles

3 samples of 100% wool jersey knit fabric (measuring 5 "x 5" in size; test fabric type 532) were each incubated in a launder-ometer at 65 ℃ and one of the following conditions (n =3) for 1 hour with a reaction volume of 300ml (bath ratio =32: 1):

1) buffer (100 mM sodium phosphate buffer, pH 7),

2) buffer + 5. mu.l/ml PGDA + 5. mu.l/ml H₂O₂(50% w/w), or

3) Buffer + 5. mu.l/ml PGDA + 5. mu.l/ml H₂O₂(50% w/w) +0.8ppm perhydrolase.

Representative results are shown in fig. 6. Samples treated with perhydrolase showed a comparison with buffer alone or with buffer + PGDA + H₂O₂The treated samples showed significantly less shrinkage. These results demonstrate that treatment of wool fibers with perhydrolase reduces shrinkage under washing conditions.

Example 4: shrinkage reduction of treated wool knit fabrics in Unimac

5 100% wool interlock samples (Britannia mills ltd, type 7061) were each treated in a Unimac (i.e. 50lb laboratory roller washer) at 60 ℃ and under one of the following conditions (n =5) in a reaction volume of 30L (bath ratio = about 51: 1) with very gentle stirring (7RPM) for 1 hour:

1) buffer +5ml/L PGDA +5ml/L H₂O₂(50%w/w)

2) Buffer +5ml/L PGDA +5ml/L H₂O₂(50% w/w) +0.8ppm of perhydrolase

The buffer was 100mM sodium phosphate buffer (pH 7) to which 0.05ml/L Triton X-100(10% w/w) was added as a wetting agent. Before treatment, each knit sample (30 cm x 40 cm; longer in the machine direction) was marked with a plurality of nubs of cotton/polyester thread (as shown in FIG. 7). These marks serve as reference points to measure shrinkage after simulated washing.

After washing, the wool swatches were subjected to three rinsing cycles (water in), then air dried flat, and then tested for felting shrinkage. A representative sample is shown in fig. 8. After washing, the samples treated with the perhydrolase enzyme were much larger than the control samples, demonstrating that treatment with the perhydrolase enzyme reduced the shrinkage of wool (even in a 50lb scale washer).

Scanning electron micrographs of the control sample and the sample treated with the perhydrolase are shown in fig. 9 and 10, respectively. The fibers of the samples treated with the perhydrolase exhibited significantly altered morphology (e.g., reduced scaling) as compared to the fibers of the control samples. For untreated fibers, from the application of buffer + PGDA + H₂O₂Fibers from treated samples and fibers from treated samples with buffer + PGDA + H₂O₂FTIR/ATR analysis of fibers of + perhydrolase treated samples again showed modification by treatment with perhydrolase.

The relaxation shrinkage was measured according to IWS TM31, 1X7A for control samples and wool interlock samples treated with perhydrolase and the felted shrinkage was measured according to the modified IWS TM31, 5X5A method. The treatment was performed using a Unimac (model UY230, 50lb laboratory roller dyeing machine with microprocessor).

The treated samples were spread out and dried and then percent shrinkage was calculated according to the method set forth in IWS TM 31. These shrinkage calculations are summarized below:

shrinkage calculation：

Relaxation shrinkage (%) = (o.m. -r.m.)/O.M. } × 100

Felting shrinkage (%) = { (r.m. -F.M.)/r.m. } × 100

Wherein: O.M. = initial measurement value

R.m. = relaxation measurement

F.M. = felting measurement

As shown in the table in fig. 13, wool knit fabrics treated with perhydrolase exhibited significantly lower felting and area shrinkage than the control treatments, which were not treated with perhydrolase. The negative number indicates that the length of the fabric increased after the treatment.

Example 5: enzyme method treatment combined with sodium sulfite treatment on wool knitted fabric

4 samples of 100% wool jersey knit (29 "× 24"; test fabrics, type 532) were each treated in a Unimac 50lb tumbler washer at 65 ℃ and under the following conditions (n =4) with a reaction volume of 30L (bath ratio = about 75: 1) with very gentle agitation (7RPM) for 1 hour:

1) buffer +5ml/L PGDA +5ml/L H₂O₂(50%w/w)

2) Buffer +5ml/L PGDA +5ml/L H₂O₂(50% w/w) +0.8ppm perhydrolase.

The buffer was 100mM sodium phosphate buffer (pH 7) to which 0.05ml/L Triton X-100(10% w/w) was added as a wetting agent. A 30cm x 40cm rectangle (longer in the longitudinal direction) was marked with a plurality of nubs of cotton/polyester thread on each knit sample, as shown in fig. 7.

After treatment, three rinses were performed, and the samples were then spread out and dried. The control and enzymatically treated samples, plus two additional samples of untreated jersey fabric, were incubated in Unimac for 1 hour at 40 ℃ and pH 7 (50 mM sodium phosphate) using 4g/L sodium sulfite (along with two untreated jersey fabrics as controls). After sodium sulfite treatment, the samples were rinsed three times, then air dried to level and then tested for felting shrinkage. The wool jersey treated in the different ways was washed according to IWS TM31 (5X 5A) to determine the felting shrinkage.

FIG. 12 shows a(1) Non-enzymatic (i.e. with PGDA + H only)₂O₂) FTIR/ATR spectra of (2) aryl esterase, (3) sodium sulfite, (4) non-enzymatic followed by sodium sulfite and (4) aryl esterase followed by sodium sulfite treated wool jersey. Samples treated with sodium sulfite were observed at about 1023cm^-1A new peak appeared indicating the formation of the Bunte salt. As before, at 1040cm^-1And 1170cm^-1The signal at (A) is the result of aryl esterase treatment.

As shown in the table in fig. 13, both the aryl esterase treated, and aryl esterase + sodium sulfite treated knit fabrics showed significantly reduced felting shrinkage compared to the other treated fabric samples. These results indicate that the combination of aryl esterase treatment and sodium sulfite treatment produced fabrics exhibiting even less shrinkage and softer hand than aryl esterase treatment alone.

Although the foregoing compositions and methods have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the description should not be construed as limiting the scope of the invention, which is defined solely by the appended claims.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Claims (17)

1. A method for reducing felting of wool or another textile formed from keratinous fibers, the method comprising:

contacting the textile with an aqueous composition comprising an enzyme having perhydrolase activity, an ester substrate for said enzyme and a hydrogen peroxide source,

wherein the enzyme generates peracids in situ in an aqueous medium, and

wherein the peracid modifies the textile, thereby reducing the felting tendency of the textile.

2. The method of claim 1, wherein the enzyme generates peracids in situ prior to contacting the textile with the aqueous composition.

3. The method of claim 1, wherein the enzyme generates peracids in situ after the textile is contacted with the aqueous composition.

4. The method of claim 1, wherein contacting the textile with the aqueous composition and the enzyme generating peracids in situ occur simultaneously.

5. A method according to any preceding claim, wherein the textile is wool.

6. The method according to any one of the preceding claims, wherein the keratinous fibers are obtained from sheep.

7. The method according to any one of the preceding claims, wherein the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence which has at least 70% identity with the amino acid sequence set forth in seq id No. 1 or seq id No. 2.

8. The method according to any one of the preceding claims, wherein the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence which has at least 80% identity with the amino acid sequence set forth in seq id No. 1 or seq id No. 2.

9. The method according to any one of the preceding claims, wherein the enzyme having perhydrolase activity is a polypeptide having an amino acid sequence which has at least 90% identity with the amino acid sequence set forth in seq id No. 1 or seq id No. 2.

10. The method according to any one of the preceding claims, wherein the enzyme having perhydrolase activity is derived from Mycobacterium smegmatis perhydrolase.

11. The method according to any one of the preceding claims, wherein the enzyme having perhydrolase activity is a variant of Mycobacterium smegmatis perhydrolase having the substitution S54V.

12. The method of any one of the preceding claims, wherein the ester substrate is Propylene Glycol Diacetate (PGDA), triacetin, ethyl acetate, tributyrin, and/or Ethylene Glycol Diacetate (EGDA).

13. The method of any one of the preceding claims, wherein the source of hydrogen peroxide is hydrogen peroxide, perborate, or percarbonate.

14. The method according to any of the preceding claims, further comprising the step of:

treating said keratinous fibres or textile fabrics comprising said keratinous fibres with sodium sulphite to further reduce felting of said textile fabrics.

15. The method according to any of the preceding claims, further comprising the step of:

treating the keratinous fibers or textile comprising the keratinous fibers with transglutaminase to improve textile strength.

16. A kit of reagents for performing the method according to any preceding claim, comprising:

an enzyme having a perhydrolase activity and a process for producing the same,

an ester substrate of the enzyme(s),

a source of hydrogen peroxide, and

and (5) instructions for use.

17. A textile prepared by the method of any preceding claim.