REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of glycosyl hydrolase family 61 polypeptides and cutinase in the treatment of polyester textile, as well as a textile composition comprising glycosyl hydrolase family 61 polypeptides and cutinase.

BACKGROUND OF THE INVENTION

Polyethylene terephthalate (abbreviated as PET) fibers accounts for the main part of the polyester applied by the textile industry. The fibers are produced by e.g. poly-condensation of terephthalic acid and ethylene glycol, and drawing of fibers from a melt.

Polyester has certain key advantages including high strength, soft hand, stretch resistance, stain resistance, machine washability, wrinkle resistance and abrasion resistance. However, polyester is not so optimal in terms of its hydrophobicity, pilling, static, dyeability, inactive surface as a medium for adhering, i.e., softening or wettability enhancing compounds, lack of breathability and undesirable high shine or luster appearance.

Because of its strength, polyester fabrics and/or garments are subject to pill formation, and possibly the most important of the cloth finishing processes applied to polyester staple-fibre materials are those designed for control of pilling. All staple-fibre materials tend to form small balls or "pills" of entangled fibres at the cloth surface, when subjected to mild abrasion during wash and wear. If the fabric contains a substantial proportion of fibres having high resistance to flexural abrasion, the pills may be retained on the surface of the cloth in sufficient numbers to produce an unpleasant handle and appearance.

Another problem with polyester is that during synthesis of PET, cyclic or linear oligomers of poly (ethylene terephthalate), such as terephtalic acid-bis-2-benzoyloxy-ethylesther (abbreviated as BETEB) and/or cyclic tri(ethylene terephthalate) are formed. These oligomers are partly deposited on machinery and partly staying on and/or in the fibers. Oligomers tend to give fabrics a grayish appearance. This is due to deposits of oligomers on the surface of the fabric, which is particularly outspoken after high temperature wet processes like high temperature dyeing. The oligomers can be removed by severe alkaline treatment, which results in a significant loss of fiber material. Organic extraction of the oligomers is a technical possibility, but not industrially feasible. The industry has made great efforts to improve the characteristics of polyester, in particular the reduction of pill formation.

WO 99/001604 discloses a method of reducing the pilling propensity of polyester fabrics and/or garments with a terephtalic acid diethyl ester hydrolytic enzyme (ETE hydrolytic enzyme) and/or an ethyleneglycol dibenzyl ester hydrolytic enzyme (BEB hydrolytic enzyme).

WO 2001/34899 discloses a method for modifying polyester comprising treating said polyester with a polyesterase enzyme.

WO 97/27237 discloses the enzymatic hydrolysis of cyclic oligomers of poly (ethylene terephthalate), which comprises subjecting the cyclic oligomer to the action of one or more carboxylic ester hydrolases.

WO 2001/092502 discloses the treatment of polyester textile with Humicola insolens cutinase variants.

However, there is still a need for improved benefit of enzymatic polyester fabric and/or garment treatment, including enhancing the efficiency of the enzymes to their substrates. In par- ticular, there is a continuous need for more efficient enzyme composition to improve the economics of the process. The present invention aims to meet these needs.

SUMMARY OF THE INVENTION

The present invention relates to a method for treating polyester textile with a glycosyl hydrolase family 61 (GH61 ) polypeptide in the presence of a cutinase in an aqueous solution.

The present invention also relates to a textile composition comprising a glycosyl hydrolase family 61 polypeptide and a cutinase.

In some embodiments, the polyester textile treatment process may further comprise one or more enzymes selected from the group consisting of lipases, esterases, laccases, peroxidases and peroxygenase and transferases.

In preferred embodiment, the polyester textile is a PET textile.

In the present invention, GH61 polypeptides can enhance the efficiency of the cutinase to its substrate with at least one of the following benefits: reduction of oligomer in the polyester textile, reduction of pill formation , and without substantial weight loss of fabric in a biopolishing process, improvement on the wettability / hydrophilicity and antistatic properties of polyester fabric.

In one embodiment, a number of enzymes can be used together with cutiase and GH61 for polyester treatment process, which comprises one or more enzymes selected from the group consisting of lipases, esterases, laccase, peroxidase, peroxygenase and transferases.

In one embodiment, the method and composition of the present invention may further comprise a co-substance, such as cysteine and ascorbate. In some embodiments, the method for manufacturing polyester textile is provided. In some embodiments, the textile is manufactured from fabric to garment.

In some embodiments, the cutinase used in the present invention is a cutianse having BETEB hydrolysis activity.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

As used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise.

Polyester Textile

"Polyester" as used herein means a linear polymeric molecule containing in-chain ester groups which are derived from condensation of a diacid with a diol or from the polymerization of hydroxy acids. The present invention applies to both aliphatic and aromatic polyesters. Particularly preferred polyesters are aromatic polyester articles which are used to produce fiber and resin and that comprise a synthetically produced long chain polymer comprising at least 85%, preferably at least 90% and most preferably at least 95%, by weight of an ester of a substituted aromatic carboxylic acid, such as substituted terephthalic acid or parasubstituted hydroxybenzoate or a mixture thereof. Other useful polyester articles include those made of bulk polymer, yarns, fabrics, films, resins and powders. The principal polyesters in industrial usage include polyethylene terephthalate (PET), tetramethylene terephthalate (PTMT), polybutylene terphthalate (PBT), polytrimethylene terephthalate (PTT) and polyethylenenaphthalate (PEN), polycyclohexanedimethylene terephthalate (CHDMT), polyethylene-4-oxybenzoate, A-Tell, polyglycolide, PHBA and 2GN. However, PET is the most common linear polymer produced and accounts for a majority of the polyester applied in industry today.

The polyester textile used herein is meant to include fibers, yarns, fabrics and garments comprising polyester. The polyester yarn or fabric or garment may be any yarn or fabric or garment that is made from pure poly (ethylene terephthalate), or that is made from blends of poly (ethylene terephthalate) fibers and any other materials conventionally used for making textile such as wool, cotton, viscose and silk.

In a preferred embodiment the polyester fabric is a fabric blend comprising more than 35% (w/w) of polyester, in particular more than 50%, more than 65%, more than 90%, or more than 95% of polyester. In a most preferred embodiment, the process of the invention is applied to fabrics or garments consisting essentially of poly (ethylene terephthalate) polyester material, i.e. pure poly (ethylene terephthalate) polyester material.

Cutinase

Cutinases are lipolytic enzymes classified as EC 3.1.1.74 according to Enzyme Nomenclature. Reference is made to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992

For purposes of the present invention, cutinase activity is determined using oligomer Te- rephtalic acid-bis-2-benzoyloxy-ethylesther (BETEB) as substrate according to Example 1 of the present invention. BETEB is a by-product during the PET synthesis and is generally remained in the fabric or garment during textile manufacturing. BETEB is produced by e.g. condensation of terephthalic acid, benzoic acid and ethylene glycol, which has the same unit of benzoyloxy- ethylester as PET.

The enzyme in question qualifies as a cutinase for use according to the present inven- tion if transparent zones are shown after testing in Example 1.

Cutinases are known from various fungi, such as a filamentous fungal cutinase, e.g. native to a strain of Humicola or Fusarium or Magnaporthe or Pseudomonas, specifically H. insolens or F. solani pisi or Magnaporthe grisea or Pseudomonas mendocina, more specifically H. insolens strain DSM 1800 (US 5,827,719), or F. solani pisi {WO 90/09446 Fig 1 ; WO 94/14964 FigI D, WO 94/03578 Fig 1 D, all hereby incorporated by reference) or Magnaporthe grisea (W010/107560 SEQ ID NO: 1 , hereby incorporated by reference) or Pseudomonas mendocina ATCC 53552 (US 5,389,536, claim 1 , hereby incorporated by reference).

SEQ ID NO: 1 is the amino acid sequence of the Humicola insolens cutinase (corresponding to the mature part of SEQ ID NO: 2 of US 5,827,719).

In one embodiment, the cutinase of the present invention has at least 70%, or 75%, or

85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% identity to SEQ ID NO: 1.

In some embodiments, the cutinase can be variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of SEQ ID NO: 1. Preferably, the total number of amino acid substitutions, deletions and/or insertions of the SEQ ID NO: 1 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8 or 9. The Humicola insolens cutinase variants described in WO 2001/092502 are hereby incorporated by reference. The cutinase enzyme may also be a variant of a parent cutinase such as those described in WO 00/34450, hereby incorporated by reference.

The fungal cutinase may also be derived from other fungal strains such as a strain of Rhizoctonia, e.g. R. solani, or a strain of Alternaria, e.g. A. brassicicola (WO 94/03578). Preferably the cutinase has a pH optimum within 1 pH unit of the pH of the process, e.g. if the processss is run at pH 8, the cutinase preferably has a pH optimum between 7 and 9.

Sequence Identity

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Glycoside hydrolase family 61 (GH61 ) polypeptides

The term "glycoside hydrolase family 61 " or "GH61 " is defined herein as a polypeptide falling into the glycoside hydrolase family 61 according to Henrissat B., 1991 , Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Biochem. J. 316: 695-696.

The present invention relates to the use of isolated GH61 polypeptides in general. A GH61 polypeptide useful in the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source in which it is naturally present or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

A GH61 polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypep- tide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide; or a Strep- tomyces polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide.

A GH61 polypeptide of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyc- es, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Chaetomium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neu- rospora, Paecilomyces, Penicillium, Piromyces, Poronia, Schizophyllum, Talaromyces, Ther- moascus, Thielavia, Tolypocladium, Trichoderma or Verticillium polypeptide.

In the present invention, any GH61 polypeptide having cutinase enhancing activity can be used.

In one embodiment, for purposes of the present invention, cutinase enhancing activity is determined by the reduction of oligmer in the PET, i.e. by measuring the increase in OD 254 absorbance under conditions as specified in Example 4, by hydrolyzing BETEB with cutinase and GH61 at a dosage of 0.05 mg protein/ml at 70°C, pH 8.0 for 40 minutes. In a preferred embodiment of the present invention, the OD is increased by at least 0.25, preferably at leat 0.28, more preferably at least 0.3, more preferably at least 0.33, more preferably at least 0.35, more preferably at least 0.38, more preferably at least 0.40, even more preferably at least 0.43, and most preferably at least 0.45 as compared to the OD result when the cutinase is used without GH61 .

In some embodiments, cutinase enhancing activity is determined by measuring the reduction of pill formation under conditions as specified in Example 6, by treating PET in Launder- O-Meter with cutinase and GH61 at a dosage of 2.8 mg protein/ gram of fabric at 70°C, pH 8.0 for 2 hours. In a preferred embodiment of the present invention, it shows the pilling note increased by at least 0.125, more preferably at least 0.250, more preferably at least 0.375, more preferably at least 0.500, more preferably at least 0.625, even more preferably at least 0.750 as compared to the pilling note when the cutinase is used without GH61 .

In a first aspect, GH61 polypeptides having cutinase enhancing activity, comprise the following motifs: [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and [FW]-[TF]-K-[AIV], wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4) is any amino acid at 4 contiguous positions.

The isolated polypeptide comprising the above-noted motifs may further comprise:

H-X(1 ,2)-G-P-X(3)-[YW]-[AILMV],

[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or

H-X(1 ,2)-G-P-X(3)-[YW]-[AILMV] and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], wherein X is any amino acid, X(1 ,2) is any amino acid at 1 position or 2 contiguous positions, X(3) is any amino acid at 3 contiguous positions, and X(2) is any amino acid at 2 contiguous positions. In the above motifs, the accepted lUPAC single letter amino acid abbreviation is employed.

In a preferred embodiment, the isolated GH61 polypeptide having cutinase enhancing activity further comprises H-X(1 ,2)-G-P-X(3)-[YW]-[AILMV]. In another preferred embodiment, the isolated GH61 polypeptide having cutinase enhancing activity further comprises [EQ]-X-Y- X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. In another preferred embodiment, the isolated GH61 polypeptide having cutinase enhancing activity further comprises H-X(1 ,2)-G-P-X(3)-[YW]- [AILMV] and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, isolated polypeptides having cutinase enhancing activity, comprise the following motif:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A-[HNQ],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguous positions. In the above motif, the accepted lUPAC single letter amino acid abbreviation is employed.

In a third aspect, the GH61 polypeptide having cutinase enhancing activity comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47.

In an embodiment, the mature polypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 3, amino acids 20 to 258 of SEQ ID NO: 4, amino acids 19 to 226 of SEQ ID NO: 5, amino acids 20 to 304 of SEQ ID NO: 6, amino acids 16 to 317 of SEQ ID NO: 7, amino acids 22 to 249 of SEQ ID NO: 8, amino acids 20 to 249 of SEQ ID NO: 9, amino acids 18 to 232 of SEQ ID NO: 10, amino acids 16 to 235 of SEQ ID NO: 1 1 , amino acids 19 to 323 of SEQ ID NO: 12, amino acids 16 to 310 of SEQ ID NO: 13, amino acids 20 to 246 of SEQ ID NO: 14, amino acids 22 to 354 of SEQ ID NO: 15, amino acids 22 to 250 of SEQ ID NO: 16, amino acids 22 to 322 of SEQ ID NO: 17, amino acids 24 to 444 of SEQ ID NO: 18, amino acids 26 to 253 of SEQ ID NO: 19, amino acids 18 to 246 of SEQ ID NO: 20, amino acids 20 to 334 of SEQ ID NO: 21 , amino acids 18 to 227 of SEQ ID NO: 22, amino acids 20 to 223 of SEQ ID NO: 23, amino acids 22 to 368 of SEQ ID NO: 24, amino acids 25 to 330 of SEQ ID NO: 25, amino acids 17 to 236 of SEQ ID NO: 26, amino acids 19 to 250 of SEQ ID NO: 27, amino acids 23 to 478 of SEQ ID NO: 28, amino acids 17 to 230 of SEQ ID NO: 29, amino acids 20 to 257 of SEQ ID NO: 30, amino acids 23 to 251 of SEQ ID NO: 31 , amino acids 19 to 349 of SEQ ID NO: 32, amino acids 24 to 436 of SEQ ID NO: 33, amino acids 21 to 344 of SEQ ID NO: 34, amino acids 26 to 400 of SEQ ID NO: 35, amino acids 21 to 389 of SEQ ID NO: 36, amino acids 22 to 406 of SEQ ID NO: 37, amino acids 20 to 427 of SEQ ID NO: 38, amino acids 18 to 267 of SEQ ID NO: 39, amino acids 21 to 273 of SEQ ID NO: 40, amino acids 21 to 322 of SEQ ID NO: 41 , amino acids 18 to 234 of SEQ ID NO: 42, amino acids 24 to 233 of SEQ ID NO: 43, amino acids 17 to 237 of SEQ ID NO: 44, amino acids 20 to 484 of SEQ ID NO: 45, amino acids 22 to 320 of SEQ ID NO: 46, or amino acids 21 to 330 of SEQ ID NO: 47.

Preferably, the GH61 polypeptide having cutinase enhancing activity comprises or consists of an amino acid sequence having at least 90% identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47. More preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47. Most preferably at least 100% identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO:

22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:

39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47

In a sixth aspect, the GH61 polypeptide having cutinase enhancing activity is a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO:

23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:

40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47 comprising a substitution, deletion, and/or insertion at one or more

{e.g., several) positions.

Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about

20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids

(arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for cutinase enhancing activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display {e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DM4 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47, is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In an embodiment, the mature polypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO: 22, amino acids 18 to 239 of SEQ ID NO: 3, amino acids 20 to 258 of SEQ ID NO: 4, amino acids 19 to 226 of SEQ ID NO: 5, amino acids 20 to 304 of SEQ ID NO: 6, amino acids 16 to 317 of SEQ ID NO: 7, amino acids 22 to 249 of SEQ ID NO: 8, amino acids 20 to 249 of SEQ ID NO: 9, amino acids 18 to 232 of SEQ ID NO: 10, amino acids 16 to 235 of SEQ ID NO: 1 1 , amino acids 19 to 323 of SEQ ID NO: 12, amino acids 16 to 310 of SEQ ID NO: 13, amino acids 20 to 246 of SEQ ID NO: 14, amino acids 22 to 354 of SEQ ID NO: 15, amino acids 22 to 250 of SEQ ID NO: 16, amino acids 22 to 322 of SEQ ID NO: 17, amino acids 24 to 444 of SEQ ID NO: 18, amino acids 26 to 253 of SEQ ID NO: 19, amino acids 18 to 246 of SEQ ID NO: 20, amino acids 20 to 334 of SEQ ID NO: 21 , amino acids 18 to 227 of SEQ ID NO: 22, amino acids 20 to 223 of SEQ ID NO: 23, amino acids 22 to 368 of SEQ ID NO: 24, amino acids 25 to 330 of SEQ ID NO: 25, amino acids 17 to 236 of SEQ ID NO: 26, amino acids

19 to 250 of SEQ ID NO: 27, amino acids 23 to 478 of SEQ ID NO: 28, amino acids 17 to 230 of SEQ ID NO: 29, amino acids 20 to 257 of SEQ ID NO: 30, amino acids 23 to 251 of SEQ ID NO: 31 , amino acids 19 to 349 of SEQ ID NO: 32, amino acids 24 to 436 of SEQ ID NO: 33, amino acids 21 to 344 of SEQ ID NO: 34, amino acids 26 to 400 of SEQ ID NO: 35, amino acids 21 to 389 of SEQ ID NO: 36, amino acids 22 to 406 of SEQ ID NO: 37, amino acids 20 to 427 of SEQ ID NO: 38, amino acids 18 to 267 of SEQ ID NO: 39, amino acids 21 to 273 of SEQ ID NO: 40, amino acids 21 to 322 of SEQ ID NO: 41 , amino acids 18 to 234 of SEQ ID NO: 42, amino acids 24 to 233 of SEQ ID NO: 43, amino acids 17 to 237 of SEQ ID NO: 44, amino acids

20 to 484 of SEQ ID NO: 45, amino acids 22 to 320 of SEQ ID NO: 46 or amino acids 1 to 20 of SEQ ID NO: 47.

Co-substance

The addition of a co-substance together with GH61 polypeptides can enhance the enzymatic efficiency even further. In one aspect, the GH61 polypeptide having cutinase enhancing activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043. In a preferred aspect, the soluble activating divalent metal cation is selected from the alkali metals or transition metals in the periodic table. In a more preferred aspect, the soluble activating divalent metal cation is selected from the group consisting of Mn++, Co++, Mg++, Ca++, and a combination thereof. In a more preferred aspect, the soluble activating divalent metal cation is Mn++. In another more preferred aspect, the soluble activating divalent metal cation is Co++. In another more preferred aspect, the soluble activating divalent metal cation is Mg++. In another more preferred aspect, the soluble activating divalent metal cation is Ca++. In another more preferred aspect, the soluble activating divalent metal cation is two or more (several) cations selected from the group consisting of Mn++, Co++, Mg++, and Ca++. In a most preferred aspect the soluble activating divalent metal cation is in the form of manganese sulfate.

In one aspect, the GH61 polypeptide having cutinase enhancing activity is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen- containing compound, or a sulfur-containing compound.

The dioxy compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl moiety as described herein. The dioxy compounds may comprise one or more {e.g., several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl de- rivatives. Non-limiting examples of the dioxy compounds include pyrocatechol or catechol; caffe- ic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1 ,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sina- pinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1 ,2-benzenediol; 4-nitro-1 ,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1 ,4-diol; (croconic acid; 1 ,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1 ,2-propanediol; 2,4,4'- trihydroxybenzophenone; cis-2-butene-1 ,4-diol; 3,4-dihydroxy-3-cyclobutene-1 ,2-dione; dihy- droxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and methyl- 3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ring system as described herein. The compounds may comprise one or more {e.g., several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally substituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof. Non-limiting examples of the bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyi moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyi moiety or optionally substituted heteroaryl moiety is an optionally substituted 5-membered heterocycloalkyi or an optionally substituted 5-membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyi or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyi moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl. Non-limiting examples of the heterocyclic compounds include (1 ,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy- 5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone; [1 ,2-dihydroxyethyl]furan-2,3,4(5H)-trione; o hydroxy-Y-butyrolactone; ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconic acid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)- furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate thereof.

The nitrogen-containing compound may be any suitable compound with one or more {e.g., several) nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety. Non-limiting examples of the nitrogen- containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2- aminophenol; 1 ,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8- tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinone moiety as described herein. Non-limiting examples of the quinone compounds include 1 ,4-benzoquinone; 1 ,4-naphthoquinone; 2-hydroxy-1 ,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1 ,4-benzoquinone or coenzyme Q ₀ ; 2, 3, 5, 6-tetramethyl-1 ,4-benzoquinone or duroquinone; 1 ,4- dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5- methoxy-1 ,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprising one or more {e.g., several) sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of the sulfur-containing compounds include ethanethiol; 2- propanethiol; 2-propene-1 -thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1 ,2- dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.

In one aspect, the amount of such a compound described above to polyester textile material as a molar ratio to glucosyl units of cellulose is about 10 ^"6 to about 10, e.g., about 10 ^"6 to about 7.5, about 10 ^"6 to about 5, about 10 ^"6 to about 2.5, about 10 ^"6 to about 1 , about 10 ^"5 to about 1 , about 10 ^"5 to about 10 ^"1 , about 10 ^"4 to about 10 ^"1 , about 10 ^"3 to about 10 ^"1 , or about 10 ^"3 to about 10 ^"2 . In another aspect, the amount of such a compound described above is about 0.1 μΜ to about 1 M, e.g., about 0.5 μΜ to about 0.75 M, about 0.75 μΜ to about 0.5 M, about 1 μΜ to about 0.25 M, about 1 μΜ to about 0.1 M, about 5 μΜ to about 50 mM, about 10 μΜ to about 25 mM, about 50 μΜ to about 25 mM, about 10 μΜ to about 10 mM, about 5 μΜ to about 5 mM, or about 0.1 mM to about 1 mM.

The term "liquor" means the solution phase, either aqueous, organic, or a combination thereof.

Polyester Fabric Manufacturing Process

Polyester such as poly (ethylene terephthalate) is synthesized by condensation, drawn into fibers from a melt, possibly cut to stables, possibly mixed with other fiber types, and spun to yarn.

After yarn is knitted or woven into fabric, the fabric is normally treated to remove spin finish oil, for example in a process where the fabric will first be heat setted at 180°C and then be pretreated with surfactants (sometimes also with addition of alkali) at 80-100°C and then option- ally followed by the weight reduction process by using severe alkali at up to 130°C to hydrolyze polyester fabric to make it more soft and luster appearance. Then the polyester fabric will be heat setted and dyed with disperse dyestuffs at pH 4.5-6 at up to 130°C, followed by reduction clearing with sodium hyposulphite at 60-80°C, pH 10. If necessary, these processes can be followed by finishing (post treatment) steps to further improve the textile properties, such as anti- pilling, wettability improvement or anti-static treatment.

During synthesis and drawing, cyclic or linear oligomers of polyethylene terephthalate are formed on and in the fibers. Removal of cyclic and/or linear oligomers can be accomplished by hydrolysis with one or more cutinase enzymes. The cutinase breaks the ring structure of the cyclic oligomer and break the BETEB chain to produce benzonic acid, terephathalate acid and ethelene glycol by hydrolyzing an ester bond. The resulting product can be removed under gentle conditions. The method of the present invention of treating polyester textile with a GH61 polypeptide and a cutinase takes place during one or more of the subsequent steps of pretreatment, weight reduction, disperse dyeing or post finishing to endow the polyeseter fabric with at least one of the following effects: reduction of oligomer in the polyester textile, reduction of pill formation , improvment of hydrophilicity and antistatic properties etc. The method of the present invention may take place either as a separate step or in combination with any of the existing polyester processing steps.

The process of the invention is readily applicable in the textile industry as it can be carried out using existing wet processing apparatus, such as in a beam dyer, a Pad-Roll, a Jigger/Winch, a J-Box, or Pad-Steam types of apparatus. The process preferably takes place during the finishing (post treatment) step.

As used herein, the term "biopolishing", "depilling", "reduction of pill formation" and "anti- pilling" are interchangeable.

Polyester fabrics have a handle appearance that is rather hard and stiff without the ap- plication of finishing components. Some fabric surface is not smooth because small fuzzy microfibrils protrude from it. In addition, after a relatively short period of wear, pilling appears on the fabric surface thereby giving it an unappealing, worn look.

Biopolishing is a method to treat polyester fabrics during their manufacturing, which improves fabric quality with respect to "reducuction of pill formation". The most important effects of biopolishing can be characterised by less fuzz and pilling, increased gloss/luster, improved fabric handle, increased durable softness, anti-static property and/or improved water absorbency. In the present context, the term "reduction of pill formation" is intended to mean a resistance to formation of pills on the surface of the treated fabric surface according to the method of the present invention.

For the purpose of the present invention, the pill formation may be tested according to the description of "pilling notes test" in the material and method section. The results of the test is expressed in terms of "pilling notes" which is a rating on a scale from pilling note 1 (heavy pill formation) to pilling note 5 (no pill formation), allowing 1/4 pilling notes.

Since the method of biopolishing catalyze hydrolysis of the polyester fibre surface, the enzymatic action will eventually result in a weight loss of fibre or fabric. In a prefered embodiment, the biopolishing is carried out in such a way so as to obtain a controlled, partial hydrolysis of the fibre surface, i.e. a proper polishing effect without excessive loss of fabric strength.

For the purpose of the present invention, the biopolishing effect is measured under conditions as specified in Example 6, by treating PET in Launder-O-Meter with cutinase of 2.8 mg protein/ gram and GH61 of 2.8 mg protein/ gram of fabric at 70°C, pH 8.0 for 2 hours. In a preferred embodiment of the present invention, the treatment with cutinase and GH61 results in a pilling note of at least 2.00, preferably at least 2.25, and even more preferably at least 2.5, while preferably at the same time shows weight loss of less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2% and most preferably less than 1 %. In preferred embodiment, compared with the treatment by cutinase of 2.8 mg protein/ gram of fabric without GH61 , PET treatment in Launder-O-Meter with cutinase of 2.8 mg protein/ gram and GH61 of 2.8 mg protein/ gram under conditions as specified in Example 6 results in increase of pilling note of 0.25.

PROCESS CONDITION GH61 polypeptides in combination with cutinase can be used during polyester textile manufacturing process, either as a separate step after any of the existing polyester manufacturing steps, or in combination with any of the existing polyester manufacturing steps like pretreatment, weight reduction, disperse dyeing or post finishing.

It is advised that a suitable liquor/textile ratio to be used in the present method may be in the range of from about 20:1 to about 1 :1 , preferably in the range of from about 15:1 to about 3:1 , more preferably in the range of from 15:1 to 5:1 (Volumn/weight, ml/mg).

The reaction time for the present invention is usually in the range of from about 10 minutes to about 8 hours. Preferably the reaction time is within the range of from about 20 minutes to about 180 minutes, more preferably the reaction time is within the range of from about 30 mi- nutes to about 150 minutes, most preferably the reaction time is within the range of from about 45 minutes to about 120 minutes.

The pH of the reaction medium greatly depends on the enzyme(s) in question. Preferably the process of the invention is carried out at +/- 1 pH unit from the pH optimum of the cutinase. Preferably, the process of the invention is carried out at a pH in the range of from about pH 3 to about pH 1 1 , preferably in the range of from about pH 4 to about pH 10, or within the range of from about pH 6 to about pH 9.

The process temperature of the present invention is preferably selected according to the optimal temperature of the cutinase +/- 10°C. Preferably the process is able to function at a temperature below 100°C, preferably below 90°C, more preferably below 80°C, and even more preferably below 75°C.

In some embodiments, the process of the present invention is conducted at the temperature range of 40-100°C, preferably 50-90°C, preferably 60-85°C, more preferably 65-80°C, and even more preferably 70-80°C.

Enzyme dosage greatly depends on the enzyme reaction time, i.e. a relatively short en- zymatic reaction time necessitates a relatively increased enzyme dosage, and vice versa. In general, enzyme dosage may be stipulated in accordance with the reaction time available. The amount of GH61 polypeptide to be used according to the method of the present invention depends on many factors and should preferably be optimized by the skilled person. According to the present invention the preferred concentration of the of GH61 polypeptide in the aqueous medium is from about 0.01 to about 50 milligram protein per gram of polyester textile, preferably 0.05-20 milligram (mg) of protein per gram (g) of polyester textile, preferably 0.1-15 milligram of protein per gram of polyester textile, more preferably 0.2 - 8 milligram of protein per gram of polyester textile, and even more preferably 0.2-5 milligram of protein per gram of polyester textile.

The amount of cutinase to be used according to the method of the present invention depends on many factors and should preferably be optimized by the skilled person. According to the present invention the preferred concentration of the cutinase enzyme in the aqueous medium is from about 0.01 to about 50 milligram enzyme protein per gram of polyester textile, preferably 0.05-20 milligram of enzyme protein per gram of polyester textile, more preferably 0.1-15 milligram of enzyme protein per gram of polyester textile, and even more preferably 0.2-5 milligram of enzyme protein per gram of polyester textile. Preferably, the dosage ratio between cutinase and GH61 is 1 : 1 to 1 : 0.5.

The process of the invention may further comprise the addition of one or more chemicals capable of improving the enzyme-substrate interaction (in order to improve the substrate's accessibility and/or dissolve reaction products), which chemicals may be added prior to, or simultaneously with the enzymatic treatment. Such chemicals may in particular be co-substance as described above, surfactants, wetting agents, anti-pilling agents and dispersing agents, or mixtures hereof.

The process of the invention may optionally comprise a rinsing step during which the hydrolyzed oligomers are subjected to rinsing, in particular to rinse with alkali solution. Alkal solution dissolves linear fragments of the oligomers, and may to some extent further hydrolyze these linear fragments.

The aqueous composition used in the method of the invention may further comprise one or more enzymes selected from the group consisting of lipases, esterases, laccases, peroxidase and etc. Composition for treating textile

The present invention also encompasses a composition suitable for treating textile where the composition comprises a GH61 polypeptide and a cutinase.

The use of the composition of the present invention can provide the polyester fabric with at least one of the following effects: reduction of oligomer in the polyester textile, reduction of pill formation, improvement of hydrophilicity and antistatic properties etc. The textile composition of the present invention is adapted for one or more of the polyester manufacturing processes such as pretreatment, weight reduction, disperse dyeing and post finishing, either in a separate step or in combination with any of those steps.

In the present invention, GH61 polypeptide enhances the cutinase activity by reducing the amount of cutinase required to reach the same degree of depilling.

In some embodiments of the invention, the composition containing a GH61 polypeptide and a cutinase further comprises other components, including without limitation other enzymes, as well as one or more of surfactants, bleaching agents, antifoaming agents, builder systems, and the like.

Enzymes suitable for use in the present invention include without limitation lipases, esterases, laccases, peroxidases, peroxygenase and transferases.

In one embodiment, the textile composition comprises one or more of the GH 61 polypeptides selected from the group consisting of an amino acid sequence that has a degree of identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In an even more preferred aspect, the textile composition further comprises a co- substance as describedin the "Co-substance" section above. In a preferred embodiment, the co- substance is cysteine.

The textile composition can be in any form, such as a solid, liquid, paste, gel or any combination thereof.

Surfactant

In the treatment of polyester textile, a conventional surfactant may be used to improve the contact with the enzyme.

The textile composition of the present invention may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. The surfactant(s) is typically present at a level of from about 0.001% to 20% by weight of composition, such as about 0.005% to about 10%, or about 0.01 % to about 5%, or about 0.02% to about 1 %.

More specifically, the surfactant used in the process or the composition of the present invention comprises a non-ionic surfactant. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkox- ylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, al- kylphenol ethoxylates (APE), Triton, nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (gluca- mides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

Other enzymes

The enzymatic polyester manufacturing process as well as the textile composition may comprise one or more additional enzymes such as a lipase, esterase, laccase, peroxidase, peroxygenase and transferases.

Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. The lipase may for example be triacylglycerol lipase (EC3.1 .1 .3), phospholipase A2 (EC 3.1.1.4), Lysophospholipase (EC 3.1 .1 .5), Monoglyceride lipase (EC 3.1 .1 .23), galactolipase (EC 3.1 .1.26), phospholipase A1 (EC 3.1.1.32), Lipoprotein lipase (EC 3.1.1.34). Examples include lipase from Thermo myces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258 068 and EP 305 216, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois et al, 1993, Biochemica et Biophysica Acta, 1 131 : 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, WO 00/060063, WO2007/087508 and WO 2009/109500.

Preferred commercially available lipase enzymes include Lipolase™, Lipolase Ultra™, and Lipex™; Lecitase™, Lipolex™; Lipoclean™, Lipoprime™ (Novozymes A/S). Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist- Brocades/Genencor Int Inc) and Bacillus sp lipase from Solvay. Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

Peroxygenase: The term "peroxygenase" means an "unspecific peroxygenase" activity according to EC 1 .1 1 .2.1 , that catalyzes insertion of an oxygen atom from H ₂ 0 ₂ into a variety of substrates, such as nitrobenzodioxole. Examples of useful peroxygenase include peroxygenase described in WO 2008/1 19780.

The present methods and compositions are further described in the following numbered paragraphs.

1 . A method for treating polyester textile with a glycosyl hydrolase family 61 polypeptide in the presence of a cutinase in an aqueous solution.

2. In some embodiments of the method of paragraph 1 , wherein the textile is yarn, fabric or garment.

3. In some embodiments of the method of paragraph 1 or 2, wherein the polyester is

PET.

4. In some embodiments of the method of paragraph 1 , wherein the aqueous solution further comprises one or more enzymes selected from the group consisting of lipases, esterases, laccases, peroxidases, peroxygenase and transferases.

5. In some embodiments of the method of any of the preceding paragraphs, wherein a co-substance is used together with a glycosyl hydrolase family 61 ; preferably the co-substance is cysteine.

6. In some embodiments of the method of any of the preceding paragraphs, wherein the glycosyl hydrolase family 61 polypeptide is applied in the range of from 0.01 to about 50 milligram protein per gram of polyester textile, preferably 0.05-20 milligram of protein per gram of polyester textile, preferably 0.1-15 milligram of protein per gram of polyester textile, more preferably 0.2-8 milligram of protein per gram of polyester textile, and even more preferably 0.25-5 milligram of protein per gram of polyester textile.

7. In some embodiments of the method of any of the preceding paragraphs, wherein the cutinase is applied in the range of from about 0.01 to about 50 milligram enzyme protein per gram of polyester textile, preferably 0.05-20 milligram of enzyme protein per gram of polyester textile, more preferably 0.1-15 milligram of enzyme protein per gram of polyester textile, and even more preferably 0.2-5 milligram of enzyme protein per gram of polyester textile. 8. In some embodiments of the method of any of the preceding paragraphs, , wherein the method is conducted in the pH range of from about pH 3 to about pH 1 1 , preferably in the range of from about pH 4 to about pH 10, or within the range of from about pH 6 to about pH 9.

9. In some embodiments of the method of any of the preceding paragraphs, wherein the method is conducted in the temperature range of 40-100°C, preferably 50-90°C, preferably

60-85°C, more preferably 65-80°C, and even more preferably 70-80°C.

10. In some embodiments of the method of any of the preceding paragraphs, wherein the method is conducted for about 10 minutes to about 8 hours, preferably about 20 minutes to about 180 minutes, more preferably about 30 minutes to about 150 minutes, more preferably about 45 minutes to about 120 minutes.

1 1. In some embodiments of the method of any of the preceding paragraphs, wherein the method for treating polyester textile is the manufacture of a polyester textile, especially manufacture of a polyester fabric.

12. In some embodiments of the method of paragraph 1 1 , wherein the method is com- bined with any of the existing polyester fabric manufacturing steps.

13. In some embodiments of the method of any of the preceding paragraphs, wherein the cutinase has BETEB hydrolysis activity.

14. In some embodiments of the method of any of the preceding paragraphs, wherein the cutinase is at least 90% sequence identity to SEQ ID NO:1 , or comprising a substitution, de- letion, and/or insertion of one or more (or several) amino acids of SEQ ID NO: 1 .

15. In some embodiments of the method of any of the preceding paragraphs, wherein the glycosyl hydrolase family 61 polypeptide has having cutinase enhancing activity when measured according to the conditions of Example 4.

16. In some embodiments of the method of any of the preceding paragraphs, wherein the glycosyl hydrolase family 61 polypeptide is at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47.

17. A composition for treating a textile comprising a glycosyl hydrolase family 61 polypeptide and a cutinase. 18. In some embodiments of the composition of paragraph 17, wherein the composition further comprises one or more enzymes selected from the group consisting of lipases, esterases, laccases, peroxidases, peroxygenase and transferases.

19. In some embodiments of the composition of paragraph 17 or 18, wherein the com- position further comprises a co-substance; preferably the co-substance is cysteine.

20. In some embodiments of the composition of any of the paragraph 17-19, wherein the composition further comprises a surfactant, preferably a non-ionic surfactant.

21. Use of a glycosyl hydrolase family 61 polypeptide to boost the effect of a cutinase on a polyester textile.

22. In some embodiments of the use of paragraph 21 , wherein the effect is to reduce pill formation on a polyester textile.

23. In some embodiments of the use of paragraph 21 or 22, wherein the pilling note increase by at least 0.125, more preferably at least 0.250, more preferably at least 0.375, more preferably at least 0.500, more preferably at least 0.625, even more preferably at least 0.750 as compared to the pilling note when the cutinase is used without GH61 under conditions as specified in Example 6.

24. In some embodiments of the use of paragraph 21 , wherein the effect is to reduce depositing of cyclic or linear oligomers of polyethylene terephthalate on machinery and/or textile when compared to the same process run under the same conditions without GH61 .

25. In some embodiments of the use of paragraph 24, wherein the oligomers are acid- bis-2-benzoyloxy-ethylesther and/or triethylene terephthalate.

EXAMPLES

Materials & Methods

Proteins

Cutinase A: variant of cutinase from Humicola. Insolens, with substitutions E6Q+A14P+E47K+R51 P+E179Q+G8D+N15D+S48E+A88H+N91 H+A130V+R189V on the parent H. insolens cutinase of SEQ ID NO: 1 (cutinase A described in WO 2001/092502)

Cutinase B: variant of cutinase from Humicola. Insolens, with substitutions E6Q+A14P+E47K+R51 P+E179Q+G8D+N15D+T29M+S48E+A88H+N91 H+A130V+T166I+L16 7P+R189V on the parent H. insolens cutinase of SEQ ID NO: 1 (cutinase B described in WO 2001/092502)

Mature polypeptide of Af GH61 : Aspergillus fumigatus GH61 B polypeptide shown as amino acids of 22 to 250 of SEQ ID NO: 16 (described in US 2010124769)

Mature polypeptide of Ta GH61 : Thermoascus aurantiacus GH61A polypeptide shown as amino acids 22 to 249 of SEQ ID NO: 8 (described in WO 2005/074656) Mature polypeptide of Nc GH61 : Neurospora crassa GH61 polypeptide shown as amino acid 21 -330 of SEQ ID NO: 47 (described in WO201 1080267)

Mature polypeptide of Ts GH61 : Talaromyces stipitatus GH61 polypeptide shown as amino acids of 22 to 320 SEQ ID NO: 46 (UNIPROT: B8M2G3)

Chemicals

Triton X-100 (Beijing Kehaoze Biotechnology Co., Ltd. China)

BETEB (Terephtalic acid-bis-2-benzoyloxy-ethylesther)

PET (polyethylene terephthalate, 100% Dacron ^® Type 64 style, Staple woven PET fabric, commercially available from SDL.)

Reagents/substrates

Britton-Robinson Buffer: Titrate the acidic mixture of 0.04 M H ₃ B0 ₃ , 0.04 M H ₃ P0 ₄ and 0.04 M CH ₃ COOH to the desired pH with 0.2 M NaOH.

4mM Britton-Robinson buffer is obtained by 10-time dilution of the Britton-Robinson buffer above and then titrate the solution with NaOH to desired pH.

2.5% BETEB substrate: 2.5 g BETEB+100 ml deionized water+0.5 ml 1 % Triton-X 100

OD absorbance and pH Measurement

Cutinases A and B were used to hydrolyze PET or BETEB in eppendorf tubes. The hydrolysis products were terephthalate and its esters which had characteristic absorbance peaks around 254 nm (UV). Therefore the OD absorbance at 254 nm reflects the hydrolytic activity of enzymes towards polyesters. The higher the OD absorbance at 254 nm is, the stronger is the enzyme activity towards PET or BETEB. OD at 254 nm is read in SpectraMax M2 Microplate Reader (Molecular Devices, LLC). If the absorbance is beyond the effective range of the Reader of 1 .5, the solution will be diluted. Dilution x15 means the solution has been diluted by 15 times.

The hydrolysis product terephthalate is acidic and will thus decrease the pH of solution, therefore the pH change before and after the reaction is a parameter for testing the activity of enzymes. Weight loss determination

The swatches were placed in the conditioned room (65%+/-5% humidity, 20+/-1 °C) for 24 hours before they were numbered, weighed by the analytical balance (for samples below 10Og) or a precision balance (for samples over 10Og) and recorded. After treatment, all samples were tumbled dried (AEG, LAVATHERM 37700, Germany) for 1 hour and conditioned for 24 hours in the same conditioned room as above. For each sample, the weight loss was defined as below: Weight loss = (weight before treatment - weight after treatment)/weight before treatment X (100%)

Pilling Notes test

Fabrics including treated and untreated which had been pre-conditioned in norm climate

(65% humidity, 20°C) for at least 24 hours were tested for the pilling notes with Nu-Martindale Tester (James H. Heal Co. Ltd, England), with untreated fabrics of the same type as the abraded fabrics. A standard pilling test (Swiss Norm (SN) 198525) was carried out after 2000 Revolutions by marking from 1-5, with the meaning defined as below, where 1 shows poor anti- pilling and 5 shows excellent anti-pilling property. Thus the higher the Martindale pilling notes score the more effective the biopolishing treatment.

Note 5 No pilling

Note 4 Slight Pilling

Note 3 Moderate Pilling

Note 2 Distinct Pilling

Note 1 Heavy Pilling

1/2, 1/4 notes are allowed

To make the test result more reliable, 3 separate readings were carried out by different persons for each sample, and the average of the 3 readings was adopted as the final result of pilling notes.

Protein Content

The protein concentration in an enzyme product or polypeptide product used in the present examples can be measured with BCA™ Protein Assay Kit (product number 23225, commercial available from Thermo Fisher Scientific Inc.) according to the product manual.

Example 1 : BETEB-Agar plate for evaluation of the cutinase activity

BETEB was hydrolyzed by cutinase into more soluble agents. Thus, after hydrolysis by enzyme, there were transparent zones on the plates poured with the mixture of Agar and BE- TEB.

BETEB molecule structure Hydrolysis of BETEB will produce

Cutinase activity was measured by the below process:

a) BETEB solution preparation: 5 ml 100% ethanol was added into a glass bottle with a plug, 20 mg BETEB was added into the ethanol and then the bottle was placed in a 60°C water bath to dissolve the BETEB.

b) 1.5% agar solution was prepared by adding 0.75 g agar into 45 ml Tris-HCI buffer (25mM, pH 7.0), and then placing the baker in a Microwave oven heating twice for 30 seconds to dissolve the Agar.

c) The agar solution was cooled down to 60°C and mixed with the BETEB solution prepared in step a. The mixture was poured into a petri dish.

d) Small holes were dug in the petri dish with a tip of 6 mm diameter or puncher.

e) Enzyme sample of 30 microgram /ml was added into the petri dish by a tip with 75 microliter (ul) enzyme sample for each hole. The petri dish was placed at 37°C overnight.

Both cutinase A and cutinase B showed transparent zones in the area around the holes, as BETEB was hydrolyzed by the cutinase.

Example 2: Cutinase A with GH61 s for PET treatment.

In this example, two GH61 s of Af GH61 and Ta GH61 were used respectively in combination with cutinase A to hydrolyze PET dots in 1.5 ml Eppendorf tubes. PET fabric was cut into small pieces of 0.5 cm diameter with 0.005 g per piece, and two pieces were added into each Eppendorf tube. Britton-Robinson buffer (4mM, pH 8) and 1 % Triton X100 were placed in a thermomixer at 70°C for 5 minutes to warm-up. After warm-up, cutinase and GH61 were added into the tube to make a total volume of 1 ml, wherein the final con- centration of Triton X100 was 0.2g/l, and the final concentration of cutinase and GH61 in the solution was as shown in Table 1 . OD254 absorbance and pH were tested, as decribed in the Materials & Methods section, immediately shown as data for 0 hour in Table 1. The tubes were placed in a thermomixer to start the reaction at 1000 rpm and at 70°C. After reaction for a certain period of time as indicated in Table 1 , the reaction was stopped by transferring the eppen- dorf tubes to an ice bath for 10 minutes. Then the eppendorf tubes were centrifuged at 13000 g/min for 10 seconds to get the supernatant for OD and pH determination. The supernatants were diluted 5 times for OD testing.

Table 1. Results of Cutinase A with two GH61 s for PET treatment (70°C, pH 8.0, 10OOrpm, 0 - 4 hours)

As can be seen from Table 1 , after 2 hours reaction the absorbance at 254 nm is 0.629 for cutinase A alone and 0.716 for cutinase A combined with Af GH61 . The pH change ("pH change" means the difference between the initial pH at 0 hour and the final pH after reaction), after 2 hours, when using cutinase A alone is 0.03 (i.e, 6.91 minus 6.88); while cutinase A and Af GH61 when used together, result in a pH change of 0.07 (i.e, 6.91 minus 6.84). The addition of the same dosage of Ta GH61 increases the absorbance at 254 nm from 0.629 to 0.774 and slightly increases the pH change from 0.03 to 0.06 (i.e, 6.85 minus 6.79).

When the reaction time was extended to 4 hour, the combination of Af GH61 and cuti- nase A increases the absorbance at 254 nm from 0.806 to 0.899 and increases the pH change from 0.08 to 0.1 1 . After 4 hours with with cutinase A and Ta GH61 the absorbance increases from 0.806 to 0.909 and the pH change from 0.08 to 0.10.

It is also found that with GH61 alone the absorbance at 254 nm would vary slightly. However the absorbance change from GH61 s alone are less than the values when combining GH61 s with cutinase A. For example, after 4 hours reaction withAf GH61 alone slightly increases the absorbance at 254 nm by 0.01 (i.e, 0.384 minus 0.374), and Cutinase A increases the absorbance by 0.388 (i.e, 0.806 minus 0.418) and the combination of Af GH61 and cutinase A lead to a significant absorbance increase of 0.505 (i.e, 0.899 minus 0.394). Therefore, the addition of GH61 to cutinase results in a synergistic effect on the increase of PET hydrolysis during PET treatment.

Example 3: Cutinase B with GH61 s for PET treatment.

In this example, two GH61 s were used in combination with Cutinase B respectively to hydrolyze PET fabric in Eppendorf tubes. The treatment protocol was the same as that de- scribed in example 2.

Table 2. Results of Cutinase B with GH61 s for PET treatment (70°C, pH 7.0, l OOOrpm, 0-4 hours)

Note: average of triple samples for each enzyme combination in Table 2.

As can been seen from Table 2, at 2 hours, the addition of Af GH61 or Ta GH61 to Cutinase B increases the absorbance at 254 nm by 0.057 and 0.028, respectively and at 4 hours the absorbance increases by 0.082 and 0.058, respectively. Over the 2 to 4 hours there is also a slightly increase in the pH change before and after reaction. In conclusion, GH61 s show a boosting effect on Cutinase B.

Example 4: Cutinase A with two GH61 s for oligomer treatment.

BETEB is produced during PET synthesis and the treatment of PET as a kind of oligomer, which might remain in the textile fabric.

GH61 s (AfGH61 or TaGH61 ) were used in combination with cutinase A in 1.5 ml eppen- dorf tube. 40 mM Britton-Robinson buffer (pH 8) was added to make the enzyme and GH61 at a concentration of 0.05 mg enzyme protein/ml solution as shown in Table 3. The tubes were placed in a thermomixer at 70°C for 5 minutes for warm-up. After warm-up, 100 ul 2.5% BETEB substrate was added into the enzyme solution to start the reaction. Eppendorf tubes were placed in the thermomixer at 70°C, 1000 rpm, for the time indicated in Table 3. The reaction was stopped by transferring the eppendorf tubes to ice bath for 10 minutes. The eppendorf tubes were centrifuged at 13000 g/min for 10 seconds to get the supernatant for OD and pH determination. The supernatants derived from Oh, 20 minutes and 40 minutes reaction were diluted 5 times, while the supernatant derived from 1 hour reaction was diluted 75 times for OD testing. The data for sampling time at 0 hour in Table 3 means data tested before BETEB addition.

Table 3. Results of Cutinase A with two GH61 s for BETEB treatment (70°C, pH 8.0, 1000 rpm, 0-1 hour)

Note: average of triple samples for each enzyme combination As shown in Table 3, after 20 min reaction the absorbance at 254 nm is 1.126 for cutinase A alone and 1 .200 for cutinase A combined with Af GH61. The pH change after 20 min when using cutinase A alone is 0.16 (i.e, 7.51 minus 7.35); while cutinase A and Af GH61 used together results in a pH change of 0.17 (i.e, 7.52 minus 7.35). The addition of the same dosage of Ta GH61 increase the absorbance at 254 nm from 1 .126 to 1.312, and slightly increases the pH change from 0.16 to 0.19 (i.e, 7.5 minus 7.31 ).

After 40 min reaction, the absorbance at 254 nm is 2.293 for cutinase A alone and 2.760 for cutinase A combined with Af GH61. The pH change after 40 min when using cutianse A alone is 0.34 (i.e, 7.51 minus 7.17); while cutinase A and Af GH61 used together results in a pH change of 0.37 (i.e, 7.52 minus 7.15). The addition of the same dosage of Ta GH61 increases the absorbance at 254 nm from 2.293 to 2.840 and the pH change from 0.34 to 0.67 (i.e, 7.5 minus 6.83). Significant boosting effect of GH61 s could be detected with BETEB as substrate when combined with cutinase A.

Example 5: Cutinase B with four GH61 s for oligomer treatment.

Four GH61 s were combined with cutinase B to hydrolyze oligomer BETEB at the dosage of 0.01 mg enzyme protein/ml solution and 0.01 mg GH61/ml solution. The treatment protocol was the same as described in example 4. The supernatants derived from Oh and 20 minutes were diluted 5 times, while the supernatant derived from 40 minutes and 1 hour reaction was diluted 75 times for OD testing.

Table 4. Results of cutinase B with four GH61 s for BETEB treatment (70°C, pH 8.0, l OOOrpm, 0- 1 hour)

Note: average σ triple samples for each enzyme combination.

As can be seen from Table 4, after 20 minutes reaction, the absorbance at 254 nm is 1.713 for cutinase B alone and 1 .787 for cutinase B combined with Af GH61 .

After 40 minutes reaction, the absorbance at 254 nm is 0.507 for cutinase B alone and 0.559 for cutinase B combined with Af GH61. The pH change after 40 minutes, when using cutinase B alone is 0.28 (i.e, 7.93 minus 7.65); while cutinase B and Af GH61 when used together results in a pH change of 0.35 (i.e, 7.93 minus 7.58). Similar results are obtained after 1 hour reaction, with OD absorbance increased from 0.748 to 0.832, pH change from 0.35 to 0.47 (i.e, 7.93 minus 7.46).

In conclusion, 4 different GH61 s show boosting effect for hydrolyzing BETEB when used together with cutinase B.

Example 6: Cutinase A with two GH61 s for PET biopolishing in LOM

PET biopolishing was carried out in a Launder-O-Meter (LOM, SDL-Atlas LP2) with cutinase and GH61 s.

PET fabric was cut into rectangular pieces 5 cm wide and 10 cm long and a weight of about 1 g. The fabric was side-locked by sewing. The pieces were placed in a conditioned room (65% relative humidity, 20°C) for 24 hours before they were numbered, weighed by the analytical balance and recorded. One conditioned piece was placed in each beaker. For each beaker, 10 small steel balls (M6M-SR-A4-80, acid proof) were used to supply the mechanical aids. Then the buffer (Britton-Robinson Buffer, pH=8) and the enzyme solutions were added according to Table 5, based on the calculation of actual fabric weights, with a liquid to fabric ratio of 10:1 (v/w). OD absorbance at 254nm and the initial pH of solution were measured, data indicated for sampling time at 0 hour.

The LOM machine was started after the temperature was chosen. The macine was set to pause when the temperature reached 70°C. Each beaker was fitted with a lid lined with 2 neoprin gaskets and close tightly with the metal clamping device. The beakers were loaded into the preheated LOM. Metal racks were used to accommodate and secure 5 beakers, in the vertical position, in each of the 4 drum positions. The LOM lid was closed and the washing program was continued and the timing was initiated. 2 hours later, all beakers were removed and the PET samples were transferred to the inactivation solution (2g/L sodium carbonate) at 95°C for 10 minutes. Then the fabrics were rinsed 2 times in hot water and 2 times in cold water. The PET samples were tumble-dried (AEG, LAVATHERM 37700, Germany) for 1 hour, and then the samples were conditioned for 24 hours at 20°C, 65% relative humidity prior to evaluation.

The solution from the treatment bath from each beaker was also collected and centri- fuged at 13000rpm for 1 minute, to further collect the supernatant for pH measurement and ab- sorbance assay at 254 nm. The fabric evaluation includes weight loss and pilling note.

Table 5: Results of cutinase A with two GH61 s for PET treatment in LOM

Average

Cutinase A (mg Ta GH61 (mg Af GH61 (mg

Weight pilling PH OD 254 change protein/g fabric) protein/g fabric) protein/g fabric)

loss note change (dilution x15)

2.8 - - 0.29% 3.000 - 0.73

5.6 - - 0.41 % 3.250 0.26 0.79

- 5.6 - 0 2.750 0.06 0.10

5.6 5.6 - 0.80% 3.500 0.32 0.82

- - 2.8 0 2.500 0.04 0.04

2.8 - 2.8 0.56% 3.625 0.28 0.83

From the table above, it is apperant that when using cutinase in combination with TaGH61 or AfGH61 , the application performance in LOM in terms of pilling note has been improved significantly, compared with using cutinase alone. Meanwhile, the weight loss is still in a low level of 0.8% or 0.56% when compared to fabric treated without cutinase and GH61. Consequently, there is synergy between Cutinase A and TaGH61 or AfGH61 for PET biopolishing.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.