WO2025140526A1

WO2025140526A1 - A method of treating a textile comprising a polyester with low crystallinity

Info

Publication number: WO2025140526A1
Application number: PCT/CN2024/143106
Authority: WO
Inventors: Chu Wang; Hong Xiao; Lipan DONG
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-12-28
Filing date: 2024-12-27
Publication date: 2025-07-03
Anticipated expiration: 2026-06-28

Abstract

The present invention relates to a method for treating a textile comprising a polyester with low crystallinity by contacting the textile with a cutinase, as well as a textile produced by such method.

Description

A METHOD OF TREATING A TEXTILE COMPRISING A POLYESTER WITH LOW CRYSTALLINITY

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 a method for treating a textile comprising a polyester with low crystallinity, as well as a textile produced by such method.

BACKGROUND OF THE INVENTION

Fibers of poly (ethylene terephthalate) abbreviated as PET account 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 pilling, or fuzz score.

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 fibre materials are those designed for control of pilling. All polyester 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.

In recent years, antimicrobial finishing of textiles has become extremely important in the production of protective, decorative and technical textile products. However, antimicrobial agents attached to a textile surface or incorporated within the fiber substantially reduce their activity and limits their availability. Furthermore, the biocide can be gradually lost during the use and washing of the textile. For these reasons, large amounts of these biocides need to be applied to textiles to effectively control bacterial growth and to sustain durability (Textile Research Journal Vol 78 (1) : 60–72) .

Another problem with polyester is that the existing technology for desizing of polyester size is a high energy consumption process, which relies on high temperature and alkaline treatment, for example, 100℃*30min with 0.1-5g/L NaOH and follows by neutralization and several rinsing bathes.

A further problem with polyester is that during synthesis of PET, oligomers are formed. Oligomers tend to give fabrics a grayish appearance. 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 treat polyester in order to improve the characteristics, amongst others by way of applying cutinases.

However, there is always a need in textile industry for treating polyester textile to improve its property. The property can be selected from the group consisting of handfeel improvement, anti-pilling, fuzz score improvement, polyester size desizing, oligomer removing, and anti-bacteria.

SUMMARY OF THE INVENTION

It has now surprisingly been found that cutinase is capable of improving a property of polyester textile consistently and greatly when the polyester has low crystallinity. The property can be selected from the group consisting of handfeel improvement, anti-pilling, fuzz score improvement, polyester size desizing, oligomer removing, and anti-bacteria. The method of the present invention can be applied to all kinds of textile comprising a polyester with low crystallinity, which makes the treatment of textile comprising a polyester with low crystallinity industrially applicable.

Accordingly, in one aspect, the present invention relates to a method for treating a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

In a further aspect, the present invention relates to a method of biopolishing a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

In a further aspect, the present invention relates to a method of desizing polyester size of a textile, comprising contacting the polyester size with a cutinase.

In a further aspect, the present invention relates to a method of removing polyester oligomer of a textile, comprising contacting the polyester oligomer with a cutinase.

In a further aspect, the present invention relates to a method of improving handfeel of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

In a further aspect, the present invention relates to a method of improving anti-bacterial effect of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

In a further aspect, the present invention relates to a textile produced according to the method of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the color depth of raw fabric without treatment by a cutinase.

Figure 2 shows the color depth of fabric in exhausting desizing without treatment by a cutinase.

Figure 3 shows the color depth of fabric in exhausting desizing with treatment by a cutinase at 1 g/L.

Figure 4 shows the color depth of fabric in exhausting desizing with treatment by a cutinase at 10 g/L.

Figure 5 shows the color depth of fabric in pad-batch without treatment by a cutinase.

Figure 6 shows the color depth of fabric in pad-batch with treatment by a cutinase at 1 g/L.

Figure 7 shows the color depth of fabric in pad-batch with treatment by a cutinase at 10 g/L.

OVERVIEW OF SEQUENCES LISTING

SEQ ID NO: 1 is the amino acid sequence of an endoglucanase from Sordaria fimicola.

SEQ ID NO: 2 is the amino acid sequence of the wild-type cutinase/lipase of Humicola insolens DSM 1800.

SEQ ID NO: 3 is amino acid sequence of the parent cutinase of cutinase B.

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 with Low Crystallinity

“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 with low crystallinity. Particularly preferred polyesters with low crystallinity 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 with low crystallinity 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.

In a preferred embodiment, the polyester textile with low crystallinity is PET with low crystallinity. In a further preferred embodiment, low crystallinity means a crystallinity of less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0; for example, 0-38%, 10%-35%, or 20%-30%, preferably 1%-33%, more preferably 1%-29%.

In a preferred embodiment, the crystallinity is determined by differential scanning calorimetry (DSC) . Differential scanning calorimetry is a thermos-analytical technique that measures physical and chemical changes within a material in response to temperature. Differential scanning calorimeter provides qualitative and quantitative information about endothermic (heat flows into the sample) and exothermic (heat flows out of the sample) process, or changes in heat capacity. Sample types include films, fibers, powders, solutions and composites. DSC measures the energy absorbed or released by a sample as it is heated or cooled. DSC also measures the difference in heat flow between the sample and reference. This differential heat is the heat required for the sample melting, which is displayed on the recorder in form of electrical power.

In a preferred embodiment, the crystallinity is determined by differential scanning calorimetry (DSC) according to GB/T 40271-2021 (National Standard of the People’s Republic of China) .

In a further preferred embodiment, the melting peak temperature of polyethylene terephthalate is 250-255 ℃, and the melting enthalpy is 37-60 J g^-1. Metals indium and tin closed to the measured sample temperature range is selected as standard samples to calibrate the DSC instrument. Two empty aluminium Tzero pans are used to test the instrument baseline.

The fiber samples of 2-3 mm long and about 3-5 mg weight are encapsulated into aluminum pans. Empty aluminium Tzero pans with a Tzero lid is set as reference. Then the sample and reference are heated from 40 ℃ to 300℃ at a constant rate of 0.5-20℃/min under nitrogen flow of 20-50 mL/min to protect the internal components of the calorimeter. The DSC curve is recorded. The area between the baseline and the melting peak is the melting enthalpy. The percentage crystallinity is determined on the first heating scan using the enthalpies of melting and cold crystallization. The following equation is used to calculate percentage crystallinity within the fiber:
%crystallinity= [ (ΔH_m-ΔH_cc) / (ΔH_m*) ] ×100%

Where ΔH_m is the enthalpies of melting (J g^-1) , ΔHcc is the enthalpies of cold recrystallization (J g^-1) , and ΔH_m*is the enthalpies of melting for 100%crystalline sample. The percentage crystallinity was calculated using the TRIOS software package provided with DSC.

In a preferred embodiment, the polyester is PET, preferably recycled PET (rPET) or modified PET.

Recycled PET (rPET)

PET is the most closed-loop recycled plastic worldwide, which is produced with fewer resources and carbon emissions than new polyester fibers. Generally speaking, PET wastes are subjected to successive treatments leading to recycled PET (rPET) . PET wastes (mainly bottles) are collected, sorted, pressed into bales, crushed, washed, chopped into flakes, melted and extruded in pellets and offered for sale. Then, recycled PET may be used to create fabrics for the textile industry or new packaging such as bottles or blister packs, etc. According to the present invention, the crystallinity of recycled polyester is lower than that of virgin polyester. Recycled PET is a polyester with low crystallinity.

Modified PET

In the present invention, PET with low crystallinity is obtained by modifying PET through at least one means of chemical, physical and/or mechanical conversion. In one embodiment, PET with low crystallinity is a titanium containing PET. In a further embodiment, the PET with low crystallinity comprises TiO₂ in an amount of 1-25%, 5-20%, 8-15%by weight.

In another aspect, the present invention relates to a textile comprising a polyester with low crystallinity produced by the method of present invention. The textile comprising a polyester with low crystallinity used herein is meant to include fibers, yarns, fabrics and garments comprising a polyester with low crystallinity. The polyester yarn or fabric or garment is made from pure poly (ethylene terephthalate) , or 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 textile comprising a polyester with low crystallinity is a polyester blend comprising more than 5% (w/w) of polyester, in particular more than 10%, more than 15%, more than 20%, more than 30%, more than 35%, more than 50%, more than 65%, more than 90%, or more than 95%of polyester. In an even preferred embodiment, such textile blend is polyester/cotton blend. In an even preferred embodiment, the process of the invention is applied to textile consisting essentially of polyester with low crystallinity, i.e., pure polyester textile (100%polyester) , such as pure PET textile. In another embodiment, the process of the invention is applied to polyester blends.

In a preferred embodiment, the polyester blends are a blend of a polyester with natural fibers or a blend of a polyester with man-made fibers. In a further embodiment, the natural fibers are selected from the group consisting of cellulosic fibers, animal fibers, and mineral fibers, and man-made fibers are selected from regenerated fibers and synthetic fibers. In a further preferred embodiment, the cellulosic fibers are selected from cotton or hemp; and the animal fibers are selected from wool or silk. In a further preferred embodiment, the regenerated fibers are selected from the group consisting of viscose, rayon, lyocell, modal, triacetate, and diacetate; and the synthetic fibers are selected from the group consisting of polyamides such as nylon 6, nylon 6.6, nylon 11, polyacrylonitrile such as acrylic or modacrylic, and polyurethanes such as Spandex, Lycra, and Elastane.

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.

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 Fig1D, WO 94/03578 Fig 1D, all hereby incorporated by reference) or Magnaporthe grisea (WO10/107560 SEQ ID NO: 1, hereby incorporated by reference) or Pseudomonas mendocina ATCC 53552 (US 5,389,536, claim 1, hereby incorporated by reference) .

In a preferred embodiment, the cutinase is selected from the group consisting of:

(a) a polypeptide having a TM-score of at least 0.60 to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold;

(b) a polypeptide having at least 60%sequence identity to SEQ ID NO: 2 or SEQ ID NO:3 or a mature polypeptide of SEQ ID NO: 3;

(c) a polypeptide derived from SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, by having 1-30 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(d) a polypeptide derived from the polypeptide of (a) , or (b) , wherein the N-and/or C-terminal end has been extended by addition of one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

(e) a fragment of the polypeptide of (a) , (b) , (c) or (d) ;

wherein the polypeptide has cutinase activity.

In some embodiments, the cutinase is a polypeptide having a TM-score of at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold.

In some embodiments, the cutinase variant has at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 2 or a mature polypeptide of SEQ ID NO: 3.

In an embodiment, the cutinase is variant of a parent fungal cutinase, which variant:

a) comprises substitution of at least one amino acid residue corresponding to position A4, T29, A88, N91, A130, Q139, I169, I178 or R189 in the cutinase of Humicola insolens strain DSM 1800 (H. insolens cutinase numbering) , and

b) is more thermostable than the parent cutinase.

In a further embodiment, the cutinase is a variant comprising the substitution A4V, T29M/I/C, A88H/L/V, N91H, A130V, Q139R, I169A/G/T/V, I178V or R189A/H/V in the cutinase of Humicola insolens strain DSM 1800.

In a further embodiment, the cutinase is a variant of a parent fungal cutinase, which variant:

a) comprises substitution of at least one amino acid residue corresponding to Q1C/L, L2K/Q/V, G8D, S11T, N15D, A16T, V38H, S48E/K, H49Y, L66I, S116K, S119P, G120D, T164S, T166M/I or L167P in the cutinase of Humicola insolens strain DSM 1800 (H. insolens cutinase numbering) , and

b) is more thermostable than the parent cutinase.

In a further embodiment, the cutinase is a variant comprising substitutions selected from the group consisting of:

a) S48E +A88H +N91H +R189V

b) Q1L +L2K +G8D +N15D

c) N44D +A130V

d) Q1C +L2V +G120D

e) A88L +R189A

f) S48E +L66I +A88L +I169A +R189H

g) A88V +S116K +S119P +Q139R +I169V +R189V

h) A88V +R189A

i) S48K +A88H +I169G +R189H

j) Q1L +L2Q +A4V +S11T

k) T164S

l) L174F

m) H49Y

n) Q1L +L2K +G8D +N15D +S48E +A88H +N91H +R189V

o) Q1L +L2K +G8D +N15D +N44D +A130V

p) Q1L +L2K +G8D +N15D +S48E +A88H +N91H +A130V +R189V

q) G8D +N15D +A16T

r) A130V

s) Q1C +L2V

t) G8D +N15D +A16T

u) G8D +N15D +S48E +A88H +N91H +A130V +R189V

v) G8D +N15D +T29M +S48E +A88H +N91H +A130V +R189V

w) G8D +N15D +T29I +S48E +A88H +N91H +A130V +R189V and/or

x) G8D +N15D +T29C +S48E +A88H +N91H +A130V +R189V

y) G8D +N15D +S48E +A88H +N91H +A130V +L174F +I178V +R189V

z) G8D +N15D +S48E +A88H +N91H +A130V +T166M +I168F +R189V

aa) G8D +N15D +S48E +A88H +N91H +A130V +T166I +L167P +R189V

bb) G8D +N15D +V38H +S48E +A88H +N91H +A130V +I169T + R189V

cc) G8D +N15D +V38H +S48E +A88H +N91H +A130V +R189V

dd) G8D +N15D +T29M +S48E +A88H +N91H +A130V +T166I +L167P +R189V.

In a further embodiment, the variant further comprises at least one amino acid substitution at positions corresponding to Q1, L2, E6, E10, S11, A14, N15, F24, L46, E47, R51, D63, L138 and/or E179 (H. insolens cutinase numbering) .

In a further embodiment, the variant further comprises at least one substitution corresponding to Q1P, L2V, E6Q, E10Q, S11C, A14P, N15T, F24Y, L46I, E47K, R51P, D63N, L138I and/or E179Q (H. insolens cutinase numbering) .

In a further embodiment, the variant further comprises substitutions corresponding to E6Q +A14P +E47K +R51P +E179Q.

In a preferred embodiment, the cutinase is a cutinase disclosed in WO 2001/092502, which is hereby incorporated by reference. It is a variant derived from the wild-type cutinase/lipase of Humicola insolens DSM 1800 (SEQ ID NO: 1 in WO 2001/092502 and SEQ ID NO: 2 herein) , which comprises the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q and R189V.

In a further embodiment, the cutinase is a variant with cutinase activity of a parent cutinase, comprising an alteration at one or more (e.g., several) positions corresponding to positions: 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO: 3 herein, wherein the alteration is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1, 2 and 182, and wherein the variant has at least 75%, but less than 100%sequence identity to the mature polypeptide of SEQ ID NO: 3 herein.

The amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3. As SEQ ID NO: 3 has signal-peptide and pro-peptide from amino acid residues 1-35, the amino acid position 1 begins with amino acid residue 36 of SEQ ID NO: 3. For example, 181 corresponds to 216; and 182 corresponds to 217 in SEQ ID NO: 3.

In a further embodiment, the cutinase comprises one or more (e.g., several) alterations selected from the group consisting of R181P, G182*, V115I, A161L, Q1*, L2*, A43C, I55C, N79A, and I5V.

In a further embodiment, the cutinase is a variant comprising or consisting of alterations selected from the group consisting of:

a. V115I +R181P +G182*

b. A161L +R181P +G182*

c. Q1*+L2*+I5V +A43C +I55C +N79A +V115I +R181P +G182*

d. Q1*+L2*+A43C +I55C +N79A +V115I +R181P +G182*

e. I5V +A43C +I55C +N79A +V115I

f. A43C +I55C +N79A

g. I5V +A43C +I55C +N79A +V115I +R181P +G182*

h. Q1*+L2*

i. I5V

j. A43C +I55C

k. N79A

l. V115I

m. A161L

n. R181P +G182*

wherein the amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3.

In a further embodiment, the cutinase is a variant further comprising an N-terminal extension.

In a further embodiment, the cutinase is a variant comprising the N-terminal extension selected from the group consisting of:

o. AAVDSNHTPAVPELVAR

p. AVDSNHTPAVPELVAR

q. VDSNHTPAVPELVAR

r. DSNHTPAVPELVAR

s. SNHTPAVPELVAR

t. NHTPAVPELVAR

u. HTPAVPELVAR

v. TPAVPELVAR

w. PAVPELVAR

x. AVPELVAR

y. VPELVAR

z. PELVAR

aa. ELVAR

bb. LVAR

cc. VAR

dd. AR

ee. R .

In a preferred embodiment, the cutinase is a cutinase disclosed in WO 2015/085920, which is hereby incorporated by reference. It is a variant with cutinase activity of a parent cutinase having mature polypeptide of SEQ ID NO: 2 in WO 2015/085920 and mature polypeptide of SEQ ID NO: 3 herein, which comprises the alterations selected from the group consisting of:

A161L +R181P +G182*.

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.

Variant

The term “variant” means a polypeptide having cutinase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%of the cutinase activity of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 3.

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “T226A” . Multiple mutations are separated by addition marks ( “+” ) or by commas, e.g., “G205R, S411 F” or “G205R + S411 F” , representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) , respectively. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted may be indicated with X, e.g., X226A.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of the amino acid at position 195 is designated as “X195*” . Multiple deletions are separated by addition marks ( “+” ) or by commas, e.g., “X195*+X411*” or “X195*, X411*” .

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after the amino acid at position 195 is designated “X195XK” . An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc. ] . For example, the insertion of lysine and alanine after the amino acid at position 195 is indicated as “X195XKA” .

In such cases, the inserted amino acid residue (s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue (s) . In the above example, the sequence would thus be:

Alternatively, an insertion of an amino acid residue such as lysine after the amino acid at position 195 may be indicated by “195aK” , and the insertion of two or more additional amino acid residues such as lysine and alanine after the amino acid at position 195 may be indicated by “195aK, 195bA” .

Preparation of Variants

The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

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; US 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 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.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

AlphaFold structure prediction

AlphaFold 2 is a computational method for calculating the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., 2021, Nature 596: 583-589) . Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 algorithm (Varadi et al., 2021, Nucleic Acids Res. 50 (D1) : D439-D444) . In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and predicting structures of new polypeptides at source code repositories such as Github. com under deepmind/alphafold/, using notebooks/AlphaFold. ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github. com under sokrypton/ColabFold using v1.5.2 or newer, using AlphaFold2. ipynb. For technical details, please see Jumper et al. (vide supra) .

AlphaFold 2 produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the lDDT-Cα metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better) . Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites) . Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction.

Structural Similarity

For purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity” .

A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g., X-ray crystallography or using in silico methods such as AlphaFold 2 (vide supra) . The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang &Skolnick, 2004, Proteins 57: 702–710) :

where L_N is the length of the native structure, L_T is the length of the aligned residues to the template structure, d_i is the distance between pair i of aligned residues and d₀ is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.

For the purposes of the present invention, L_N is the length of the reference polypeptide:

A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, 1998, Protein Eng., 11: 739-747) , DALI (Holm and Sander, 1995, Trends Biochem. Sci., 20:478-480) , or TM-align (Zhang and Skolnick, 2005, Nucleic Acids Res. 33 (7) : 2302-2309) .

For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website (zhanggroup. org/TM-score/) . The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:

TMalign <query. pdb> <reference. pdb> -L <length of reference>

where <query. pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference. pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

The maximal TM-score is 1, e.g., 1.0, corresponding to identical three-dimensional structures.

Sequence Identity

The relatedness between two amino acid sequences is described by the parameter “sequence identity” .

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” 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 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100) / (Length of Alignment –Total Number of Gaps in Alignment) .

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and/or C-terminal processing (e.g., removal of signal peptide) . In one aspect, the mature polypeptide is amino acids 22 to 294 of SEQ ID NO: 1, based on SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 21 of SEQ ID NO: 1 are a signal peptide. It is further confirmed by the N-terminal sequencing, showing mature peptide begins with ASGSGK, which is consistent with the prediction that amino acids 1 to 21 of SEQ ID NO: 1 are a signal peptide. In another aspect, the mature polypeptide is amino acids 36 to 229 of SEQ ID NO: 3, amino acids 1 to 23 of SEQ ID NO: 3 are a signal-peptide, and amino acids 24 to 35 of SEQ ID NO: 3 are a pro-peptide.

Biopolishing effect

As used herein, the term “biopolishing” , “depilling” , and “anti-pilling” are interchangeable.

Most polyester fabrics and polyester blend fabrics have a handle appearance that is rather hard and stiff without the application of finishing components. The fabric surface also 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.

In one aspect, the preset invention provides a method of biopolishing a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, for example crystallinity of less than 38%.

In the present invention, biopolishing is a method of treating textile comprising a polyester with low crystallinity by an enzyme such as a cutinase, which improves fabric quality with respect to “reduced pilling formation” . The most important effects of biopolishing can be characterized by less fuzz and anti-pilling, and improved fabric handle. Biopolishing usually takes place in the wet processing of the manufacture of knitted or woven fabrics or garments. Wet processing comprises such steps as e.g., desizing, scouring, bleaching, washing and finishing. Biopolishing could be performed as a separate step after any of the wetting steps or in combination with any of those wetting steps, such as in combination with catalase bleaching step and/or in combination with dyeing step.

In a preferred embodiment, the treatment of textile comprising a polyester with low crystallinity by an enzyme such as a cutinase is carried out during the manufacture of the textile. Biopolishing normally is combined with mechanical force. For example, it can be applied in overflow/airflow dyeing machine, dye jigger, sand washing machine, or other machines in textile wet process.

A balance between biopolishing performance and weight loss should be considered in mill applications. In a preferred embodiment, weight loss%of the biopolished textile is 0.01-5, preferably 0.05-4, more preferably 1-3.

Desizing effect

Polyester (such as PET based size) is widely used for yarns sizing, it enables the yarns to a right strength for weaving. Desizing is a necessary step for woven dyeing and finishing. The fabrics become hydrophilic after desizing which are easier to be dyed. As used herein, the terms “desize” and “desizing” refer to the process of eliminating/removing size from a textile.

Polyester size can be a type of PET with lower crystallinity than PET fiber. In the present invention, polyester size is a polyester with low crystallinity.

In one aspect, the present invention relates to a method of desizing polyester size of a textile, comprising contacting the polyester size with a cutinase. Cutinases can hydrolyze the PET based size from textile efficiently and replace conventional high temperature and alkaline treatment in a convenient way.

Oligomer removing effect

During synthesis of PET, cyclic or linear oligomers of poly (ethylene terephthalate) , such as terephtalic acid-bis-2-benzoyloxy-ethylesther (BETEB) and/or cyclic tri (ethylene terephthalate) are formed. These oligomers are partly deposited on machinery and partly staying on/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.

In the present invention the PET based oligomers are a polyester with low crystallinity.

In one aspect, the present invention relates to a method of removing polyester oligomer of a textile, comprising contacting the polyester oligomer with a cutinase.

Anti-bacterial effect

The method of present invention results in the antimicrobial efficacy. The term “antimicrobial” means inhibit or reduce the microbe grown on the PET fabric or PET blend fabric (for example, PET/cotton blend) comprising a polyester with low crystallinity. The microbe on the fabric can be bacterial or fungi.

In some embodiments, the method of the present invention inhibits the bacterial growth on the PET fabric or PET blend fabric (for example PET/cotton blend) comprising a polyester with low crystallinity.

A number of test methods have been developed to determine the efficacy of antimicrobial on fabric. The bacterial species Klebsiella Pneumoniae is recommended in most test methods. The species is potentially pathogenic and therefore requires proper physical containment facilities for handling (e.g., a biosafety cabinet) . Many studies have used the innocuous Escherichia coli as a test microorganism which can be cultured and handled in a standard laboratory with minimal health risk.

For the purpose of the present invention, the antimicrobial efficacy is measured according to any one of the standard test method ASTM E 2149-01 (American Society for Testing and Materials) , testing Method AATCC 100-2004 (American Association of Textile Chemists and Colorists) , and Agar plate test method.

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 set at 180℃ and then be pretreated with surfactants (sometimes also with addition of alkali) at 80-100℃ and then optionally followed by the weight reduction process by using severe alkali at up to 130℃ to hydrolyze polyester fabric to make it more soft and luster appearance. Then the polyester fabric will be heat set and dyed with disperse dyestuffs at pH 4.5-6 at up to 130℃, followed by reduction clearing with sodium hyposulphite at 60-80℃, pH 10. If necessary, these processes can be followed by finishing (post treatment) steps to further improve the textile properties, such as anti-pilling or fuzz score improvement. For PET/cotton blend fabric, there will be a soaping step after finishing.

In a preferred embodiment, the method of the present invention takes place in an aqueous solution during one or more of the subsequent steps of pretreatment, weight reduction, disperse dyeing, post finishing and soaping step. 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.

In some embodiments, it has been optimized to treat the textile comprising a polyester with low crystallinity by cutinase integrated into the finishing and dyeing process of the textile and maintain the highest performance. Cellulase and cutinase can be combined in a dyeing process; cellulase and cutinase can be combined in one bath of soaping step; separately applied with cellulase treatment combined in dyeing process, cutinase treatment in one bath of soaping or after soaping.

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 jet 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.

In another embodiment, the method of treating polyester textile comprising a polyester with low crystallinity is manufacturing the polyester textile, especially manufacturing the polyester fabric.

In another embodiment, the method of the present invention is in combination with any of the existing polyester fabric manufacturing steps.

Process condition

Cutinase can be used during the manufacturing process for textile comprising a polyester with low crystallinity, 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, post finishing or soaping.

In one embodiment, the textile is contacted with the cutinase in an aqueous solution.

It is advised that a suitable liquor/textile ratio to be used in the present method may be in the range of from about 100: 1 to about 1: 1, preferably in the range of from about 80: 1 to about 3:1, more preferably in the range of from 60: 1 to 5: 1 (Volumn/weight, ml/g) .

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 minutes 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 11, 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℃. Preferably the process is able to function at a temperature below 100℃, preferably below 90℃, more preferably below 80℃, and even more preferably below 75℃.

In some embodiments, the process of the present invention is conducted at the temperature range of 40-100℃, preferably 50-90℃, preferably 60-88℃, more preferably 65-85℃, and even more preferably 70-85℃.

Enzyme dosage greatly depends on the enzyme reaction time, i.e., a relatively short enzymatic 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 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 0.5-10 milligram of enzyme protein per gram of polyester textile.

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. Alkali solution dissolves linear fragments of the oligomers, and may to some extent further hydrolyze these linear fragments.

In another embodiment, the method of the present invention further comprises contacting the textile with one or more enzymes selected from the group consisting of cellulases, amylases, proteases, lipases, esterase, laccase, peroxidase, peroxygenase and transferase.

In another embodiment, the method of the present invention comprises treatment of the textile comprising a polyester with low crystallinity by a cutinase for 1-20, 1-15, 1-10, or 1-5, for example, 1, 2, 3, 4, or 5 cycles. In the present invention, the treatment effect is improved when the textile comprising a polyester with low crystallinity is treated with a cutinase for one cycle. The property of the textile comprising a polyester with low crystallinity is further improved when treatment cycles are further increased.

Composition for treating textile

In one aspect, the present invention relates to a composition suitable for treating textile where the composition comprises a cutinase.

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 some embodiments of the invention, the composition containing 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 cellulases, amylases, proteases, lipases, esterase, laccase, peroxidase, peroxygenase and transferase.

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) , alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol 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 (glucamides, 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 cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxygenase and transferase.

Cellulase:

In the present context, the term “cellulase” or “cellulolytic enzyme” refers to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and other cellooligosaccharides. Cellulose is a polymer of glucose linked by beta-1, 4-glucosidic bonds. Cellulose chains form numerous intra-and intermolecular hydrogen bonds, which result in the formation of insoluble cellulose microfibrils. Microbial hydrolysis of cellulose to glucose involves the following three major classes of cellulases: endo-1, 4-beta-glucanases (EC 3.2.1.4) , which cleave beta-1, 4-glucosidic links randomly throughout cellulose molecules; cellobiohydrolases (EC 3.2.1.91) (exoglucanases) , which digest cellulose from the nonreducing end; and beta-glucosidases (EC 3.2.1.21) , which hydrolyse cellobiose and low-molecular-mass cellodextrins to release glucose. Most cellulases consist of a cellulose-binding domain (CBD) and a catalytic domain (CAD) separated by a linker rich in proline and hydroxy amino acid residues. In the specification and claims, the term “endoglucanase” is intended to denote enzymes with cellulolytic activity, especially endo-1, 4-beta-glucanase activity, which are classified in EC 3.2.1.4 according to the Enzyme Nomenclature (1992) and are capable of catalysing (endo) hydrolysis of 1, 4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans including 1, 4-linkages in beta-D-glucans also containing 1, 3-linkages. Any cellulase suitable for use in alkaline solutions can be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens. Especially suitable cellulases are the cellulases having colour care benefits. Examples of such cellulases are cellulases described in European patent application No. 0 495 257, WO 91/17243 and WO 96/29397.

Commercially available cellulases include Celluzyme^TM and Denimax^TM produced by a strain of Humicola insolens, (Novo Nordisk A/S) , and KAC-500 (B) ^TM (Kao Corporation) .

Cellulases are normally incorporated in the composition at a level of from 0.00001%to 2%of enzyme protein by weight of the composition, preferably at a level of from 0.0001%to 1%of enzyme protein by weight of the composition, more preferably at a level of from 0.001%to 0.5%of enzyme protein by weight of the composition, even more preferably at a level of from 0.01%to 0.2%of enzyme protein by weight of the composition.

Amylase:

Any amylase (α and/or β) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, α-amylases obtained from a special strain of B. licheniformis, described in more detail in GB 1, 296, 839. Commercially available amylases are Duramyl^TM, Termamyl^TM, Fungamyl^TM and BAN^TM (available from Novo Nordisk A/S) and Rapidase^TM and Maxamyl P^TM (available from Genencor) .

The amylases are normally incorporated in the composition at a level of from 0.00001%to 2%of enzyme protein by weight of the composition, preferably at a level of from 0.0001%to 1%of enzyme protein by weight of the composition, more preferably at a level of from 0.001%to 0.5%of enzyme protein by weight of the composition, even more preferably at a level of from 0.01%to 0.2%of enzyme protein by weight of the composition.

Protease:

Polypeptides having protease activity, or proteases, are sometimes also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Proteases may be of the exo-type that hydrolyses peptides starting at either end thereof, or of the endo-type that act internally in polypeptide chains (endopeptidases) . Endopeptidases show activity on N-and C-terminally blocked peptide substrates that are relevant for the specificity of the protease in question.

Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.

Examples of useful proteases are the variants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263, WO11/036264, or WO 2019/042306.

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 Thermomyces, 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, 1131: 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^TM, Lipolase Ultra^TM, and Lipex^TM; Lecitase^TM, Lipolex^TM; Lipoclean^TM, Lipoprime^TM (Novozymes A/S) .

Esterases:

An “esterase” , also referred to as a carboxylic ester hydrolases, refers to enzymes acting on ester bonds, and includes enzymes classified in EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (available at http: //www, chem, qmw. ac. uk/iubmb/enzyme or from Enzyme Nomenclature 1992, Academic Press, San Diego, California, with Supplement 1 (1993) , Supplement 2 (1994) , Supplement 3 (1995) , Supplement 4 (1997) and Supplement 5, in Eur. J. Biochem. 1994, 223, 1-5; Eur. J, Biochem. 1995, 232, 1-6; Eur. J. Bio-chem. 1996, 237, 1-5; Eur. J, Biochem. 1997, 250; 1-6, and Eur. J, Biochem, 1999, 264, 610-650: respectively) . Non-limiting examples of esterases include arylesterase, triacylglycerol lipase, acetylesterase, acetylcholinesterase, cholinesterase, tropinesterase, pectinesterase, sterol esterase, chlorophyllase, L-arabinonolactonase, gluconolactonase, uronolactonase, tannase, retinyl-palmitate esterase, hydroxybutyrate-dimer hydrolase, acylglycerol lipase, 3-oxoadipate enol-lactonase, 1, 4-lactonase, galactolipase, 4-pyridoxolactonase, acylcarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinonolactonase, 6-phosphogluconolactonase, phospholipase A1, 6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoumarin lipase, limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase, actinomycin lactonase, orsellinate-depside hydrolase, cephalosporin-C deacetylase, chlorogenate hydrolase, alpha-amino-acid esterase, 4-methyloxaloacetate esterase, carboxymethylenebutenolidase, deoxylimonate A-ring-lactonase, 2-acetyl-1-alkylglycerophosphocholine esterase, fusarinine-C ornithinesterase, sinapine esterase, wax-ester hydrolase, phorboldiester hydrolase, phosphatidylinositol deacylase, sialate O-acetylesterase, acetoxybutynylbithiophene deacetylase, acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase, 2-pyrone-4, 6-dicarboxylate lactonase, N-acetylgalactosaminoglycan deacetylase, juvenile-hormone esterase, bis (2-ethylhexyl) phthalate esterase, protein-glutamate methylesterase, 11-cis-retinyl-palmitate hy-drolase, all-trans-retinyl-palmitate hydrolase, L-rhamnono-1, 4-lactonase, 5- (3, 4-diacetoxybut-1 -ynyl) -2, 2'-bithiophene deacetylase, fatty-acylethyl-ester synthase, xylono-1, 4-lactonase, N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxate benzyles-terase, acetylalkylglycerol acetyl hydrolase, and acetylxylan esterase.

Preferred esterases for use in the present invention are lipolytic enzymes, such as, lipases (as classified by EC 3.1.1.3, EC 3.1.1.23 and/or EC 3.1.1.26) , phospholipases (as classified by EC 3.1.1.4 and/or EC 3.1.1.32, including lysophospholipases as classified by EC 3.1.1.5) , and cutinases classified as EC 3, 11.74.

An esterase enzyme may in an embodiment of the invention be dosed in the range from 7.5-25 LU per dry gram of substrate.

Laccases:

The term “laccase” means a benzenediol: oxygen oxidoreductase (E. C. 1.10.3.2) that catalyzes the following reaction: 1, 2-or 1, 4-benzenediol + O₂ = 1, 2-or 1, 4-benzosemiquinone +2 H₂O.

Laccase activity can be determined by measuring the oxidation of syringaldazine (4, 4′-[azinobis (methanylylidene) ] bis (2, 6-dimethoxyphenol) ) to the corresponding quinone 4, 4′-[azobis (methanylylidene] ) bis (2, 6-dimethoxycyclohexa-2, 5-dien-1-one) .

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^TM (Novozymes A/S) .

Peroxygenase:

The term “peroxygenase” means an “unspecific peroxygenase” activity according to EC 1.11.2.1, that catalyzes insertion of an oxygen atom from H₂O₂ into a variety of substrates, such as nitrobenzodioxole. Examples of useful peroxygenase include peroxygenase described in WO 2008/119780.

Transferases:

Preferred transferases are transferases in any of the following sub-classes:

a) Transferases transferring one-carbon groups (EC 2.1) ;

b) Transferases transferring aldehyde or ketone residues (EC 2.2) ; acyltransferases (EC 2.3) ;

c) Glycosyltransferases (EC 2.4) ;

d) Transferases transferring alkyl or aryl groups, other than methyl groups (EC 2.5) ; and

e) Transferases transferring nitrogenous groups (EC 2.6) .

A preferred type of transferase in the context of the invention is a transglutaminase (protein-glutamine γ-glutamyltransferase; EC 2.3.2.13) .

Further examples of suitable transglutaminases are described in WO 96/06931 (Novo Nordisk A/S) .

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

Materials &Methods

Materials

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Enzymes

Cellulase: a mature polypeptide of endoglucanase from Sordaria fimicola which is shown as SEQ ID NO: 2 in WO 2014/026630 and SEQ ID NO: 1 herein.

Cutinase A: a cutinase disclosed in WO 2001/092502. It is a variant derived from the wild-type cutinase/lipase of Humicola insolens DSM 1800 (SEQ ID NO: 1 in WO 2001/092502 and SEQ ID NO: 2 herein) , which comprises the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q and R189V.

Cutinase B: a cutinase disclosed in WO 2015/085920. It is a variant with cutinase activity of a parent cutinase having mature polypeptide of SEQ ID NO: 2 in WO 2015/085920 and mature polypeptide of SEQ ID NO: 3 herein, which comprises the alterations selected from the group consisting of:

A161L +R181P +G182*.

The amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3. As SEQ ID NO: 3 has signal-peptide and pro-peptide from amino acid residues 1-35, the amino acid position 1 begins with amino acid residue 36 of SEQ ID NO: 3. For example, A161 corresponds to A196; R181 corresponds to R216; and G182 corresponds to G217 in SEQ ID NO: 3.

Method

Protein content

The enzyme protein was measured with BCA^TM Protein Assay Kit (product number 23225, commercially available from Thermo Fisher Scientific Inc., USA) according to the product manual. Below table showed the protein content of enzymes tested by BCA^TM Protein Assay Kit.

Weight loss%determination

The swatches were placed in the conditioned room (65%humidity, 21℃) for 24 hours before they were numbered, weighed by the analytical balance (for samples below 100 g) recorded. After treatment, all samples were tumbled dried (AEG, LAVATHERM 37700, Germany) for 1 hour and conditioned for 24 hours in the conditioned room mentioned as above. For each sample, the weight loss was defined as below:

Pilling Note test

Swatches including treated and untreated which had been pre-conditioned in norm climate (65%humidity, 21℃) 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, three separate readings were carried out by different persons for each sample, and the average of the three readings was adopted as the final result of pilling notes.

Fuzz determination

The target swatches were cut into rectangular pieces with about 13 cm tall and 13 cm long and placed in norm climate (65%humidity, 21℃) for at least 24 hours before measurement. Fuzz score of the target swatch was read by PillGrade^TM (Automated 3D Pilling &Fuzz Grading System, SDL ATLAS, LineTech Industries, Inc., USA) , and this machine is grading in compliance with both ASTM and ISO test methods. The height and the amount of fuzz can be measured by scanning the horizon of the fabric, meanwhile, the patterns and weave/knit structure of the fabric is disregarded. To make the test result more reliable, all four directions of the square fabric were measured and the average of the 4 values was recorded as the Fuzz score about this piece of fabric. The lower the Fuzz score, the more effective the biopolishing treatment.

Differential Scanning Calorimetry (DSC) test (GB/T 40271-2021)

Differential Scanning Calorimeter 25 (DSC25, TA Instrument Waters^TM Inc, USA) was used to determine the percentage crystallinity of the PET fibers. The nitrogen flow rate was 50 mL/min. The heating rate was 10 ℃ /min. Fabric or fiber was cut into 2 mm or 3 mm long. PET fibers (3-5mg) were placed in the aluminium Tzero pans with a Tzero solid sample lid. Detail process was as below:

Samples were first heated from 40℃ to 300℃ at 10℃ /min, held at 300℃ for 1 min in a DSC25. The percentage crystallinity was determined on the first heating scan using the enthalpies of melting and cold crystallization. The following equation was used to calculate percentage crystallinity within the PET fiber:

Where ΔH_m was the enthalpies of melting (J g^-1) , ΔH_cc was the enthalpy of cold recrystallization (J g^-1) , and ΔH_m*was the enthalpy of melting for a 100%crystalline PET sample, which was 140.1 J g^-1. ΔH_m and ΔH_cc were measured by integrating from 60-70℃ to roughly 275℃ with a linear baseline. The percentage crystallinity was calculated using the TRIOS software package provided with DSC25.

In the heating process, some materials would appear cold recrystallization which had not crystallization entirely in room temperature. The recrystallization peak often appeared at 150℃. For some materials recrystallization could not happen, so the ΔH_cc may be equal to 0 J g^-1.

Example 1. Biopolishing performance with a cutinase on PET/spandex fabric

The PET/spandex fabric (containing about 13%TiO₂, 95%PET &5%spandex, Siro spinning yarn) was used, and de-oil was needed for raw fabric. The de-oil program in Wascator (Electrolux FOM71CLS, Sweden) was as below.

The de-oiled fabrics were tumble-dried and conditioned for 24 hours at 65%relative humidity, 21℃ prior to next step.

Then, the de-oiled PET/spandex fabric was bio-polished with cutinase B or cutinase A in Wascator for 1 time or more times under certain dosage, temperature, pH, time, liquid to fabric ratio (LR) and surfactant. The bio-polishing (BP) program in Wascator was as below.

The bio-polished fabrics were tumble-dried and conditioned for 24 hours at 65%relative humidity, 21℃ prior to evaluate fuzz score and pilling notes.

As summarized in Table 1, cutinases, including cutinase B and cutinase A, delivered a good biopolishing performance on PET/spandex fabric (containing about 13%TiO₂, Siro spinning yarn) , which was indicated by a significant improvement on both fuzz score and pilling notes. The improvement level was more obvious along with the washing cycle increased from 1 to 3 times. Compared with cutinase A, cutinase B was more efficient on PET biopolishing application, the BP performance resulted by cutinase B for 1 washing cycle was almost close to the level got from cutinase A by 3 washing cycles.

Table 1. Biopolishing of PET/Spandex knits in Wascator

Example 2. Dosage response on bio-polishing performance in Laundry-O-Meter with a cutinase

The PET/spandex fabric (containing about 13%TiO₂, Siro spinning yarn) after de-oiled was used, the de-oil program was the same as that in Example 1.

Biopolishing was carried out in a Launder-O-Meter (LOM_M228AA, SDL ATLAS, LineTech Industries, Inc., USA) . The PET/spandex fabrics were cut into rectangular pieces with 15 cm tall and 15 cm long and weight about 4-5 g. The fabrics were placed in the conditioned room (65%relative humidity, 21oC) for 24 hours before they were numbered, weighed by the analytical balance and recorded. Two conditioned pieces were placed in each beaker. For each beaker, 20 steel balls (about 220g in total) were used to supply the mechanical aids. Then the pH 8.0 buffer (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L de-ionized water) containing 0.2%Triton X-100 was used here. The buffer and the cutinase A or cutinase B were added according to Table 2, based on the calculation of actual fabric weights, with a liquid to fabric ratio of 10: 1 (v/w) .

The LOM machine was started after the required program was chosen, and it would be hold when the temperature reached 60℃ or 80℃. Each beaker was fitted with a lid lined with 2 neoprene 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 then the washing program was continued, and the timing was initiated. After 1.5 hours the fabrics were rinsed in cold water for 3 times. The fabrics were tumble-dried (AEG, LAVATHERM 37700, Germany) for 1 hour, and then the samples were conditioned for 24 hours at 21 ℃, 65%relative humidity prior to evaluation. fuzz score, pilling note and weight loss%of the fabric was evaluated.

Table 2 showed that increasing the dosage of cutinase B and cutinase A resulted in improved bio-polishing properties, including fuzz score, pilling notes and weight loss%relatively. The improvement level brought by dosage increasing was limited. For example, there was only 0.25 grades up even for quadruple dosage. Increasing the washing cycle as listed in Table 1 was more efficient in improving biopolishing than increasing the enzyme dosage as listed in table 2.

Table 2. Biopolishing of PET/Spandex fabric in Launder-O-Meter, pH 8 for 90min.

Example 3. The biopolishing performance of a cutinase on recycled PET (rPET) fabrics and the fabrics containing rPET

Eight different rPET fabrics was collected from Chinese market, the information was listed in below table. 100%virgin PET fabric was selected as reference and noted with Fabric 0.

De-oil for fabric 0, 1, 2, 3, 4, 5, 6, 7 or 8 was conducted in Wascator, and the program was the same as that in Example 1. Then, fabric 1, 2, 3 and 4 were pretreated by a cellulase in Wascator to remove the fuzz caused by cellulosic fibers, the treatment program was as below.

The pre-treated fabrics were tumble-dried and conditioned for 24 hours at 65%relative humidity, 21℃ prior to evaluate fuzz score and pilling notes.

Cutinase B treatment in Wascator was followed for all the fabrics, and the progress refed to Example 1. The dosage of cutinase B was 3g/L for 1 cycle, and the BP cycles with cutinase B were showed in Table 3.

As summarized in Table 3, cutinase B showed consistent outstanding BP performance on rPET fabrics and the fabrics containing rPET, including kints and woven, which indicated by the significant drop on fuzz scores after cutinase treatment and the final pilling notes were above 4 for all fabrics, meaning the fabrics were almost no pills after abrased by Nu-Martindale Tester for 2000r. The commonality of cutinase for biopolishing application on rPET fabrics was validated.

Table 3. BP performance on 9 different PET fabrics in Wascator

Example 4. The weight loss of PET fabrics after bio-polishing by cutinase

Three different rPET fabrics were used, which were the same as fabric 6, 7 and 8 in Example 3.

De-oil for fabric 6, fabric 7 and fabric 8 was conducted in Wascator, and the program was the same as that in Example 1. Then, each fabrics was treated by cutinase B in a Launder-O-Meter (LOM) , and the program was the same as that in Example 2. The dosage of cutinase B was added according to Table 4, based on the calculation of actual fabric weights, with a liquid to fabric ratio of 10: 1 (v/w) . The BP cycle was also run according to Table 4.

As showed in Table 4, the weight loss%was increased with the increase of the bio-polishing performance. The weight loss%of the three pure rPET fabrics was less than 4 when an outstanding bio-polishing performance was achieved, which was indicated by a significant improvement on fuzz score and the best score on pilling notes (pilling notes of 5) . At the same time, the fabrics after biopolishing treatment were still strong enough to be used in the following processing. A balance between bio-polishing performance and weight loss should be considered in mill applications. For example, bio-polishing of fabric 6, 7 and 8 with 0.5-1g/L cutinase B for 2 cycles was good enough with a good pilling note (>3) and an acceptable weight loss% (around 1-2) .

Table 4. Weight loss and BP performance in LOM

Example 5. PET crystallinity test by DSC 25

The crystallinity of virgin polyester fabric/fiber and recycled polyester fabric/fiber was tested according to standard test method of GB/T 40271-2021 (described in method part) and the results were shown in Table 5.

Table 5 showed that the crystallinity of recycled polyester was lower than that of virgin polyester. The crystallinity of recycled PET fabric/fiber was from 25%to 29%, while the crystallinity of virgin PET fabric/fiber was from 39%to 43%. As combined with the results in example 3, cutinase was more suitable for rPET treatment.

When virgin PET was deeply modified by TiO₂, it also obtained good BP performance as described in Table 1 and 2 with a crystallinity of about 32%. All the results showed that the crystallinity of PET was highly related to BP performance. Not wishing to be bound by any theory, it is believed that the cutinase is easier to get to the hydrolysis site of the PET fiber with lower crystallinity. The PET fabric/fiber with crystallinity below 33%was easy to be treated by cutinase.

Table 5. Various types of PET’s crystallinity

Example 6. Polyester size desizing with Cutinase

A woven fabric sized with polyester during weaving was used. Exhausting desizing was carried out in Lab-O-Mat (Mathis LABOMAT, Typ-Nr. BFA24 201205, Switzerland) , while pad-batch desizing was padded with Padder (Typ-Nr. VFM41497, Mathis U. S. A. Inc. Switzerland) .

Exhausting desizing. The woven fabric sized with polyester was cut into rectangular pieces with 15.5 cm tall and 17.5 cm long and weight about 10 g. The fabrics were placed in the conditioned room (65%relative humidity, 21 oC) for 24 hours before they were numbered. One conditioned piece was placed in each beaker. Then 100mL pH 8.0 buffer (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L de-ionized water) was added to each beaker containing 0.2%Triton X-100 . The cutinase B was added according to Table 6. The beakers were loaded into the Lab-O-Mat machine symmetrically, then the Lab-O-Mat was started after the required program was chosen, and it would be hold when the temperature reached to 80℃. After 10 min the fabrics were rinsed in hot water (95oC) for 2 times, rinsed in cold water (room temperature) for 2 times, spined and laid flat to dry.

Pad-batch desizing. The woven fabric sized with polyester was cut into rectangular pieces with 15.5 cm tall and 20.0 cm long and weight about 13 g. The fabrics were placed in the conditioned room (65%relative humidity, 21oC) for 24 hours before they were numbered. 200mL pH 8.0 buffer (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L de-ionized water) was added to each beaker containing 0.2%Triton X-100. The cutinase B was added according to Table 6. One conditioned piece was dipped in one beaker for 30 seconds then padded by Padder to maintain the liquid pick-up to 90%. Each fabric was double dipped and double padded before putting into a resealable bag. All the fabrics were incubated in room temperature (20-25 oC) for 20 hours (overnight) . The fabrics were rinsed in hot water (95oC) for 2 times, rinsed in cold water (room temp. ) for 2 times, spined and laid flat to dry.

The residual PET size on fabric was combined with methylene blue at certain conditions, and it was determined as follows. Fabrics listed in Table 6 were cut into 5cm x 5cm squares and put together into one beaker with 250 mL of methylene blue solution, the concentration of methylene blue was 1g/L. The beaker was loaded into Lab-O-Mat machine, then started after the required program was chosen. It would be hold when the temperature reached to 70℃. After 10min the fabrics were rinsed in cold water (room temp. ) for 2 times, spined and laid flat to dry. The fabrics were placed in the conditioned room (65%relative humidity, 21 oC) for 24 hours before the K/S values were measured by Datacolor (Datacolor 500, serial No. 8811258, USA) . The color depth of the dyed fabrics was characterized by K/S value, the higher, the deeper. The raw fabric after dyeing was set as standard.

Table 6 showed that the polyester staples sized on the woven fabrics were significantly removed after treated by cutinase. The color depth of fabrics treated by 1 g/L or 10 g/L cutinase were lighter than raw fabric and the fabrics treated with 0 g/L enzyme following the same process (the smaller the K/S value was, the less residual PET on the fabric) . Cutinase showed great adaptability to temperature and process, both exhausting and pad-batch desizing processes.

Table 6. Treatment of polyester size by a cutinase

The invention is further defined by the following numbered paragraphs:

1. A method for treating a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

2. The method of paragraph 1, wherein the treated textile exhibits at least one property selected from the group consisting of handfeel improvement, anti-pilling, fuzz score improvement, polyester size desizing, oligomer removing, anti-bacteria, relative to an untreated textile and/or wherein weight loss%of the treated textile is 0.01-5, preferably 0.05-4, more preferably 1-3.

3. A method of biopolishing a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

4. The method of paragraph 3, wherein the biopolishing is anti-pilling, and/or fuzz score improvement.

5. A method of desizing polyester size of a textile, comprising contacting the polyester size with a cutinase.

6. A method of removing polyester oligomer of a textile, comprising contacting the polyester oligomer with a cutinase.

7. A method of improving handfeel of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

8. A method of improving anti-bacterial effect of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.

9. The method of any of paragraphs 1-8, wherein the PET has crystallinity of less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0; for example, 0%-38%, 10%-35%, or 20%-30%, preferably 1%-33%, more preferably 1%-29%.

10. The method of paragraph 9, wherein the crystallinity is determined by differential scanning calorimetry, preferably according to GB/T 40271-2021.

11. The method of any of paragraphs 1-10, wherein the polyester is PET, preferably rPET or modified PET, preferably modified by TiO₂.

12. The method of any of paragraphs 1-11, wherein the textile is fiber, yarn, fabric or garment.

13. The method of any of paragraphs 1-12, wherein the textile is pure polyester textile or polyester blends.

14. The method of paragraph 13, wherein polyester blend comprises more than 5% (w/w) of polyester, in particular more than 10%, more than 15%, more than 20%, more than 30%, more than 35%, more than 50%, more than 65%, more than 90%, or more than 95%of polyester.

15. The method of paragraph 14, wherein the polyester blends are a blend of a polyester with natural fibers or a blend of a polyester with man-made fibers.

16. The method of paragraph 15, wherein the natural fibers are selected from the group consisting of cellulosic fibers, animal fibers, and mineral fibers, and man-made fibers are selected from regenerated fibers and synthetic fibers.

17. The method of paragraph 16, wherein the cellulosic fibers are selected from cotton or hemp; and the animal fibers are selected from wool or silk; or wherein the regenerated fibers are selected from the group consisting of viscose, rayon, lyocell, modal, triacetate, and diacetate; and the synthetic fibers are selected from the group consisting of polyamides such as nylon 6, nylon 6.6, and nylon 11, polyacrylonitrile such as acrylic or modacrylic, and polyurethanes such as Spandex, Lycra, and Elastane.

18. The method of any paragraphs 1-17, wherein the cutinase is selected from the group consisting of:

(b) a polypeptide having at least 60%sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3;

(e) a fragment of the polypeptide of (a) , (b) , (c) or (d) ;

wherein the polypeptide has cutinase activity.

19. The method of paragraph 18, wherein the polypeptide has a TM-score of at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold.

20. The method of paragraph 18, wherein the polypeptide has at least 65%, at least 70%, at least 75%, or at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%sequence identity to SEQ ID NO: 2 or a mature polypeptide of SEQ ID NO: 3.

21. The method of any of paragraphs 1-18, wherein the cutinase is a variant of a parent fungal cutinase, which variant:

b) is more thermostable than the parent cutinase.

22. The method of paragraph 21, wherein the cutinase is a variant comprising the substitution A4V, T29M/I/C, A88H/L/V, N91H, A130V, Q139R, I169A/G/T/V, I178V or R189A/H/V in the cutinase of Humicola insolens strain DSM 1800.

23. The method of any of paragraphs 1-18, wherein the cutinase is a variant of a parent fungal cutinase, which variant:

b) is more thermostable than the parent cutinase.

24. The method of any of paragraphs 1-18, wherein the cutinase is a variant comprising substitutions selected from the group consisting of:

a) S48E +A88H +N91H +R189V

b) Q1L +L2K +G8D +N15D

c) N44D +A130V

d) Q1C +L2V +G120D

e) A88L +R189A

f) S48E +L66I +A88L +I169A +R189H

g) A88V +S116K +S119P +Q139R +I169V +R189V

h) A88V +R189A

i) S48K +A88H +I169G +R189H

j) Q1L +L2Q +A4V +S11T

k) T164S

l) L174F

m) H49Y

n) Q1L +L2K +G8D +N15D +S48E +A88H +N91H +R189V

o) Q1L +L2K +G8D +N15D +N44D +A130V

p) Q1L +L2K +G8D +N15D +S48E +A88H +N91H +A130V +R189V

q) G8D +N15D +A16T

r) A130V

s) Q1C +L2V

t) G8D +N15D +A16T

u) G8D +N15D +S48E +A88H +N91H +A130V +R189V

v) G8D +N15D +T29M +S48E +A88H +N91H +A130V +R189V

w) G8D +N15D +T29I +S48E +A88H +N91H +A130V +R189V and/or

x) G8D +N15D +T29C +S48E +A88H +N91H +A130V +R189V

y) G8D +N15D +S48E +A88H +N91H +A130V +L174F +I178V +R189V

z) G8D +N15D +S48E +A88H +N91H +A130V +T166M +I168F +R189V

aa) G8D +N15D +S48E +A88H +N91H +A130V +T166I +L167P +R189V

bb) G8D +N15D +V38H +S48E +A88H +N91H +A130V +I169T + R189V

cc) G8D +N15D +V38H +S48E +A88H +N91H +A130V +R189V

dd) G8D +N15D +T29M +S48E +A88H +N91H +A130V +T166I +L167P +R189V.

25. The method of any of paragraphs 1-18, wherein the cutinase is a variant further comprising at least one amino acid substitution at positions corresponding to Q1, L2, E6, E10, S11, A14, N15, F24, L46, E47, R51, D63, L138 and/or E179 (H. insolens cutinase numbering) .

26. The method of any of paragraphs 1-18, wherein the cutinase is a variant further comprising at least one substitution corresponding to Q1P, L2V, E6Q, E10Q, S11C, A14P, N15T, F24Y, L46I, E47K, R51P, D63N, L138I and/or E179Q (H. insolens cutinase numbering) .

27. The method of any of paragraphs 1-18, wherein the cutinase is a variant further comprises substitutions corresponding to E6Q +A14P +E47K +R51P +E179Q.

28. The method of any of paragraphs 1-18, wherein the cutinase is a variant with cutinase activity of a parent cutinase, comprising an alteration at one or more (e.g., several) positions corresponding to positions: 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO: 3, wherein the alteration is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1, 2 and 182, and wherein the variant has at least 75%, but less than 100%sequence identity to the mature polypeptide of SEQ ID NO: 3; wherein the amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3.

29. The method of paragraph 28, wherein the cutinase comprises one or more (e.g., several) alterations selected from the group consisting of R181P, G182*, V115I, A161L, Q1*, L2*, A43C, I55C, N79A, and I5V.

30. The method of paragraph 29, wherein the cutinase is a variant comprising or consisting of alterations selected from the group consisting of:

a. V115I +R181P +G182*

b. A161L +R181P +G182*

c. Q1*+L2*+I5V +A43C +I55C +N79A +V115I +R181P +G182*

d. Q1*+L2*+A43C +I55C +N79A +V115I +R181P +G182*

e. I5V +A43C +I55C +N79A +V115I

f. A43C +I55C +N79A

g. I5V +A43C +I55C +N79A +V115I +R181P +G182*

h. Q1*+L2*

i. I5V

j. A43C +I55C

k. N79A

l. V115I

m. A161L

n. R181P +G182*

31. The method of any of paragraphs 1-18, wherein the cutinase is a variant derived from a parent cutinase having the polypeptide of SEQ ID NO: 2, comprising the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q and R189V and wherein the amino acid position number corresponds to amino acid residue of SEQ ID NO: 2; or wherein the cutinase is a variant of a parent cutinase having the mature polypeptide of SEQ ID NO: 3, comprising the alterations: A161L +R181P +G182*, and wherein the amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3.

32. The method of any of paragraphs 1-31, wherein the textile is contacted with the cutinase in an aqueous solution.

33. The method of any of paragraphs 1-32, further comprising contacting the textile with one or more enzymes selected from the group consisting of cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxygenase and transferase.

34. The method of any of paragraphs 1-33, wherein the cellulase is an endoglucanase.

35. The method of paragraph 34, wherein the endoglucanase is selected from the group consisting of:

(a) a polypeptide having a TM-score of at least 0.60 to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold;

(b) a polypeptide having at least 60%sequence identity to SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1;

(c) a polypeptide derived from SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1, by having 1-30 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(e) a fragment of the polypeptide of (a) , (b) , (c) or (d) ;

wherein the polypeptide has endoglucanase activity.

36. The method of paragraph 35, wherein the polypeptide has a TM-score of at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold.

37. The method of paragraph 35, wherein the polypeptide has at least 65%, at least 70%, at least 75%, or at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%sequence identity to SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1.

38. The method of any of the preceding paragraphs, wherein the cutinase is applied in the range of 0.01 to 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 0.5-10 milligram of enzyme protein per gram of polyester textile.

39. 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 11, 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.

40. The method of any of the preceding paragraphs, wherein the method is conducted in the temperature range of 40-100℃, preferably 50-90℃, preferably 60-88℃, more preferably 65-85℃, and even more preferably 70-85℃.

41. 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.

42. The method of any of the preceding paragraphs, wherein the method comprises the treatment of the textile by a cutinase for 1-20, 1-15, 1-10, or 1-5, for example, 1, 2, 3, 4, or 5 cycles.

43. The method of any of the preceding paragraphs, wherein the treating polyester textile is manufacturing the polyester textile, especially manufacturing the polyester fabric.

44. The method of paragraph 43, wherein the method is in combination with any of the existing polyester fabric manufacturing steps.

45. A textile produced according to the method of any of the preceding paragraphs.

Claims

A method for treating a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.
The method of claim 1, wherein the treated textile exhibits at least one property selected from the group consisting of handfeel improvement, anti-pilling, fuzz score improvement, polyester size desizing, oligomer removing, anti-bacteria, relative to an untreated textile and/or wherein weight loss%of the treated textile is 0.01-5, preferably 0.05-4, more preferably 1-3.
A method of biopolishing a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.
The method of claim 3, wherein the biopolishing is anti-pilling, and/or fuzz score improvement.
A method of desizing polyester size of a textile, comprising contacting the polyester size with a cutinase.
A method of removing polyester oligomer of a textile, comprising contacting the polyester oligomer with a cutinase.
A method of improving handfeel of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.
A method of improving anti-bacterial effect of a textile, comprising contacting the textile with a cutinase, wherein the textile comprises a polyester with crystallinity lower than that of virgin polyester, preferably the textile comprises a polyester with crystallinity of less than 38%.
The method of any of claims 1-8, wherein the PET has crystallinity of less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0; for example, 0-38%, 10%-35%, or 20%-30%, preferably 1%-33%, more preferably 1%-29%.
The method of any of claims 1-9, wherein the polyester is PET, preferably rPET or modified PET, preferably modified by TiO₂.
The method of any of claims 1-10, wherein the textile is pure polyester textile or polyester blends.
The method of any claims 1-11, wherein the cutinase is selected from the group consisting of:

(a) a polypeptide having a TM-score of at least 0.60, e.g., at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold;

(b) a polypeptide having at least 60%sequence identity, preferably at least 65%, at least 70%, at least 75%, or at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3;

(c) a polypeptide derived from SEQ ID NO: 2 or SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3, by having 1-30 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(d) a polypeptide derived from the polypeptide of (a) , or (b) , wherein the N-and/or C-terminal end has been extended by addition of one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

(e) a fragment of the polypeptide of (a) , (b) , (c) or (d) ;

wherein the polypeptide has cutinase activity.
The method of any of claims 1-12, wherein the cutinase is a variant derived from a parent cutinase having the polypeptide of SEQ ID NO: 2, comprising the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51 P, A88H, N91 H, A130V, E179Q and R189V and wherein the amino acid position number corresponds to amino acid residue of SEQ ID NO: 2; or wherein the cutinase is a variant of a parent cutinase having the mature polypeptide of SEQ ID NO: 3, comprising the alterations: A161 L +R181 P +G182*, and wherein the amino acid position number corresponds to amino acid residue of mature polypeptide of SEQ ID NO: 3.
The method of any of claims 1-13, further comprising contacting the textile with one or more enzymes selected from the group consisting of cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxygenase and transferase.
The method of claim 14, wherein the cellulase is an endoglucanase, preferably the endoglucanase is selected from the group consisting of:

(a) a polypeptide having a TM-score of at least 0.60, e.g., at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold;

(b) a polypeptide having at least 60%sequence identity, preferably at least 65%, at least 70%, at least 75%, or at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%sequence identity to SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1;

(c) a polypeptide derived from SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1, by having 1-30 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(d) a polypeptide derived from the polypeptide of (a) , or (b) , wherein the N-and/or C-terminal end has been extended by addition of one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

(e) a fragment of the polypeptide of (a) , (b) , (c) or (d) ;

wherein the polypeptide has endoglucanase activity.
A textile produced according to the method of any of the preceding claims.