Method of treating a textile comprising a polyester having low crystallinity

Abstract

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

Description

References to sequence lists

[0001] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for processing textiles comprising polyester having low crystallinity, and textiles produced by such method. Background Technology

[0003] Poly(ethylene terephthalate) (PET) fibers constitute the majority of polyesters used in the textile industry. These fibers are produced through the polycondensation of, for example, terephthalic acid and ethylene glycol, and by stretching fibers from the melt.

[0004] Polyester possesses certain core advantages, including high strength, a soft hand feel, tensile resistance, stain resistance, machine washability, wrinkle resistance, and abrasion resistance. However, polyester is not the best in terms of pilling or fuzzing.

[0005] Due to their strength, polyester fabrics and / or clothing are susceptible to pilling, and arguably, the most important finishing processes applied to polyester materials are those designed to control pilling. All polyester materials, when subjected to minor abrasion during washing and wear, tend to form small balls or "beads" with entangled fibers on the fabric surface. If the fabric contains a significant proportion of fibers highly resistant to flexural abrasion, these beads can remain on the fabric surface in numbers sufficient to produce an unpleasant feel and appearance.

[0006] In recent years, antimicrobial finishing of textiles has become extremely important in the production of protective, decorative, and technical textile products. However, antimicrobial agents adhering to the surface of textiles or incorporated into fibers significantly reduce their activity and limit their usability. In addition, biocides may gradually leach out during the use and washing of textiles. For these reasons, large amounts of these biocides need to be applied to textiles to effectively control bacterial growth and maintain durability (Textile Research Journal, Vol. 78, No. (1): 60–72).

[0007] Another problem with polyester is that existing technologies for desizing polyester slurries are energy-intensive processes that rely on high temperatures and alkali treatments, such as at 100°C with 0.1-5 g / L NaOH. The treatment lasts 30 minutes, followed by neutralization and several rinses.

[0008] Another problem with polyester is the formation of oligomers during PET synthesis. These oligomers tend to give fabrics a light gray appearance. These oligomers can be removed through harsh alkali treatment, resulting in a significant loss of fibrous material. Organic extraction of these oligomers is a technical possibility, but not industrially feasible.

[0009] Among other things, the industry has made great efforts to improve the properties of polyester by applying keratinase to treat it.

[0010] However, in the textile industry, there is always a need to treat polyester textiles to improve their properties. These properties can be selected from the following group: improved hand feel, anti-pilling, improved nap score, polyester sizing desizing, oligomer removal, and antibacterial properties. Summary of the Invention

[0011] It has now been surprisingly discovered that when polyester has low crystallinity, keratinase can consistently and significantly improve the properties of polyester textiles. These properties can be selected from the group consisting of: improved hand feel, anti-pilling, improved fuzz score, polyester sizing desizing, oligomer removal, and antibacterial properties. The method of this invention can be applied to all kinds of textiles containing polyesters with low crystallinity, making the treatment of textiles containing polyesters with low crystallinity industrially applicable.

[0012] Therefore, in one aspect, the present invention relates to a method for treating textiles, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

[0013] In another aspect, the present invention relates to a method for biopolishing textiles, the method comprising contacting the textiles with a keratinase, wherein the textiles comprise a polyester with a crystallinity lower than that of the original polyester, preferably the textiles comprising a polyester with a crystallinity of less than 38%.

[0014] In another aspect, the present invention relates to a method for desizing polyester sizing of textiles, the method comprising contacting the polyester sizing with a keratinase.

[0015] In another aspect, the present invention relates to a method for removing polyester oligomers from textiles, the method comprising contacting the polyester oligomers with a keratinase.

[0016] In another aspect, the present invention relates to a method for improving the hand feel of a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprising a polyester with a crystallinity of less than 38%.

[0017] In another aspect, the present invention relates to a method for improving the antibacterial properties of textiles, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

[0018] In another respect, the present invention relates to a textile produced by a method according to the present invention. Attached Figure Description

[0019] Figure 1 The color depth of the original fabric that was not treated with keratinase is shown.

[0020] Figure 2 The color depth of fabrics undergoing desizing without keratinase treatment is shown.

[0021] Figure 3 The color depth of fabrics undergoing desizing after treatment with 1 g / L keratinase is shown.

[0022] Figure 4 The color depth of fabrics undergoing desizing after treatment with 10 g / L keratinase is shown.

[0023] Figure 5 The color depth of fabrics undergoing desizing in pad-batch processing without keratinase treatment is shown.

[0024] Figure 6 The color depth of fabrics treated with 1 g / L keratinase during pad-batch desizing is shown.

[0025] Figure 7 The color depth of fabrics treated with 10 g / L keratinase during pad-batch desizing is shown. Sequence List Overview

[0026] SEQ ID NO: 1 is the amino acid sequence of an endoglucanase from Sordaria fimicola.

[0027] SEQ ID NO: 2 is the amino acid sequence of wild-type keratinase / lipase of Humicola insolens DSM 1800.

[0028] SEQ ID NO: 3 is the amino acid sequence of the parent keratinase of keratinase B. Detailed Implementation

[0029] The present invention will now be described in detail by reference using the following definitions and examples. All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

[0030] Unless the context clearly indicates otherwise, as used herein, the singular terms “a / an” and “the” include plural references.

[0031] Polyester textiles with low crystallinity

[0032] As used herein, "polyester" refers to a linear polymeric molecule containing lactone groups derived from the condensation of diacids and glycols or the polymerization of hydroxy acids. This invention applies to both aliphatic and aromatic polyesters with low crystallinity. Particularly preferred polyesters with low crystallinity are aromatic polyester articles used to produce fibers and resins, and which comprise a synthetically produced long-chain polymer containing 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 a substituted terephthalic acid or a para-substituted hydroxybenzoic acid ester or a mixture thereof. Other available polyester articles with low crystallinity include those made from bulk polymers, yarns, fabrics, films, resins, and powders. These basic polyesters used in industrial applications include polyethylene terephthalate (PET), tetramethylene terephthalate (PTMT), polybutylene terephthalate (PBT), polypropylene terephthalate (PTT), polyethylene naphthalate (PEN), polycyclohexanedimethyl terephthalate (CHDMT), polyethylene-4-oxybenzoate, A-Tell, polyglycolic acid lactide, PHBA, and 2GN. However, PET is the most commonly produced linear polymer and accounts for the majority of polyesters used in industry today.

[0033] In a preferred embodiment, the polyester textile with low crystallinity is PET with low crystallinity. In another preferred embodiment, low crystallinity means crystallinity 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%.

[0034] In a preferred embodiment, crystallinity is determined by differential scanning calorimetry (DSC). Differential scanning calorimetry is a thermal analysis technique used to measure physical and chemical changes within a material in response to temperature. A differential scanning calorimeter provides qualitative and quantitative information about endothermic (heat flowing into the sample) and exothermic (heat flowing out of the sample) processes or changes in heat capacity. Sample types include films, fibers, powders, solutions, and composites. DSC measures the energy absorbed or released by the sample when it is heated or cooled. DSC also measures the heat flow difference between the sample and a reference. This heat difference is the amount of heat required to melt the sample, which is displayed on the recorder as electrical power.

[0035] 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).

[0036] In another preferred embodiment, the melting peak temperature of polyethylene terephthalate is 250°C-255°C, and the enthalpy of fusion is 37-60 J / g. -1 Indium and tin, metals close to the sample temperature range to be measured, were selected as standard samples to calibrate the DSC instrument. Two empty Tzero aluminum disks were used to test the instrument baseline.

[0037] Encapsulate a 2-3 mm long fiber sample weighing approximately 3-5 mg into an aluminum disk. Set an empty Tzero aluminum disk with a Tzero cap as a reference. Then, heat the sample and reference from 40°C to 300°C at a constant rate of 0.5°C-20°C / min under a nitrogen flow of 20-50 mL / min to protect the internal components of the calorimeter. Record the DSC curves. The area between the baseline and the melting peak is the enthalpy of fusion. Determine the percentage of crystallinity using the enthalpy of fusion and the enthalpy of cold crystallization during the first heating scan. Calculate the percentage of crystallinity within the fiber using the following equation:

[0038] % crystallinity = [( H m - H cc ) / ( H m )] × 100%

[0039] in H m It is the enthalpy of fusion (J g) -1 ), Hcc is the enthalpy of cold recrystallization (J g). -1 ),and Hm This is the enthalpy of melting for a 100% crystallized sample. The percentage of crystallinity is calculated using the TRIOS software package provided with DSC.

[0040] In a preferred embodiment, the polyester is PET, preferably recycled PET (rPET) or modified PET.

[0041] Recycled PET (rPET)

[0042] PET is the world's most closed-loop recycled plastic, requiring fewer resources and generating less carbon emissions compared to virgin polyester fibers. Generally, PET waste undergoes continuous processing to produce recycled PET (rPET). PET waste (mainly bottles) is collected, sorted, bundled, crushed, washed, shredded, melted, and extruded into granules, and then offered for sale. Recycled PET can then be used to produce fabrics for the textile industry or new packaging (such as bottles or blister packs). 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.

[0043] Modified PET

[0044] In this invention, PET is modified by at least one chemical, physical, and / or mechanical transformation method to obtain PET with low crystallinity. In one embodiment, the PET with low crystallinity is titanium-containing PET. In another embodiment, the PET with low crystallinity contains 1%-25%, 5%-20%, or 8%-15% TiO2 by weight.

[0045] On the other hand, the present invention relates to a textile comprising a polyester having low crystallinity, which is produced by the method of the present invention. The textile comprising a polyester having low crystallinity, as used herein, is intended to include fibers, yarns, fabrics, and garments comprising a polyester having low crystallinity. The polyester yarn, fabric, or garment is made of pure polyethylene terephthalate (PET), or a blend of PET fibers and any other materials conventionally used in the manufacture of textiles, such as wool, cotton, viscose fibers, and silk.

[0046] In a preferred embodiment, the textile containing polyester with low crystallinity is a polyester blend containing more than 5% (w / w) of polyester, particularly 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% polyester. In even more preferred embodiments, such textile blends are polyester / cotton blends. In even more preferred embodiments, the method of the invention is applied to textiles composed essentially of polyester with low crystallinity, i.e., pure polyester textiles (100% polyester), such as pure PET textiles. In another embodiment, the method of the invention is applied to polyester blends.

[0047] In a preferred embodiment, the polyester blend is a blend of polyester and natural fibers or a blend of polyester and man-made fibers. In another embodiment, the natural fibers are selected from the group consisting of cellulose fibers, animal fibers, and mineral fibers, and the man-made fibers are selected from regenerated fibers and synthetic fibers. In another preferred embodiment, the cellulose fibers are selected from cotton or hemp; and the animal fibers are selected from wool or silk. In another 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, and nylon 11, polyacrylonitrile such as acrylic or modified polyacrylonitrile, and polyurethanes such as Spandex, Lycra, and elastane.

[0048] Keratinase

[0049] Keratinase is a lipase classified as EC 3.1.1.74 according to enzyme nomenclature. See Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992.

[0050] Keratinases are known to originate from various fungi, such as filamentous fungal keratins, for example, strains from the genera *Humicola*, *Fusarium*, *Magnaporthe*, or *Pseudomonas*, particularly *Humicola*, *F. solani pisi*, *Magnaporthegrisea*, or *Pseudomonas mendocina*, and more particularly *Humicola* strain DSM 1800 (US 5,827,719) or *F. solani pisi* (WO 90 / 09446). Figure 1 ;WO 94 / 14964 Figure 1 D, WO 94 / 03578 Figure 1 D, all of which are hereby incorporated by reference) or Inospora oryzae (WO 10 / 107560 SEQ ID NO: 1, hereby incorporated by reference) or Pseudomonas mendoza ATCC 53552 (US 5,389,536, claim 1, hereby incorporated by reference).

[0051] In a preferred embodiment, the keratinase is selected from the group consisting of:

[0052] (a) A polypeptide having a TM-score of at least 0.60 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold;

[0053] (b) A polypeptide having at least 60% sequence identity with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 3;

[0054] (c) A polypeptide derived from a mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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, particularly substitutions;

[0055] (d) A polypeptide derived from (a) or (b) wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and

[0056] Fragments of polypeptides of (e), (a), (b), (c), or (d);

[0057] This polypeptide possesses keratinase activity.

[0058] In some embodiments, the keratinase 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 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold.

[0059] In some embodiments, the keratinase 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 with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0060] In this embodiment, the keratinase is a variant of the parent fungal keratinase, which is:

[0061] a) Contains a substitution of at least one amino acid residue at position A4, T29, A88, N91, A130, Q139, I169, I178, or R189 in the keratinase corresponding to the specific *Porcine* strain DSM 1800 (specific *Porcine* keratinase number), and

[0062] b) It has greater thermal stability than the parental keratinase.

[0063] In another embodiment, the keratinase is a variant of the keratinase containing the specific *Pythium spp.* strain DSM 1800, which substitutes A4V, T29M / I / C, A88H / L / V, N91H, A130V, Q139R, I169A / G / T / V, I178V, or R189A / H / V.

[0064] In another embodiment, the keratinase is a variant of the parent fungal keratinase, which:

[0065] a) Contains a substitution of at least one amino acid residue (specifically, the *Porcine keratinase* number) in the keratinase corresponding to the specific *Porcine spp.* strain DSM 1800, namely Q1C / L, L2K / Q / V, G8D, S11T, N15D, A16T, V38H, S48E / K, ​​H49Y, L66I, S116K, S119P, G120D, T164S, T166M / I, or L167P.

[0066] b) It is more thermally stable than the parent keratinase.

[0067] In another embodiment, the keratinase is a variant comprising a substitution selected from the group consisting of:

[0068] a)S48E +A88H +N91H +R189V

[0069] b)Q1L +L2K +G8D +N15D

[0070] c) N44D + A130V

[0071] d)Q1C +L2V +G120D

[0072] e)A88L +R189A

[0073] f)S48E +L66I +A88L +I169A +R189H

[0074] g)A88V +S116K +S119P +Q139R +I169V +R189V

[0075] h)A88V +R189A

[0076] i)S48K +A88H +I169G +R189H

[0077] j)Q1L +L2Q +A4V +S11T

[0078] k)T164S

[0079] l)L174F

[0080] m)H49Y

[0081] n)Q1L +L2K +G8D +N15D +S48E +A88H +N91H +R189V

[0082] o)Q1L +L2K +G8D +N15D +N44D +A130V

[0083] p) Q1L + L2K + G8D + N15D + S48E + A88H + N91H + A130V + R189V

[0084] q) G8D + N15D + A16T

[0085] r) A130V

[0086] s) Q1C + L2V

[0087] t) G8D + N15D + A16T

[0088] u) G8D + N15D + S48E + A88H + N91H + A130V + R189V

[0089] v) G8D + N15D + T29M + S48E + A88H + N91H + A130V + R189V

[0090] w) G8D + N15D + T29I + S48E + A88H + N91H + A130V + R189V and / or

[0091] x) G8D + N15D + T29C + S48E + A88H + N91H + A130V + R189V

[0092] y) G8D + N15D + S48E + A88H + N91H + A130V + L174F + I178V + R189V

[0093] z) G8D + N15D + S48E + A88H + N91H + A130V + T166M + I168F + R189V

[0094] aa) G8D + N15D + S48E + A88H + N91H + A130V + T166I + L167P + R189V

[0095] bb) G8D + N15D + V38H + S48E + A88H + N91H + A130V + I169T + R189V

[0096] cc) G8D + N15D + V38H + S48E + A88H + N91H + A130V + R189V

[0097] dd)G8D +N15D +T29M +S48E +A88H +N91H +A130V +T166I +L167P +R189V.

[0098] In another embodiment, the variant further includes at least one amino acid substitution (specific humic mold keratinase number) at positions corresponding to Q1, L2, E6, E10, S11, A14, N15, F24, L46, E47, R51, D63, L138 and / or E179.

[0099] In another embodiment, the variant further comprises at least one substitution (specific humic mold keratinase number) corresponding to Q1P, L2V, E6Q, E10Q, S11C, A14P, N15T, F24Y, L46I, E47K, R51P, D63N, L138I and / or E179Q.

[0100] In another embodiment, the variant further includes a substitution corresponding to E6Q +A14P +E47K +R51P +E179Q.

[0101] In a preferred embodiment, the keratinase is the keratinase disclosed in WO 2001 / 092502 (which is hereby incorporated by reference). This keratinase is a variant of the wild-type keratinase / lipase derived from *Pseudomonas spp.* DSM 1800 (SEQ ID NO: 1 in WO 2001 / 092502 and SEQ ID NO: 2 herein), which contains the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q, and R189V.

[0102] In another embodiment, the keratinase is a variant having the keratinase activity of the parental keratinase, the variant comprising alterations 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 alterations are substitutions for positions 181, 115, 161, 43, 55, 79, and 5, and deletions for positions 1, 2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 3 herein.

[0103] The amino acid position numbers correspond to the amino acid residues of the mature polypeptide in SEQ ID NO: 3. Since SEQ ID NO: 3 has a signal peptide and a propeptide from amino acid residues 1-35, amino acid position 1 begins with amino acid residue 36 of SEQ ID NO: 3. For example, 181 corresponds to 216 in SEQ ID NO: 3; and 182 corresponds to 217 in SEQ ID NO: 3.

[0104] In another embodiment, the keratinase comprises one or more (e.g., several) variations selected from the group consisting of: R181P, G182 V115I, A161L, Q1 L2 A43C, I55C, N79A, and I5V.

[0105] In another embodiment, the keratinase is a variation comprising a group of changes selected from or consisting of these changes:

[0106] a.V115I +R181P +G182

[0107] b.A161L +R181P +G182

[0108] c.Q1 +L2 +I5V +A43C +I55C +N79A +V115I +R181P +G182

[0109] d.Q1 +L2 +A43C +I55C +N79A +V115I +R181P +G182

[0110] e.I5V +A43C +I55C +N79A +V115I

[0111] f.A43C +I55C +N79A

[0112] g.I5V +A43C +I55C +N79A +V115I +R181P +G182

[0113] h.Q1 +L2

[0114] i.I5V

[0115] j.A43C +I55C

[0116] k.N79A

[0117] l.V115I

[0118] m.A161L

[0119] n.R181P +G182

[0120] The amino acid position number corresponds to the amino acid residue of the mature polypeptide in SEQ ID NO: 3.

[0121] In another embodiment, the keratinase is a variant that further includes an N-terminal extension.

[0122] In another embodiment, the keratinase is a variant comprising an N-terminal extension selected from the group consisting of:

[0123] o.AAVDSNHTPAVPELVAR

[0124] p.AVDSNHTPAVPELVAR

[0125] q.VDSNHTPAVPELVAR

[0126] r.DSNHTPAVPELVAR

[0127] s.SNHTPAVPELVAR

[0128] t.NHTPAVPELVAR

[0129] u.HTPAVPELVAR

[0130] v.TPAVPELVAR

[0131] w.PAVPELVAR

[0132] x.AVPELVAR

[0133] y.VPELVAR

[0134] z.PELVAR

[0135] aa.ELVAR

[0136] bb.LVAR

[0137] cc.VAR

[0138] dd.AR

[0139] ee.R.

[0140] In a preferred embodiment, the keratinase is the keratinase disclosed in WO 2015 / 085920 (which is hereby incorporated by reference). This keratinase is a variant having the keratinase activity of a parent keratinase having the mature polypeptide of SEQ ID NO: 2 of WO 2015 / 085920 and the mature polypeptide of SEQ ID NO: 3 herein, the variant comprising changes selected from the group consisting of:

[0141] A161L +R181P +G182 .

[0142] Keratinases can also be variants of the parental keratinase, such as those described in WO 00 / 34450 (which are hereby incorporated by reference).

[0143] Fungal keratinase can also be derived from other fungal strains, such as strains of the genus Rhizoctonia, for example Rhizoctonia solani, or strains of the genus Alternaria, for example Alternaria brassicicola (WO 94 / 03578).

[0144] Preferably, the keratinase has an optimal pH value within one pH unit of the pH of the process; for example, if the process is operated at pH 8, the keratinase preferably has an optimal pH value between 7 and 9.

[0145] variants

[0146] The term "variant" refers to a polypeptide having keratinase activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) sites. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and 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 keratinase activity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0147] In describing variations of the invention, the nomenclature described below has been adjusted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0148] replaceFor amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is represented as "T226A". Multiple mutations are separated by a plus sign ("+") or a comma, for example, "G205R, S411F" or "G205R + S411F" represents the substitution of glycine (G) for arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) for phenylalanine (F). Since the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted can be represented by X, for example, X226A.

[0149] Missing For amino acid deletions, use the following nomenclature: original amino acid, position, Accordingly, the deletion of the amino acid at position 195 is represented as "X195". Multiple missing items are separated by a plus sign ("+") or a comma, for example, "X195". + X411 "or "X195 X411 ".

[0150] insert For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, inserting lysine after the amino acid at position 195 is represented as "X195XK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting lysine and alanine after the amino acid at position 195 is represented as "X195XKA".

[0151] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0152]

[0153] Alternatively, inserting an amino acid residue (such as lysine) after the amino acid at position 195 can be represented as "195aK", and inserting two or more additional amino acid residues (such as lysine and alanine) after the amino acid at position 195 can be represented as "195aK, 195bA".

[0154] Preparation of variants

[0155] 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.

[0156] Site-directed mutagenesis is a technique that introduces one or more mutations at one or more designated sites in a polynucleotide encoding the parent.

[0157] Site-directed mutagenesis in vitro can be achieved through PCR involving the use of primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis in vitro can also be performed via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent, followed by ligation of the mutated oligonucleotide into the polynucleotide. Typically, the restriction enzymes digesting the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0158] In vivo site-directed mutagenesis can also be achieved using methods known in the art. See, for example, 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, FungalGenet. Newslett. 43: 15-16.

[0159] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0160] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode target peptides. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al., 2004, Nature [Nature] 432: 1050-1054, and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0161] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested. These relevant screening procedures include 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).

[0162] Mutagenesis / recombination methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenesis-encoded DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0163] Semi-synthetic gene construction is achieved through a combination of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes a combination of the process of synthesizing polynucleotide fragments with PCR technology. Therefore, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0164] AlphaFold structural prediction

[0165] AlphaFold 2 is a computational method for calculating the three-dimensional structure of peptides based on their amino acid sequences (Jumper et al., 2021, Nature 596: 583-589). The predicted structures of millions of peptides in the UniProt database are stored 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 peptide can be obtained by searching for its UniProt accession number.

[0166] In addition to many publicly available 3D structures, the code can be used to reproduce and predict the structures of novel peptides in source code repositories, such as notebooks / AlphaFold.ipynb at deepmind / alphafold / Github.com using AlphaFold v2.3.1 or later. Alternatively, it can be found at sokrypton / ColabFold / Github.com using v1.5.2 or later (using AlphaFold2.ipynb). For technical details, see Jumper et al. (see above).

[0167] AlphaFold 2 generates per-residue estimates of its confidence level on a scale of 0 to 100. This confidence level metric is called pLDDT and corresponds to the model's predicted score on the lDDT-Cα index. It is stored in the B-factor field of the downloadable mmCIF and PDB files (although, unlike the B-factor, a higher pLDDT is better). Regions with pLDDT scores above 90 are expected to be modeled with high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with pLDDT scores between 70 and 90 are expected to be modeled well, corresponding to generally good main-chain predictions.

[0168] Structural similarity

[0169] For the purposes of this invention, the correlation between two polypeptide three-dimensional structures is described by the parameter "structural similarity".

[0170] The three-dimensional structure of any polypeptide can be obtained experimentally, for example via X-ray crystallography or using computational methods such as AlphaFold 2 (see above). The structural similarity between the three-dimensional structures can then be determined using a TM-score, which is calculated using the following general formula (Zhang and Skolnick, 2004, Proteins 57:702–710):

[0171]

[0172] Where L N It is the length of the natural structure, L T It is the length of the residues compared with the template structure, d i d0 is the distance between residues in the i-th pair, and d0 is the scale of the normalized matching difference. 'Max' represents the maximum value after optimal spatial superposition.

[0173] For the purposes of this invention, L N The length of the reference polypeptide is:

[0174]

[0175] Before the TM-score can be calculated, structural alignment of the three-dimensional structures of the two peptides is necessary. This is achieved via an algorithm that optimizes 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).

[0176] For the purposes of this invention, TM-align is used. For convenience, the TM-score is integrated into the TM-align software, which is available from the authors' website (zhanggroup.org / TM-score / ). The version of TM-align is preferably the latest version from August 22, 2019, or later, and the TM-score between the reference protein and the query protein is determined by running the following command:

[0177] TMalign<query.pdb><reference.pdb> -L<length of reference>

[0178] in<query.pdb> It is the name of the PDB file containing the coordinates of the queried peptide.<reference.pdb> This is the name of the PDB file containing the coordinates of the reference peptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0179] The maximum TM-score is 1, for example 1.0, corresponding to the same three-dimensional structure.

[0180] Sequence identity

[0181] The correlation between two amino acid sequences is described by the parameter "sequence identity".

[0182] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows:

[0183] (identical residues x 100) / (alignment length – total number of vacancies in the alignment).

[0184] Mature polypeptidesThe term "mature polypeptide" refers to a polypeptide that has undergone N-terminal and / or C-terminal processing (e.g., removal of the signal peptide) to reach its mature form. In one aspect, based on the SignalP 3.0 procedure predicting that amino acids 1 to 21 of SEQ ID NO: 1 are the signal peptide (Bendtsen et al., 2004, J. Mol. Biol. [Journal of Molecular Biology] 340: 783-795), the mature polypeptide is amino acids 22 to 294 of SEQ ID NO: 1. This was further confirmed by N-terminal sequencing, which showed that the mature peptide begins with ASGSGK, consistent with the prediction that amino acids 1 to 21 of SEQ ID NO: 1 are the signal peptide. In another aspect, the mature polypeptide is amino acids 36 to 229 of SEQ ID NO: 3, where amino acids 1 to 23 of SEQ ID NO: 3 are the signal peptide, and amino acids 24 to 35 of SEQ ID NO: 3 are the propeptide.

[0185] Biopolishing

[0186] As used in this article, the terms “biopolishing,” “de-pilling,” and “anti-pilling” are interchangeable.

[0187] Without finishing components, most polyester fabrics and polyester blends have a rather stiff and hard hand feel. The fabric surface is also not smooth because small, fuzzy microfibers protrude from it. Additionally, pilling occurs on the fabric surface after relatively short periods of wear, giving it an unattractive, worn appearance.

[0188] In one aspect, the present invention provides a method for biopolishing a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, for example, a crystallinity of less than 38%.

[0189] In this invention, biopolishing is a method of treating textiles containing polyesters with low crystallinity using enzymes such as keratinase, which improves fabric quality regarding "reduction of pilling." The most important role of biopolishing can be characterized by less fuzz and anti-pilling properties, as well as improved fabric hand feel. Biopolishing is typically performed in the wet processing of knitted or woven fabrics or garments. Wet processing includes, for example, the following steps: desizing, scouring, bleaching, washing, and finishing. Biopolishing can be performed as a separate step after any of the wetting steps or can be combined with any of those wetting steps, such as combined with a catalase bleaching step and / or combined with a dyeing step.

[0190] In a preferred embodiment, textiles containing polyesters with low crystallinity are treated with enzymes (such as keratinase) during the textile manufacturing process. Biopolishing is typically combined with mechanical force. For example, biopolishing can be applied in overflow / airflow dyeing machines, tumbling machines, sand washing machines, or other machines in the wet processing of textiles.

[0191] In abrasive applications, a balance between biopolishing performance and weight loss should be considered. In a preferred embodiment, the weight loss % of the biopolished textile is 0.01-5%, preferably 0.05-4%, and more preferably 1-3%.

[0192] Desizing

[0193] Polyesters (such as PET-based sizing agents) are widely used in yarn sizing, which gives the yarn the appropriate weave strength. Desizing is a necessary step in the dyeing and finishing of woven fabrics. After desizing, the fabric becomes hydrophilic, making it easier to dye. As used herein, the term "desizing" refers to the process of removing / removing sizing agents from textiles.

[0194] The polyester slurry can be a type of PET with a lower crystallinity than PET fiber. In this invention, the polyester slurry is a polyester with low crystallinity.

[0195] In one aspect, the present invention relates to a method for desizing polyester sizing of textiles, the method comprising contacting the polyester sizing with a keratinase. The keratinase can efficiently hydrolyze PET-based sizing from textiles and conveniently replace conventional high-temperature and alkaline treatments.

[0196] Oligomer removal

[0197] During the synthesis of PET, cyclic or linear oligomers of poly(ethylene terephthalate) are formed, such as bis-2-benzoyloxyethyl terephthalate (BETEB) and / or cyclic tri(ethylene terephthalate). These oligomers are partially deposited mechanically and partially remain on / within these fibers. The oligomers tend to give the fabric a light gray appearance. This is attributed to the deposition of the oligomers on the fabric surface, specifically after high-temperature wet processing such as high-temperature dyeing.

[0198] In this invention, the PET-based oligomer is a polyester with low crystallinity.

[0199] In one aspect, the present invention relates to a method for removing polyester oligomers from textiles, the method comprising contacting the polyester oligomers with a keratinase.

[0200] Antibacterial effect

[0201] The method of this invention produces antimicrobial effects. The term "antimicrobial" means inhibiting or reducing the growth of microorganisms on PET fabrics or PET blends (e.g., PET / cotton blends) containing polyesters with low crystallinity. The microorganisms on the fabric can be bacteria or fungi.

[0202] In some embodiments, the method of the present invention inhibits bacterial growth on PET fabrics or PET blends (e.g., PET / cotton blends) containing polyesters with low crystallinity.

[0203] Numerous testing methods have been developed to determine the efficacy of antimicrobial agents on fabrics. The bacterial species *Klebsiella pneumoniae* is recommended in most testing methods. This species is potentially pathogenic, therefore appropriate physical safeguards (e.g., biosafety cabinets) are required when handling it. Many studies have used harmless *Escherichia coli* as a test microorganism, which can be cultured and handled in standard laboratories with minimal health risks.

[0204] For the purposes of this invention, antimicrobial efficacy is measured according to any one of the following standard test methods: ASTM E 2149-01 (American Society for Testing and Materials), AATCC 100-2004 (American Association of Textile Chemists and Dyers), and agar plate test method.

[0205] Polyester fabric manufacturing process

[0206] Polyesters such as polyethylene terephthalate are synthesized by condensation, stretching from a melt into fibers, cutting into short fibers if possible, blending with other fiber types if possible, and spinning into yarns.

[0207] After knitting yarns or weaving fabrics, the fabric is generally treated to remove spin finish oil. For example, in a process where the fabric is first heat-set at 180°C and then pre-treated at 80°C-100°C with a surfactant (sometimes with the addition of an alkali), and then optionally followed by hydrolysis of the polyester fabric at up to 130°C using a strong alkali to induce a weight reduction process, resulting in a softer and shinier appearance. The polyester fabric is then heat-set and dyed with disperse dyes at pH 4.5-6 and up to 130°C, followed by a reduction wash with sodium thiosulfate at 60°C-80°C and pH 10. If desired, these processes can be followed by finishing (post-treatment) steps to further improve textile properties, such as anti-pilling or improved fuzz score. For PET / cotton blends, a soaping step is performed after finishing.

[0208] In a preferred embodiment, the method of the present invention is carried out in an aqueous solution during one or more subsequent steps, including pretreatment, weight reduction, dispersion dyeing, finishing, and soaping. The method of the present invention can be carried out as a separate step or in combination with any of the existing polyester processing steps.

[0209] In some embodiments, optimization has been performed to treat textiles containing polyesters with low crystallinity by incorporating keratinase into the finishing and dyeing processes of textiles and to maintain optimal performance. Cellulase and keratinase can be combined during the dyeing process; cellulase and keratinase can be combined in a single bath during the soaping step; or keratinase treatment can be applied separately from cellulase treatment combined during the dyeing process, either in a single bath during the soaping process or after the soaping process.

[0210] The process of this invention is readily applicable to the textile industry because it can be carried out using existing wet processing equipment, such as jet dyeing machines, pad-roll, jigger / winch, J-box, or pad-steam type equipment. This process preferably occurs during the finishing (post-treatment) step.

[0211] In another embodiment, a method of processing polyester textiles comprising polyester with low crystallinity is to manufacture polyester textiles, particularly polyester fabrics.

[0212] In another embodiment, the method of the present invention is combined with any of the existing polyester fabric manufacturing steps.

[0213] Process conditions

[0214] Keratinase can be used during the manufacturing process of textiles containing polyester with low crystallinity, either as a standalone step following any of the existing polyester manufacturing steps, or in combination with any of the existing polyester manufacturing steps such as pretreatment, weight reduction, dispersion dyeing, finishing, or soaping.

[0215] In one embodiment, the textile is contacted with keratinase in an aqueous solution.

[0216] It is suggested that a suitable liquid / textile ratio to be used in this method may be in the range of about 100:1 to about 1:1, preferably in the range of about 80:1 to about 3:1, and more preferably in the range of 60:1 to 5:1 (volume / weight, ml / mg).

[0217] The reaction time of the present invention is generally in the range of about 10 minutes to about 8 hours. Preferably, the reaction time is in the range of about 20 minutes to about 180 minutes, more preferably in the range of about 30 minutes to about 150 minutes, and most preferably in the range of about 45 minutes to about 120 minutes.

[0218] The pH of the reaction medium depends largely on one or more enzymes in question. Preferably, the process of the present invention is carried out at a pH range of + / - 1 pH unit from the optimal pH value of the keratinase. Preferably, the process of the present invention is carried out at a pH range of about pH 3 to about pH 11, preferably from about pH 4 to about pH 10, or from about pH 6 to about pH 9.

[0219] The process temperature of the present invention is preferably selected based on the optimal temperature of the keratinase plus or minus 10°C. Preferably, the process can be operated at temperatures below 100°C, preferably below 90°C, more preferably below 80°C, and even more preferably below 75°C.

[0220] In some embodiments, the process of the present invention is carried out in a temperature range of 40°C-100°C, preferably 50°C-90°C, preferably 60°C-88°C, more preferably 65°C-85°C, and even more preferably 70°C-85°C.

[0221] Enzyme dosage is largely dependent on enzyme reaction time; that is, a relatively short enzyme reaction time necessitates a relatively increased enzyme dosage, and vice versa. Typically, enzyme dosage can be determined based on the available reaction time.

[0222] The amount of keratinase to be used according to the method of the present invention depends on many factors and should preferably be optimized by a technician. According to the present invention, the preferred concentration of keratinase in an aqueous medium is from about 0.01 to about 50 mg enzyme protein / g polyester textile, preferably 0.05-20 mg enzyme protein / g polyester textile, more preferably 0.1-15 mg enzyme protein / g polyester textile, and even more preferably 0.5-10 mg enzyme protein / g polyester textile.

[0223] The process of the present invention may optionally include a rinsing step, during which the hydrolyzed oligomers are rinsed, specifically with an alkaline solution. The alkaline solution dissolves the linear fragments of the oligomers and can further hydrolyze these linear fragments to some extent.

[0224] In another embodiment, the method of the present invention further includes contacting the textile with one or more enzymes selected from the group consisting of cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxygenase, and transferase.

[0225] In another embodiment, the method of the present invention includes treating a textile containing a polyester with low crystallinity with a keratinase for 1-20, 1-15, 1-10, or 1-5 cycles, for example, 1, 2, 3, 4, or 5 cycles. In the present invention, the treatment effect is improved when the textile containing the polyester with low crystallinity is treated with a keratinase for one cycle. When the number of treatment cycles is further increased, the properties of the textile containing the polyester with low crystallinity are further improved.

[0226] Compositions for treating textiles

[0227] In one aspect, the present invention relates to a composition suitable for treating textiles, wherein the composition comprises a keratinase.

[0228] The textile compositions of the present invention are suitable for one or more of the polyester manufacturing processes such as pretreatment, weight reduction, dispersion dyeing and finishing, whether as separate steps or in combination with any of these steps.

[0229] In some embodiments of the invention, the composition containing keratinase further comprises other components, including but not limited to other enzymes, and one or more surfactants, bleaching agents, defoamers, building block systems, and similar substances.

[0230] Suitable enzymes for use in this invention include, but are not limited to, cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxyase, and transferase.

[0231] The textile composition can be in any form, such as solid, liquid, paste, gel, or any combination thereof.

[0232] surfactants

[0233] In the treatment of polyester textiles, conventional surfactants can be used to improve contact with enzymes.

[0234] The textile compositions of the present invention may contain one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. The one or more surfactants are typically present at levels from about 0.001% to 20% by weight of the composition, such as about 0.005% to about 10%, or about 0.01% to about 5%, or about 0.02% to about 1%.

[0235] More specifically, the surfactants used in the processes or compositions of the present invention include nonionic surfactants. Non-limiting examples of nonionic 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), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA), or fatty acid glucosamide (FAGA)), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0236] Other enzymes

[0237] The enzymatic polyester manufacturing process and the textile composition may contain one or more additional enzymes, such as cellulase, amylase, protease, lipase, esterase, laccase, peroxidase, peroxygenase and transferase.

[0238] Cellulase :

[0239] In the context of this invention, the term "cellulase" or "cellolytic enzyme" refers to an enzyme that catalyzes the degradation of cellulose into glucose, cellobiose, triose, and other cellooligosaccharides. Cellulose is a polymer of glucose linked by β-1,4-glycosidic bonds. Cellulose chains form numerous intramolecular and intermolecular hydrogen bonds, which leads to the formation of insoluble cellulose microfibrils. The microbial hydrolysis of cellulose into glucose involves three main classes of cellulases: endoglucanases (EC 3.2.1.4), which randomly cleave β-1,4-glycosidic bonds throughout the cellulose molecule; cellobiose hydrolases (EC 3.2.1.91) (exoglucanases), which digest cellulose from the non-reducing end; and β-glucosidases (EC 3.2.1.21), which hydrolyze cellobiose and low molecular weight cellodextrin to release glucose. Most cellulases consist of a cellulose-binding domain (CBD) and a catalytic domain (CAD), separated by linkers rich in proline and hydroxy amino acid residues. In the specification and claims, the term "endoglucanase" is intended to refer to an enzyme having cellulolytic activity, particularly endo-1,4-β-glucanase activity, which is classified under EC 3.2.1.4 according to enzyme nomenclature (1992) and is capable of catalyzing the (endo)hydrolysis of 1,4-β-D-glycosidic bonds in cellulose, lichen polysaccharides, and cereal β-D-glucans, including 1,4-linked (and also containing 1,3-linked) β-D-glucans. Any cellulase suitable for use in alkaline solutions may be used. Suitable cellulases include those of bacterial or fungal origin. This includes chemically or genetically modified mutants. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced by specific *Pythium* fungi. Particularly suitable cellulases are those with color care benefits. Examples of such cellulases are those described in European patent applications 0 495 257, WO 91 / 17243 and WO 96 / 29397.

[0240] Commercially available cellulases include Celluzyme™ and Denimax™ (Novo Nordisk A / S) and KAC-500(B)™ (Kao Corporation) produced by specific strains of *Pyrophyte*.

[0241] Cellulase is normally incorporated into the composition at a level of 0.00001% to 2% enzyme protein by weight of the composition, preferably at a level of 0.0001% to 1% enzyme protein by weight of the composition, more preferably at a level of 0.001% to 0.5% enzyme protein by weight of the composition, and even more preferably at a level of 0.01% to 0.2% enzyme protein by weight of the composition.

[0242] amylase :

[0243] Any amylase (α and / or β) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. This includes chemically or genetically modified mutants. Amylases include, for example, α-amylases obtained from specific strains of Bacillus licheniformis (described in more detail in GB 1,296,839). Commercially available amylases include Duramyl. TM Terminyl TM Fungayl TM and BAN TM (Available from Novo Nordisk), and Rapidase TM and Maxamyl P TM (Available from Genencor).

[0244] Amylase is normally incorporated into the composition at a level of 0.00001% to 2% enzyme protein by weight of the composition, preferably at a level of 0.0001% to 1% enzyme protein by weight of the composition, more preferably at a level of 0.001% to 0.5% enzyme protein by weight of the composition, and even more preferably at a level of 0.01% to 0.2% enzyme protein by weight of the composition.

[0245] protease :

[0246] Peptides or proteases exhibiting protease activity are sometimes also designated as peptidases, prions, peptide hydrolases, or proteolytic enzymes. A protease can be an exoprotease that begins hydrolyzing peptides at either end or an endoprotease (endopeptidase) that functions within the polypeptide chain. Endopeptidases exhibit activity against N- and C-terminated peptide substrates, the specificity of which depends on the protease in question.

[0247] Suitable proteases include those derived from bacteria, fungi, plants, viruses, or animals, such as those from plants or microorganisms. Microbial sources are preferred. This includes chemically modified mutants or protein-engineered mutants.

[0248] Examples of available proteases are variants described in the following: WO 92 / 19729, WO 96 / 034946, WO98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO 11 / 036264, or WO 2019 / 042306.

[0249] Lipase :

[0250] Suitable lipases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of suitable lipases include triacylglycerol lipase (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), monoacylglycerol lipase (EC 3.1.1.23), galactosidase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32), and lipoprotein lipase (EC 3.1.1.34). Examples include lipases from the genus *Thermomyces*, such as *T. lanuginosus* (formerly named *Humicola lanuginosa*) as described in EP 258 068 and EP 305 216; and lipases from the genus *Pseudomonas*, such as those from *P. alcaligenes* or *P. pseudoalcaligenes* (EP 218 272), *P. cepacia* (EP 331 376), *P. stutzeri* (GB 1,372,034), *P. fluorescens*, and *Pseudomonas* sp. strains SD 705 (WO 95 / 06720 and WO...). 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus lipases, such as those from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta [Chinese Journal of Biochemistry and Biophysics], 1131: 253-360), B. stearothermophilus (JP 64 / 744992) or B. pumilus (WO 91 / 16422).

[0251] Other examples are lipase variants, such as those described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260105, 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, WO 2007 / 087508, and WO 2009 / 109500.

[0252] Preferred commercially available lipases include Lipolase. TM Lipolase Ultra TM And Lipex™; Lecitase TM Lipolex TM Lipoclean TM Lipoprime TM (Novozymes A / S).

[0253] esterase :

[0254] "Esterase," also known as carboxylate hydrolase, refers to enzymes that act on ester bonds, and includes enzymes classified as EC 3.1.1 carboxylate hydrolases according to enzyme nomenclature (available at: http: / / www.chem.qmw.ac.uk / iubmb / enzyme, or Enzyme Nomenclature 1992, Academic Press, San Diego, California, with appendices 1 (1993), 2 (1994), 3 (1995), 4 (1997) and 5, respectively 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. [European Journal of Biochemistry] 1997, 250; 1-6, and Eur. J, Biochem [European Journal of Biochemistry], 1999, 264, 610-650.Non-restrictive examples of esterases include aryl esterases, triacylglycerol lipases, acetylesterases, acetylcholinesterases, cholinesterases, tropinesterase, pectin esterases, sterol esterases, chlorophyllases, L-arabinonolactonase, gluconolactone, uronolactonase, tannic acid esterases, retinyl palmitate esterases, hydroxybutyrate dimer hydrolases, acylglycerol lipases, 3-oxoadipate enol-lactone esterases, and 1 4-Lactonease, galactosyllipase, 4-pyridoxine lactonease, acylcarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinose lactonease, 6-phosphoglucuronide lactonease, phospholipase A1, 6-acetylglucosamine deacetylase, lipoprotein lipase, dihydrocoumarin lipase, limonene-D-cyclic lactonease, steroid lactonease, triacetyl lactonease, actinomycin lactonease, serotonin-phenolic acid hydrolase, cephalosporin-C deacetylase, chlorogenic acid hydrolase, α-amino acid esterase, 4-methyloxaloacetyl esterase, carboxymethyl esterase Carboxymethylenebutenolidase, deoxycitrate A-cyclic lactonease, 2-acetyl-1-alkylglycerol phosphocholinesterase, falciparine-C-ornithine esterase, sinigrin esterase, wax ester hydrolase, phorbol diester hydrolase, phosphatidylinositol deacetase, sialic acid O-acetylesterase, acetoxybutynyl-thiophene deacetylase, acetylsalicylic acid deacetylase, methyl umbelliferone acetate deacetylase, 2-pyranone-4,6-dicarboxylate lactonease, N-acetylgalactosamine Deacetylase, juvenile hormone esterase, bis(2-ethylhexyl)phthalate esterase, protein-glutamate methyl esterase, 11-cis-retinyl palmitate hydrolase, all-trans-retinyl palmitate hydrolase, L-rhamnolate-1,4-lactone esterase, 5-(3,4-diacetoxybut-1-ynyl)-2,2'-bithiophene deacetylase, fatty acyl ethyl ester synthase, xylan acid-1,4-lactone esterase, N-acetylglucosamine phosphatidylinositol deacetylase, cetriate benzyl esterase, acetylalkyl glycerol acetyl hydrolase, and acetylxylan esterase.

[0255] Preferred esterases used in this invention are lipases, such as lipases (e.g., classified as EC3.1.1.3, EC 3.1.1.23 and / or EC 3.1.1.26), phospholipases (e.g., classified as EC 3.1.1.4 and / or EC 3.1.1.32, including lysophospholipase classified as EC 3.1.1.5), and keratinase classified as EC 3.11.74.

[0256] In embodiments of the present invention, the esterase can be administered in the range of 7.5-25 LU / kg substrate.

[0257] Laccase :

[0258] The term "laccase" refers to phenylene glycol:oxygen oxidoreductase (EC1.10.3.2), which catalyzes the following reaction: 1,2- or 1,4-phenylene glycol + O2 = 1,2- or 1,4-benzosemiquinone + 2 H2O.

[0259] Laccase activity can be determined by measuring the oxidation of syringaldehyde azodicarbonyl (4,4'-[azodi(methyl)]bis(2,6-dimethoxyphenol)) to the corresponding quinone 4,4'-[azodicarbonyl(methyl)]bis(2,6-dimethoxycyclohexane-2,5-dien-1-one).

[0260] Peroxidase / oxidase :

[0261] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of available peroxidases include those from the genus *Coprinus*, such as those from *C. cinereus*, and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0262] Commercially available peroxidases include Guardzyme™ (Novozymes).

[0263] peroxygenase :

[0264] The term "peroxygenase" refers to "non-specific peroxygenase" activity as defined in EC 1.11.2.1, which catalyzes the insertion of an oxygen atom from H₂O₂ into a variety of substrates, such as nitrobenzide. Examples of available peroxygenases include those described in WO 2008 / 119780.

[0265] transferase :

[0266] Preferred transferases are those from any of the following subclasses:

[0267] a) Transferases that transfer one-carbon groups (EC 2.1);

[0268] b) Transferases that transfer aldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3);

[0269] c) Glycosyltransferase (EC 2.4);

[0270] d) Transferases that transfer alkyl or aryl groups other than methyl groups (EC 2.5); and

[0271] e) Transferases that transfer nitrogen-containing groups (EC 2.6).

[0272] In the context of this invention, the preferred type of transferase is transglutaminase (protein-glutamine γ-glutamyltransferase; EC 2.3.2.13).

[0273] Other examples of suitable transglutaminases are described in WO 96 / 06931 (Novo Nordisk).

[0274] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention. Example

[0275] Materials and methods

[0276] Material

[0277] The chemicals used as buffers and substrates are at least reagent-grade commercial products.

[0278] enzymes

[0279] Cellulase: A mature polypeptide of an endoglucanase derived from coccidioidomycetes, shown as SEQ ID NO: 2 in WO2014 / 026630 and SEQ ID NO: 1 herein.

[0280] Keratinase A: The keratinase disclosed in WO 2001 / 092502. This keratinase is a variant of the wild-type keratinase / lipase derived from *Pyrophyllus* DSM1800 (SEQ ID NO: 1 in WO 2001 / 092502 and SEQ ID NO: 2 herein), which contains the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q, and R189V.

[0281] Keratinase B: The keratinase disclosed in WO 2015 / 085920. This keratinase is a variant having the keratinase activity of a parent keratinase having the mature polypeptide of SEQ ID NO: 2 in WO 2015 / 085920 and the mature polypeptide of SEQ ID NO: 3 herein, the variant comprising changes selected from the group consisting of:

[0282] A161L +R181P +G182 .

[0283] The amino acid position numbers correspond to the amino acid residues of the mature polypeptide in SEQ ID NO: 3. Since SEQ ID NO: 3 has a signal peptide and a propeptide from amino acid residues 1-35, 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.

[0284] method

[0285] Protein content

[0286] According to the product manual, use BCA TM The protein assay kit (product number 23225, commercially available from Thermo Fisher Scientific Inc.) measures enzyme proteins. The following table shows the results obtained using BCA... TM The protein content of an enzyme is tested using a protein assay kit.

[0287]

[0288] Weight loss % determined

[0289] Small fabric samples were placed in a controlled room (65% relative humidity, 21°C) for 24 hours before numbering, and weighed (for samples less than 100 g) using an analytical balance and the weight recorded. After treatment, all samples were rolled dry (AEG, LAVATHERM 37700, Germany) for 1 hour and conditioned for 24 hours in the controlled room as mentioned above. Weight loss was defined for each sample as follows:

[0290]

[0291] Balling record test

[0292] Small fabric samples (both treated and untreated) that had been pre-conditioned for at least 24 hours under standard climatic conditions (65% humidity, 21°C) were tested against pilling records using a Nu-Martindale tester (James H. Hill Heal Co. Ltd, England) with untreated fabric of the same type as the abraded fabric. A standard pilling test (Swiss Standard (SN) 198525) was performed after 2000 revolutions, using a scale of 1-5 with the following meanings: 1 indicating poor pilling resistance and 5 indicating excellent pilling resistance. Therefore, a higher Martindale pilling record score indicates a more effective bio-polishing treatment.

[0293] Record 5: No pilling

[0294] Record 4: Slight pilling

[0295] Record 3: Moderate pilling

[0296] Record 2: Obvious pilling

[0297] Record 1: Severe pilling

[0298] Records of 1 / 2 or 1 / 4 are allowed.

[0299] To make the results more reliable, three separate readings were taken for each sample by different people, and the average of these three readings was used as the final result of the pilling record.

[0300] downy identification

[0301] Cut the target fabric sample into rectangular pieces approximately 13 cm high and 13 cm long, and place them in a standard climate (65% humidity, 21°C) for at least 24 hours before measurement. (via PillGrade) TM (Automatic 3D Pilling and Fluff Grading System, SDL ATLAS, LineTech Industries, USA) This system reads the fluff score of a target small fabric sample and grades it according to ASTM and ISO test methods. The height and amount of fluff are measured by scanning the horizontal lines of the fabric, ignoring the fabric pattern and weave / knit structure. To make the test results more reliable, all four directions of a square fabric are measured, and the average of the four values ​​is recorded as the fluff score for that fabric. The lower the fluff score, the more effective the biopolishing treatment.

[0302] Differential Scanning Calorimetry (DSC) Test (GB / T 40271-2021)

[0303] Differential scanning calorimetry 25 (DSC25, TA Instrument Waters) was used. TM The company (USA) determined the crystallinity percentage of PET fibers. Nitrogen flow rate was 50 mL / min. Heating rate was 10°C / min. Fabrics or fibers were cut into 2 mm or 3 mm lengths. PET fibers (3–5 mg) were placed in a Tzero aluminum dish with a Tzero solid sample cap. Detailed procedures are as follows:

[0304] The sample was first heated from 40°C to 300°C at a rate of 10°C / min and held at 300°C for 1 min in a DSC25. The percentage of crystallinity was determined using the enthalpy of melting and the enthalpy of cold crystallization during the first heating scan. The percentage of crystallinity within the PET fibers was calculated using the following equation:

[0305]

[0306] in H m It is the enthalpy of fusion (J g) -1 ), H cc It is the enthalpy of cold recrystallization (J g) -1 ),and H m The melting enthalpy of a 100% crystalline PET sample is 140.1 J g. -1 Measurements were taken by integrating from 60°C–70°C to approximately 275°C using a linear baseline. H m and H cc The percentage of crystallinity was calculated using the TRIOS software package provided with the DSC25.

[0307] During heating, some materials will undergo cold recrystallization, meaning they haven't fully crystallized at room temperature. The recrystallization peak often appears at 150°C. For some materials, recrystallization does not occur, therefore... H cc It can be equal to 0 J g -1 .

[0308] Example 1. Biopolishing properties of keratinase on PET / Spandex fabrics

[0309] Use PET / spandex fabric (containing approximately 13% TiO2, 95% PET, and 5% spandex, Siro-spun yarn), and the original fabric needs to be de-oiled. The de-oiling procedure in Wascator (Electrolux FOM71CLS, Sweden) is as follows.

[0310]

[0311] Before the next step, roll the degreased fabric to dry and acclimatize it for 24 hours at 65% relative humidity and 21°C.

[0312] Then, the deoiled PET / spandex fabric is biopolished once or multiple times in a Wascator using keratinase B or keratinase A at specific dosages, temperatures, pH, times, liquid-to-fabric ratios (LR), and surfactants. The biopolishing (BP) procedure in the Wascator is as follows.

[0313]

[0314] Before evaluating the pile score and pilling record, the biopolished fabric was tumble-dried and conditioned for 24 hours at 65% relative humidity and 21°C.

[0315]

[0316] Before evaluating the pile score and pilling record, the biopolished fabric was tumble-dried and conditioned for 24 hours at 65% relative humidity and 21°C.

[0317] As summarized in Table 1, keratinases (including keratinase B and keratinase A) provided good biopolishing performance on PET / spandex fabrics (containing approximately 13% TiO2, Sirospun yarn), indicated by significant improvements in both nap score and pilling record. The level of improvement became more pronounced as the number of wash cycles increased from 1 to 3. Keratinase B was more effective for biopolishing applications on PET compared to keratinase A, with BP performance from keratinase B in 1 wash cycle almost approaching the level obtained from keratinase A in 3 wash cycles.

[0318] Table 1. Biopolishing of PET / Spandex Knitted Fabrics in Wascator

[0319]

[0320] Example 2. Dose-response relationship of biopolishing performance of keratinase in Laundry-O-Meter

[0321] Using de-oiled PET / spandex fabric (containing approximately 13% TiO2, Sirospun yarn), the de-oiling procedure was the same as in Example 1.

[0322] Biopolishing was performed in a Launder-O-Meter (LOM_M228AA, SDL ATLAS, LineTech Industries, USA). PET / Spandex fabric was cut into rectangular blocks 15 cm high and 15 cm long, weighing approximately 4–5 g. These fabrics were placed in a controlled chamber (65% RH, 21°C) for 24 hours before numbering, and weighed and recorded using an analytical balance. Two conditioned blocks were placed in each beaker. For each beaker, 20 steel balls (approximately 220 g in total) were used to provide mechanical assistance. A pH 8.0 buffer containing 0.2% Triton X-100 (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L of deionized water) was then used. Based on calculations of the actual fabric weight, this buffer and either keratinase A or keratinase B were added at a liquid-to-fabric ratio of 10:1 (v / w), according to Table 2.

[0323] After selecting the desired program, start the LOM machine and maintain the temperature at 60°C or 80°C. Each beaker is fitted with a lid lined with two neoprene gaskets and tightly closed using a metal clamp. Load these beakers into the preheated LOM. In the vertical position, use a metal frame to hold and secure five beakers in each of the four drum positions. Close the LOM lid and continue the washing program while starting the timer. After 1.5 hours, rinse the fabric three times in cold water. Tumble dry the fabric (AEG, LAVATHERM 37700, Germany) for 1 hour, and then condition the samples at 21°C and 65% relative humidity for 24 hours before evaluation. Evaluate the fabric's nap score, pilling record, and weight loss percentage.

[0324] Table 2 shows that increasing the dosage of keratinase B and keratinase A relatively produces improved biopolishing properties, including vellum score, pilling record, and weight loss percentage. The level of improvement caused by dosage increases is limited. For example, even for a fourfold increase in dosage, it is only grade 0.25. Increasing the number of wash cycles as listed in Table 1 is more effective in improving biopolishing than increasing the enzyme dosage as listed in Table 2.

[0325] Table 2. Biopolishing of PET / Spandex fabrics in Launder-O-Meter (pH 8) for 90 min.

[0326]

[0327] Example 3. Biopolishing properties of keratinase on recycled PET (rPET) fabrics and fabrics containing rPET.

[0328] Eight different rPET fabrics were collected from the Chinese market, and the information is listed in the table below. A 100% virgin PET fabric was selected as a reference and recorded as fabric 0.

[0329]

[0330] In the Wascator, fabrics 0, 1, 2, 3, 4, 5, 6, 7, or 8 are deoiled using the same procedure as in Example 1. Then, fabrics 1, 2, 3, and 4 are pretreated in the Wascator with cellulase to remove fuzz caused by cellulose fibers, as follows.

[0331]

[0332] Before evaluating the pile score and pilling record, the pretreated fabric was rolled dry and conditioned for 24 hours at 65% relative humidity and 21°C.

[0333] For all fabrics, keratinase B treatment was performed in the Wascator, following the procedure described in Example 1. The dose of keratinase B was 3 g / L, for one cycle, and the BP cycle of keratinase B is shown in Table 3.

[0334] As summarized in Table 3, keratinase B exhibited consistently excellent BP performance on rPET fabrics and fabrics containing rPET (including knitted and woven fabrics), indicated by a significant decrease in the pile score after keratinase treatment, and all fabrics showed a final pilling record above 4, meaning that the fabrics showed almost no pilling after 2000 rpm abrasion on a Nu-Martindale tester. This validates the versatility of the biopolishing application of keratinase on rPET fabrics.

[0335] Table 3. BP performance on 9 different PET fabrics in Wascator

[0336]

[0337] Example 4. Weight loss of PET fabric after biopolishing with keratinase

[0338] Three different rPET fabrics were used, which were the same as fabrics 6, 7 and 8 in Example 3.

[0339]

[0340] Fabrics 6, 7, and 8 were deoiled in the Wascator using the same procedure as in Example 1. Each fabric was then treated with keratinase B in a Launder-O-Meter (LOM) using the same procedure as in Example 2. The dosage of keratinase B was added at a liquid-to-fabric ratio of 10:1 (v / w) based on calculations of the actual fabric weight, according to Table 4. The BP cycle was also run according to Table 4.

[0341] As shown in Table 4, the weight loss % increases with increasing biopolishing performance. When excellent biopolishing performance is achieved, the weight loss % for all three pure rPET fabrics is less than 4, indicated by a significant improvement in the nap score and the best pilling record (5). Simultaneously, the biopolished fabrics remain sufficiently strong for subsequent processing. A balance between biopolishing performance and weight loss should be considered in abrasive applications. For example, two cycles of biopolishing with 0.5–1 g / L keratinase B on fabrics 6, 7, and 8 are sufficiently good, with a good pilling record (>3) and an acceptable weight loss % (approximately 1–2).

[0342] Table 4. Weight loss and BP performance in LOM

[0343]

[0344] Example 5. PET crystallinity test using DSC 25

[0345] The crystallinity of virgin polyester fabrics / fibers and recycled polyester fabrics / fibers was tested according to the standard test method of GB / T 40271-2021 (described in the Methods section), and the results are shown in Table 5.

[0346] Table 5 shows that the crystallinity of recycled polyester is lower than that of virgin polyester. The crystallinity of recycled PET fabrics / fibers ranges from 25% to 29%, while that of virgin PET fabrics / fibers ranges from 39% to 43%. Combined with the results in Example 3, keratinase is more suitable for rPET treatment.

[0347] When virgin PET was deeply modified with TiO2, it also achieved good BP properties as described in Tables 1 and 2, with a crystallinity of approximately 32%. All results show a strong correlation between PET crystallinity and BP properties. Without being bound by any theory, it is believed that keratinase can more easily reach the hydrolysis sites of PET fibers with lower crystallinity. PET fabrics / fibers with a crystallinity below 33% are readily treated with keratinase.

[0348] Table 5. Crystallinity of various types of PET

[0349]

[0350] Example 6. Desizing of polyester sizing agents using keratinase

[0351] Woven fabrics sized with polyester during weaving were used. Exhaustion desizing was performed in Lab-O-Mat (Mathis LABOMAT, Typ-Nr. BFA24 201205, Switzerland), while pad dyeing of the pad-batch desizing material was performed in Padder (Typ-Nr. VFM41497, Mathis, USA, Switzerland).

[0352] Exhaustion desizing. Cut the polyester-sized woven fabric into rectangular blocks 15.5 cm high and 17.5 cm long, weighing approximately 10 g. Place these fabrics in a controlled chamber (65% relative humidity, 21°C) for 24 hours before numbering. Place one conditioned block in each beaker. Add 100 mL of pH 8.0 buffer (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L of deionized water) to each beaker containing 0.2% Triton X-100. Add keratinase B according to Table 6. Load the beakers symmetrically into the Lab-O-Mat machine, start the Lab-O-Mat after selecting the desired program, and hold it at 80°C. After 10 min, rinse the fabric twice in hot water (95°C), twice in cold water (room temperature), spin, and lay flat to dry.

[0353] Padding and desizing. Cut the polyester-sized woven fabric into rectangular blocks 15.5 cm high and 20.0 cm long, weighing approximately 13 g. Place these fabrics in a controlled room (65% relative humidity, 21°C) for 24 hours before numbering. Add 200 mL of pH 8.0 buffer (16.95 g Na₂HPO₄·12H₂O and 0.42 g NaH₂PO₄·2H₂O in 1 L of deionized water) to each beaker containing 0.2% Triton X-100. Add keratinase B according to Table 6. Immerse one conditioned block in a beaker for 30 seconds, then pad-dye using a Padder to maintain 90% liquid uptake. Double-dye and double-pad-dye each fabric, then place them in resealable bags. Incubate all fabrics at room temperature (20°C–25°C) for 20 hours (overnight). The fabric is rinsed twice in hot water (95°C) and twice in cold water (room temperature), then spun and laid flat to dry.

[0354] Under certain conditions, residual PET sizing on the fabric was combined with methylene blue, and the following determination was made: Fabrics listed in Table 6 were cut into 5 cm x 5 cm squares and placed in a beaker with 250 mL of methylene blue solution at a concentration of 1 g / L. The beaker was loaded into the Lab-O-Mat machine, and the desired program was selected and started. The temperature was maintained at 70°C. After 10 min, the fabric was rinsed twice in cold water (room temperature), spun, and laid flat to dry. These fabrics were placed in a control room (65% relative humidity, 21°C) for 24 hours, and then the K / S value was measured using a Datacolor (Datacolor 500, serial number 8811258, USA). The color depth of the dyed fabric was characterized by the K / S value; the higher the value, the deeper the color. The original dyed fabric was used as a standard.

[0355] Table 6 shows that sized polyester staple fibers on woven fabrics were significantly removed after treatment with keratinase. Fabrics treated with 1 g / L or 10 g / L keratinase had a lighter color depth than the original fabric and fabrics treated with the same process using 0 g / L enzyme (the lower the K / S value, the less residual PET on the fabric). Keratinase showed great adaptability to temperature and process (both exhaustion desizing and pad-batch desizing).

[0356] Table 6. Polyester slurry treated with keratinase

[0357]

[0358] The invention is further defined by the following numbered paragraphs:

[0359] 1. A method for treating textiles, the method comprising contacting the textiles with a keratinase,

[0360] The textile contains polyester with a crystallinity lower than that of the original polyester, and preferably the textile contains polyester with a crystallinity of less than 38%.

[0361] 2. The method as described in paragraph 1, wherein the treated textile exhibits at least one property selected from the group consisting of: improved hand feel, anti-pilling, improved pile score, polyester sizing desizing, oligomer removal, antibacterial properties, and / or wherein the weight loss % of the treated textile is 0.01-5, preferably 0.05-4, more preferably 1-3.

[0362] 3. A method for biopolishing a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

[0363] 4. The method as described in paragraph 3, wherein the biopolishing is an improvement in anti-pilling and / or fuzz score.

[0364] 5. A method for desizing a polyester sizing agent for textiles, the method comprising contacting the polyester sizing agent with a keratinase.

[0365] 6. A method for removing polyester oligomers from textiles, the method comprising contacting the polyester oligomers with a keratinase.

[0366] 7. A method for improving the hand feel of a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

[0367] 8. A method for improving the antimicrobial properties of a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

[0368] 9. The method as described in any one of paragraphs 1-8, wherein the crystallinity of PET is 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%.

[0369] 10. The method as described in paragraph 9, wherein the crystallinity is determined by differential scanning calorimetry, preferably according to GB / T40271-2021.

[0370] 11. The method as described in any one of paragraphs 1-10, wherein the polyester is PET, preferably rPET or modified PET, preferably modified by TiO2.

[0371] 12. The method as described in any one of paragraphs 1-11, wherein the textile is a fiber, yarn, fabric, or garment.

[0372] 13. The method as described in any one of paragraphs 1-12, wherein the textile is a pure polyester textile or a polyester blend.

[0373] 14. The method as described in paragraph 13, wherein the polyester blend comprises more than 5% (w / w) of polyester, particularly 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.

[0374] 15. The method as described in paragraph 14, wherein the polyester blends are blends of polyester with natural fibers or blends of polyester with man-made fibers.

[0375] 16. The method as described in paragraph 15, wherein the natural fibers are selected from the group consisting of cellulose fibers, animal fibers and mineral fibers, and the man-made fibers are selected from regenerated fibers and synthetic fibers.

[0376] 17. The method as described in paragraph 16, wherein the cellulose 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 modified polyacrylonitrile, and polyurethanes such as spandex, Lycra, and elastic fibers.

[0377] 18. The method as described in any paragraph 1-17, wherein the keratinase is selected from the group consisting of:

[0378] (a) A polypeptide having a TM-score of at least 0.60 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold;

[0379] (b) A polypeptide having at least 60% sequence identity with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 3;

[0380] (c) A polypeptide derived from a mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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, particularly substitutions;

[0381] (d) A polypeptide derived from (a) or (b) wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and

[0382] Fragments of polypeptides of (e), (a), (b), (c), or (d);

[0383] This polypeptide possesses keratinase activity.

[0384] 19. The method as described in 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 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold.

[0385] 20. The method as described in 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 with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0386] 21. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant of the keratinase of the parent fungus, the variant being:

[0387] a) Contains a substitution of at least one amino acid residue at position A4, T29, A88, N91, A130, Q139, I169, I178, or R189 in the keratinase corresponding to the specific *Porcine* strain DSM 1800 (specific *Porcine* keratinase number), and

[0388] b) It is more thermally stable than the parent keratinase.

[0389] 22. The method as described in paragraph 21, wherein the keratinase is a variant of the keratinase comprising the specific *Pythium spp.* strain DSM 1800, substituted with A4V, T29M / I / C, A88H / L / V, N91H, A130V, Q139R, I169A / G / T / V, I178V, or R189A / H / V.

[0390] 23. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant of the keratinase of the parent fungus, the variant being:

[0391] a) Contains a substitution of at least one amino acid residue (specifically, the *Porcine keratinase* number) in the keratinase corresponding to the specific *Porcine spp.* strain DSM 1800, namely Q1C / L, L2K / Q / V, G8D, S11T, N15D, A16T, V38H, S48E / K, ​​H49Y, L66I, S116K, S119P, G120D, T164S, T166M / I, or L167P.

[0392] b) It is more thermally stable than the parent keratinase.

[0393] 24. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant comprising a substitute selected from the group consisting of:

[0394] a)S48E +A88H +N91H +R189V

[0395] b)Q1L +L2K +G8D +N15D

[0396] c) N44D + A130V

[0397] d)Q1C +L2V +G120D

[0398] e)A88L +R189A

[0399] f)S48E +L66I +A88L +I169A +R189H

[0400] g)A88V +S116K +S119P +Q139R +I169V +R189V

[0401] h)A88V +R189A

[0402] i)S48K +A88H +I169G +R189H

[0403] j)Q1L +L2Q +A4V +S11T

[0404] k)T164S

[0405] l) L174F

[0406] m) H49Y

[0407] n) Q1L + L2K + G8D + N15D + S48E + A88H + N91H + R189V

[0408] o) Q1L + L2K + G8D + N15D + N44D + A130V

[0409] p) Q1L + L2K + G8D + N15D + S48E + A88H + N91H + A130V + R189V

[0410] q) G8D + N15D + A16T

[0411] r) A130V

[0412] s) Q1C + L2V

[0413] t) G8D + N15D + A16T

[0414] u) G8D + N15D + S48E + A88H + N91H + A130V + R189V

[0415] v) G8D + N15D + T29M + S48E + A88H + N91H + A130V + R189V

[0416] w) G8D + N15D + T29I + S48E + A88H + N91H + A130V + R189V and / or

[0417] x) G8D + N15D + T29C + S48E + A88H + N91H + A130V + R189V

[0418] y) G8D + N15D + S48E + A88H + N91H + A130V + L174F + I178V + R189V

[0419] z) G8D + N15D + S48E + A88H + N91H + A130V + T166M + I168F + R189V

[0420] aa) G8D + N15D + S48E + A88H + N91H + A130V + T166I + L167P + R189V <0bb)G8D +N15D +V38H +S48E +A88H +N91H +A130V +I169T + R189V

[0422] cc)G8D +N15D +V38H +S48E +A88H +N91H +A130V +R189V

[0423] dd)G8D +N15D +T29M +S48E +A88H +N91H +A130V +T166I +L167P +R189V.

[0424] 25. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant that further comprises at least one amino acid substitution (specific humic mold keratinase number) at positions corresponding to Q1, L2, E6, E10, S11, A14, N15, F24, L46, E47, R51, D63, L138 and / or E179.

[0425] 26. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant further comprising at least one substitution (specific humic mold keratinase number) corresponding to Q1P, L2V, E6Q, E10Q, S11C, A14P, N15T, F24Y, L46I, E47K, R51P, D63N, L138I and / or E179Q.

[0426] 27. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant further comprising a substitution corresponding to E6Q + A14P + E47K + R51P + E179Q.

[0427] 28. The method of any one of paragraphs 1-18, wherein the keratinase is a variant having the keratinase activity of the parent keratinase, the variant comprising alterations 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 alterations are substitutions for positions 181, 115, 161, 43, 55, 79, and 5, and deletions for positions 1, 2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 3; wherein the amino acid position numbers correspond to amino acid residues of the mature polypeptide of SEQ ID NO: 3.

[0428] 29. The method as described in paragraph 28, wherein the keratinase 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.

[0429] 30. The method as described in paragraph 29, wherein the keratinase is a variation comprising or composed of a group of the following:

[0430] a.V115I +R181P +G182

[0431] b.A161L +R181P +G182

[0432] c.Q1 +L2 +I5V +A43C +I55C +N79A +V115I +R181P +G182

[0433] d.Q1 +L2 +A43C +I55C +N79A +V115I +R181P +G182

[0434] e.I5V +A43C +I55C +N79A +V115I

[0435] f.A43C +I55C +N79A

[0436] g.I5V +A43C +I55C +N79A +V115I +R181P +G182

[0437] h.Q1 +L2

[0438] i.I5V

[0439] j.A43C +I55C

[0440] k.N79A

[0441] l.V115I

[0442] m.A161L

[0443] n.R181P +G182

[0444] The amino acid position number corresponds to the amino acid residue of the mature polypeptide in SEQ ID NO: 3.

[0445] 31. The method as described in any one of paragraphs 1-18, wherein the keratinase is a variant of the parental keratinase derived from the polypeptide having SEQ ID NO: 2, the variant comprising the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q, and R189V, and wherein the amino acid position numbers correspond to the amino acid residues of SEQ ID NO: 2; or wherein the keratinase is a variant of the parental keratinase of the mature polypeptide having SEQ ID NO: 3, the variant comprising the following changes: A161L + R181P + G182 And the amino acid position number therein corresponds to the amino acid residue of the mature polypeptide of SEQ ID NO:3.

[0446] 32. The method as described in any one of paragraphs 1-31, wherein the textile is in contact with the keratinase in an aqueous solution.

[0447] 33. The method of any one 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, peroxyase, and transferase.

[0448] 34. The method as described in any one of paragraphs 1-33, wherein the cellulase is an endoglucanase.

[0449] 35. The method as described in paragraph 34, wherein the endoglucanase is selected from the group consisting of:

[0450] (a) A polypeptide having a TM-score of at least 0.60 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold;

[0451] (b) A polypeptide having at least 60% sequence identity with the mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 1;

[0452] (c) A polypeptide derived from a mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 1 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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, particularly substitutions;

[0453] (d) A polypeptide derived from (a) or (b) wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and

[0454] Fragments of polypeptides of (e), (a), (b), (c), or (d);

[0455] This polypeptide possesses endoglucanase activity.

[0456] 36. The method as described in 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 compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold.

[0457] 37. The method as described in 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 with the mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 1.

[0458] 38. The method as described in any of the preceding paragraphs, wherein the keratinase is applied in the range of 0.01 to 50 mg enzyme protein / g polyester textile, preferably 0.05-20 mg enzyme protein / g polyester textile, more preferably 0.1-15 mg enzyme protein / g polyester textile, and even more preferably 0.5-10 mg enzyme protein / g polyester textile.

[0459] 39. The method as described in any of the preceding paragraphs, wherein the method is carried out in a pH range from about pH 3 to about pH 11, preferably from about pH 4 to about pH 10, or from about pH 6 to about pH 9.

[0460] 40. The method as described in any of the preceding paragraphs, wherein the method is carried out in a temperature range of 40°C-100°C, preferably 50°C-90°C, preferably 60°C-88°C, more preferably 65°C-85°C, and even more preferably 70°C-85°C.

[0461] 41. The method as described in any of the preceding paragraphs, wherein the method is carried out 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, and even more preferably about 45 minutes to about 120 minutes.

[0462] 42. The method as described in any of the preceding paragraphs, wherein the method comprises treating the textile with a keratinase for 1-20, 1-15, 1-10, or 1-5 cycles, for example, 1, 2, 3, 4, or 5 cycles.

[0463] 43. The method as described in any of the preceding paragraphs, wherein the treatment of the polyester textile is for manufacturing polyester textiles, particularly for manufacturing polyester fabrics.

[0464] 44. The method as described in paragraph 43, wherein the method is combined with any of the existing polyester fabric manufacturing steps.

[0465] 45. A textile product produced according to any of the methods described in any of the preceding paragraphs.

Claims

1. A method for treating textiles, the method comprising contacting the textiles with a keratinase, The textile contains polyester with a crystallinity lower than that of the original polyester, and preferably the textile contains polyester with a crystallinity of less than 38%.

2. The method of claim 1, wherein the treated textile exhibits at least one property selected from the group consisting of: improved hand feel, anti-pilling, improved pile score, polyester sizing desizing, oligomer removal, antibacterial properties, and / or wherein the weight loss % of the treated textile is 0.01-5, preferably 0.05-4, more preferably 1-3.

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

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

5. A method for desizing a polyester sizing agent for textiles, the method comprising contacting the polyester sizing agent with a keratinase.

6. A method for removing polyester oligomers from textiles, the method comprising contacting the polyester oligomers with a keratinase.

7. A method for improving the hand feel of a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

8. A method for improving the antimicrobial properties of a textile, the method comprising contacting the textile with a keratinase, wherein the textile comprises a polyester with a crystallinity lower than that of the original polyester, preferably the textile comprises a polyester with a crystallinity of less than 38%.

9. The method according to any one of claims 1-8, wherein the crystallinity of PET is 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 any one of claims 1-9, wherein the polyester is PET, preferably rPET or modified PET, preferably modified by TiO2.

11. The method according to any one of claims 1-10, wherein the textile is a pure polyester textile or a polyester blend.

12. The method of any one of claims 1-11, wherein the keratinase is selected from the group consisting of: (a) A polypeptide having a TM-score of at least 0.60, such as 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, compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or the 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 with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 3, 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; (c) A polypeptide derived from a mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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, particularly substitutions; (d) A polypeptide derived from (a) or (b) wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and Fragments of polypeptides of (e), (a), (b), (c), or (d); This polypeptide possesses keratinase activity.

13. The method of any one of claims 1-12, wherein the keratinase is a variant of the parental keratinase derived from the polypeptide having SEQ ID NO: 2, the variant comprising the following 12 mutations: E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, N91H, A130V, E179Q, and R189V, and wherein the amino acid position numbers correspond to the amino acid residues of SEQ ID NO: 2; or wherein the keratinase is a variant of the parental keratinase of the mature polypeptide having SEQ ID NO: 3, the variant comprising the following changes: A161L + R181P + G182 And the amino acid position number therein corresponds to the amino acid residue of the mature polypeptide of SEQ ID NO:

3.

14. The method of any one 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, peroxyase, and transferase.

15. The method of claim 14, wherein the cellulase is an endoglucanase, preferably selected from the group consisting of: (a) A polypeptide having a TM-score of at least 0.60, such as 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, compared to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 or the 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 with the mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 1, 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; (c) A polypeptide derived from a mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 1 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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, particularly substitutions; (d) A polypeptide derived from (a) or (b) wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and Fragments of polypeptides of (e), (a), (b), (c), or (d); This polypeptide possesses endoglucanase activity.

16. A textile product produced according to the method as described in any one of the preceding claims.

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