WO2019042306A1 - Méthode d'inactivation de cellulase - Google Patents

Méthode d'inactivation de cellulase Download PDF

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WO2019042306A1
WO2019042306A1 PCT/CN2018/102880 CN2018102880W WO2019042306A1 WO 2019042306 A1 WO2019042306 A1 WO 2019042306A1 CN 2018102880 W CN2018102880 W CN 2018102880W WO 2019042306 A1 WO2019042306 A1 WO 2019042306A1
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cellulase
protease
subtilisin
bacillus
seq
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PCT/CN2018/102880
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Weijian Lai
Elmar JANSER
Andreu Colomera CEBA
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Novozymes A/S
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/40—Specific cleaning or washing processes
    • C11D2111/44—Multi-step processes

Definitions

  • the present invention relates to a method for inactivating a cellulase and a method for treating a textile.
  • Cellulase or “cellulolytic enzyme” is a group of glycoside hydrolase enzymes that catalyze the hydrolysis of beta-1, 4-glycosidic linkages in the cellulose polymer. Such enzymes include endoglucanase (s) , cellobiohydrolase (s) , beta-glucosidase (s) , or combinations thereof.
  • cellulases are widely used to improve the appearance and softness of cellulose-containing fabrics.
  • a widespread application of cellulase enzymes is to remove cotton fuzz and loose surface fibers in or on the fabric. This process referred to as “biopolishing” smoothes the surface of the fabric, which in turn improves its softness and appearance.
  • Cellulase treatment also aids in the prevention of subsequent formation of fiber pills that make the garments appear worn. During biopolishing it is desirable to minimize weight loss and/or strength loss of the fabric due to the hydrolytic action of the cellulases.
  • biostoning Another industrial application of cellulase enzymes is for treating denim fabrics so as to impart to them a “stone-washed” appearance. Such a process is known in the industry as “biostoning” .
  • biostoning was adopted, as pumice stones were traditionally used to treat the fabric.
  • Cellulases have largely replaced pumice stones in recent years. Biostoning aims to remove colour from denim and control its re-deposition on the fabric.
  • the present invention relates to a method for inactivating a cellulase, comprising treating the cellulease with a serine protease.
  • the present invention further relates to a method for treating a textile, comprising (a) treating the textile with a cellulase; and (b) treating the textile with a serine protease to inactivate the cellulase.
  • Figure 1 shows a result of SDS-PAGE gel.
  • the lanes from left to right are marker; protease-1; protease-2; cellulase-A; cellulase-A and protease-1; cellulase-A and protease-2; cellulase-B; cellulase-B and protease-1; cellulase-B and protease-2; cellulase-C; cellulase-C and protease-1; cellulase-C and protease-2; cellulase-D; cellulase-D and protease-1; cellulase-D and protease-2.
  • Figure 2 shows a result of SDS-PAGE gel.
  • the lanes from left to right are marker; protease-3; protease-4; cellulase-A; cellulase-A and protease-3; cellulase-A and protease-4; cellulase-B; cellulase-B and protease-3; cellulase-B and protease-4; cellulase-C; cellulase-C and protease-3; cellulase-C and protease-4; cellulase-D; cellulase-D and protease-3; cellulase-D and protease-4.
  • Figure 3 shows a result of SDS-PAGE gel.
  • the lanes from left to right are marker; protease-5; protease-6; cellulase-A; cellulase-A and protease-5; cellulase-A and protease-6; cellulase-B; cellulase-B and protease-5; cellulase-B and protease-6; cellulase-C; cellulase-C and protease-5; cellulase-C and protease-6; cellulase-D; cellulase-D and protease-5; cellulase-D and protease-6.
  • the present invention relates to a method for inactivating a cellulase, comprising treating the cellulease with a serine protease.
  • a serine protease can effectively inactivate a cellulase.
  • the protese is a family S8 protease.
  • the cellulase is GH45 cellulase.
  • the cellulase is an acid cellulase or neutral cellulase.
  • the present invention relates to a method for treating a textile, comprising:
  • the textile is contacted with a serine protease to inactivate the cellulase.
  • the method of the present invention reduces chemicals, saves energy and resources, and reduces a waste stream.
  • the method of the present invention has a reduced weight loss of textile, and/or a reduced loss in textile strength, relative to a method without step (b) .
  • the method of the present invention has reduced about 5%to about 100%, preferably from about 10%to about 100%, more preferably from about 20%to about 90%weight loss of textile, and/or a loss in textile strength, relative to a method without step (b) .
  • the textile is treated with the cellulase for a period of time to allow the cellulase work sufficiently on the textile in step (a) .
  • the textile is treated with the protease for a period of time to allow the protease inactivate the cellulase in step (b) .
  • Step (a) and step (b) can be carried out sequentially.
  • the term "sequential" with reference to a plurality of enzymatic treatments of a textile means that a second specified enzymatic treatment is performed after a first specified enzymatic treatment is performed. Sequential treatments may be separated by intervening wash steps.
  • sequential enzymatic treatments may be performed "in a same bath, " meaning in the substantially the same liquid medium without intervening wash steps.
  • Single-bath sequential treatment may include pH adjustments, temperature adjustment, and/or the addition of salts, activators, mediators, and the like, but should not include washes or rinses.
  • step (a) and step (b) are carried out in a single bath or two sequential baths.
  • Polypeptides having protease activity, or proteases are sometimes also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes.
  • Proteases may be of the exo-type that hydrolyses peptides starting at either end thereof, or of the endo-type that act internally in polypeptide chains (endopeptidases) . Endopeptidases show activity on N-and C-terminally blocked peptide substrates that are relevant for the specificity of the protease in question.
  • prote includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof) .
  • the EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively.
  • the nomenclature is regularly supplemented and updated; see e.g. the World Wide Web (WWW) at http: //www. chem. qmw. ac. uk/iubmb/enzyme/index. html.
  • Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate.
  • a given protease is a serine protease, and a family S8 protease
  • the database is described in Rawlings, N.D., Barrett, A.J. &Bateman, A. (2010) MEROPS: the peptidase database. Nucleic Acids Res 38, D227-D233.
  • the family S8 proteases contain the catalytic triad in the order Asp, His, Ser. Mutation of any of the amino acids of the catalytic triad will result in change or loss of enzyme activity.
  • amino acids of the catalytic triad of the S8 protease 1 from Bacillus sp-13231 are probably positions Asp32, His62 and Ser215.
  • the serine protease is a family S8 protease.
  • Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question.
  • Assay-pH and assay-temperature are likewise to be adapted to the protease in question.
  • assay-pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
  • assay-temperatures are 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C.
  • protease substrates are casein, such as Azurine-Crosslinked Casein (AZCL-casein) , or suc-AAPF-pNA.
  • Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • subtilases refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • the serine protease is subtilase, especially subtilisin.
  • subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140) .
  • the subtilisin comprises or consists of Bacillus Lentus protease shown in SEQ ID NO: 1 of the present invention.
  • WO92/19729 examples include the variants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263, WO11/036264, especially the variants with mutations in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255,
  • subtilase variants may comprise one or more of the mutations: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M
  • the protease variants are preferably variants of the Bacillus lentus protease shown in SEQ ID NO: 1 of the present invention, the Bacillus amylolichenifaciens protease (BPN’) shown in SEQ ID NO: 2 of WO2016/001449.
  • the protease variants preferably have at least 60%, at least 70%, at least 80%, 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%, or at least 99%sequence identity to SEQ ID NO: 1 of the present invention.
  • Suitable commercially available protease enzymes include those sold under the trade names Duralase Tm , Durazym Tm , Ultra, Ultra, Ultra, Ultra, Blaze 100T, Blaze 125T, Blaze 150T, and (Novozymes A/S) , those sold under the tradename Purafect Purafect Excellenz P1000 TM , Excellenz P1250 TM , Preferenz P100 TM , Purafect Preferenz P110 TM , Effectenz P1000 TM , Effectenz P1050 TM , Purafect Effectenz P2000 TM , and (Danisco/DuPont) , Axapem TM (Gist-Brocases N.V. ) , BLAP (sequence shown in Figure 29 of US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.
  • Cellulase or “cellulolytic enzyme” is a group of glycoside hydrolase enzymes that catalyze the hydrolysis of beta-1, 4-glycosidic linkages in the cellulose polymer.
  • the cellulase is GH45 cellulase.
  • the GH Family 45 cellulase enzymes (formerly Family K) act with inversion of anomeric configuration to generate the alpha-D anomer of the oligosaccharide as a product. It has been elucidated that, in the active site, one aspartic acid amino acid acts as a general acid and another as a general base.
  • the three dimensional structure of Family 45 enzymes has been elucidated (see, for example, the structure of Humicolainsolens in Davies et al, 1996, ActaCrystallographica Section D-Biological Crystallography 52: 7-17 Part 1) .
  • the enzymes contain a six-stranded beta-barrel to which a seventh strand is appended.
  • the structure contains both parallel and anti-parallel beta-strands.
  • the active center is located in an open substrate-binding groove.
  • GH45 cellulase As used herein, the term “GH45 cellulase” , “Family 45 cellulase” or “Cel45” means a carbohydrate active cellulase enzyme that contains a glycoside hydrolase Family 45 catalytic domain that is classified under EC 3.2.1.4.
  • the term encompasses a carbohydrate active enzyme that hydrolyzes cellulose and cello-oligosaccharides using an inverting mechanism, and has either of the following two signature sequences in the vicinity of the catalytic aspartic acid amino acids: (i) both a first conserved signature sequence of A/S/T -T -R/N/T -Y/F/T -X -D -X -X -X -X -C/A-A/G/S-W/C and a second conserved signature sequence of H/Q/D/N - F/L -D -I/L/F; or (ii) has the second conserved signature sequence of H/Q/D/N -F/L -D -I/L/F but lacks said first conserved sequence.
  • the second conserved signature sequence is H-F-D-I.
  • Organism Abreviated Name GenBank Accession Number Trichoderma reesei TrCel45A CAA83846.1 Trichomderma viride TvEGV AAQ21385.1 Penicillium decumbens PdCel 45A ACF33814.1 Aspergillus nidulans AnAN6786.2 EAA58604.1 Hadiotis discus discus HddEG1 ABO26608.1 Ampullaria crossean AcEG27I ABR92637.1 Ampullaria crossean AcEG27II ABR92638.1 Mytilus edulis MeEG CAC59695.1 Phanerochaete chrysosporium PcCel45A BAG68300.1
  • the cellulase of the present invention may be obtained from microorganisms or plants or animals of any genus.
  • the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • the cellulase may be a bacterial polypeptide.
  • the cellulase may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having cellulase activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
  • the cellulase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.
  • the cellulase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.
  • the cellulase is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.
  • the cellulase may be a fungal polypeptide.
  • the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Mycelioph
  • the cellulase is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.
  • the cellulase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fu
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the cellulase may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc. ) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc. ) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
  • the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra) .
  • the cellulase has at least 60%, at least 70%, at least 80%, 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%, or at least 99%sequence identity to the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7, or the mature polypeptide of SEQ ID NO: 8 of the present invention.
  • the cellulase comprises or consists of the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7, or the mature polypeptide of SEQ ID NO: 8.
  • the cellulase can be an acid cellulase, neutral cellulase, or alkaline cellulase.
  • the term “acid cellulase” means a cellulase which has an optimum activity at a pH below 7, preferably from 2 to 6, more preferably from 3.5 to 5.5.
  • neutral cellulase means a cellulase which has an optimum activity at a pH around 7, for example 5.5 to 8.5, preferably from 6 to 8.
  • alkaline cellulase means a cellulase which has an optimum activity at a pH above 8, for example 8 to 12, preferably from 8 to 10.
  • the cellulase is an acid cellulase, or neutral cellulase.
  • the acid cellulase is an enzyme mixture composed of three major enzymes (Trichoderma reesei cellobiohydrolase I, II and endoglucanase II) expressed in Trichoderma reesei cellulase background.
  • Trichoderma reesei cellobiohydrolase I is a 54.1 kDa enzyme (EC 3.2.1.176) .
  • Trichoderma reesei cellobiohydrolase II is a 49.6 kDa enzyme (EC 3.2.1.91) .
  • Trichoderma reesei endoglucanase II is a 44.1 kDa enzyme (EC 3.2.1.21) .
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity” .
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) , preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the term “textile” refers to fibers, yarns, fabrics, garments, and non-wovens.
  • the term encompasses textiles made from natural, synthetic (e.g., manufactured) , and various natural and synthetic blends. Textiles may be unprocessed or processed fibers, yarns, woven or knit fabrics, non-wovens, and garments and may be made using a variety of materials, some of which are mentioned, herein.
  • the process of the invention is most beneficially applied to cellulose-containing textile or cellulosic fabrics, such as cotton, viscose, rayon, ramie, linen, Tencel, or mixtures thereof, or mixtures of any of cellulose-containing fibres, or mixtures of any of these fibres together with synthetic fibres such as mixtures of cotton and spandex (stretch-denim) .
  • the fabric is dyed fabric.
  • the fabric is denim.
  • the denim fabric may be dyed with vat dyes such as indigo, or indigo-related dyes such as thioindigo.
  • textile is cotton-containing textile or man-made cellulose-containing textile.
  • biofinishing refers to the treatment of textile using cellulases and includes, but not limited to, biopolishing and biostoning.
  • biofinishing activity is determined as set forth in Examples.
  • the biopolishing effectiveness of the cellulases can be measured by the activity in removing fuzz, or small balls of fuzz (referred to as pills) , from fabric.
  • the depilling can be expressed as the depilling activity per unit of protein (i.e., specific depilling activity) .
  • an assay that measures biofinishing activity is a pilling note test for biopolishing activity.
  • the processing of a fabric, such as of a cellulosic material, into material ready for garment manufacture involves several steps: spinning of the fiber into a yarn; construction of woven or knit fabric from the yarn; and subsequent preparation processes, dyeing/printing and finishing operations.
  • Preparation processes are necessary for removing natural and man-induced impurities from fibers and for improving their aesthetic appearance and processability prior to for instance dyeing/printing and finishing.
  • Common preparation processes comprise desizing (for woven goods) , scouring, and bleaching, which produce a fabric suitable for dyeing or finishing.
  • Woven fabric is constructed by weaving “filling” or “weft” yarns between warp yarns stretched in the longitudinal direction on the loom.
  • the warp yarns must be sized before weaving in order to lubricate and protect them from abrasion at the high speed insertion of the filling yarns during weaving.
  • Common size agents are starches (or starch derivatives and modified starches) , poly (vinyl alcohol) , carboxyl methyl cellulose (i.e., CMC) where starches are dominant. Paraffin, acrylic binders and variety of lubricants are often included in the size mix.
  • the filling yarn can be woven through the warp yarns in a “over one -under the next” fashion (plain weave) or by “over one -under two” (twill) or any other myriad of permutations.
  • dresses, shirts, pants, sheeting’s, towels, draperies, etc. are produced from woven fabric. After the fabric is made, size on the fabric must be removed again (i.e., desizing) .
  • Knitting is forming a fabric by joining together interlocking loops of yarn.
  • weaving which is constructed from two types of yarn and has many “ends”
  • knitted fabric is produced from a single continuous strand of yarn.
  • Desizing is the degradation and/or removal of sizing compounds from warp yarns in a woven fabric. Starch is usually removed by an enzymatic desizing procedure. In addition, oxidative desizing and chemical desizing with acids or bases are sometimes used.
  • the desizing enzyme is an amylolytic enzyme, such as an alpha-amylase, a beta-amylase, a mannanase, a glucoamylase, or a combination thereof.
  • Suitable alpha and beta-amylases include those of bacterial or fungal origin, as well as chemically or genetically modified mutants and variants of such amylases.
  • Suitable alpha-amylases include alpha-amylases obtainable from Bacillus species.
  • Suitable commercial amylases include but are not limited to NEXT, FLEX and COOL (all from Genencor International Inc. ) , and DURAMYL TM , ERMAMYL TM , FUNGAMYL TM TERMAMYL TM , AQUAZYME TM and BAN TM (all available from Novozymes A/S, Bagsvaerd, Denmark) .
  • amylolytic enzymes include the CGTases (cyclodextrin glucanotransferases, EC 2.4.1.19) , e.g., those obtained from species of Bacillus, Thermoanaerobactoror Thermoanaero-bacterium.
  • CGTases cyclodextrin glucanotransferases, EC 2.4.1.19
  • Scouring is used to remove impurities from the fibers, to swell the fibers and to remove seed coat. It is one of the most critical steps.
  • the main purposes of scouring is to a) uniformly clean the fabric, b) soften the motes and other trashes, c) improve fabric absorbency, d) saponify and solubilize fats, oils, and waxes, and e) minimize immature cotton.
  • Sodium hydroxide scouring at about boiling temperature is the accepted treatment for 100%cotton, while calcium hydroxide and sodium carbonate are less frequently used.
  • Synthetic fibers are scoured at much milder conditions. Surfactant and chelating agents are essential for alkaline scouring. Enzymatic scouring has been introduced, wherein cellulase, hemicellulase, pectinase, lipase, and protease are all reported to have scouring effects.
  • Bleaching is the destruction of pigmented color and/or colored impurities as well as seed coat fragment removal. Bleaching is performed by the use of oxidizing or reducing chemistry. Oxidizing agents can be further subdivided into those that employ or generate: a) hypochlorite (OCl - ) , b) chloride dioxide (ClO 2 ) , c) permanganate (MnO 4 -) , d) ozone, and hydroperoxide species (OOH - and/or OOH) . Reducing agents are typical sulfur dioxide, hydrosulfite salts, etc. Enzymatic bleaching using glucose oxidase or peroxidase (for example, see WO 2013/040991) has been reported. Traditionally, hydrogen peroxide is used in this process.
  • Printing and dyeing of textiles is carried out by applying dyes to the textile by any appropriate method for binding the dyestuff to the fibres in the textiles.
  • the dyeing of textiles may for example be carried out by passing the fabric through a concentrated solution of dye, followed by storage of the wet fabric in a vapour tight enclosure to permit time for diffusion and reaction of the dye with the fabric substrate prior to rinsing off un-reacted dye.
  • the dye may be fixed by subsequent steaming of the textile prior to rinsing.
  • the dyes include synthetic and natural dyes.
  • Typical dyes are those with anionic functional groups (e.g., acid dyes, direct dyes, Mordant dyes and reactive dyes) , those with cationic groups (e.g., basic dyes) , those requiring chemical reaction before application (e.g., vat dyes, sulphur dyes and azoic dyes) , disperse dyes and solvent dyes.
  • anionic functional groups e.g., acid dyes, direct dyes, Mordant dyes and reactive dyes
  • cationic groups e.g., basic dyes
  • those requiring chemical reaction before application e.g., vat dyes, sulphur dyes and azoic dyes
  • disperse dyes and solvent dyes e.g., disperse dyes and solvent dyes.
  • Biopolishing is a method to treat cellulosic fabrics during their manufacture by enzymes such as cellulases, which improves fabric quality with respect to “reduced pilling formation” .
  • the most important effects of biopolishing can be characterized by less fuzz and pilling, increased gloss/luster, improved fabric handle, increased durable softness and/or improved water absorbency.
  • Biopolishing usually takes place in the wet processing of the manufacture of knitted and woven fabrics or garments. Wet processing comprises such steps as e.g., desizing, scouring, bleaching, washing, dying/printing and finishing. Biopolishing could be performed as a separate step after any of the wetting steps or in combination with any of those wetting steps. In the present invention, the step of biofinishing is carried out before, during or after step of desizing, bleaching, or printing/dyeing.
  • CMCU/ml concentration of enzyme in an aqueous solution
  • Determination of suitable temperature or pH of the the aqueous solution can be achieved using routine experimentation by establishing a matrix of conditions and testing different points in the matrix.
  • the enzyme concentration, the temperature or pH at which the contacting occurs, and the time of contact can be varied, after which the resulting fiber or textile is evaluated for (a) one or more biopolished properties, such as, e.g., fabric handle, appearance, or pilling resistance, and, optionally, (b) potential loss in fabric strength and/or weight.
  • Fabric handle and appearance are evaluated by panel testing, using a rating of 1-3 (worst to best) .
  • Pilling can be measured using any conventional method, such as, e.g., according to American Society for Testing and Materials protocol ASTM D 4970-89, using a Martindale Abrasion and Pilling Tester (James H. Heal &Co, UK) . In this method, pilling is evaluated visually on a scale of 1 to 5, where 1 signifies severe pilling and 5 signifies no pilling.
  • Fabric strength is measured using any conventional method, such as, e.g., according to ASTM protocol D 3786-87, using a Mullen Burst tester (Model C, B. F. Perkins, Chicopee MA) .
  • biopolishing is carried out at a temperature of between about 20°C and about 75 °C, preferably between about 25 °C to about 70 °C, and most preferably between about 25 °C and about 60 °C; and at a pH of between about 4 and 12, preferably between about 5 and 10, and most preferably between about 5 and 8.
  • residual cellulases are inactivated by an increase of treatment temperature and/or an adjustment of pH by harsh chemicals, for example, Na 2 CO 3 , and washed with a large amount of water.
  • the temperature can be raised to a temperature higher than the biopolishing temperature. In one embodiment, the temperature is raised to between about 75°C and about 100 °C.
  • the pH can be adjusted to pH lower or higher than the pH for biopolishing. In one embodiment, pH is adjusted to between about 0 and 3 or about 10 and 14.
  • high treatment temperature or adjustment of pH may need a lot of energy, and harsh chemials, and it may cause low fabric handle and softness, and change the color tone.
  • the present invention can effectively inactivate a cellulase by the action of a serine protease. It is mild and environmentally friendly by saving energy and water, and reducing comsumption of harsh chemials. It can work at biopolishing temperature and pH, and lead to a better fabric handle and softness and brighter fabric color.
  • residual cellulase after biopolishing step further delivers about 1%, about 5%, about 10%, about 15%, about 20%, 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or about 100%, for example, from about 10%to about 100%, preferably from about 20%to about 90%, more preferably from about 30%to about 80%, lower cellulase activity with a serine protease inactivation than the cellulases without a serine protease inactivation after biopolishing.
  • a serine protease inactivates or inhibits from about 10%to about 100%, preferably from about 20%to about 90%, more preferably from about 30%to about 80%cellulase activity.
  • a residual cellulase after biopolishing step further delivers about 0.1, about 0.2, about 0.5, about 0.8, about 1.0 pilling note lower than residual cellulase without a serine protease inactivation.
  • the yarns are dyed before weaving.
  • the warp yarns are dyed for example with indigo, and sized, before weaving.
  • the dyeing of the denim yarn is a ring-dyeing.
  • a preferred embodiment of the invention is ring-dyeing of the yarn with a vat dye such as indigo, or an indigo-related dye such as thioindigo, or a sulfur dye, or a direct dye, or a reactive dye, or a naphthol.
  • the yarn may also be dyed with more than one dye, e.g., first with a sulphur dye and then with a vat dye, or vice versa.
  • the yarns undergo scouring and/or bleaching before they are dyed, in order to achieve higher quality of denim fabric.
  • the dyed fabric or garment proceeds to a desizing stage, preferably followed by a stoning or abrasion step and/or a color modification step.
  • the desizing process as used herein is the same process as mentioned above in the text.
  • the dyed fabric undergoes a biostoning step.
  • the biostoning step can be performed with enzymes or pumice stones or both.
  • biostoning “stone washing” and “abrasion” are interchangeable, which means agitating the denim in an aqueous medium containing a mechanical abrasion agent such as pumice, an abrading cellulase or a combination of these, to provide a “stone-washed” look.
  • mechanical action is needed to remove the dye, and the treatment is usually carried out in washing machines, like drum washers, belly washers. As a result of uneven dye removal there are contrasts between dyed areas and areas from which dye has been removed.
  • Treatment with cellulase can completely replace treatment with pumice stones.
  • cellulase treatment can also be combined with pumice stone treatment, when it is desired to produce a heavily abraded finish.
  • biofinishing includes “biostoning” .
  • CMCU/ml concentration of enzyme in an aqueous solution
  • Determination of suitable temperature or pH of the the aqueous solution can be achieved using routine experimentation by establishing a matrix of conditions and testing different points in the matrix.
  • the enzyme concentration, the temperature or pH at which the contacting occurs, and the time of contact can be varied, after which the resulting fiber or textile is evaluated for (a) one or more biostoned properties, such as, e.g., “stone-washed” look, and, optionally, (b) potential loss in fabric strength and/or weight.
  • biostoning is carried out at a temperature of between about 20°C and about 75 °C, preferably between about 25 °C to about 70 °C, and most preferably between about 25 °C and about 60 °C; and at a pH of between about 4 and 12, preferably between about 5 and 10, and most preferably between about 5 and 8.
  • the present invention can effectively inactivate a cellulase by the action of a serine protease. It is mild and environmentally friendly by saving energy and water, and reducing harsh chemials. It can work at biostoning temperature and pH, and lead to a better fabric handle and softness and brighter fabric color.
  • a residual cellulase further delivers about 1%, about 5%, about 10%, about 15%, about 20%, 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or about 100%, for example, from about 10%to about 100%, preferably from about 20%to about 90%, more preferably from about 30%to about 80%, lower cellulase activity than a cellulase without a serine protease inactivation after biostoning.
  • a serine protease inactivates or inhibits from about 10%to about 100%, preferably from about 20%to about 90%, more preferably from about 30%to about 80%cellulase activity.
  • the abrasion is followed by a color modification step.
  • color modification or “color adjustment” are used without distinction to refer to any change to the color of a textile resulting from the destruction, modification, or removal of a dyestuff associated with the textile.
  • color modification results from the modification of chromaphores associated with a textile material, thereby changing its visual appearance.
  • the chromophores may be naturally-associated with the material used to manufacture a textile (e.g., the white color of cotton) or associated with special finishes, such as dying or printing.
  • Color modification encompasses chemical modification to a chromophore as well as chemical modification to the material to which a chromophore is attached.
  • a method for inactivating a cellulase comprising treating the cellulease with a serine protease.
  • subtilase is derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and protease PD138.
  • Bacillus lentus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and protease PD138.
  • subtilase comprises or consists of SEQ ID NO: 1 or a variant thereof having at least 60%, at least 70%, at least 80%, 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%, or at least 99%sequence identity to SEQ ID NO: 1.
  • the variant is comprises mutations in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269 wherein the positions correspond to the positions of the Bacillus Lentus protease shown in SEQ ID NO: 1.
  • the variant comprises one or more of the mutations: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V19
  • cellulase is a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide, or a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
  • the cellulase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.
  • the cellulase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusa
  • cellulase comprises or consists of the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7, or the mature polypeptide of SEQ ID NO: 8.
  • a method for treating a textile comprising:
  • step (a) and step (b) are carried out in a single bath or in two sequential baths.
  • protease is a subtilase, preferably a subtilisin.
  • subtilase is derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and protease PD138.
  • Bacillus lentus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and protease PD138.
  • subtilase comprises or consists of SEQ ID NO: 1 or the variants thereof having at least 60%, at least 70%, at least 80%, 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%, or at least 99%sequence identity to SEQ ID NO: 1.
  • the variant comprises mutations in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269 wherein the positions correspond to the positions of the Bacillus Lentus protease shown in SEQ ID NO: 1.
  • the variant comprises one or more of the mutations: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V19
  • cellulase is a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide, or a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
  • the cellulase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.
  • cellulase is Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrich
  • the cellulase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusa
  • cellulase comprises or consists of the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7, or the mature polypeptide of SEQ ID NO: 8.
  • step (a) and/or step (b) is carried out at a temperature of between about 20°C and about 75 °C, preferably between about 25 °C to about 70 °C, and most preferably between about 25 °C and about 60 °C.
  • step (a) and/or step (b) is carried out at a pH of between about 4 and 12, preferably between about 5 and 10, and most preferably between about 5 and 8.
  • Buffer-1 2.135g of potassium dihydrogen phosphate and 0.37g sodium hydroxide dissolved in 1L de-ionized water to pH 7
  • Buffer-2 3.052g of potassium dihydrogen phosphate and 0.102g sodium hydroxide dissolved in 1L de-ionized water to pH 6
  • Buffer-3 1g/l sodium acetate adjusted with acetic acid to pH 5
  • the swatches were placed in the conditioned room (65%+/-5%humidity, 20+/-1°C) for 24 hours before they were numbered, weighed by the analytical balance (for samples below 100 g) or a precision balance (for samples over 100 g) and recorded. After treatment, all samples were tumbled dried for 1 hour and conditioned for 24 hours in the conditioned room mentioned as above. For each sample, the weight loss was defined as below:
  • the Bursting Strength Tester is used for determination of strength referring to GB/T19976-2005 or BS ISO 13938 -2: 1999. The area of the sample to be tested is clamped between steel rings. An increasing steel ball pressure is applied until the specimen burst. Test is conducted with Marble Bursting Strength Tester (commercially available from Darong Textile Instrument) and TruBurst (commercially available from James Heal) .
  • the enzyme protein in an enzyme product can be measured with BCA TM Protein Assay Kit (product number 23225, commercial available from Thermo Fisher Scientific Inc. ) according to the product manual.
  • the substrate carboxymethyl cellulose (CMC) was hydrolyzed with cellulase at pH 7.5, 50°C for 30 min.
  • the reaction is stopped by an alkaline reagent containing PAHBAH and bismuth which forms complexes with reducing sugar.
  • the complex formation results in color production which can be read at 405 nm by a spectrophotometer.
  • the produced color is proportional to the cellulase activity.
  • Enzymatic reaction and measurement of absorbance proceed automatically in the Konelab analyzer. Cellulase activity is determined relative to a Novozymes enzyme standard.
  • Enzyme samples were diluted to a concentration of 1 mg/ml enzyme.
  • each enzyme sample was filled into a 96-well Cell Culture Plate (commercially available from Eppendorf) .
  • the plate was sealed and transferred to a pre-heated Thermomixer Comfort and kept stirring at 600rpm for 60min at the temperature specified: cellulase-A, cellulase-B and cellulase-C at 55°C, and cellulase-D at 40°C.
  • SDS-PAGE gels ( Gel 1.5mm*15well from Invitrogen) were run according to Technical Guide. Marker ( Plus 2 Prestained, commercially available from Invitrogen) had a molecular weight of 198KDa, 98KDa, 62KDa, 49KDa, 38KDa, 28KDa, 17KDa, 14KDa, 6KDa, 3KDa.
  • protease-1 and protease-6 degraded all tested cellulases, while protease-2 and protease-3 degraded cellulase-D, protease-4 and protease-5 could not degrade cellulase-A.
  • the plate was sealed and transferred to a pre-heated Thermomixer Comfort and kept stirring at 600 rpm for 60min at the temperature specified: cellulase-A, cellulase-B and cellulase-C at 55°C, and cellulase-D at 40°C.
  • One Cellazyme C tablet (commercially available from Megazyme) was filled in and the tube was capped and whirl mixed strongly.
  • Fabric swatches were weighted to 5g each after balanced in the constant temperature and humidity environment.
  • Buffer and cellulase sample were drained and 2 g/l Sodium carbonate were added to the beaker with boiling water (>80°C) and stayed for 10min, drained, rinsed with cold water and spinned off the water on the fabrics and tumble dryer.
  • step 3-1 Blank and cellulase sample were run continuously for 1 hour at 40°C. After that, step 3-1 was carried out.
  • step 3-3 Protease was added to the cellulase sample and then run for 1 hour at 40°C. After that, step 3-1 was carried out.
  • protease-1 and protease-6 had a better inactivating effect on a cellulase-D than protease-2, protease-3, protease-4 and protease-5, indicating by less weight loss and strength loss of fabric.
  • %weight/strength loss to ref. 100* (weight/strength loss-sample -weight/strength loss-1h) / (weight/strength loss-2h -weight/strength loss-1h)
  • protease-1 and protease-6 had a better inactivating effect on a cellulase-B than protease-2, protease-3, protease-4 and protease-5, indicating by less weight loss of fabric.
  • protease-1 and protease-6 had a better inactivating effect on a cellulase-E than protease-2, protease-3, protease-4 and protease-5, indicating by less weight loss of fabric.
  • Step 1 biopolishing with 0.2 %cellulase-D on weight of fabric in Wascator:
  • Step 2 Fabrics were divided into 3 groups and cellulase inactivation was done in 3 ways. For group 1, 8 pieces of fabrics were rinsed twice for 10 and 5 min respectively at room temperature; drained; centrifuged.
  • Step 3 the fabrics from step 2 were dried immediately, and 1 piece of fabric from each group was set as the reference of 100%bursting strength for each group.
  • Step 4 7 pieces of fabrics from each group were post washed separately at 40 °C for 10 h, drained by water and re-filled every 2 h.
  • Step 5 fabric conditioning and evaluation for bursting strength was conducted with TruBurst.

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Abstract

L'invention concerne une méthode d'inactivation d'une cellulase avec une sérine protéase et une méthode de traitement d'un textile.
PCT/CN2018/102880 2017-08-31 2018-08-29 Méthode d'inactivation de cellulase WO2019042306A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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