EP2658968A1 - Procédé de traitement d'un textile avec l'endoglucanase - Google Patents

Procédé de traitement d'un textile avec l'endoglucanase

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Publication number
EP2658968A1
EP2658968A1 EP11853772.9A EP11853772A EP2658968A1 EP 2658968 A1 EP2658968 A1 EP 2658968A1 EP 11853772 A EP11853772 A EP 11853772A EP 2658968 A1 EP2658968 A1 EP 2658968A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
textile
seq
cbm
fabric
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11853772.9A
Other languages
German (de)
English (en)
Other versions
EP2658968A4 (fr
Inventor
Weijian Lai
Guifang Wu
Ye Liu
Yucheng Zhou
Yang HAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP2658968A1 publication Critical patent/EP2658968A1/fr
Publication of EP2658968A4 publication Critical patent/EP2658968A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • 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
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/13—Fugitive dyeing or stripping dyes
    • D06P5/137—Fugitive dyeing or stripping dyes with other compounds
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/15—Locally discharging the dyes
    • D06P5/158—Locally discharging the dyes with other compounds
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
    • D06P1/22—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using vat dyestuffs including indigo
    • D06P1/228—Indigo

Definitions

  • the present invention relates to the method for manufacturing textile, by treating textile with an isolated polypeptide having endoglucanase activity, especially in biostoning and bio- polishing process.
  • cel- lulases are used in denim finishing to create a fashionable stone washed appearance on denim cloths using a biostoning process.
  • Cellulases are also used, for instance, to clean fuzz and prevent formation of pills on the surface of cotton garments using a biopolishing process.
  • a general problem associated with enzymatic stone washing is the backstaining caused by redeposition of removed Indigo dye during or after abrasion.
  • the "backstaining" or “redeposi- tion” of Indigo dye reduces the desired contrast between the white and indigo dyed yarns and it can be most easily distinguished on the reverse side of denim and the interior pockets (as increased blueness). On the face side of the denim this may be seen as reduced contrast between dyed areas and areas from which dye has been removed during biostoning.
  • the denim manufacturers use large amounts of surfactants to make the redeposited parts white again for example to increase the contrast between abraded parts and non abraded parts of the denim in a soaping process.
  • the heavy wash condition causes colour change or colour-fading problems for finished denim products. Also additional water has to be used in the subsequent soaping process.
  • anti-redeposition chemicals such as surfactants or other agents into the cellulase wash.
  • WO 97/09410 describes that the addition of a certain type of cellulase to another cellulase having abrading activity reduces backstaining.
  • the additional cellulase belongs to glycosyl hydrolase family 5 or 7, but it has no significant abrading effect by itself.
  • WO 01/92453 discloses backstaining reduction by treating textile with a cutinase.
  • WO 91/17243 and WO 95/09225 describe a process using a single-component endoglucanase denoted EGV with a molecular weight of 43 kD derived from Humicola insolens strain DSM 1800.
  • WO 94/21801 describes the use in "stone washing" of a single-component endoglucanase called EGIII derived from Trichoderma longibrachia- turn.
  • WO 95/16782 suggests the use of other single-component endoglucanases derived from Trichoderma in "stone washing”.
  • WO 2009/103237 discloses the use of an endoglucanase derived from Aspergillus fumigates in "stone washing".
  • Biopolishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance.
  • 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. Wet processing comprises such steps as e.g. desizing, scouring, bleaching, washing, dying/printing and finishing.
  • the present invention aims to meet these needs.
  • the present invention relates to a method for treating textile, by treating textile with an isolated polypeptide which has endoglucanase activity but does not comprise a functional CBM, selected from the group consisting of:
  • polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 , or (iii) the full-length complementary strand of (i) or (ii);
  • the method may be applied to a biostoning process to form localized variation of color density in the surface of a dyed cellulosic or cellulose-containing textile.
  • the dyed cellulosic or cellulose-containing fabric is a denim fabric, more preferabley in- digo dyed denim fabric.
  • the biostoning process shows abrasion effect of at least 0.5 Delta L * unit, preferably at least 1 , more preferably 1.5, and more preferably 2 and even more preferably 2.5 Delta L * unit.
  • the biostoning process may further comprise one or more en- zymes selected from the group consisting of proteases, lipases, cutinases, amylases, pectinas- es, hemicellulases and cellulases.
  • the method may be applied to a biopolishing process.
  • the biopolishing process shows pilling note of at least 3, more preferably at least 3.4, more preferably at least 3.8, and most preferably at least 4. More preferably, the biopolishing process fur- ther shows weight loss of less than 10%, preferably less than 5%, more preferably less than 4%, more preferably less than 3.5%, and most preferably less than 3%.
  • the method for manufacturing textile is provided.
  • the textile is manufactured from fabric to garment.
  • the textile is cellulose-containing or cellulosic textile. In some embo- diments, the textile is denim.
  • the polypeptide of the present invention when used alone, i.e. without other enzymes, especially without other cellulases, can provide both increased abrasion effect and low backstaining level during a biostoning process.
  • the method of the present invention can obtain a cellulosic textile fabric with strongly reduced pilling formation but without substantial weight loss of fabric in a biopolishing process, especially in the presence of surfactant.
  • the further advantage of the present invenetion is that the method can be conducted in low temperature, such as below 50°C, so as to save energy in textile manufacturing process.
  • Figure 1 shows a restriction map of pRenoCBD.
  • Figure 2 shows a restriction map of pRenoCore.
  • Endoglucanase means an endo-1 ,4-(1 ,3;1 ,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1 .4), which catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452- 481 ).
  • endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of part VI in page 264 of Ghose, 1987, Pure and Appl. Chem. 59: 257-268.
  • the endoglucanase has at least two functional domains, a carbohydrate-binding module (CBM) and a catalytic module.
  • CBM carbohydrate-binding module
  • the catalytic module is defined as an amino acid sequence that is capable of enzymatically cleaving cellulose, e.g. has endoglucanase activity.
  • the catalytic module is not considered to be a carbohydrate-binding module.
  • a "linker sequence" connects the two functional modules.
  • Carbohydrate-binding module is defined as an amino acid sequence that binds to a substrate. CBMs are for example described in Boraston et al, Biochem. J. (2004) 382, 769-781 and in Tomme et al., John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1995. It is believed that the CBM binding to the substrate which increases the efficacy of the catalytic active part of the enzyme.
  • CBM cellulose-binding domain
  • CBD cellulose-binding domain
  • Binding activity can be determined, for example, by binding to microcrystalline cellulose such as Avicel and showing that the putative binding module is removed from solution.
  • a polypeptide having endoglucanase activity but lacking a fuc- tional CBM can either lack a CBM sequence entirely, or may contain a residue CBM sequence that has been modified to destroy its cellulose-binding activity, by deletion, addition, and/or substitution of one or more amino acid residues or by any chemical or enzymatic modification of the intact protein; such a modified sequence is also referred to as a polypeptide having endoglucanase activity with a non-functional CBM.
  • a polypeptide having endoglucanase activity but lacking a fuctional CBM can be identified using, for example, the method describe in Example 6 below, which involves incubation of the enzyme with cellulose substrate Avicel to allow binding, followed by centrifugation and detection of the protein content in the supernant.
  • a polypeptide having endoglucanase activity but lacking a fuctional CBM has a low affinity for Avicel in the assay, which leads to high protein content remained in the supernant.
  • a polypeptide having endoglucanase activity but lacking a fuctional CBM shows no more than 15% absorption, more preferably no more than 14% absorption, more preferably no more than 13% absorption, more preferably no more than 12% absorption, more preferably no more than 1 1 % absorption, even more preferably no more than 10% absorption as defined in Example 6.
  • a polypeptide having endoglucanase activity but lacking a CBM sequence entirely can be a native (wild-type) endoglucanse.
  • CBM is a polypeptide having endoglucanase activity with CBM truncated by any of a variety of techniques, including biochemical or genetic engineering techniques.
  • Isolated polypeptide The terms "isolated” and “purified” mean a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated.
  • a polypeptide may be at least 1 % pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE and a polynucleotide may be at least 1 % pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose electrophoresis.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 22 to 237 of SEQ ID NO: 2 and amino acids 1 to 21 of SEQ ID NO: 2 are a signal peptide based on the program SignalP (Nielsen et al., 1997, Protein Engineering 10: 1 -6).
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having endoglucanase activity.
  • the mature polypeptide coding sequence is nucleotides 64 to 858 of SEQ ID NO: 1 and nucleotides 1 to 63 of SEQ ID NO: 1 encode a signal peptide based on the program SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6).
  • Sequence Identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
  • fragment means a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has endoglucanase activity.
  • Subsequence means a polynucleotide having one or more (several) nucleotides deleted from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having endoglucanase activity.
  • allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA.
  • the coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
  • Control sequences means all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • Operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
  • Expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to additional nucleotides that provide for its expression.
  • Host cell means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • variant means a polypeptide having endoglucanse activity compris- ing an alteration, i.e., a substitution, insertion, and/or deletion of one or more (several) amino acid residues at one or more (several) positions.
  • a substitution means a replacement of an amino acid occupying a position with a different amino acid;
  • a deletion means removal of an amino acid occupying a position; and
  • an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
  • Textile The term “textiles” used herein is meant to include fibers, yarns, fabrics and garments.
  • Fabric can be constructed from fibers by weaving, knitting or non-woven operations. Weaving and knitting require yarn as the input whereas the non-woven fabric is the result of random bonding of fibers (paper can be thought of as non-woven).
  • the term "fabric" is also intended to include fibers and other types of processed fabrics.
  • the method of the invention may be applied to any textile known in the art (woven, knitted, or non-woven).
  • the process of the present invention may be applied to cellulose-containing or cellulosic textile, such as cotton, viscose, rayon, ramie, linen, lyocell ⁇ e.g., Tencel, produced by Courtaulds Fibers), or mixtures thereof, or mixtures of any of these fibers together with synthetic fibres ⁇ e.g., polyester, polyamid, nylon) or other natural fibers such as wool and silk., such as viscose/cotton blends, lyocel l/cotton blends, viscose/wool blends, lyocell/wool blends, cotton/wool blends; flax (linen), ramie and other fabrics based on cellulose fibers, including all blends of cellulosic fibers with other fibers such as wool, polyamide, acrylic and polyester fibers, e.g., viscose/
  • the present invention relates to a method for treating textile, by treating textile with an isolated polypeptide which has endoglucanase activity but does not comprise a functional CBM, selected from the group consisting of:
  • polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 , or (iii) the full-length complementary strand of (i) or (ii);
  • polypeptide which can be obtained from SEQ ID NO: 2 by substitution, deletion, and/or insertion of one or more (or several) amino acids.
  • the isolated polypeptides having endoglucanase activity but without functional CBM have sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the polypeptides differ by no more than ten amino acids, e.g., by ten amino acids, nine amino acids, eight amino acids, seven amino acids, six amino acids, five amino acids, four amino acids, three amino acids, two amino acids, or one amino acid from the mature polypeptide of SEQ ID NO: 2.
  • the polypeptide identified above has a histidine at the position corresponding to position 141 when aligned with SEQ ID NO: 2. More preferably, the isolated polypeptide is a catalytic module of endoglucanse. Even more preferably, the isolated polypeptide of the present invention is a mature polypeptide of SEQ ID NO: 2.
  • the present invention also relates to isolated polypeptides having endoglucanse activity that are encoded by polynucleotides that hybridize under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 , or (iii) the full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • the polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having endoglucanase activity from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 14, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
  • the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having endoglucanse activity.
  • Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is preferably used in a Southern blot.
  • hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1 ; the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 , or the mature polypeptide coding sequence of SEQ ID NO: 1 ; its full-length complementary strand; or a subsequence thereof; under very low to very high stringency conditions.
  • Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.
  • the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1.
  • the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or a fragment thereof.
  • the nucleic acid probe is SEQ ID NO: 1.
  • very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
  • the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C (very low stringency), at 50°C (low stringency), at 55°C (medium stringency), at 60°C (medium-high stringency), at 65°C (high stringency), and at 70°C (very high stringency).
  • stringency conditions are defined as prehybridization and hybridization at about 5°C to about 10°C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1 X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.
  • the carrier material is finally washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T m .
  • the present invention also relates to isolated polypeptides having endoglucanse activity encoded by polynucleotides having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the polypeptide can be variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 2, or a homologous sequence thereof.
  • amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • amino acid residue corresponding to position 141 of SEQ ID NO: 2 is histidine.
  • SEQ ID NO: 2 can be obtained by substituting the glutamine residue (Q) with histidine (H) in the catalytic module of wild-type Thielavia terrestris endoglucanse in position corresponding to position 141 of SEQ ID NO: 2.
  • conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • the most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • One or more of the mutations like G43K, N71 E and Q168R could be introduced into SEQ ID NO: 2 of the present invention.
  • amino acid substitution the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of Glycine with Lysine at position 43 is designated as "Gly43Lys" or "G43K”.
  • Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for endoglucanse activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent polypeptide.
  • D33 and D143 in SEQ ID NO: 2 are essential amino acid, which is unchangeable in order to maintain its endoglucanse activity.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display ⁇ e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8 or 9.
  • the polypeptide may be hybrid polypeptide in which a portion of one polypeptide is fused at the N-terminus or the C-terminus of a portion of another polypeptide.
  • the polypeptide may be a fused polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
  • a fused polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
  • cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.
  • a polypeptide having endoglucanse activity but without a functional CBM, of the present invention may be obtained from microorganisms 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 polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a bacterial polypeptide.
  • Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having endoglucanase activity, or a Gram-negative bacterial polypeptide such as a
  • polypeptide is a Dictyoglomus thermophilum, or Dictyoglomus turgidum polypeptide.
  • the polypeptide may be an Archaea peptide.
  • the polypeptide may be a Pyrococcus polypeptide.
  • the polypeptide is Pyrococcus furiosus, Pyrococcus abyssi, Pyrococcus endeavori, Pyrococcus glycovorans, Pyrococcus horikoshii, Pyrococcus woesei polypeptide.
  • the polypeptide 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, Myceliophthora, Neocallimastix, Neurospora, Pae
  • the polypeptide 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, Fusa
  • 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. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the polypeptide 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 bulk solution containing the polypeptide having endoglucanase activity further comprises other components, including without limitation other enzymes, as well as one or more of surfactants, bleaching agents, antifoaming agents, build- er systems, and the like, that enhance the biopolishing and/or biostoning process and/or provide superior effects related to, e.g., dyeability and/or wettability.
  • the aqueous solution may also contain dyeing agents.
  • Enzymes suitable for use in the present invention include without limitation proteases, lipases, cutinases, amylases, hemicellulases, pectinases, and cellulases.
  • proteases are used in the present invention. Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may for example be a metalloprotease (EC 3.4.17 or EC 3.4.24) or a serine protease (EC 3.4.21 ), preferably an alkaline microbial protease or a trypsin-like protease.
  • proteases are subtilisins (EC 3.4.21.62), especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • subtilisin-like proteases are trypsin ⁇ e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Preferred commercially available protease enzymes include Alcalase ® , Savinase ® , Pri- mase ® , Duralase ® , Esperase ® , and Kannase ® (Novozymes A/S), Maxatase ® , Maxacal ® , Max- apem ® , Properase ® , Purafect ® , Purafect OxP ® , FN2TM, and FN 3TM (Genencor International Inc.).
  • Lipases In other embodiments of the present invention, lipases are used in the present invention. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants of such lipases are included in this connection.
  • the lipase may for example be triacylglyce- rol lipase (EC3.1 .1.3), phospholipase A2 (EC 3.1.1 .4), Lysophospholipase (EC 3.1.1 .5), Monog- lyceride lipase (EC 3.1.1 .23), galactolipase (EC 3.1 .1 .26), phospholipase A1 (EC 3.1.1.32), Lipoprotein lipase (EC 3.1 .1 .34).
  • triacylglyce- rol lipase EC3.1 .1.3
  • phospholipase A2 EC 3.1.1 .4
  • Lysophospholipase EC 3.1.1 .5
  • Monog- lyceride lipase EC 3.1.1 .23
  • galactolipase EC 3.1 .1 .26
  • phospholipase A1 EC 3.1.1.32
  • Lipoprotein lipase EC 3.1 .1
  • useful lipases include a Humicola lanuginosa lipase, e.g., as described in EP 258 068 and EP 305 216; a Rhizomucor miehei lipase, e.g., as described in EP 238 023 or from H. insolens as described in WO 96/13580; a Candida lipase, such as a C. antarctica lipase, e.g., the C.
  • antarctica lipase A or B described in EP 214 761 a Pseudomonas lipase, such as one of those described in EP 721 981 ⁇ e.g., a lipase obtainable from a Pseudomonas sp. SD705 strain having deposit accession number FERM BP-4772), in PCT/JP96/00426, in PCT/JP96/00454 ⁇ e.g., a P. solanacearum lipase), in EP 571 982 or in WO 95/14783 ⁇ e.g., a P. mendocina lipase), a P. alcaligenes or P.
  • a Pseudomonas lipase such as one of those described in EP 721 981 ⁇ e.g., a lipase obtainable from a Pseudomonas sp. SD705 strain having deposit accession number FERM BP
  • pseudoalcaligenes lipase e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in GB 1 ,372,034, or a P. fluorescens lipase; a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al. (1993) Biochemica et Biophysica Acta 1131 :253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
  • a Bacillus lipase e.g., a B. subtilis lipase (Dartois et al. (1993) Biochemica et Biophysica Acta 1131
  • Suitable commercially available lipases include Lipex ® , Lipolase ® and Lipolase Ultra ® , Lipo- lex ® , Lipoclean ® (available from Novozymes A/S), M1 LipaseTM and LipomaxTM (available from Genencor Inc.) and Lipase P "Amano” (available from Amano Pharmaceutical Co. Ltd.).
  • Commercially available cutinases include LumafastTM from Genencor Inc.
  • cutinases are used in the present invention.
  • Potentially useful types of lipolytic enzymes include cutinases (EC 3.1 .1.74), e.g., a cutinase derived from Pseudo- monas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (described, e.g.,, in WO 90/09446). Due to the lipolytic activity of cutinases they may be effective against the same stains as lipases.
  • Commercially available cutinases include LumafastTM from Genencor Inc.
  • amylases are used in the present invention.
  • Amylases comprise e.g., alpha-amylases (EC 3.2.1.1 ), beta-amylases (EC 3.2.1 .2) and/or glucoamylases (EC 3.2.1 .3) of bacterial or fungal origin. Chemically or genetically modified mutants of such amylases are included in this connection.
  • Alpha-amylases are preferred in relation to the present invention.
  • Relevant alpha-amylases include, for example, oamylases obtainable from Bacillus spe- cies, in particular a special strain of B. licheniformis, described in more detail in GB 1296839.
  • alpha-amylases derived from Bacillus sp. he AA560 alpha-amylase derived from Bacillus sp. DSM 12649 disclosed as SEQ ID NO: 2 in WO 00/60060 (hereby incorporated by reference) and the variants of the AA560 alpha-amylase, including the AA560 variant disclosed in Example 7 and 8 (hereby incorporated by reference).
  • amylases include Natalase ® , Stainzyme ® , Duramyl ® , Termamyl ® , TermamylTM Ultra, Fungamyl ® and BAN ® (all available from Novozymes A/S, Bags- vaerd, Denmark), and Rapidase ® and Maxamyl ® P (available from DSM, Holland) and Puras- tar ® , Purastar OxAm and PoweraseTM (available from Danisco A/S).
  • CGTases cyclodextrin glucanotransferases, EC 2.4.1.19
  • CGTases cyclodextrin glucanotransferases, EC 2.4.1.19
  • EC 2.4.1.19 cyclodextrin glucanotransferases, EC 2.4.1.19
  • Hemicellulases are used in the present invention.
  • Hemicelluloses are the most complex group of non-starch polysaccharides in the plant cell wall. They consist of polymers of xylose, arabinose, galactose, mannose and/or glucose which are often highly branched and connected to other cell wall structures. Hemicellulases of the present invention therefore include enzymes with xylanolytiactivity, arabinolytic activity, galactolytic activity and/or mannolytic activity.
  • the hemi-cellulases of the present invention may for example be selected from xylanases (EC 3.2.1.8, EC 3.2.1.32, and EC 3.2.1.136), xyloglucanases (EC 3.2.1 .4 and EC 3.2.1.151 ), arabinofuranosidases (EC 3.2.1 .55), acetylxylan esterases (EC EC 3.1.1.72), glucuronidases (EC 3.2.1.31 , EC 3.2.1.56, 3.2.1 .128 and 3.2.1.139), glucanohydrolase (EC 3.2.1 .1 1 , EC 3.2.1.83 and EC 3.2.1.73), ferulic acid esterases (EC 3.1.1.73), coumaric acid es- terases (EC 3.1.1 .73), mannanases (EC 3.2.1.25; EC 3.2.1 .78 and EC 3.2.1 .101 ), arabinosi- dase (EC 3.2.1 .88), arab
  • Mannananase is a preferred hemicellulase in relation to the present invention.
  • Manna- nases hydrolyse the biopolymers made up of galactomannans.
  • Mannan containing stains often comprise guar gum and locust bean gum, which are widely used as stabilizers in food and cosmetic products.
  • Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • the mannanase is derived from a strain of the genus Bacillus, especially Bacillus sp.
  • I633 disclosed in positions 31-330 of SEQ ID NO:2 or in SEQ ID NO: 5 of WO 99/64619 (hereby incorporated by reference) or Bacillus agaradhaerens, for example from the type strain DSM 8721 .
  • a suitable commercially available mannanase is Mannaway ® produced by Novozymes A/S or PurabriteTM produced by Genen- cor a Danisco division.
  • Xylanase is a preferred hemicellulase in relation to the present invention.
  • a suitable commercially available xylanase is Pulpzyme ® HC (available from Novozymes A/S).
  • pectinases are used in the present invention.
  • the term pectinase or pectolytic enzyme is intended to include any pectinase enzyme defined according to the art where pectinases are a group of enzymes that catalyze the cleavage of glycosidic linkages. Basically three types of pectolytic enzymes exist: pectinesterase, which only removes methoxyl residues from pectin, a range of depolymerizing enzymes, and protopectinase, which solubilizes protopectin to form pectin (Sakai et al., (1993) Advances in Applied Microbiology vol 39 pp 213- 294).
  • Example of a pectinases or pectolytic enzyme useful in the invention is pectate lyase (EC 4.2.2.2 and EC 4.2.2.9), polygalacturonase (EC 3.2.1.15 and EC 3.2.1 .67), polymethyl galactu- ronase, pectin lyase (EC 4.2.2.10), galactanases (EC 3.2.1 .89), arabinanases (EC 3.2.1.99) and/or pectin esterases (EC 3.1.1 .1 1 ).
  • Suitable pectinolytic enzymes include those described in WO 99/27083, WO 99/27084, WO 00/55309 and WO 02/092741 .
  • Suitable pectate lyases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • the pectate lyase is derived from a strain of the genus Bacillus, especially a strain of Bacillus substilis, especially Bacillus subtilis DSM14218 disclosed in SEQ ID NO:2 or a variant thereof disclosed in Example 6 of WO 02/092741 (hereby incorporated by reference) or a variant disclosed in WO 03/095638 (hereby incorporated by reference).
  • the pectate lyase is derived from a strain of Bacillus licheniformis, especially the pectate lyases disclosed as SEQ ID NO: 8 in WO 99/27083 (hereby incorporated by reference) or variants thereof as described in WO 02/06442.
  • Suitable commercially available pectate lyases are Pectaway ® or X Pect ® produced by No- vozymes A/S.
  • the method of the present invention may further include other cellulase different from the polypeptide with endoglucanse activity defined in the present invention.
  • the term "cellulase” or “cellulolytic enzyme” refers to an enzyme which catalyzes the degradation of cellulose to glucose, cellobiose, triose and other cello-oligosaccharides which enzyme is understood to include a mature protein or a precursor form thereof or a functional fragment thereof, e.g., a catalytic active module, which essentially has the activity of the full-length enzyme.
  • the term “cellulolytic” enzyme is intended to include homologues or analogues of said enzyme. Suitable cellulases include those of animal, vegetable or microbial origin.
  • the cellulolytic enzyme may be a component occurring in a cellulase system produced by a given microorganism, such a cellulase system mostly comprising several different cellulase enzyme components including those usually identified as, e.g., cellobiohydrolases, endogluca- nases, and beta-glucosidases.ln a preferred embodiment the cellulase is an endoglucanase.
  • Examples of commercially available cellulase enzyme products useful in the method of the present invention are: Cellusoft CR ® ' Cellusoft L ® , Novoprime A 378 ® all available from Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark); Indiage TM , Primafast TM (both from Genencor International Inc., U.S.A.); Powerstone TM (from logen, Canada); Ecostone TM (from Alko, Finland); Rocksoft TM (from CPN, U.S.A.), and Sanko Bio TM (from Meiji/Rakuto Kasei Ltd., Japan). Textile Manufacturing Process
  • the processing of a fabric, such as of a cellulosic material, into material ready for garment manufacturing 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 fi- nishing 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), polyvinyl 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 mannanases, a glucoamylases, 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 OPTISIZE ® NEXT, OPTISIZE ® FLEX and OPTISIZE ® COOL (all from Genencor International Inc.), and DURAMYLTM, ERMAMYLTM, FUNGAMYLTM TERMAMYLTM, AUQAZYMETM and BANTM (all available from Novozymes A S, Bagsvaerd, Denmark).
  • amylolytic enzymes include the CGTases (cyclodextrin glucanotransfe- rases, EC 2.4.1 .19), e.g., those obtained from species of Bacillus, Thermoanaerobactor or Thermoanaero-bacterium.
  • CGTases cyclodextrin glucanotransfe- rases, EC 2.4.1 .19
  • Suitable amylolytic enzymes include the CGTases (cyclodextrin glucanotransfe- rases, EC 2.4.1 .19), e.g., those obtained from species of Bacillus, Thermoanaerobactor or Thermoanaero-bacterium.
  • 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. It is the most critical chemical treatment since a balance between the degrees of whiteness with fiber damage must be maintained. 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 (OCI " ), b) chloride dioxide (CI0 2 ), and hydroperoxide species (OOH " and/or OOH). Reducing agents are typical sulfur dioxide, hydrosulfite salts, etc. Enzymatic bleaching using glucose oxidase has been reported. Traditionally, hydrogen peroxide is used in this process.
  • Printing or dyeing of textiles is carried out by applying dyes to the textile by any appro- priate method for binding the dyestuff to the fibres in the textiles.
  • the dyeing of textiles is for example 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 natu- ral 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 e.g.
  • biopolishing As used herein, the term “biopolishing”, “depilling” and “anti-pilling” are interchangeable. Most cotton fabrics and cotton blend fabrics have a handle appearance that is rather hard and stiff without the application of finishing components. The fabric surface also is not smooth because small fuzzy microfibrils protrude from it. In addition, after a relatively short period of wear, pilling appears on the fabric surface thereby giving it an unappealing, worn look.
  • Biopolishing is a method to treat cellulosic fabrics during their manufacturing by enzymes such as cellulases, which improves fabric quality with respect to "reduced pilling formation".
  • the most important effects of biopolishing can be characterised 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 per- formed as a separate step after any of the wetting steps or in combination with any of those wetting steps.
  • the method for manufacturing textile of the present invention by treating textile with an isolated polypeptide having endoglucanase activity but without functional CBM as defined in the present invention can be applied to a biopolishing process.
  • the invention provides a method for obtaining a cellulosic or cellulose-containing textile having a reduced pilling formation, the method comprising treating textile with a polypeptide having endoglucanase activity in an aqueous solution.
  • the method of biopolishing can be applied to yarn, fabric or garment.
  • the term “reduced pilling formation” is intended to mean a resistance to formation of pills on the surface of the treated (biopolished) fabric surface according to the method of the present invention, in comparison with fabric without enzymatic treatment.
  • the pilling formation may be tested according the description of "pilling notes test” in the material and method section. The results of the test is expressed in terms of "pilling notes” which is a rating on a scale from pilling note 1 (heavy pill formation) to pilling note 5 (no pill formation), allowing 1/4 pilling notes.
  • the enzymes of the present invention catalyze hydrolysis of the cellulosic fibre surface, the enzymatic action will eventually result in a weight loss of fibre or fabric.
  • the biopolishing is carried out in such a way so as to obtain a controlled, partial hydrolysis of the fibre surface, a proper polishing effect without excessive loss of fabric strength has hitherto been obtained.
  • the biopolishing effect is measured under condition as specified in Example 5, by treatment of polypeptide of the present invention in Launder-O-Meter (LOM) at 55°C, pH 6.5 for 1 hour, with enzyme dosage of 0.63 mg/g fabric and LAS concentration of 0.5 g/L.
  • LOM Launder-O-Meter
  • the polypeptide of the present invention shows pilling note of at least least 3, preferably at least 3.2, more preferably at least 3.4, more preferably at least 3.6, more preferably at least 3.7, more preferably at least 3.8, more preferably at least 3.9, most preferably at least 4, while preferably at the same time shows weight loss of less than 10%, preferably less than 8%, more preferably less than 7%, more preferably less than 6%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3.8%, more preferably less than 3.6%, more preferably less than 3.5%, more preferably less than 3.4%, more preferably less than 3.3%, more preferably less than 3.2%, more preferably less than 3.1 %, most preferably less than 3%.
  • the method of the invention can be carried out in any conventional wet textile processing step, preferably after the desizing or bleaching of the textile fabric, either simultanously with a conventional process or as an additional process step.
  • the method will typically be accomplished in high-speed circular systems such as jet-overflow dyeing machines, high-speed winches and jiggers.
  • An example of a useful High-speed system is the "Aero 1000" manufactured by Biancalani, Italy.
  • the method of the present invention can be carried out in a batch, continuous or semi-continuous apparatus, such as a J-Box, on a Pad- Roll or in a Pad-Bath.
  • 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 biostoning step and/or a color modification step.
  • the dyed fabric undergoes a biostoning step.
  • the biostoning step can be performed with enzymes or pumice stones or both.
  • biostoning stone washing
  • 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 (i.e. a localized variation of colour density in the denim surface).
  • mechanical action is needed to remove the dye, and the treatment is usually carried out in washing machines, like drum washers, belly washers.
  • abrasion level is used to indicate the localized variation of colour density, which is measured under condition as specified in Example 4, by treatment with polypeptide of the present invention in Launder-O-Meter (LOM) at 30°C, pH 6.5 for 2 hour, with enzyme dosage of 0.05 mg/g fabric.
  • LOM Launder-O-Meter
  • the endoglucanse having abrasion effect shows at least 0.5 Delta L * unit, preferably at least 1 , more preferably at least 1.2, more preferably at least 1 .3, more preferably at least 1 .5, more preferably at least 1.6, more preferably at least 1 .7, more preferably at least 1.8, more preferably at least 1.9, more preferably at least 2, more preferably at least 2.1 , more preferably at least 2.2, more preferably at least 2.3, more preferably at least 2.4, more preferably at least 2.5, more preferably at least 2.6, even more preferably at least 2.7, even most preferably at least 2.8 Delta L * unit.
  • Delta L * unit is defined in the material and method section under colour measurement.
  • the dyestuff removed from the denim material after the treatment with cellulase or by a conventional washing process may cause "backstaining" or "redeposition” of indigo onto the denim material, e.g. re-colouration of the blue threads and blue coloration of the white threads, resulting in a less contrast between the blue and white threads.
  • backstaining or "redeposition” of indigo onto the denim material
  • redeposition e.g. re-colouration of the blue threads and blue coloration of the white threads
  • the higher abrasion level will lead to higher backstaining level as more dyestuff is removed and redeposited into the fabric.
  • the process which causes high abrasion level but low backstaining level is desirable for the textile manufacture.
  • the delta L * unit from one process shall be compared with a control process when both process reach the similar abrasion level (i.e. similar Delta L * unit), because the similar abrasion level general means similar amount of dyestuff removed by the process.
  • low backstaining level is measured under condition as specified in Example 4, by treatment with polypeptide of the present invention in LOM at 30°C, pH 6.5 for 2 hour, with enzyme dosage of 0.025-0.1 mg/g fabric to achieve an equiva- lent abrasion level represented by delta L * on the textile surface, when compared with the results from using the control enzyme of mature polypeptide of Tt Cel45a (CBM+) as in Example 4.
  • Curves shall be drawn with horizontal coordinate as Delta L * unit and vertical coordinate as Delta b * unit for the data obtained in the presention invention and the control process respec- tively.
  • the backstaining level (Delta b * ) of the present invention shall be compared with that of the control process under the same Delta L * unit in the curves.
  • the endoglucanse having low backstaining effect shows at least 0.1 Delta b * unit increase, preferably at least 0.2, more preferably at least 0.3, even more preferably at least 0.4 Delta b * unit increase, when compared with the result from using control enzyme of mature polypeptide of Tt Cel45a (CBM+) as in Example 4.
  • Delta b * unit is defined in the material and method part of the present invention.
  • Abrasion is generally followed by the third step, after-treatment which generally includes washing and rinsing steps during which detergents, optical brighteners, bleaching agents or sof- teners may be used.
  • the method for manufacturing textile of the present invention by treating textile with an isolated polypeptide having endoglucanase activity but without a functional CBM as defined in the present invention can be applied to a biostoning process.
  • the invention provides a method for introducing into the surface of dyed fabric or garment, localized variations in colour density in which the method comprises the step of contacting the fabric or garment with a polypeptide having endoglucanase activity as defined in the present invention.
  • the dyed fabric or garment is cellulosic or cellulose- containing fabric or garment. More preferably, the dyed fabric is a denim fabric, even more preferably, indigo dyed denim fabric.
  • the invention provides a denim manufacturing process, which comprises: a) desizing of the denim fabric; b) biostoning the denim with a polypeptide having endoglucanase activity but without a functional CBM; c) rinsing.
  • the process of the invention may be carried out at conventional conditions in a washing machine conventionally used for stone-washing, e.g., a washer-extractor, belly washer, etc.
  • the enzyme of the invention should be added in an effective amount.
  • the present invention further relates to enzyme composition for textile comprising one or more polypeptide as defined in the presention invention.
  • the textile composition may be adapted for specific uses, such as biostoning or biopolishing, which can provide at least one of the textile benefits as reduced pilling formation, reduced weight loss of fabric, increased abrasion effect, and low backstaing level.
  • the textile composition may further include one or more of the enzymes selected from the group consisting of cellulase, proteases, lipases, cutinases, amylases, pectinases, hemicel- lulases, oxidoreductases, peroxidases, laccases, and transferases.
  • the enzymes selected from the group consisting of cellulase, proteases, lipases, cutinases, amylases, pectinases, hemicel- lulases, oxidoreductases, peroxidases, laccases, and transferases.
  • the textile composition typically comprises conventional ingredients including without limitation other enzymes, as well as surfactants, stabilizer, wetting agent, dispersing agents, anti- foaming agents, lubricants, builder systems, and the like, or a mixture thereof, that provide superior effects related to, e.g., strength, resistance to pilling, water absorbency, and dyeability.
  • the textile composition can be in any form, such as a solid, liquid, paste, gel or any combination thereof.
  • the method of treating textile with an isolated polypeptide having endoglucanase activity but without a functional CBM is applied in a biostoning or a biopolishing process. All process conditions below are applicable for both biotoning process and biopolishing process.
  • a suitable liquor/textile ratio to be used in the present method may be in the range of from about 20:1 to about 1 :1 , preferably in the range of from about 15:1 to about 3:1 , more preferably in the range of from 15:1 to 5:1 (Volumn/weight, ml/mg).
  • the reaction time is usually in the range of from about 10 minutes to about 8 hours. Preferably the reaction time is within the range of from about 20 minutes to about 180 minutes, more preferably the reaction time is within the range of from about 30 minutes to about 120 minutes.
  • the pH of the reaction medium greatly depends on the enzyme(s) in question.
  • the process of the invention is carried out at a pH in the range of from about pH 3 to about pH 1 1 , preferably in the range of from about pH 4 to about pH 8, or within the range of from about pH 4.5 to about pH 7.5.
  • the process of the present invention is able to function at a temperature below 90°C, preferably below 75°C, more preferably below 65°C, more preferably below 50°C, more preferably below 40°C, even more preferably below 30°C.
  • the process of the present invention is conducted at the temperature range of 5-90°C, preferably 10-80°C, more preferably 10-75°C, more preferably 15-65°C, more preferably 20-50°C, more preferably 20-40°C, and even more preferably 20-30°C.
  • Enzyme dosage greatly depends on the enzyme reaction time and enzyme activity, i.e. a relatively short enzymatic reaction time or low enzymatic activity necessitates a relatively increased enzyme dosage, and vice versa.
  • enzyme dosage may be stipulated in accordance with the reaction time available.
  • the amount of polypeptide with endoglucanse activity but without a functional CBM to be used according to the method of the present invention depends on many factors.
  • the concentration of the polypeptide of the present invention in the aqueous medium may be from about 0.001 to about 10 milligram (mg) enzyme protein per gram (g) of fabric, preferably 0.02-5 milligram of enzyme protein per gram of fabric, more preferably 0.05-2 milligram of enzyme protein per gram of fabric.
  • the mothod of the present invention can provide the textile benefits of increased abrasion effect and/or low backstaing level during biostoning process.
  • the method of the present invention can provide the textile benefits of low pilling formation but without substantial weight loss of fabric during in the presence of anionic surfactant, during a biopolishing process.
  • Novoprime A 868® (a mono-component Humicola insolens GH45 endoglucanase product with CBM truncated, commercially available from Novozymes A/S)
  • the abrasion level and backstaining level of the denim samples were determined by measuring the reflectance with pre-calibrated DataColor SF450X, alternatively an equivalent apperatus can be used. Four readings were taken for each sample, and the average of the readings were used. The abrasion level was evaluated with the index CIE L * on the blue side (front side) of the sample, and the backstaining level was evaluated with the index CIE b * on the back side of the sample.
  • L * indicates the change in white/black on a scale from 0 to 100, and a decrease in L * means an increase in black colour (decrease in white colour) and an increase in L * means an increase in white colour (decrease in black colour).
  • Delta L * unit L * of the swatch treated with a certain celllulase - L * of the swatch before cellulase treatment. The larger the Delta L * unit is the higer is the denim abrasion level, e.g. a Delta L * unit of 4 has higher abrasion level than Del- ta l_ * unit of 3.
  • b * indicates the change in blue/yellow, and a decrease in b * means an increase in blue colour (decrease in yellow colour), and an increase in b * means an increase in yellow colour (decrease in blue colour).
  • Delta b * units b * of the swatch treated with a certain celllulase - b * of the swatch before cellulase treatment.
  • a larger Delta b * unit corresponds to a lower backstaining level, e.g. a Delta b * unit of -1 .5 has lower backstaining level than the Delta b * unit of -2.5.
  • 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 100g) or a precision balance (for samples over 100g) and recorded. After treatment, all samples were tumbled dried (AEG, LAVATHERM 37700, Germany) for 1 hr and conditioned for 24 hr in conditioned room same as above. For each sample, the weight loss was defined as below:
  • Weight loss (weight before treatment - weight after treatment)/weight before treatment X(100%)
  • the enzyme protein in an enzyme product can be measured with BCATM Protein Assay Kit (product number 23225, commercial available from Thermo Fisher Scientific Inc.) according to the product manual.
  • BCATM Protein Assay Kit product number 23225, commercial available from Thermo Fisher Scientific Inc.
  • the wild type GH45 endoglucanase gene was cloned from the genomic DNA of Thiela- via terrestris NRRL 8126 as described in Example 1A of WO 96/29397 (hereby incorporated by reference).
  • Thielavia terristris was grown in PDA agar plate at 37°C for 4-5 days. Mycelia were collected directly from the agar plate into a sterilized mortar and frozen under liquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to a fine powder, and genomic DNA was isolated using a DNeasy® Plant Mini Kit (QIAGEN Inc., Valencia, CA, USA).
  • Oligonucleotide primers sense primerl and antisense primerl , were designed to amplify the Tt Cel45a(CBM+) while sense primerl and antisense primer2 were to amplify Tt Cel45a(CBM-) gene.
  • An ln-fusion CF Dry-down Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to clone the fragment directly into the expression vector pPFJ0355, without the need for restriction digestion and ligation.
  • Sense primerl (SEQ ID NO: 5): 5' acacaactggggatcC ACC atqcqctctactcccqttcttc 3'
  • Antisense primerl (SEQ ID NO: 6): 5' GTCACCCTCTAGATCT GACGAAGTTGACGGT CTCCTTG 3'
  • Antisense primer2 (SEQ ID NO: 7): 5' GTCACCCTCTAGATCT CACCTCGTGCGAAAA GCTGTTTAGA 3'
  • the expression vector pPFJ0355 contained the TAKA-amylase promoter derived from
  • pPFJ0355 had pUC18 derived sequences for selection and propagation in E. coli, and a pyrG gene, which encoded an orotidine decarboxylase derived from Aspergillus nidulans for selection of a transformant of a pyrG mutant Aspergillus strain.
  • Tt Cel45a(CBM- ' ) gene Twenty picomoles of each of Sense primerl and Antisense primer2 were used in a PCR reaction composed of plasmid DNA Plasmidl , 10 ⁇ of 5X GC Buffer, 1 .5ul of DMSO, 1.5ul of 10mM dNTP and 0.6 unit of PhusionTM High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 ⁇ .
  • the amplification was performed using a Peltier Thermal Cycler (M J Research Inc., South San Francisco, CA, USA) programmed for denaturing at 98°C for 1 minute; 8 cycles of denaturing at 98°C for 15 seconds, annealing at 68°C for 30 seconds, with 1 °C increasing per cycle and elongation at 72°C for 75 seconds; and another 22 cycles each at 98°C for 15 seconds, 65C for 30 seconds and 72°C for 75 seconds; final extension at 72°C for 5 minutes.
  • the heat block then went to a 4°C soak cycle.
  • reaction products were isolated by 1.0% agarose gel electrophoresis using 90mM
  • Tweenty picomoles of Sense primerl and Antisense primerl were used in a PCR reaction composed of plasmid DNA Plasmidl , 10 ⁇ of 5X GC Buffer, 1 .5ul of DMSO, 1 ul of 10mM dNTP and 0.6 unit of PhusionTM High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 ⁇ .
  • the amplification was performed using a Peltier Thermal Cycler (M J Research Inc., South San Francisco, CA, USA) programmed for denaturing at 98°C for 40 seconds; 8 cycles of denaturing at 98°C for 15 seconds, annealing at 70°C for 30 seconds, with 1 °C increasing per cycle and elongation at 72°C for 80 seconds; and another 23 cycles each at 98°C for 15 seconds, 62°C for 30 seconds and 72°C for 80 seconds; final extension at 72°C for 5 minutes.
  • the heat block then went to a 4°C soak cycle.
  • reaction products were isolated by 1.0% agarose gel electrophoresis using 90mM Tris-borate and 1 mM EDTA (TBE) buffer where a ⁇ 1.0kb product band was excised from the gel, and purified using an illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.
  • Plasmid pPFJ0355 was digested with Bam I and Bgl II, isolated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using an illustra GFX PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.
  • Tt Cel45a(CBM+) or Tt Cel45a(CBM-) The gene fragments, Tt Cel45a(CBM+) or Tt Cel45a(CBM-), and the digested vector were ligated together using an ln-fusion CF Dry-down PCR Cloning resulting in pRenoCBD and pRenoCore ( Figure 1 and 2, repctively).
  • the transcription of the Tt Cel45a(CBM+) and Tt Cel45a(CBM-) genes were under the control of a TAKA-amylase promoter from the gene for Aspergillus oryzae alpha-amylase. The cloning operation was performed according to the manufacturer's instruction.
  • Tt Cel45a(CBM+) gene inserted in pRenoCBD and Tt Cel45a(CBM-) inserted in pRenoCore were confirmed by DNA sequencing using 3730XL DNA Analyzers (Applied Biosystems Inc, Foster City, CA, USA).
  • the genomic DNA sequences of Tt Cel45a(CBM-) and Tt Cel45a(CBM+) genes were shown as SEQ ID NO: 1 and 3 respectively.
  • the deduced protein sequences of Cel45a(CBM-) genes was shown as SEQ ID NO: 2, wherein amino acids 1 -21 constitue the signal pepetide and amino acids 22-237 constitue the mature polypeptide of the catalytic module.
  • the deduced protein sequences of Cel45a(CBM+) genes was shown in SEQ ID NO: 4, wherein amino acids 1-21 constitue the signal pepetide and amino acids 22-299 constitue the mature polypeptide with catalytic module, linker (amino acids 238- 261 ) and CBM (amino acids 262-299).
  • Aspergillus oryzae HowB101 (described in W09535385 example 1 , hereby incorporated by reference) protoplasts were prepared according to the method described in Christensen et a/., 1988, Bio/Technology 6: 1419-1422. Three ⁇ g of pRenoCBD or pRenoCore were used to transform Aspergillus oryzae HowB101 .
  • the culture was harvested on day 3 by filtering the culture against MIRACLOTH® (CALBIOCHEM, Inc. La Jolla, CA, USA). The filtered culture broth was then again filtered using a 0.45 ⁇ DURAPORE Membrane (Milli- pore, Bedford, MA, USA).
  • Example 3 Purification of mature polypeptides of Tt Cel45a(CBM+) and Tt Cel45a(CBM-) 3000 ml supernatant of the transformant-2 of Strain-1 as described in Example 2 was precipitated with ammonium sulfate (80% saturation) and re-dissolved in 100 ml 25mM Tris-HCI buffer, pH7.0, then dialyzed against the same buffer and filtered through a 0.45 mm filter, the final volume was 200 ml. The solution was applied to a 50 ml Q FF column (Phamacia) equilibrated in 25 mM Tris-HCI buffer, pH7.0, and the proteins were eluted with a linear NaCI gradient (0-0.4M).
  • Tt Cel45a(CBM+) i.e. amino acids 22-299 of SEQ ID NO: 4
  • Tt Cel45a(CBM-) i.e. amino acids 22-237 of SEQ ID NO:2
  • LOM Launder-O-Meter
  • Abrasion level and backstaing level were compared.
  • Raw denim was desized and cut to 12.5 cm tall and 23 cm long. The denim was cut and sewn, forming a tube with height of 12.5 cm and weight of about 14g.
  • the tubes were placed in a conditioned room (65% relative humidity, 20°C) for 24 hours before they were numbered, weighed by the analytical balance and recorded. One conditioned tube was placed in each 500 ml beaker, with the blue side facing inward.
  • LOM Launder-O-Meter
  • the swatches were rinsed in hot water for 2 times and in cold water for 2 times.
  • the denim samples were tumble- dried (AEG, LAVATHERM 37700, Germany) and then conditioned for 24 hours at 20°C, 65% relative humidity prior to evaluation.
  • the abrasion and backstaining level of the denim samples were determined by measuring the reflectance before and after endoglucanse treatment with pre-calibrated DataColor SF450X. For both L * and b * , four readings were conducted for each fabric and the average of the four readings was used. The abrasion level was reflected by the changes in the index CIE L * of the blue side of the sample, and the backstaining level was reflected by the changes in the index CIE b * of the back of the sample.
  • Tt Cel45a(CBM-), endoglucanase without CBM showed higher denim abrasion level of delta L * than the CBM containing endoglucanase of Tt Cel45a(CBM+) when the same amount of proteins were loaded. And the performance difference became more substantial when the comparison was done at a lower temperature, 30°C.
  • Tt Cel45a(CBM-) also showed advantage in term of less backstaining on equivalent abrasion level, as shown in Table 1 .
  • Table 1 the use of Tt Cel45a(CBM+) at 50°C reachs the delta L * of 4.7, and the use of Tt Cel45a(CBM-) at 50°C reachs the delta L * of 5.3, the use of Tt Cel45a(CBM-) shows lower backstaining level of -2.3 than that of Tt Cel45a(CBM+) of -2.9.
  • Tt Cel45a(CBM-) shows lower backstaining level than that of Tt Cel45a(CBM+) in the same abrasion level.
  • Biopolishing trials were conducted in Launder-O-Meter for the CBM truncated molecules of mature polypeptide of Tt Cel45a(CBM-) and Novoprime A 868® (a mono-component Humico- la insolens GH45 endoglucanase product with CBM truncated) with different amount of LAS (Linear Alkylbenzene Sulfonates), an anionic surfactant which was widely used in textile, detergent, etc.
  • LAS Linear Alkylbenzene Sulfonates
  • Knitted 100% cotton interlock fabric was cut into 16.5 cm * 16.5 cm, as the standard swatches.
  • the swatches were placed in the conditioned room (65% humidity, 20°C) for 24 hours before they were numbered, weighed by the analytical balance and recorded.
  • Two conditioned swatches were placed in each 500ml beaker.
  • 20 big steel balls (total weight of 220g) in each beaker were used to supply the mechanical aids.
  • the buffer 50 mM phosphate buffer, pH 6.5
  • the LOM machine was started after the required program was chosen, and it would hold when the temperature reached 55°C.
  • Tt Cel45a(CBM-) and enzyme protein from Novoprime A 868® are added at different dosage to reach the similar pilling notes.
  • different amounts of LAS were loaded into each beaker according to Table 2. The beakers were sealed up and placed in the LOM, and the enzymatic treatment was started in LOM for 1 hour.
  • Tt Cel45a(CBM-) exhibited a much better compatibility with LAS than the counterpart from Novoprime A 868®: Tt Cel45a(CBM-) retained its original performance at the presence of 0.5g/L LAS (in term of weight loss performance) or as high as 0.8g/L LAS (in term of pilling notes).
  • the enzyme from Novoprime A 868® significantly lost its performance if the LAS concentration was 0.5g/L or higher, as the drop of weight loss at 0.5g/l LAS concentration implied the deactivation of the enzyme.
  • Tt Cel45a(CBM-) also shows advantage in term of less weight loss on equivalent anti-pilling effect: for example, when reaching the pilling note of 4.4, Tt Cel45a(CBM-) results in the weight loss of 2.8% while Novoprime A 868® resulted in the weight loss of 3.1 %.
  • the enzyme protein in Novoprime A 868® can be measured by BCATM Protein Assay
  • the following method is used to measure the functional CBM of a polypeptide, in order to identify a polypeptide having endoglucanase activity but lacking a fuctional CBM.

Abstract

La présente invention a pour objet le procédé de fabrication d'un textile, par le traitement du textile avec un polypeptide isolé ayant une activité endoglucanase, spécialement dans un procédé de biostoning et de biopolissage.
EP11853772.9A 2010-12-30 2011-12-14 Procédé de traitement d'un textile avec l'endoglucanase Withdrawn EP2658968A4 (fr)

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US8802423B2 (en) 2014-08-12
US20130295651A1 (en) 2013-11-07
EP2658968A4 (fr) 2017-05-03
BR112013016491A2 (pt) 2018-05-22
WO2012089024A1 (fr) 2012-07-05
MX2013007048A (es) 2013-07-29

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