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WO1997040127A1 - Blanchissage de tissus a l'aide d'une enzyme hybride contenant une enzyme oxydant un phenol qui est fusionnee a un domaine de liaison de la cellulose - Google Patents

Blanchissage de tissus a l'aide d'une enzyme hybride contenant une enzyme oxydant un phenol qui est fusionnee a un domaine de liaison de la cellulose Download PDF

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Publication number
WO1997040127A1
WO1997040127A1 PCT/DK1997/000165 DK9700165W WO9740127A1 WO 1997040127 A1 WO1997040127 A1 WO 1997040127A1 DK 9700165 W DK9700165 W DK 9700165W WO 9740127 A1 WO9740127 A1 WO 9740127A1
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Prior art keywords
fabric
enzyme
process according
alkyl
esters
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PCT/DK1997/000165
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English (en)
Inventor
Anders Hjelholt Pedersen
Jesper Vallentin Kierulff
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Novo Nordisk A/S
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Priority to AU23812/97A priority Critical patent/AU2381297A/en
Publication of WO1997040127A1 publication Critical patent/WO1997040127A1/fr

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Classifications

    • 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
    • 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
    • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
    • 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
    • D06P5/02—After-treatment

Definitions

  • the present invention relates to a process for providing a bleached look in the colour density of the surface of dyed fabric, especially cellulosic fabric such as denim.
  • the most usual method of providing a bleached stone- washed look in denim fabric or jeans is by washing the denim or jeans made from such fabric in the presence of pumice stones to provide the desired localized lightening of the colour of the fabric. This is then followed by a bleaching process where the fabric is treated with sodium hypochlorite typically at 60°C and pH 11-12 for up to 20 min. , followed by a neutralisation step and a rinsing.
  • hypochlorite is undesirable, both because chlorite itself is undesirable and because the neutralisation subsequently generates high amounts of salts leading to disposal and pollution problems.
  • Bleaching enzymes such as peroxidases together with hydrogen peroxide or oxidases together with oxygen have also been suggested for bleaching of dyed textiles (see WO 92/18683) , either alone or together with a phenol such as p-hydroxycinnamic acid, 2,4-dichlorophenol, p-hydroxybenzene sulphonate, vanillin or p-hydroxybenzoic acid.
  • a phenol such as p-hydroxycinnamic acid, 2,4-dichlorophenol, p-hydroxybenzene sulphonate, vanillin or p-hydroxybenzoic acid.
  • the disclosed process is not efficient.
  • the problem to be solved is not easy as many VAT-dyes, especially indigo, are not soluble in water and have a very compact structure on the fibre surface, making them difficult for an enzyme to attack.
  • formula X represents (-0-) or (-S-), and the substituent groups R ⁇ -R 9 , which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, carboxy, and esters and salts hereof, carbamoyl, sulfo, and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C ⁇ C ⁇ -alkyl, Ci- Cg-alkoxy, carbonyl-C x -C 3 -alkyl, aryl-C ⁇ C. ⁇ -alkyl; which car ⁇ bamoyl, sulfamoyl, and amino groups may furthermore be un- substituted or substituted once or twice with a substituent group R 10 ; and which phenyl may furthermore be unsubstituted or substituted with one or more substituent groups R 10 ; and which C x -C 14 -alkyl, C 1 -C 3
  • the process of the invention is most beneficially applied to cellulose-containing fabrics, such as cotton, viscose, rayon, ramie, linen, Tencel, or mixtures thereof, or mixtures of any of these fibres, or mixtures of any of these fibres together with synthetic fibres such as mixtures of cotton and spandex (stretch-denim) .
  • the fabric is denim.
  • the fabric may be dyed with vat dyes such as indigo, or indigo-related dyes such as thioindigo.
  • the fabric is indigo-dyed denim, including clothing items manufactured therefrom.
  • an enzyme hybrid comprises a phenol oxidizing enzyme fused to an amino acid sequence having a cellulose-binding domain.
  • a phenol oxidizing enzyme is meant an enzyme, which by using hydrogen peroxide or molecular oxygen, is capable of oxidizing organic compounds containing phenolic groups. Examples of such enzymes are peroxidases and oxidases.
  • Enzyme hybrids are known in the art, (for reference see e.g. WO 90/00609 and WO 95/16782) : They may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose-binding domain ligated, with or without a linker, to a DNA sequence encoding the enzyme of interest and growing the host cell to express the fused gene.
  • the enzyme hybrids may be described by the following formula:
  • CBD - MR - X wherein:
  • CBD can be either the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the cellulose-binding domain;
  • MR is the middle region (the linker) , and may be a bond, or a short linking group of from about 2 to about 100 carbon atoms, in particular of from 2 to 40 carbon atoms, or typically from about 2 to about 100 amino acids, in particular of from 2 to 40 amino acids, and
  • X can be either the N-terminal or the C-terminal region and is a phenol oxidizing enzyme.
  • cellulose-binding domain is intended to be understood as defined by Peter Tomme et al .
  • Cellulose- Binding Domains: Classification and Properties in “Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.) , ACS Symposium Series, No. 618, 1996.
  • This definition classifies more than 120 cellulose- binding domains (CBDs) into 10 families (I-X) , and it demonstrates that CBDs are found in various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases .
  • CBDs have also been found in algae, e.g., the red alga Porphyra purpurea as a non- hydrolytic polysaccharide-binding protein, for reference see Peter Tomme et al . , supra.
  • CBDs are from cellulases and xylanases. CBDs are found at the N or C termini of proteins or are internal .
  • Cellulases useful for preparation of Cellulose-Binding Domains The techniques used in isolating a cellulase gene are well known in the art .
  • cellulase refers to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and other cello-oligosaccharides .
  • the cellulase to be used in the method is an endoglucanase (EC 3.2.1.4) , preferably a monocomponent (recombinant) endogluc ⁇ anase.
  • the cellulase is a microbial cellulase, more preferably a bacterial or fungal cellulase.
  • bacterial cellulases are cellul ⁇ ases derived from or producible by bacteria from the group consisting of Pseudomonas, Bacillus, Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellum and Actinomycets such as Streptomyces, Termomonospora and Acidothemus, in particular from the group consisting of Pseudomonas cellulolyticus.
  • the cellulase may be an acid, a neutral or an alkaline cellulase, i.e. exhibiting maximum cellulolytic activity in the acid, neutral or alkaline range, respectively.
  • a useful cellulase is an acid cellulase, preferably a fungal acid cellulase, which is derived from or producible by fungi from the group of genera consisting of Trichoderma, Myrothecium, Aspergillus, Phanaerochaete, Neurospora, Neo- callimastix and Botrvtis.
  • a preferred useful acid cellulase is derived from or producible by fungi from the group of species consisting of Trichoderma viride, Trichoderma reesei, Trichoderma longibra- chiatum, Myrothecium verrucaria. Aspergillus niger, Aspergil ⁇ lus oryzae, Phanaerochaete chrysosporium, Neurospora crassa, Neocallimastix partriciarum and Botrytis cinerea.
  • Another useful cellulase is a neutral or alkaline cellulase, preferably a fungal neutral or a fungal alkaline cellulase, which is derived from or producible by fungi from the group of genera consisting of Aspergillus, PeniciIlium, Myceliophthora, Humicola, Irpex. Fusarium. Stachybotrvs, Scopulariopsis. Chaetomium. Mycogone, Verticillium, M ⁇ rothe- cium, Papulospora, Gliocladium, Cephalosporium and Acremonium.
  • a preferred alkaline cellulase is derived from or producible by fungi from the group of species consisting of Humicola insolens, Fusarium oxysporum, Myceliopthora ther ⁇ mophila, Penicillium ianthinellum and Cephalosporium sp. , preferably from the group of species consisting of Humicola insolens, DSM 1800, Fusarium oxysporum, DSM 2672, Mvceliopt- hora thermophila, CBS 117.65, and Cephalosporium sp. , RYM-202.
  • Other examples of useful cellulases are variants having, as a parent cellulase, a cellulase of fungal or bacterial origin, e.g. a cellulase derivable from a strain of the fungal genus Humicola, Trichoderma or Fusarium.
  • Isolation of a Cellulose-Binding domain In order to isolate the cellulose binding domain of e.g. a cellulase, several genetic approaches may be used.
  • One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment.
  • Another method involves the use of exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5 ' and the 3 ' ends of the DNA or internally from a restricted gap within the gene.
  • exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5 ' and the 3 ' ends of the DNA or internally from a restricted gap within the gene.
  • These gene deletion methods result in a mutated gene encoding a shortened gene molecule which may then be evaluated for substrate binding ability.
  • Appropriate substrates for evaluating the binding activity include compounds
  • nucleotide sequence encoding the substrate binding region may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest .
  • the cellulose binding encoding fragment and the DNA encoding the enzyme of interest are then ligated with or without a linker.
  • the resulting ligated DNA may then be manipulated in a variety of ways to provide for expression.
  • Microbial hosts such as Aspergillus, e.g., A. niger and A ⁇ _ oryzae, Bacillus, E. coli or S.cerevisiae are preferred.
  • Suitable peroxidases to be fused with the sequence encoding the cellulose-binding domain may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7), or any fragment derived therefrom, exhibiting peroxidase activity.
  • the peroxidase employed in the method of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria.
  • Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hypho- mycetes, e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embel- lisia. Cladosporium or Dreschlera. in particular Fusarium oxysporum (DSM 2672) , Humicola insolens.
  • DSM 2672 Fusarium oxysporum
  • Trichoderma resii Myrothecium verrucana (IFO 6113) , Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754) , Cal ⁇ dariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes .
  • Other preferred fungi include strains belonging to the subdivision Basidiomvcotina, class Basidiomycetes, e.g. Coprinus. Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f.
  • microsporus IFO 8371
  • Coprinus macror ⁇ hizus Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus) .
  • T. versicolor e.g. PR4 28-A
  • fungi include strains belonging to the subdivision Zygomvcotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particular Mucor hiemalis.
  • Some preferred bacteria include strains of the order
  • Actinomvcetales e.g., Streptomyces spheroides (ATTC 23965) , Streptomyces thermoviolaceus (IFO 12382) or Streptoverticilium verticillium ssp. verticillium.
  • Other preferred bacteria include Bacillus pumilus
  • bacteria include strains belonging to Mvxococcus, e.g., M. virescens.
  • a recombinantly produced peroxidase is preferred, e.g., a peroxidase derived from a Coprinus sp. , in particular C. macrorhizus or C. cinereus according to WO
  • laccases and Laccase Related Enzymes Suitable laccases to be fused with the sequence encoding the cellulose-binding domain include laccases and laccase related enzymes, i.e., any laccase enzyme comprised by the enzyme classification (EC 1.10.3.2) , any chatechol oxidase enzyme comprised by the enzyme classification (EC 1.10.3.1) , any bilirubin oxidase enzyme comprised by the enzyme classification (EC 1.3.3.5) or any monophenol monooxygenase enzyme comprised by the enzyme classification (EC 1.14.18.1) .
  • the laccase enzymes are known from microbial and plant origin.
  • the microbial laccase enzyme may be derived from bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrvtis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. vil ⁇ losa and T. versicolor, Rhizoctonia. e.g., R. solani, Copri- nus, e.g. C. plicatilis and C.
  • cinereus Psatyrella, Myceliophthora, e.g. M. thermophila, Scvtalidium, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046) , or Coriolus, e.g., C. hirsutus (JP 2-238885) , in particular laccases obtainable from Trametes, Myceliophthora, Scytalidium or Polyporus.
  • Plasmids Preparation of plasmids capable of expressing fusion proteins having the amino acid sequences derived from fragments of more than one polypeptide are well known in the art, for reference see e.g. WO 90/00609.
  • the expression cassette may be included within a replication system for episomal maintenance in an appropiate cellular host or may be provided without a replication system, where it may become integrated into the host genome.
  • the DNA may be introduced into the host in accordance with known techniques such as transformation, microinjection or the like.
  • the host may be grown to express the fused gene. Normally it is desirable additionally to add a signal sequence which provides for secretion of the fused gene.
  • Typical examples of useful fused genes are :
  • the recombinant product may be glycosylated or non- glycosylated.
  • the source may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide, e.g., percarbonate or perborate, or a hydrogen peroxide generating enzyme system, e.g. an oxidase and a substrate for the oxidase, or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof .
  • Hydrogen peroxide may be added at the beginning of or during the process, e.g. in a concentration corresponding to 0.001-25 mM H 2 0 2 . If the phenol oxidizing enzyme requires molecular oxygen, molecular oxygen from the atmosphere will usually be present in sufficient quantity.
  • the concentration of the hybrid enzyme in the aqueous medium where the localized variation in the colour density of the surface of the dyed fabric is taking place may be 0.001-10000 ⁇ g of hybrid enzyme protein per g of fabric, preferably 0.1-1000 ⁇ g of hybrid enzyme protein per g of fabric, more preferably 1-100 ⁇ g of hybrid enzyme protein per g of fabric.
  • the peroxidase activity may be determined in the following way:
  • 1 peroxidase unit is the amount of enzyme that catalyzes the conversion of 1 ⁇ mole hydrogen peroxide per minute at the following analytical conditions: 0.88 mM hydrogen peroxide, 1.67 mM 2,2 ' -azinobis (3- ethylbenzothiazoline-6-sulfonate) , 0.1 M phosphate buffer, pH 7.0, incubated at 30°C, photometrically followed at 418 nm.
  • the laccase activity may be determined in the following way:
  • Laccase activity is determined from the oxidation of syringaldazin under aerobic conditions.
  • the violet colour produced is photometered at 530 nm.
  • the analytical conditions are 19 ⁇ M syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30°C, 1 min. reaction time.
  • 1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 ⁇ mole syringaldazin per minute at these conditions.
  • Enhancing Agents The enhancing agent used in the present invention may be described by the following formula:
  • formula X represents (-0-) or (-S-)
  • the substituent groups R ⁇ R 9 which may be identical or different, independently represents any of the following radicals: hydrogen, halogen, hydroxy, formyl, carboxy, and esters and salts hereof, carbamoyl, sulfo, and esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C ⁇ -C ⁇ -alkyl, C j - C 3 -alkoxy, carbonyl-C 1 -C 5 -alkyl, aryl-C ⁇ -C ⁇ alkyl; which car- bamoyl, sulfamoyl, and amino groups may furthermore be un ⁇ substituted or substituted once or twice with a substituent group R 10 ; and which phenyl may furthermore be unsubstituted or substituted with one or more substituent groups R 10 ; and which C ⁇ C ⁇ -alkyl, Ci-C ⁇ -alk
  • the enhancing agent is 10-methylphenothiazine, phenothiazine-10-propionic acid, N-hydroxysuccinimide phenothiazine-10-propionate, 10-ethyl- phenothiazine-4-carboxylic acid, 10-ethylphenothiazine, 10- propylphenothiazine, 10-isopropylphenothiazine, methyl pheno- thiazine-10-propionate, 10-phenylphenothiazine, 10-allylpheno- thiazine, 10- (3- (4-methylpiperazin-1-yl)propyl)phenothiazine, 10- (2-pyrrolidin-1-yl-ethyl)phenothiazine, 2-methoxy-10- methyl-phenothiazine, l-methoxy-10-methylphenothiazine, 3- methoxy-10-methylphenothiazine, 3, 10-dimethylphenothia
  • the enhancing agent used in the present invention may be described by the following formula: B-0
  • a in the above mentioned formula is -CO-E, in which E may be -H, -OH, -R, or -OR; R being a C x -C l ⁇ alkyl, preferably a C l -C ⁇ alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C may be the same or different and selected from C m H 2 ⁇ l ; 1 ⁇ m ⁇ 5.
  • the enhancing agent is acetosyringone, methylsyringate, ethylsyringate, propyl- syringate, butylsyringate, hexylsyringate, or octylsyringate .
  • the enhancing agent of the invention may be present in concentrations of from 0.005-1000 ⁇ mole per g of fabric, preferably 0.05-100 ⁇ mole per g of fabric, more preferably 0.05-10 ⁇ mole per g of fabric.
  • the process of the present invention is typically used in industrial machines for making fabric look bleached. Normally, the process of the invention will be performed on fabric already stonewashed, but the process may also be applied to fabric which has not undergone a stonewashing process beforehand. Most commonly the fabric is added to the machine according to the machine capacity per the manufacturer's instructions. The fabric may be added to the machine prior to introducing water or the fabric may be added after water is introduced. The hybrid enzyme, the enhancing agent, and in some cases the hydrogen peroxide source may be present in the water prior to adding the fabric or they may be added after the fabric has been wetted. The hybrid enzyme may be added simultaneously with the enhancing agent or they may be added separately. After the fabric has been contacted with the hybrid enzyme and the enhancing agent it should be agitated in the machine for a sufficient period of time to ensure that the fabric is fully wetted and to ensure the action of the enzyme system and the enhancing agent .
  • the optimum bleaching conditions may be a compromise between optimum stability of the enzyme, optimum activity of the enzyme, optimum stability of the radical of the enhancing agent, and optimum reactivity (oxidation potential) of the radical, as well as choice of buffering system (buffer capacity, buffer toxicity, costs of buffer etc.) .
  • test procedure for fabric bleaching in particular denim bleaching, may be performed as described below, or as described in Examples 2 and 3 :
  • the denim swatch is rinsed with distilled water and air dried, whereafter it is evaluated for the degree of bleaching. The evaluation is performed visually and by using a Minolta Chroma Meter CR200 or a Minolta Chroma Meter CR300.
  • a Minolta Chroma Meter CR200 or CR300 (available from Minolta Corp.) is used according to Manufacturer's instructions to evaluate the degree of bleaching as well as to estimate any discoloration using the change in the colour space coordinates L * a * b * (CIELAB-system) : L * gives the change in white/black at a scale of from 0 to 100, a * gives the change in green (-a * ) /red (+a * ) , and b * gives the change in blue (-b * ) /yellow (+b * ) .
  • a decrease in L * means an increase in black colour (decrease of white colour)
  • an increase in L * means an increase in white colour (a decrease in black colour)
  • a decrease in a * means an increase in green colour (decrease in red colour)
  • an increase in a * means an increase in red colour (a decrease in green colour)
  • a decrease in b * means an increase in blue colour (a decrease in yellow colour)
  • an increase in b * means an increase in yellow colour (a decrease in blue colour) .
  • the bleached stone washed denim swatches are compared to non-treated stone washed denim swatches.
  • the Minolta Chroma Meter CR200 or the Minolta Chroma Meter CR300 is operated in the L * a * b * colour space (coordinate system) .
  • the light source used is a CIE light standard C.
  • Each measurement is an average of 3 measurements.
  • the instrument is calibrated using a Minolta calibration plate (white) . 10 non- treated denim swatches are measured 2 times each and the average of the coordinates L * a * b * are calculated and entered as a reference. The coordinates of the samples are then calculated as the difference ( ⁇ ) of the average of 3 measurements on each swatch from the reference value of the coordinates L * a * b * .
  • CiP-CBD and mCiP842-CBD This example concerns fusion proteins comprising a CBD linked to Coprinus cinereus peroxidase (CiP) or to a mutant thereof (mCiP842) (see, e.g., WO 95/10602) .
  • Yeast expression system The pJC106/YNG344 host/vector system was chosen as the standard expression system for all CiP experiments utilizing yeast expression. Mutant mCiP842 contains the following amino acid substitutions relative to the parent CiP: V53A, E239G, M242I, Y272F.
  • CBD-CiP fusion vector JC20A or JC20D Construction of the CBD-CiP fusion vector JC20A or JC20D: CiP signal seq. -if. insolens family 45 cellulase CBD-H. insolens family 45 cellulase linker-CiP, or CiP signal seq.-H. insolens family 45 cellulase CBD-H. insolens family 45 cellulase linker-mCiP842.
  • the CBD-CiP fusion was constructed by amplifying four separate gene fragments using PCR.
  • the sequence of the H. insolens family 45 cellulase is disclosed in WO 91/17244.
  • CiPpcrdwn CCCCCTTCCCTGGCGAATTCCGCATGAGG
  • Amplified products of reactions A) and B) were purified and phosphorylated using T4 polynucleotide kinase, ligated to one another for 15 min. at room temperature, and amplified with primers 1 and 4 to generate product AB.
  • Amplified products of reactions C) and D) were purified and mixed, then PCR-amplified to generate product CD.
  • Reaction products AB and CD were purified and phosphorylated using T4 polynucleotide kinase, ligated to one another for 15 min. at room temperature, and amplified with primers 1 and 8 to generate the final product.
  • Plasmid JC20A contains the wild type CiP gene, whereas plasmid JC20D contains the peroxide-stable mutant mCiP842. Transformants were selected on minimal media lacking uridine.
  • Yeast transformants were grown on minimal media plates containing 2% galactose (to induce the GAL1 yeast promoter driving CBD-CiP expression) that had been covered with a double filter layer consisting of cellulose acetate on top of nitrocellulose. After overnight growth, both filters were washed twice with 100 ml of 20 mM phosphate buffer, pH 7.0 for 5 minutes, after which no colony debris could be detected. Filters were then assayed for bound peroxidase activity by coating them with a 100 mM phosphate buffer, pH 7.0, containing 50 ⁇ g/ml of diamino-benzidine and 1 mM hydrogen peroxide. Bound peroxidase activity appears as a brown precipitate on the filter.
  • Liquid Assay Liquid cultures of mutants demonstrating cellulose binding in the filter assay were grown overnight in minimal media containing 2% galactose. 20 ⁇ l samples of culture broth were mixed with Avicel crystalline cellulose (20 g/L) in 0.1 M phosphate buffer, pH 7, 0.01% Tween 20 in a total volume of 100 ⁇ l and incubated at 22°C for 10 minutes. The mixture was then centrifuged to pellet the insoluble cellulose fraction, and the supernatants were assayed for peroxidase activity. Binding was scored as the % activity bound to the insoluble cellulose fraction based on the decrease in soluble activity.
  • This screening process utilizes broth samples from yeast cultures grown in microtiter plates.
  • the 96-well plate screen is performed by first growing yeast transformants of a pool of mutants in 50 ⁇ L volumes of URA(-) medium, pH 6.0 in 96- well microtiter plates. Cultures are inoculated by dilution into medium and pipetting (robotic or manual autopipettor) into 96-well plates. These are placed in an incubator set at
  • CiP and mCiP842 and the related fusion proteins were subjected to a combined pH - temperature - HO stress test: After an initial activity assay, cultures are diluted to ca. 0.06 PODU/ml, and incubated in 200 ⁇ M hydrogen peroxide, 100 mM phosphate/borate buffer, pH 10.5 at 50°C. After 0, 10, 20 and 30 minutes, samples are removed and residual activity is measured using the standard ABTS assay, pH 7.0. Improved mutants are those showing higher residual activity than CiP and are expressed as percent residual activity relative to the time 0 assay result.
  • Yeast expression plasmids designed to make H. insol ens family 45 cellulase CBD-CiP fusions were constructed and sequenced. These JC20-series plasmids were transformed into S . cerevisae for expression and testing. After transformation, yeast colonies were grown on selective plates covered with a double filter layer: cellulose acetate filters on top of nitrocellulose. Wild type CiP secreted from yeast JC106 and the stable mutant mCiP842 pass through the cellulose acetate, then binds to the nitrocellulose where it can be visualized using diaminobenzidine (DAB) and H 2 0 2 .
  • DAB diaminobenzidine
  • the cellulose acetate filter does not bind any wild-type or mCiP842 peroxidase.
  • the N-terminal CBD-CiP fusions encoded by plasmids JC20A, and JC20D are all detectable on both filters using the DAB assay, indicating that the fusion proteins have both peroxidase and cellulose-binding activities.
  • the peroxidase activity bound to the cellulose acetate filter remains bound even after washing extensively with buffer at pH 7.
  • the activity bound to the lower nitrocellulose filter suggests that binding of the CBD-CiP may be incomplete, or the cellulose filter gets saturated, allowing some of the fusion protein to pass through to the lower filter, or that some percentage of the fusion protein gets truncated to include only the peroxidase domain.
  • Denim Dakota 14 oz . pure indigo denim (standard fabric from Swift, "raw” denim i.e. not stone washed) .
  • the denim 75x100 cm was sewn into "legs” (denim cylinders) weighing approximately 350-375 g each
  • CiP-CBD produced as described in Ex. 1 : approximately 30000 PODU/ml.
  • mCiP842-CBD, (produced as described in Ex. 1) approximately 37000 PODU/ml.
  • Minolta Chroma Meter CR 300 was used to quantify the bleaching.
  • CiP-CBD 2 ⁇ g enzyme protein per g denim
  • mCiP842-CBD 3.4 ⁇ g enzyme protein per g denim
  • Example 2 was repeated with methylsyringate instead of 10-phenothiazine propionic acid as enhancing agent.

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  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Detergent Compositions (AREA)

Abstract

Cette invention concerne un procédé permettant de conférer un aspect de blanchiment à la densité des couleurs sur la surface de tissus imprimés. Ce procédé, qui s'effectue dans un milieu aqueux, consiste à mettre un tissu imprimé en contact avec une enzyme hybride, laquelle contient une enzyme oxydant un phénol qui est fusionnée à une séquence d'acide aminé possédant un domaine de liaison de la cellulose. Cette enzyme hybride comprend également une source de peroxyde d'hydrogène lorsque l'enzyme oxydant le phénol consiste en une peroxydase.
PCT/DK1997/000165 1996-04-19 1997-04-17 Blanchissage de tissus a l'aide d'une enzyme hybride contenant une enzyme oxydant un phenol qui est fusionnee a un domaine de liaison de la cellulose WO1997040127A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU23812/97A AU2381297A (en) 1996-04-19 1997-04-17 Bleaching of fabric with a hybrid enzyme containing a phenol oxidizing enzyme fused to a cellulose binding domain

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK0467/96 1996-04-19
DK0466/96 1996-04-19
DK46696 1996-04-19
DK46796 1996-04-19

Publications (1)

Publication Number Publication Date
WO1997040127A1 true WO1997040127A1 (fr) 1997-10-30

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Country Status (2)

Country Link
AU (1) AU2381297A (fr)
WO (1) WO1997040127A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912405A (en) * 1994-09-27 1999-06-15 Novo Nordisk A/S Enhancers such as acetosyringone
WO2000036094A1 (fr) * 1998-12-11 2000-06-22 Unilever N.V. Enzymes de blanchiment et compositions detergentes les renfermant
US7129069B2 (en) 2003-10-28 2006-10-31 Novo Zymes Als Hybrid enzymes
US7319112B2 (en) 2000-07-14 2008-01-15 The Procter & Gamble Co. Non-halogenated antibacterial agents and processes for making same
US8652455B2 (en) 2010-12-20 2014-02-18 E I Du Pont De Nemours And Company Targeted perhydrolases
US20210214888A1 (en) * 2020-01-14 2021-07-15 Sanko Tekstil Isletmeleri San. Ve Tic. A.S. Process for dyeing textiles and enzymes used therein

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000609A1 (fr) * 1988-07-08 1990-01-25 The University Of British Columbia Proteines de fusion se liant a la cellulose
WO1992018683A1 (fr) * 1991-04-12 1992-10-29 Novo Nordisk A/S Procede de blanchiment de textiles colores
WO1994007998A1 (fr) * 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1994012621A1 (fr) * 1992-12-01 1994-06-09 Novo Nordisk Amelioration de reactions enzymatiques
WO1996023928A1 (fr) * 1995-02-01 1996-08-08 Genencor International, Inc. Procede et compositions pour traiter des tissus contenant de la cellulose, faisant appel a une cellulase tronquee

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000609A1 (fr) * 1988-07-08 1990-01-25 The University Of British Columbia Proteines de fusion se liant a la cellulose
WO1992018683A1 (fr) * 1991-04-12 1992-10-29 Novo Nordisk A/S Procede de blanchiment de textiles colores
WO1994007998A1 (fr) * 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1994012621A1 (fr) * 1992-12-01 1994-06-09 Novo Nordisk Amelioration de reactions enzymatiques
WO1996023928A1 (fr) * 1995-02-01 1996-08-08 Genencor International, Inc. Procede et compositions pour traiter des tissus contenant de la cellulose, faisant appel a une cellulase tronquee

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912405A (en) * 1994-09-27 1999-06-15 Novo Nordisk A/S Enhancers such as acetosyringone
WO2000036094A1 (fr) * 1998-12-11 2000-06-22 Unilever N.V. Enzymes de blanchiment et compositions detergentes les renfermant
US6277806B1 (en) 1998-12-11 2001-08-21 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Bleaching enzymes and detergent compositions comprising them
US7319112B2 (en) 2000-07-14 2008-01-15 The Procter & Gamble Co. Non-halogenated antibacterial agents and processes for making same
US7749744B2 (en) 2003-10-28 2010-07-06 Novozymes A/S Hybrid enzymes
US7312055B2 (en) 2003-10-28 2007-12-25 Novozymes A/S Hybrid enzymes
US7129069B2 (en) 2003-10-28 2006-10-31 Novo Zymes Als Hybrid enzymes
US8652455B2 (en) 2010-12-20 2014-02-18 E I Du Pont De Nemours And Company Targeted perhydrolases
US8815550B2 (en) 2010-12-20 2014-08-26 E I Du Pont De Nemours And Company Targeted perhydrolases
US20210214888A1 (en) * 2020-01-14 2021-07-15 Sanko Tekstil Isletmeleri San. Ve Tic. A.S. Process for dyeing textiles and enzymes used therein
CN113123144A (zh) * 2020-01-14 2021-07-16 尚科纺织企业工业及贸易公司 对纺织品进行染色的方法及用于其中的酶
EP3851574A1 (fr) * 2020-01-14 2021-07-21 Sanko Tekstil Isletmeleri San. Ve Tic. A.S. Procédé de teinture de textiles et enzymes utilisés selon ce procédé
WO2021144356A1 (fr) * 2020-01-14 2021-07-22 Sanko Tekstil Isletmeleri San. Ve Tic. A.S. Procédé de coloration de textiles et enzymes utilisées dans ce procédé
US11920290B2 (en) 2020-01-14 2024-03-05 Sanko Tekstil Isletmeleri San. Ve Tic A.S. Process for dyeing textiles and enzymes used therein

Also Published As

Publication number Publication date
AU2381297A (en) 1997-11-12

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