A PROCESS FOR COMBINED DESIZING AND "STONE-WASHING" OF DYED DENIM

Abstract: A one-step process for combined desizing and "stone-washing" of dyed denim, wherein the denim is treated with an amylolytic enzyme in combination with a first abrading monocomponent endoglucanase and a second streak-reducing monocomponent endoglucanase.

Description: A PROCESS FOR COMBINED DESIZING AND "STONE-WASHING" OF DYED DENIM The present invention relates to a desizmg and "stone- washing" one-step process whereby dyed denim having localized variation m colour density of improved uniformity is achieved by treating dyed denim, especially dyed denim garment such as denim jeans, with an amylolytic enzyme and two different endoglucanases the very same process step. BACKGROUND OF THE INVENTION During the weaving of textiles, the threads are exposed to considerable mechanical strain. Prior to weaving on mechanical looms, warp yarns are often coated with size starch or starch derivatives m order to increase their tensile strength and to prevent breaking. The most common sizing agent is starch in native or modified form, yet other polymeric compounds such as polyvinylalcohol (PVA) , polyvmylpyrrolidone (PVP) , polyacrylic acid (PAA) or derivatives of cellulose (e.g. carboxymethylcellulose (CMC) , hydroxyethylcellulose, hydroxypropylcellulose or methylcellulose), may also be abundant the size. In general, after the textiles have been woven, the fabric proceeds to a desizmg stage, followed by one or more additional fabric processing steps. Desizmg is the act of removing size from textiles. After weaving, the size coating must be removed before further processing the fabric order to ensure a homogeneous and wash-proof result. The preferred method of desizmg is enzymatic hydrolysis of the size by the action of amylolytic enzymes. For the manufacture of denim clothes, the fabric is cut and sown into garments, that is afterwards finished. In particular, for the manufacture of denim garment, different enzymatic finishing methods have been developed. The finishing of denim garment normally is initiated with an enzymatic desizmg step, during which garments are subjected to the action of amylolytic enzymes in order to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing steps . Cotton wax and other lubricants can be applied to yarns in order to increase the speed of cotton weaving. Also waxes of higher melting points are being introduced. Wax lubricants are predominantly triglyceride ester based lubricants. After desizing, the wax either remains or redeposits on the fabric and as a result, the fabric gets darker in shade, gets glossy spots, and becomes more stiff. International Patent Application No. WO 93/13256 (Novo Nordisk A/S) describes a process for the removal of hydrophobic esters from fabric, in which process the fabric is impregnated during the desiz g step with an aqueous solution of lipase. This process has been developed for use in the fabric mills only, and is carried out using existing fabric mill equipment, i.e. a pad roll, a jigger, or a J box. JP-A 2-80673 discloses a method whereby desiz g and softening are achieved by treating cellulose fibres with an aqueous solution containing both amylase and cellulase. For many years denim jeans manuf cturers have washed their garments in a finishing laundry with pumice stones to achieve a soft-hand as well as a desired fashionable "stone-washed" look. This abrasion effect is obtained by locally removing the surface bound dyestuff. Recently cellulytic enzymes have been introduced into the finishing process, turning the stone-washing process into a "bio-stonmg process". The goal of a bio-stonmg process is to obtain a distinct, but homogeneous abrasion of the garments (stone-washing appearance) . However, uneven stone-washing ("streaks" and "creases") are very frequently occurring. In consequence repair work ("after-painting") is needed on a major part (up to about 80%) of the stone-washed jeans that have been processed m the laundries . Thus, it is an object of the present invention to provide a process which reduces the problem of streaks and creases on the finished denim garments. SUMMARY OF THE INVENTION Accordingly, the present invention provides a process for the treatment of fabrics, which process improves the color distribution/uniformity, stone-wash quality, etc., and which reduces the need for after-pa tmg of the finished clothes. The invention provides a one-step process for enzymatically desizmg and stone-washmg dyed denim, which process comprises treating the denim with an amylolytic enzyme, such as an α- amylase, in combination with a first abrading monocomponent endoglucanase and a second streak-reducing monocomponent endoglucanase . DETAILED DESCRIPTION OF THE INVENTION The present invention provides a process for enzymatic treatment of fabrics, by which process it is possible to provide desized and enzymatically stone-washed dyed denim of improved visual quality. As described above, enzymatic treatment of fabrics conven¬ tionally includes the steps of desizmg the fabric by use of amylolytic enzymes, softening the garment (including the steps of bio-polishmg, bio-stonmg and/or garment wash) by use of cellulytic enzymes, optionally followed by dyeing the garment, washing the garment, and/or softening the garment with a chemical softening agent, typically a catio ic, sometimes silicone-based, surface active compound. The process of the present invention may conveniently take place during the desizmg and/or softening step of the conventional garment manufacturing steps . Accordingly, in a preferred embodiment, the process of present invention relates to a one-step process for combined desizmg and "stone-washmg" of dyed denim, wherein the denim is treated with an amylolytic enzyme, such as an oc-amylase, combination with a first abrading monocomponent endoglucanase and a second streak-reducing monocomponent endoglucanase. In the present context, the term "abrading endoglucanase (or cellulase) " is intended to mean an endoglucanase which is capable of providing the surface of dyed denim fabric (usually sown into garment, especially jeans) localized variations in colour density. Examples of abrading cellulase are those mentioned in the International Patent Application PCT/US89/03274 published as WO 90/02790 which is hereby incorporated by reference . The term "monocomponent endoglucanase" denotes an endoglucanase which is essentially free from other proteins, in particular other endoglucanases . Monocomponent endoglucanases are typically produced by recombmant techniques, i.e. by cloning and expression of the relevant gene in a homologous or a heterologous host . In the present context, the term "streak-reducing endoglucanase (or cellulase)" or "levelling" endoglucanase is intended to mean an endoglucanase which is capable of reducing formation of streaks usually present on the surface of dyed denim fabric (usually sown into garment, especially jeans) which has been subjected to a "stone-washmg" process, either an enzymatic stone-washmg process or process using pumice for providing localized variations in colour density on the denim surface. Examples of streak-reducing or levelling cellulases are those mentioned in the International Patent Application PCT/DK95/00108 published as WO 95/24471 which is hereby incorporated by reference. The first endoglucanase is preferably a fungal EG V type cellulase. Another useful endoglucanase is a fungal EG III type cellulase obtainable from a strain of the genus Trichoderma . Examples of useful fungal EG III type cellulases are those dis¬ closed in WO 92/06184, WO 93/20208 and WO 93/20209, and WO 94/21801 which are hereby incorporated by reference. Preferably, the EG V type endoglucanase is derived from or producible by a strain of Scytalidium (f. Humi cola ) , Fusari um , Mycel ioph thora , more preferably derived from or producible by Scytal idium thermophil um (f. Humicola msolens) , Fusari um oxysporum or Mycel ioph thora themophila , most preferably from Humicola msolens, DSM 1800, Fusarium oxysporum, DSM 2672, or Mycel ioph thora themophila , CBS 117.65. In one embodiment of the invention, the first endoglucanase is an endoglucanase comprising the ammo acid sequence of the Humicola msolens endoglucanase shown in SEQ ID No. 1 or is an analogue of said endoglucanase which is at least 60% homologous with the sequence shown in SEQ ID No. 1, reacts with an antibody raised against said endoglucanase, and/or is encoded by a DNA sequence which hybridizes with the DNA sequence encoding said endoglucanase. In another embodiment of the invention, the first endoglucanase is an endoglucanase comprising the ammo acid sequence of the Fusari um oxysporum endoglucanase shown in SEQ ID No. 2 or is an analogue of said endoglucanase which is at least 60% homologous with the sequence shown m SEQ ID No. 2, reacts with an antibody raised against said endoglucanase, and/or is encoded by a DNA sequence which hybridizes with the DNA sequence encoding said endoglucanase. In the present context the homology may be determined as the degree of identity between two or more ammo acid sequences by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453) . For purposes of determining the degree of identity between two ammo acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. In the present context the antibody reactivity may be determined as follows: Antibodies to be used in determining lmmunological cross- reactiv ty may be prepared by use of the relevant purified enzyme. More specifically, antiserum against the enzyme may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al . in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) . Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ( (NH 4 γ S0 ), followed by dialysis and ion exchange chromatography, e . g . on DEAE-Sephade . Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (0. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir, Ed.) , Blackwell Scientific Publications, 1967, pp. 655-706) , by crossed lmmunoelectrophoresis (N. Axelsen et al . , supra, Chapters 3 and 4), or by rocket lmmunoelectrophoresis (N. Axelsen et al . , Chapter 2) . The hybridization may be determined by allowing the DNA (or corresponding RNA) sequences to hybridize under the following conditions: Presoaking of a filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 mm, and prehybπdization of the filter in a solution of 5 x SSC, 5 x Denhardt' s solution (Sambrook et al. 1989) , 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al . 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal . Bi ochem . 132:6- 13), 32 P-dCTP-labeled (specific activity > 1 x 10 9 cpm/μg ) probe for 12 hours at ca . 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at at least 55°C, more preferably at least 60°C, even more preferably at least 65°C, ana still more preferably at least 70°C (high stringency) , even more preferably at least 75°C. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x- ray film. In a preferred embodiment of the process of the invention, the second endoglucanase has a catalytic activity on cellotriose at pH 8.5 corresponding to k cat of at least 0.01 s "1 , preferably of at least 0.1 s "1 , more preferably of at least 1 s "1 . Preferably, the second endoglucanase is obtainable by or derived from a strain of Humi cola , Tπ choderma , Mycel ioph thora , Pemcillium , Irpex, Aspergill us , Scytalidium or Fusarium, more preferably from a strain of Humicola msolens, Fusarium oxy¬ sporum or Trichoderma reesei . Preferred second endoglucanases are of the EG I type. An example of a useful second endoglucanase is an endoglucanase comprising the ammo acid sequence of the Humi cola msolens endoglucanase shown in SEQ ID No. 3 or is an analogue of said endoglucanase which is at least 60% homologous with the sequence shown in SEQ ID No. 3, reacts with an antibody raised against said endoglucanase, and/or is encoded by a DNA sequence which hybridizes w th the DNA sequence encoding said endoglucanase . In the process of the invention, the first and second endoglucanase, respectively, can be used in an amount of corre- sponding to a cellulase activity between 5 and 8,000 ECU per litre of desιzmg/"stone-washmg" liquour, preferably between 10 and 5000 ECU per litre of liquor, and more preferably between 50 and 500 ECU per litre of liquor. The first and second endoglucanase, respectively, is preferably dosed m an amount corresponding to 0.01-40 mg endoglucanase/1, more preferably 0.1-2.5 mg/1, especially 0.1-1.25 mg/1. The substrate of the process of the invention is dyed denim. The denim may be dyed with a natural or a synthetic dye. Examples of synthetic dyes are direct dyes, fiber-reactive dyes or indirect dyes. In a preferred embodiment, the denim is dyed with indigo. Typically, the denim is cut and sown into garment before subjected to the process of the present invention. Examples of garment are jeans, jackets and skirts. An especially preferred example is indigo-dyed denim jeans. In the process of the invention, conventional desizmg enzymes, in particular amylolytic enzymes, can be used in order to remove starch-containing size. Therefore, an amylolytic enzyme, preferably an α-amylase, may be added during the process of the invention. Conven- tionally, bacterial α-amylases are used for the desizmg, e.g. an α-amylases derived from a strain of Ba cill us, particularly a strain of Bacill us l i cheniformis, a strain of Ba cill us amyl ol iquefa ciens, or a strain of Ba cill us s tearothermophil us ; or mutants thereof. Am o acid sequences of such amylases are apparent from, e.g., WO 95/21247. Examples of suitable commer- cial α-amylase products are Termamyl™, Aquazy ™ Ultra and Aquazym™ (available from Novo Nordisk A/S, Denmark) . However, also fungal α-amylases can be used. Examples of fungal α- amylases are those derived from a strain of Aspergil l us . Other useful α-amylases are the oxidation-stable α-amylase mutants disclosed m WO 95/21247. For instance, an α-amylase mutant prepared from a parent α-amylase by replacing one or more of the methionme ammo acid residues with a Leu, Thr, Ala, Gly, Ser, lie, Asn, or Asp amino acid residue, preferably a Leu, Thr, Ala, or Gly ammo acid residue. Of particular interest is an α- amylase mutant prepared from the B . l icheniformi s α-amylase in which the methionme at position 197 has been replaced with any other ammo acid residue, in particular with Leu, Thr, Ala, Gly, Ser, lie, Asn, or Asp ammo acid residue, preferably a Leu, Thr, Ala, or Gly ammo acid residue. The amylolytic enzyme may be added in amounts conven¬ tionally used in desiz g processes, e.g. corresponding to an α- amylase activity of from about 10 to about 10,000 KNU/1 such as from 100 to about 10,000 KNU/1 or from 10 to about 5,000 KNU/1. Also, in the process according to the present invention, 1-10 mM of Ca ++ may be added as a stabilizing agent. The process of the present invention may be accomplished at process conditions conventionally prevailing in desizmg/ "stone-washmg" processes, as carried out by the person skilled in the art. The process of the invention may, e.g., be carried out batch-wise in a washer extractor. It is at present contemplated that a suitable liquor/textile ratio 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 5:1. In conventional desizing and "stone-washmg" processes, the reaction time is usually in the range of from about 1 hour to about 24 hours. However, in the process of the present invention the reaction time may well be less than 1 hour, i.e. from about 5 minutes to about 55 minutes. Preferably the reaction time is with the range of from about 5 or 10 to about 120 minutes. The pH of the reaction medium greatly depends on the enzyme in question. Preferably the process of the invention is carried out at a pH in the range of from about pH 3 to about pH 11, preferably in the range of from about pH 6 to about pH 9, or withm the range of from about pH 5 to about pH 8. A buffer may be added to the reaction medium to maintain a suitable pH for the enzymes used. The buffer may suitably be a phosphate, borate, citrate, acetate, adipate, triethanolamme, monoethanolamine, diethanolamme, carbonate (especially alkali metal or alkaline earth metal, in particular sodium or potassium carbonate, or ammonium and HCl salts) , diamine, especially diammoethane, lmidazole, or ammo acid buffer. The process of the invention may be carried out in the presence of conventional textile finishing agents, including wetting agents, polymeric agents, dispersing agents, etc. A conventional wetting agent may be used to improve the contact between the substrate and the enzymes used in the process. The wetting agent may be a nonionic surfactant, e.g. an ethoxylated fatty alcohol, an ethoxylated oxo alcohol, an ethoxylated alkyl phenol or an alkoxylated fatty alcohol. Examples of suitable polymers include proteins (e.g. bovine serum albumin, whey, casein or legume proteins) , protein hydrolysates (e.g. whey, casein or soy protein hydrolysate) , polypeptides, lignosulfonates, polysacchaπdes and derivatives thereof, polyethylene glycol, polypropylene glycol, polyvmyl pyrrolidone, ethylene diamine condensed with ethylene or propylene oxide, ethoxylated polyamines, or ethoxylated amine polymers . The dispersing agent may suitably be selected from nonionic, anionic, cationic, ampholytic or zwitteπonic surfactants. More specifically, the dispersing agent may be selected from carboxymethylcellulose, hydroxypropylcellulose, alkyl aryl sulphonates, long-chain alcohol sulphates (primary and secondary alkyl sulphates) , sulphonated olefins, sulphated monoglycerides, sulphated ethers, sulphosuccmates, sulphonated methyl ethers, alkane sulphonates, phosphate esters, alkyl isothionates, acylsarcosides, alkyltaurides, fluorosurfactants, fatty alcohol and alkylphenol condensates, fatty acid condensates, condensates of ethylene oxide with an amine, condensates of ethylene oxide with an amide, sucrose esters, sorbitan esters, alkyloamides, fatty amine oxides, ethoxylated monoammes, ethoxylated diammes, alcohol ethoxylate and mixtures thereof. In another preferred embodiment of the invention, the pro¬ cess may be performed using a lipolytic enzyme that is capable of carrying out lipolysis at elevated temperatures. In order to efficiently hydrolyse hydrophobic esters of high melting points, lipolytic enzymes that possess sufficient thermostability and lipolytic activity at temperatures of about 60°C or above, are preferred. Adequate hydrolysis can be obtained even above or below the optimum temperature of the lipolytic enzyme by increasing the enzyme dosage. The lipolytic enzyme may be of animal, plant or microbial origin. Examples of microorganisms producing such thermostable lipolytic enzymes are strains of Humicola , preferably a strain of Humicola brevispora , a strain of Humicola lan ug osa , a strain of Humi cola brevis var . thermoidea, a strain of Humi col a msolens , a strain of Fusarium, preferably a strain of Fusari um oxysporum, a strain of Rhizomucor, preferably a strain of Rhizomucor iehei , a strain of Chromoba cteri um, preferably a strain of Chromobacteπum viscosum, and a strain of Aspergill us , preferably a strain of Aspergill us niger . Preferred thermostable lipolytic enzymes are derived from strains of Candida or Pseudomonas, particularly a strain of Candida antarcti ca , a strain of Candida tsukubaensis , a strain of Candida a uri culaπae, a strain of Candida humicola , a strain of Candida fol iarum, a strain of Candida cyl mdracea (also called Candida rugosa ) , a strain of Pseudomonas cepa cia , a strain of Pseudomonas fl uorescens , a strain of Pseudomonas fragi , a strain of Pseudomona s s t u tzeri , or a strain of Thermomyces lanugmosus . Lipolytic enzymes from strains of Candida an tarct i ca and Pseudomonas cepa cia are preferred, in particular lipase A from Candida an tarct i ca . Such lipolytic enzymes, and methods for their production, are known from e.g. WO 88/02775, US 4,876,024, and WO 89/01032, which publications are hereby mcluded by reference . The enzyme dosage is dependent upon several factors, including the enzyme in question, the desired reaction time, the temperature, the liquid/textile ratio, etc. It is at present contemplated that the lipolytic enzyme may be dosed in an amount corresponding to of from about 0.01 to about 10,000 KLU/1, preferably of from about 0.1 to about 1000 KLU/1. Conventional finishing agents that may be present in a process of the invention include, but are not limited to pumice stones and perlite. Perlite is a naturally occurring volcanic rock. Preferably, heat expanded perlite may be used. The heat expanded perlite may e.g. be present in an amount of 20-95 w/w% based on the total weight of the composition. Cellulytic Activity The cellulytic activity may be measured m endo-cellulase units (ECU) , determined at pH 7.5, with carboxymethyl cellulose (CMC) as substrate. The ECU assay quantifies the amount of catalytic activity present in the sample by measuring the ability of the sample to reduce the viscosity of a solution of carboxy-methylcellulose (CMC) . The assay is carried out at 40°C; pH 7.5; 0. IM phosphate buffer; time 30 mm; using a relative enzyme standard for reduc- mg the viscosity of the CMC Hercules 7 LFD substrate; enzyme concentration approx . 0.15 ECU/ml . The arch standard is defined to 8200 ECU/g. Amylolytic Activity The amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue colour is formed, but during the break-down of the starch the blue colour gets weaker and gradually turns into a reddish-brown, which is compared to a coloured glass standard. One Kilo Novo alfa Amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca 2 Y and pH 5.6) dextrinizes 5.26 g starch dry substance Merck Amylum solubile. A folder AF 9/6 describing this analytical method in more detail is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference. Lipolytic Activity The lipolytic activity may be determined using tributyrine as substrate. This method is based on the hydrolysis of tributyrin by the enzyme, and the alkali consumption is registered as a function of time. One Lipase Unit (LU) is defined as the amount of enzyme which, under standard conditions (i.e. at 30.0°C; pH 7.0; with Gum Arabic as emulsifier and tributyrine as substrate) liberates 1 μmol titrable butyric acid per minute (1 KLU = 1000 LU) . A folder AF 95/5 describing this analytical method in more detail is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference. EXAMPLE 1 The following example illustrates the effect of adding a streak-reducing or levelling endoglucanase to the combined desizing-abrasion process in order to reduce the number of streaks on denim jeans or other garment and to produce denim garment, especially jeans, with a uniformly localized color variation. Wash trials were carried out under the following conditions: Textile : Blue denim DAKOTA, 14^ oz, 100 % cotton. The denim was cut and sewed into "legs" of approximately 37.5x100 cm (about 375 g each) . Two new legs and one old (used one time) leg were used in each trial (a total of approx . 1100 g textile) . Enzyme: Trial A: Amylase: TermamyY", dosage: 200 KNU/1 Endoglucanase (cellulase) : EG V (a monocomponent ~43 kD endoglucanase from Humi cola msolens , DSM 1800, having the ammo acid sequence of SEQ ID No. 1) , dosage: 10 ECU/g denim Trial B: Amylase: Termamyl ® , dosage: 200 KNU/1 Endoglucanase (cellulase) : EG V (as n trial A) , dosage: 10 ECU/g denim EG I (monocomponent endoglucanase from Humi cola msolens , DSM 1800, having the ammo acid sequence of SEQ ID No. 3) , dosage: 10 ECU/g denim Washing was carried out in a wascator (FOM71 LAB) . Wash-program: 1) Mam wash at 55°C, 20 1 water, 120 mm, buffer and enzyme added. Buffer:30 g KH 2 P0 4 + 20 g Na 2 HP0 4 , pH7 2) Dram 30 sec. 3) Rinse at 80°C, normal action, 32 1 water, 15 mm.; 20 g Na 2 C0 3 added 4) Dra 30 sec. 5) Rinse at 54°C, normal action, 32 1 water, 5 m . 6) Dra 30 sec. 7) Rinse at 14°C, normal action, 32 1 water, 5 mm . 8) Dram 30 sec. 9) Spinning 40 sec. at low speed and 50 sec. at high speed. Drying: The samples were dried in a tumble-dryer. The jeans from the two trials were abraded to almost the same level . Evaluation: 5 persons skilled in the art of evaluating denim were asked to grade the denim legs (two legs from each trial, leg "1" and "3" from trial B, leg "2" and "4" from trial A) from 1 to 4, where 1 was the least streaked denim leg and 4 was the leg with most streaks on. Grading were as shown in the table below: As can be seen from the table, the denim legs treated in the combi-process of the invention with a combination of two monocomponent endoglucanases having abrading and strak-reducing properties, respectively, e.g. an EG V type and EG I type cellulase, are all rated to have the best appearance with respect to streaking and uniformity of the localized color variation. Figures 1 and 2: To illustrate the change in uniformity that can be obtained by using a streak-reducing or levelling endoglucanse (cellulase) in the process of the invention, swatches from trial A and B were scanned (HP ScanJet II CX) into a computer and printed m black-and-white. Figure 1 show part of a denim leg from trial B and figure 2 show part of a denim leg from trial A. SEQUENCE LISTING INFORMATION FOR SEQ ID NO: 1 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 415 ammo acids (B) TYPE: ammo acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (B) STRAIN: DSM 1800 SEQUENCE DESCRIPTION: SEQ ID NO: 1 : Gin Lys Pro Gly Glu Thr Lys Glu Val His Pro Gin Leu Thr Thr Phe 1 5 10 15 Arg Cys Thr Lys Arg Gly Gly Cys Lys Pro Ala Thr Asn Phe lie Val 20 25 30 Leu Asp Ser Leu Ser His Pro lie His Arg Ala Glu G y Leu Gly Pro 35 40 45 Gly Gly Cys Gly Asp Trp Gly Asn Pro Pro Pro Lys Asp Val Cys Pro 50 55 60 Asp Val Glu Ser Cys Ala Lys Asn Cys lie Met Glu Gly lie Pro Asp 65 70 75 80 Tyr Ser Gin Tyr Gly Val Thr Thr Asn Gly Thr Ser Leu Arg Leu Gin 85 90 95 His lie Leu Pro Asp Gly Arg Val Pro Ser Pro Arg Val Tyr Leu Leu 100 105 110 Asp Lys Thr Lys Arg Arg Tyr Glu Met Leu His Leu Thr Gly Phe Glu 115 120 125 Phe Thr Phe Asp Val Asp Ala Thr Lys Leu Pro Cys Gly Met Asn Ser 130 135 140 Ala Leu Tyr Leu Ser Glu Met His Pro Tnr Gly Ala Lys Ser Lys Tyr 145 150 155 160 Asn Pro Gly Gly Ala Tyr Tyr Gly Thr Gly Tyr Cys Asp Ala Gin Cys 165 170 175 Phe Val Thr Pro Phe He Asn Gly Leu Gly Asn He Glu Gly Lys Gly 180 185 190 Ser Cys Cys Asn Glu Met Asp He Trp Glu Ala Asn Ser Arg Ala Ser 195 200 205 His Val Ala Pro His Thr Cys Asn Lys Lys Gly Leu Tyr Leu Cys Glu 210 215 220 Gly Glu Glu Cys Ala Pne Glu Gly Val Cys Asp Lys Asn Gly Cys Gly 2 5 230 235 240 Trp Asn Asn Tyr Arg Val Asn Val Thr Asp Tyr Tyr Gly Arg Gly Glu 245 250 255 Glu Phe Lys Val Asn Thr Leu Lys Pro Phe Thr Val Val Thr Gin Phe 260 265 270 Leu Ala Asn Arg Arg Gly Lys Leu Glu Lys He His Arg Phe Tyr Val 275 280 285 Gin Asp Gly Lys Val He Glu Ser Phe Tyr Thr Asn Lvs Glu Gly Val 290 295 300 Pro Tyr Thr Asn Met He Asp Asp Glu Phe Cys Glu Ala Thr Gly Ser 305 310 315 320 Arg Lys Tyr Met Glu Leu Gly Ala Thr Gin Gly Met Gly Glu Ala Leu 325 330 335 Thr Arg Gly Met Val Leu Ala Met Ser He Trp Trp Asp Gin Gly Gly 340 345 350 Asn Met Glu Trp Leu Asp His Gly Glu Ala Gly Pro Cys Ala Lys Gly 355 360 365 Glu Gly Ala Pro Ser Asn He Val Gin Val Glu Pro Phe Pro Glu Val 370 375 380 Tnr Tyr Thr Asn Leu Arg Trp Gly Glu He Gl;> Ser Thr Tyr Gin Glu 385 390 395 400 Val Gin Lys Pro Lys Pro Lys Pro Gly His Gly Pro Arg Ser Asp 405 410 1 INFORMATION FOR SEQ ID NO:2: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 409 ammo acids (B) TYPE: ammo acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Fusarium oxysporum (B) STRAIN: DSM 2672 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gin Thr Pro Asp Lys Ala Lys Glu Gin His Pro Lys Leu Glu Thr lyr 1 5 10 15 Arg Cys Thr Lys Ala Ser Gly Cys Lys Lys Gin Thr Asn Ivr He Val 20 25 30 ) Ala Asp Ala Gly He His Gly He Arg Arg Ser Ala Gl^ Cys Ciy Asp 35 40 45 Trp Gly Gin Lys Pro Asn Ala Thr Ala Cys Pro Asp Glu Ala Ser Cys 50 55 60 Ala Lys Asn Cys He Leu Ser Gly Met Asp Ser Asn Aid Tyr Lys Asn 65 70 75 80 Ala Gly He Thr Thr Ser Gly Asn Lys Leu Arg Leu Gin Gin Leu He 85 90 95 Asn Asn Gin Leu Val Ser Pro Arg Val Tyr Leu Leu Glu Glu Asn Lys 100 105 110 Lys Lys Tyr Glu Met Leu His Leu Thr Gly Thr Glu Phe Ser Phe Asp 115 120 125 Val Glu Met Glu Lys Leu Pro Cys Gly Met Asn Gly Ala Leu Tyr Leu 130 135 140 Ser Glu Met Pro Gin Asp Gly Gly Lys Ser Thr Ser Arg Asn Ser Lys 145 150 155 160 Ala Gly Ala Tyr Tyr Gly Ala Gly Tyr Cys Asp Ala Gin Cys Tyr Val 165 170 175 Thr Pro Phe He Asn Gly Val Gly Asn He Lys Gly Gin Gly Val Cys 180 185 190 Cys Asn Glu Leu Asp He Trp Glu Ala Asn Ser Arg Ala Thr His He 195 200 205 Ala Pro His Pro Cys Ser Lys Pro Gly Leu Tyr Gly Cys Thr Gly Asp 210 215 220 Glu Cys Gly Ser Ser Gly He Cys Asp Lys Ala Gly Cys Gly Trp Asn 225 230 235 240 His Asn Arg He Asn Val Thr Asp Phe Tyr Gly Arg Gly Lys Gin Tyr 245 250 255 Lys Val Asp Ser Tnr Arg Lys Phe Thr Val Thr Ser Gin Phe Val Ala 260 265 270 Asn Lys Gin Gly Asp Leu He Glu Leu His Arg His Tyr He Gin Asp 275 280 285 Asn Lys Val He Glu Ser Ala Val Val Asn He Ser Gly Pro Pro Ly; 290 295 300 He Asn Phe He Asn Asp Lys Tyr Cys Ala Ala Thr Gly Ala Asn Glu 305 310 315 320 Tyr Met Arg Leu Gly Gly Thr Lys Gin Met Gly Asp Ala Met Ser Arg 325 330 Gly Met Val Leu Ala Met Ser Val Trp Trp Ser Glu Gly Asp Phe Met 340 345 350 Ala Trp Leu Asp Gin Gly Val Ala Gj.y Pro Cys Asp A la Th r G u Gl v 355 360 365 Aso Pro Lys Asn He Val Lys Val Gin Pro Asn Pro Glu V e i l Th r P h e 370 375 380 Ser Asn He Arg He Gly Glu He Gly Ser Thr Ser Ser Val L ys A le 385 390 395 400 10 Pro Ala Tyr Pro Gly Pro His Arg Leu 405 INFORMATION FOR SEQ ID NO: 3: (l) SEQUENCE CHARACTERISTICS: H (A) LENGTH: 415 (435) ammo acids (B) TYPE: ammo acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein 20 (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola msolens (B) STRAIN: DSM 1800 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: :> SEQUENCE LISTING (May be renumbered from -21 to 415, total 435 aa) Met Ala Arg Gly Thr Ala Leu Leu Gly Leu Thr Ala Leu Leu Leu Gly 30 1 5 10 15 Leu Val Asn Gly Gin Lys Pro Gly Glu Thr Lys Glu Val His Pro Gin 20 25 30 35 Leu Thr Thr Phe Arg Cys Thr Lys Arg Gly Gly Cys Lys Pro Ala Thr 35 40 45 Asn Phe He Val Leu Asp Ser Leu Ser His Pro He His Arg Ala Glu 50 55 60 40 Gly Leu Gly Pro Gly Gly Cys Gly Asp Trp Gly Asn Pro Pro Pro Lys 65 70 75 80 Asp Val Cys Pro Asp Val Glu Ser Cys Ala Lys Asn Cys He Met Glu 85 90 95 Gly He Pro Asp Tyr Ser Gin Tyr Gly Val Thr Thr Asn Gly Thr Ser 100 105 110 Leu Arg Leu Gin His He Leu Pro Asp Gly Arg Val Pro Ser Pro Arg 115 120 125 Val Tyr Leu Leu Asp Lys Thr Lys Arg Arg Tyr Glu Met Leu His Leu 130 135 140 Thr Gly Phe Glu Phe Thr Phe Asp Val Asp Ala Thr Lys Leu Pro Cys 145 150 155 160 Gly Met Asn Ser Ala Leu Tyr Leu Ser Glu Met His Pro Thr Gly Ala 165 170 175 Lys Ser Lys Tyr Asn Ser Gly Gly Ala Tyr Tyr Gly Thr Gly Tyr Cys 180 185 190 Asp Ala Gin Cys Phe Val Thr Pro Phe He Asn Gly Leu Gly Asn He 195 200 205 Glu Gly Lys Gly Ser Cys Cys Asn Glu Met Asp He Trp Glu Val Asn 210 215 220 Ser Arg Ala Ser His Val Val Pro His Tnr Cys Asn Lys Lys Gly Leu 225 230 235 240 Tyr Leu Cys Glu Gly Glu Glu Cys Ala Phe Glu Gly Val Cys Asp Lys 245 250 255 Asn Gly Cys Gly Trp Asn Asn Tyr Arg Val Asn Val Thr Asp Tyr Tyr 260 265 270 Gly Arg Gly Glu Glu Pne Lys Val Asn Thr Leu Lys Pro Phe Tnr Val 275 280 285 Val Thr Gin Phe Leu Ala Asn Arg Arg Gly Lys Leu Glu Lys He His 290 295 300 Arg Phe Tyr Val Gin Asp Gly Lys Val He Glu Ser Pne Tyr Thr Asn 305 310 315 320 Lys Glu Gly Val Pro Tyr Thr Asn Met He Asp Asp Glu Phe Cys Glu 325 330 ' 335 Ala Thr Gly Ser Arg Lys Xyr Met Glu Leu Giy Ala Thr Gin Gly Met 340 345 35C Gly Glu Ala Leu Thr Arg Gly Met Val Leu Ala Met Ser He Trp Trp 355 360 365 Asp Gin Gly Gly Asn Met Glu Trp Leu Asp His Gly Glu Ala Gly Pro 370 375 380 Cys Ala Lys G y Glu Gly Ala Pro Ser Asn He Val Gin Val Giu Pro 385 390 395 400 Phe Pro Glu Val Thr Tyr Thr Asn Leu Arq Trp Giy Glu He Gly Ser 405 410 415 Thr Tyr Gin Glu Val Gin Lys Pro Lys Pro Lys Pro Gly His Gly Pro 420 425 430 Arg Ser Asp 435

Claims: CLAIMS 1. A one-step process for combined desiz g and "stone- washmg" of dyed denim, wherein the denim is treated with an amylolytic enzyme in combination with a first abrading monocomponent endoglucanase and a second streak-reducing monocomponent endoglucanase. 2. The process according to claim 1, wherein the amylolytic enzyme is an α-amylase, preferably a microbial α- amylase, such as a bacterial or a fungal α-amylase. 3. The process according to claim 2, wherein the α- amylase is producible by the bacterium Bacillus , or by the fungus Aspergillus . 4. The process according to claim 2, wherein the α- amylase is producible by the Bacillus licheniformis , Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus stearothermophilus; or mutants thereof. 5. The process according to claim 4, wherein the α- amylase are selected from the oxidation-stable α-amylase mutants disclosed m International Patent Application PCT/DK9 /00371 (WO 95/21247) . 6. The process according to any of the claims 1-5, wherein the first endoglucanase is a fungal EG V type cellulase, or a fungal EG III type cellulase obtainable from a strain of the genus Tπchoderma . 7. The process according to claim 6, wherein the EG V type endoglucanase is derived from or producible by a strain of Scytalidium (f. Humicola) , Fusarium, or Myceliophthora. 8. The process according to claim 7, wherein the EG V is derived from or producible by Scytalidium thermophilum (f. Humicola msolens) , Fusarium oxysporum or Myceliophthora themophila, preferably from Humicola msolens, DSM 1800, Fusarium oxysporum, DSM 2672, or Myceliophthora themophila, CBS 117.65. 9. The process according to claim 8, in which the endoglucanase comprises the ammo acid sequence of the Humicola insol ens endoglucanase shown in SEQ ID No. 1 or is an analogue of said endoglucanase which i) is at least 60% homologous with the sequence shown in SEQ ID No. 1, ii) reacts with an antibody raised against said endoglucanase, and/or iii) is encoded by a DNA sequence which hybridizes with the DNA sequence encoding said endoglucanase. 10. The process according to claim 8, in which the endoglucanase comprises the amino acid sequence of the Fusari um oxysporum endoglucanase shown in SEQ ID No. 2 or is an analogue of said endoglucanase which i) is at least 60% homologous with the sequence shown in SEQ ID No. 2, ii) reacts with an antibody raised against said endoglucanase, and/or iii) is encoded by a DNA sequence which hybridizes with the DNA sequence encoding said endoglucanase. 11. The process according to any of the claims 1 - 10, wherein the second endoglucanase has a catalytic activity on cellotriose at pH 8.5 corresponding to k cat of at least 0.01 s _1 , preferably of at least 0.1 s _1 , more preferably of at least 1 s ~ 1 12. The process according to claim 11, wherein the second endoglucanase is obtainable by or derived from a strain of Humicola , Tri choder a , Mycel iophthora , Peni cill i u , Irpex , Aspergill us , Scytal idi um or Fusarium. 13. The process according to claim 12, wherein the endoglucanase is derivable from a strain of Humi cola insol ens , Fusarium oxysporum or Trichoderma reesei . 14. The process according to claim 12, in which the endoglucanase comprises the amino acid sequence of the Humi cola insol ens endoglucanase shown in SEQ ID No. 3 or is an analogue of said endoglucanase which i) is at least 60% homologous with the sequence shown in SEQ ID No. 3, 11) reacts with an antibody raised against said endoglucanase, and/or in) is encoded by a DNA sequence which hybridizes with the DNA sequence encoding said endoglucanase. 15. The process according to any of the claims 1-14, in which the first and second endoglucanase, respectively, is used in an amount of corresponding to a cellulase activity between 5 and 8000 ECU per litre of desιzmg/"stone-washmg" liquor, pre¬ ferably between 50 and 500 ECU per litre of liquor. 16. The process according to any of the claims 1-15, in which the treatment is performed at a temperature in the range of 30-100°C, preferably 30-60°C, and a pH the range of 3-11, preferably 7-9. 17. The process according to any of the claims 1-16, wherein denim is dyed with a natural dye, preferably indigo; or a synthetic dye, preferably direct dye, indirect dye and fiber- reactive dye. 18. The process according to any of the claims 1-17, in which the denim additionally is treated with a thermostable lipolytic enzyme; preferably a thermostable lipolytic enzyme derived from a strain of Pseudomona s , more preferably a strain of Pseudomonas fragi; a strain of Pseudomonas st utzeri , a strain of Pseudomonas cepa cia , a strain of Pseudomona s fl uorescens , or a strain of Candida , preferably a strain of Candida cyl mdra cea (also called Candida rugosa ) , or a strain of Candida an tarcti ca . 19. The process according to claim 18, in which the lipolytic enzyme is dosed in an amount of from about 0.01 to about 10,000 KLU/1, preferably of from about 0.1 to about 1000 KLU/1. 20. The process according to any of the claims 1-19, in which the α-amylase is dosed in an amount of from about 100 to about 10,000 KNU/1.

Inventors: LUND HENRIK, LUND, HENRIK
Applicants: LUND HENRIK, NOVONORDISK AS, LUND, HENRIK, NOVO NORDISK A/S
Application year: 1996
Main classification: C11D3
IPC codes: C11D3, C11D7, C11D11, C12N9, D06L1, D06M16, D06P5