PT101152A - Dye-transfer-inhibiting enzyme detergent compositions - Google Patents

Abstract

Detergent compositions which prevent the transfer of dye between fabrics during washing comprising an enzyme which has a peroxidase activity, hydrogen peroxide or a hydrogen peroxide precursor or an enzyme system capable of generating hydrogen peroxide, an additional oxidizable substrate and a cellulase, compositions characterized in that the cellulase accounts for at least 10% removal of immobilized carboxymethyl cellulose radioactively labelled in accordance with the C14 CMC method at 25 x 10-6% by weight of the washing test solution are described. In accordance with this invention a preferred cellulase essentially comprises a homogeneous component of endoglucanase which is immunoreactive with the monoclonal antibody created against a partly purified cellulase 43 KD derived from Humicola insolens DM 1800.

Description

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DETERGENT COMPOSITIONS -INZIMATIC INHIBITORS OF THE TRANSFER OF COLORS

Field of the Invention The present invention relates to an enzymatic composition for inhibiting the transfer of dyes from a red tissue to another tissue during washing.

BACKGROUND OF THE INVENTION One of the more persistent and more complicated problems arising during modern fabric washing operations is the tendency for some red fabrics to release the dye in the washing solutions. The dye is thus transferred to other fabrics being simultaneously washed '

One way to overcome this problem would be to remove by bleaching the dyes from the red fabrics before they have the opportunity to fix themselves on the other articles present in the washing.

Suspended or solubilized dyes may undergo a degree of oxidation in the solution by the use of known bleaching agents. Patent E 101 167 discloses a stable liquid bleaching composition which contains a hydrogen peroxide precursor which is activated to release peroxide of hydrogen in the N-dilution ', it is important, however, that at the same time the dyes actually present in the fabrics are not whitened, i.e. what? do not damage the cSres. U.S. Patent A-077,768 describes a process for inhibiting the transfer of palm dyes from the use of an oxidizing bleaching agent together with catalytic compounds such as p-porphines

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Mod. 71-20,000 ex. - 90/08 20 25 30 fervo r. U.S. Patent 421,414 describes peroxidases and oxidases used for the oxidation of organic or inorganic substances, including dyed substances. A dye transfer inhibiting composition comprising an enzyme system capable of generating hydrogen peroxidase and iron catalysts has been described in co-pending European Patent Application 91202655.6 filed on 9 October 1991. EP 424 398-A discloses an additive detergent capable of exerting a bleaching effect comprising a peroxidase. The additive further comprises one or more enzymes, particularly a lipase, protease, amylase or a cellulase. EP-A-350 098 describes a C14CMC method which defines cellulase selection criteria that are relevant for application in detergents. It has been found that a minimum of 10% removal of radiolabeled carboxymethylcellulose labeled according to the CMC method at 25x: 10-6% by weight of cellulase protein in the test solution provides a high active cellulase. A preferred cellulase group, which fits into the definition of high activity, in accordance with the present invention was described in copending European Patent Application No. 1159/90 filed May 5, 1990. A disclosure is hereby incorporated by reference. a cellulase preparation consisting essentially of a homogeneous endoglucanase component which is immunoreactive with a monoclonal antibody raised against a partially purified 43 kD cellulase derived from Humicola insolens DM800. It has now surprisingly been found that the efficiency of the peroxidase, in terms of inhibition of dye transfer, is considerably increased by 35 1 * 5 10 15

Mod. 71-20,000. The use of said high activity cellulase and more particularly the specific cellulase preparation described in copending Danish Patent Application No. 1159/90. It is therefore an object of the present invention to provide dye transfer inhibiting compositions which have an optimum colorant transfer rate in the wash liquids by using said cellulase and peroxidase of high cellulase and peroxidase activities. In accordance with one embodiment of this invention, there is provided a dye transfer inhibiting composition comprising a cellulase preparation which is economically effective, for example with the use of, for example, recombinant DNA techniques. According to another In another preferred embodiment of the present invention, there is provided a process for washing operations involving red fabrics. Summary of the Invention The present invention provides a dye transfer inhibiting composition comprising an enzyme having peroxidase activity, a hydrogen peroxide or a hydrogen peroxide pathway or an enzymatic system capable of generating hydrogen peroxide, an additional oxidizable substrate and a cellulase, characterized in that the cellulase provides at least 10% removal of radiolabeled immobilized carboxymethylcellulose according to the C14CMC method at 25x10-65% by weight of the wash assay solution.

Wake up with & present invention, a cellulase 4 · T 1 65552

The preferred compound consists essentially of a homogenous endoglucanase component which is immunoreactive with a monoclonal antibody raised against a partially purified A3kD cellulase derived from Humicola insolens DM1800. 10 15

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Detailed Description of the Invention CELLULASE The activity of the enzymes and particularly the activity of the cellulase enzyme has been defined for various applications by different analytical methods. These methods all attempt to provide a realistic assessment of the expected performance in use, or by at least one measurement that is related to the performance in use. As has been described in detail in EP-A-35009S, many of the methods, particularly those frequently used. by the cellulase manufacturers, are not sufficiently related to the performance in use of the cellulase in detergent laundry compositions. This is due to the other conditions of use for which these methods of measuring activity have been developed. The method described in EP-A-350098 has been developed to be and to have a predictive correlation for the hierarchy of cellulase activity in detergent laundry compositions. The present invention therefore uses the method described in EP-A- 350098, to investigate cellulases in order to distinguish cellulases which are Citable in the present invention from those which will not provide the objects of the present invention. The method of arrest, hereinafter referred to as Method C1A-CMC, which has been adapted from the method described in EP-A-350098, can be described as follows.

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The principle of the C14CMC method for investigating is to measure at a defined cellulase concentration in a wash solution the removal of the immobilized carboxymethyl cellulose (CMC) from a tissue substrate. Removal of the CMC is measured by labeling The simple counting of the amount of radioactive C14 in the tissue substrate prior to and after the treatment with cellulase enables evaluation of the activity of the cellulose "

Preparation of the samples

Preparation of CMC: The base solution of the radioactive CMC is prepared according to Table 1. The radioactive CMC can be obtained by the methods referred to in EP-A-35809S

Tissue Substrates Tissue substrates are made of 5cm x 5cm Egyptian cotton swabs. They are inoculated at the center with 0.35 ml of the radioactively labeled CMC base sieving. The Egyptian cotton swabs are then air dried,

Immobilization of CMC! To immobilize radioactively labeled CMC on Egyptian cotton swabs, lavodetric equipment is used " Linitest Original -Haunau ", manufactured by Original Haunau, Germany. A vial of the metal from the laundry machine is filled with 400 ml of hard water (4 mmol / liter Ca +). A maximum of 13 wicks per jar may be used. The vat is then incubated in a progressive heating cycle of from 0øC to about 0øC for 40 minutes, in a lavandometric apparatus. After incubation, the vortexes are quenched with running city water for 1 minute. They are squeezed and allowed to air dry for at least 30 minutes. According to the samples EP-A-350Q98 of the wicks with 6 35 τ 5 10 16

65555 the immobilized radioactive CMC can also be measured as " blank samples ", without washing "

Mod. 71-20,000 ex. - 70/08 20 25 30 TREATMENT OF SAMPLES; Washing Assay Solution The wash assay solution is prepared according to the composition of Table II. έ equilibrated to a pH of 7.5. The lavage assay solution is the basis upon which a sample is added for the cellulase assay. Care should be taken not to dilute the wash assay solution by adding water to equilibrium 100H before determining the amount of cellulase to be added. The amount of cellulase that is used in this? detection assay, should be added so as to provide Ξ5 x 10-6 weight percent cellulase protein in the wash test solution (equivalent to 0.25 milligrams / liter). Washing Procedure: The wicks so inoculated with CMC, are then treated in a wash simulation process. The washing process is simulated on the laundry machine type, " Linritest, Original Haunau " made by Original Haunau, Haunau, Germany. An individual wick is placed in a glass tube The tube is filled with 10 ml of laundry test solution and then sealed in a liquid-tight manner. Up to 5 vials are placed in each wash-out bottle. The vial is filled with water as a transfer medium of the heat for the simulation of the wash. The washing simulation is carried out in the form of a progressive heating cycle from 20 ° to 60 ° C for 40 minutes. In the samples, the vials are submerged in cold water and each of the wicks is subsequently drawn off. its vial, rinsed in a beaker with water 7 35 T 5 10 15

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65555 soft chain, squeezed and allowed to air dry for at least 30 minutes "iv! Sd lion !! To measure the removal of the radiolabeled CMC, a scintillation counter is used, for example a LKB 1Ξ10 U1 trabecula Seiritillation Counter. In order to obtain more accurate results, the instructions for the optimum operation of that particular flicker should be followed by the meter counter manual. For example, for the

of Ultrabeta scintillation LKB 1 21 0.0 the following procedure should be followed. The wick to be measured is placed in a plastic bottle filled with 12 ml of scintillant liquid < for example, Packard's 299 scintillator). The sample is then allowed to stabilize for at least 30 minutes. The vial is then placed into the LKB 1210 Ultrabeta Scintillation Counter and the respective radioactivity counts for the wick are obtained. To. measuring the amount of CMC removal due only to cellulase, a measurement of a wick that has been inoculated at the same time but which has been treated in the wash solution without cellulase is required. The activity of the cellulase is then expressed as a percentage of the removal of the radiolabeled CMC. This percentage is calculated by the following% formulas of removal of the radiolabeled CMC. = XO - XC x 100 X0 where XO is the scintillation radioactivation count of a wick treated with the cellulase-free washing assay solution XC is the radioactive scintillation count of a wick treated with the wash assay solution containing cellulase to be evaluated. β 35

Gonslderasges ^ stajy ^ tic ^^ In order to provide statistically reliable results, a statistical analysis should be used. For the example given, with the use of the Ultrabeta Scintillation Counter LKB 1210, it has been found that a sample size of 3 rows can be used for each scintillation radioactivity count. In order to confirm the process by means of an internal counter-test, it is recommended to measure and calculate the " blank sample " according to EP-A-350098 This will allow detecting and eliminating errors »

Interpretation of results; The detection assay described provides a rapid, single and safe method for identifying cellulases meeting the activity criteria of the present invention against cellulases not forming part of the present invention.

It has been found that a removal of 10% or more of the radiolabeled CMC immobilized according to the above indicated CMC method indicates that the respective cellulase satisfies the requirements of the invention.

It will be obvious to those skilled in the art that percentages of removal above 10% indicate a higher activity for the respective cellulase. It is therefore envisaged that cellulase which provides more than 5% or preferably more than 50% removal of the cellulase. CMC, on protein concentration in the wash assay solution according to the Cl-CMC method, will provide an indication of even better cellulase performance in use in laundry detergents'

It was also considered that the use of 9 T τ T τ 1 10

Mod. 71-20,000 ex. Higher concentrations of cellulase for the C14CMC method would provide higher removal percentages. However, there is no proven linear relationship between the concentration of cellulase and the percentage of removal obtained therefrom.

It was also considered that the use of higher concentrations of cellulase for the C14CMCS method would provide higher percentages of removal '

Table I Radioactive labeled CMC base solution (All percentages by weight of the total solution)

1 CMC # Total (CMC should be detergent grade detergent grade with a degree of substitution of between about 0 / + 7 and about 0.7) 99, S > 10-a K 'Ethanol .17 + 985, 12 m 10-3½ 1 Deionized Aaua 84915.68> 10-3 ° I Tq te 11 ........ 1 ÔO% S; The total CMC contains both radioactive and non-radioactive CMC to provide a radioactivity that allows for sufficiently clear readings. in the scintillation counter used. For example, the radioactive CMC may have an activity of 0.7 milicuries / g and be mixed with non-radioactive CMC in a ratio of 1 to 6 p7.

Mod. 71-20,000 ex. - 90/08 T .1 65555 1 Table 11 Laundry assay solution (all percentages by weight of the total solution) 6 Coconut alkylsulfonic acid 1-O-5-O Coconut alkyl sulfate (TEA salt ) 0.040% Alcoholic ethoxylate 0.100% Coconut fatty acid 0.100% 10 Oleic acid 0.050% Citric acid 0.010% Tris-tanolamine 0.040% Ethanol 0.000% 16 Propanediol 0.015% Sodium hydroxide 0.030% Sodium formate 0.010% Protease 0.06% Water (2.5 mmol / liter ca), pH adjusting agent (solutions of HCL or NaOH> and cellulase Remaining to 100% In accordance with the present invention, the preferred cellulases are the same as those described in Danish Patent Application 1159/90 For example, a cellulase preparation useful in the compositions of the invention may essentially consist of a homogeneous endoglucanase component which is reactive with an antibody created against a c highly purified 43kD elulease, derived from Humicola insolens, DSM 1800, or which is homologous to said 11

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Mod. 71-20,000 ex. - 90/08 20 25 30 endoglucanase of 43kD. . It should be noted that all the enzymes. However, in Danish Patent Application 1159/90, additional criteria are set forth which, in combination with the present test of the invention, detection methods, enable to identify preferred cellulase enzymes. Cellulase preparations particularly useful in the compositions of the present invention are those wherein, in addition to the detection assay, the endoolucanase component exhibits a CMC endoase activity of at least about 50, Preferably at least about 60, especially about 90 CMC endoase units per mg of the total protein. A preferred endoglucanase component exhibits, in particular, a CMC endoase activity of at least 100 CMC endoase units per milligram of In the present context, the term " CMC-endo-endoase activity " refers to the endoglucanase activity of the endoglucan component in terms of its ability to degrade cellulose to glucose, cellobiose and triose as determined by the decrease in viscosity of a carboxymethylcellulose solution (CMC) after incubation with the cellulase preparation of the invention as described in detail below. . The endo-CMC activity of the endogenous enzyme can be determined from the decrease in the viscosity of the CMC, as shown below. g / 1 of (I: MC < / RTI > -cules 7 LFD) in 0.1 M t- The sample of the enzyme to be analyzed is soluble in this same buffer. 10 ml of substrate solution and 0.5 ml of enzyme solution are

IS 35 • J 65555

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Mod. 71-20,000 ex. 90 (0820 25 30) mixed and transferred to a viscometer (eg Haake VT 181, IMV sensor at 181 rpm), thermostated at 40Â ° C. Viscosity readings are obtained as soon as possible after mixing and again 30 minutes later. The amount of enzyme which reduces viscosity by half under these conditions is defined as 1 unit of CMC endoase activity. SDS polyacrylamide jelly electrophoresis ISDS ~ PABE > and isoelectric focusing with target proteins in a manner known to those skilled in the art have been used to determine the molecular weight and isoelectric point of the endoglucanase component respectively in the cellulase preparation useful in the present context. Thus, the molecular weight of a specific component endoglucanase activity was determined to be 43kD. The isoelectric point of this endoglucanase was determined to be about 5.1. The cellobiohydrolase activity can be defined with o being the activity that tends to p-nitrophenyl cellobiose. Activity is determined as nitrophenyl pmole released per minute at 37 ° C and pH 7.0. It was found that the present endoglucanase component had practically not. no cellobiohydrolase activity. The endoglucanase component in the present cellulase preparation was initially isolated by extensive purification procedures, involving, for example, reversed-phase HPLC purification of a crude mixture of H insolins according to U.S. Patent 4,435,307. This process surprisingly resulted in the isolation of a 43kD endoglucanase as the only component with unexpected properties 13 35 1 i 5 10 16

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favorable 'because of a particularly high endoglucanase activity. Likewise, in addition to the detection assay, the cellulase enzymes useful in the present compositions may further be defined as endoglucanase activity enzymes < hereinafter " endoglucanase enzymes ", which enzymes have the amino acid sequence 'represented in the attached Sequence Listing ID # 2, or a homologue thereof having endoglucanase activity. In the present context, the term " homologue " is intended to indicate a polypeptide encoded by the hybridizing DNA Î'. the same probe as coding the DNA for the endoglucanase enzyme with this sequence under certain specified conditions, such as prior embedding with SxSSC and prehybridization for 1 h at 40 ° C in a solution of 0.0% formamide , 5x Denhardt's solution, 50 mM sodium phosphate, pH 6.8 and 50 μg sonicated and denatured calf thymus DNA, followed by hybridization in the same solution, supplemented with 100 μl ATP for 18 h at 40 ° C ). The term is intended to include derivatives of the above-mentioned sequence obtained by the addition of one or more amino acid residues to one or both of the C- and N-terminal of the native sequence, substitution of one or more amino acid residues at either or both sites of the native amino acid sequence deletion of pm or more amino acid residues at either or both ends of the amino acid sequence or at one or more sites within the native sequence, or insertion of a or more amino acid residues at one or more locations in the native sequence. The present endoglucanase enzyme may be an enzyme produced by Humicola species such as Humicola 14-35 1 5 10 15

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insolens ", for example strain DSM 1800, filed on 1 October 1981 in the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1B, D-33Q0 Braunschweig, (Germany), as determined in the Budapest Treaty on the International Recognition of Deposit of Micro-organisms for the Purposes of Patent Processing (the Budapest Treaty). In yet another aspect, the cellulase enzymes usable herein may be defined, in addition to the detection assay, as endoglucanase enzymes having the amino acid sequence shown in SEQ ID No. 4 attached, or a homologue thereof (as defined above ) having an endoglucanase activity. The said endoglucanase enzyme may be one which is producible by a Fusarium species, such as Fusarium oxysporum. for example the DSM Bó2 strain, deposited. on June 6, 1983 in the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 18, D-3300 Braunschweig, Germany, according to the provisions of the Budapest Treaty. 'In addition, homologous endoglucanases may be derived from other microorganisms which produce cellulocyte enzymes, for example Trichoderma species. Mvce 1 lophthoraPhanerochaeteSchizophvl Ium Penic 111ium. For the industrial preparation of the present cellulase preparation, however, it is preferred to employ recombinant ADIM techniques or other techniques involving fermentation or mutation adjustments of the involved microorganisms to ensure the overproduction of the enzymatic activities desired. Such methods and techniques are known 15 35 1 5 10 15

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in industry and can easily be performed by branch technicians. The endozylucanase component may thus be one which can be produced by a method consisting of culturing a host cell transformed by a recombinant DNA vector carrying a DNA sequence encoding said endoglucanase component, or a precursor of said endoglucanase component as well as DNA sequences encoding functions which allow the expression of the DNA sequence encoding the endoglucanase component or a precursor thereof in a culture medium and under conditions permitting the expression of the endoglucanase component or its precursor and DNA constructs comprising a DNA sequence encoding an endoglucanase enzyme as described above, or a precursor form of the enzyme, include those DNA constructs having the sequence of DNA as depicted in Sequence Listings ID # 1 and JD # 3 appended, or one ua modification. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to the endogenous amino acid sequence of endoglucanase but which correspond to the codon usage of the host organism in which the DNA construct is introduced or the nucleotide substitutions that originate a different amino acid sequence and therefore possibly a different protein structure which may, give rise to a mutant endoglucanase with different properties of the native enzyme. Further examples of possible modifications are the insertion of one or more nucleotides at each end of the sequence, or the deletion of one or more nucleotides at either or both ends of the sequence.

Mod. 71-20,000 ex. The DNA constructs encoding the endoglucanase enzymes usable herein may be prepared synthetically by established standard methods, for example the phosphoamidite method described for 5. L Beaucage et al., H. Caruthers, Tetrahedron Letters, 1981, pp. 1859-1869, or the method described by Matthes et al., EiiBO Journa 1983, pp. According to the phosphoamidite method, the oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, extinguished, ligated and cloned into suitable vectors. A DNA construct encoding the endoglucanase enzyme or a precursor thereof may, for example, be isolated by the establishment of a cDNA or genomic library of a cell culture producing microorganism such as Humico inso lens . DSM 1800 and clone detection. positive cells by conventional methods, such as by hybridization using oligonucleotide probes synthesized on the basis of a complete or partial amino acid sequence of the endoglucanase according to standard techniques (cf. Sambrook et al., Molecular Cloning s Or by selection of clones expressing the appropriate enzymatic activity (i.e., CMC endoase activity as defined above) or by selection of clones producing the protein which reacts with an antibody against a native cellulase (endoglucanase). Finally, the DNA construct may be of mixed synthetic and genomic, mixed synthetic and cDNA origin prepared by linking the fragments of synthetic, genomic and cDNA origin (as appropriate), the fragments corresponding to the various parts

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Mod. 71 · 20,000 et. - 90/08 20 25 30 of the entire DNA construction of wakes up with standardized techniques. The DNA construct may also be prepared by reacting the polymer chain using specific primers, for example. as described in U.S. Patent 4,683,202 or R.K.Saiki et al., Science 539, 1988, pp. 487-491. Recombinant expression vectors in which the above DNA constructs are inserted include any vector that may conveniently be subjected to recombinant DNA processes and the choice of vector will often depend on the host cell in which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, for example a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome (s) into which it has been integrated. In the vector, the DNA sequence encoding endoglucanase should be operably linked to a suitable promoter and terminator sequence. The promoter sequence may be any DNA sequence which exhibits an activity. transcriptional in the chosen host cell and may be derived from genes encoding proteins, both homologous and heterologous of the host cell. The processes used to bind DNA sequences encoding endo-g-lucanase, the promoter sequence and the terminator respectively and to insert them into suitable vectors are well known to those skilled in the art (cf., for example, Sambrook et al., quot; quot;). Host cells which are transformed with

Mod. 71-20,000 c.

The above ADIM constructs or expression vectors may, for example, belong to a species of Aspergillus. More preferably Asperacillus prvzae or Asperacillus nioer. Fungal cells can be transformed by a process which involves the formation and protoplastic transformation of the protoplast, followed by regeneration of the cell wall in a manner already known. The use of Aspergillus as a host microorganism is described in EP 238 023 (Novo Industri A / S), the contents of which are hereby incorporated by reference. The host cell may also be a yeast cell, for example, a strain of Saccharomyces cerevisae. Alternatively, the host organism may be a bacterium, in particular strains of S-treotomy and Bac11lus and E.co1i. The transformation of the bacterial cells can be performed according to conventional methods, for example, as described in Sambrook et al., Molecular Cloninqs A Laboratorv Manual ,. Screening of the appropriate DNA sequences and construction of the vectors may also be performed by standard procedures, see Sambrook et al., Quoted. The medium used to grow the transformed host cells may be any conventional medium suitable for the development of the host cells in question. The expressed endoglucanase can be conveniently secreted into the culture medium and can be recovered therefrom. By well-known procedures, which include separation of the cells from the medium by centrifugation or filtration, precipitation of the protein components from the medium by means of a slurry, such as 19-sulfate sulfate

ammonium followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like. Recombinant DNA techniques, as indicated above, include protein purification techniques, fermentation and mutation techniques, and other techniques which are well known in the art, it is possible to provide high purity endoglucanases. The level of cellulase of the present composition described above should be such that the amount of the enzyme protein to be released in the wash solution is between 0.005 and 40 mg / liter of washing solution, preferably 0.01 to 10 mg / liter of washing solution.

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FEROXIPASES 25

Peroxidases which may be employed for the present purpose may be isolated from and produced by plants (for example, horseradish peroxidase) or micro-organisms such as fungi or bacteria. Some preferred fungi include strains belonging to the Deuteromycotina subdivision, class Hyphomycetes, for example, Fusarium, Humicola, Trichoderma, Myrothecium, Verticillum, Anthromyces, Caldaryomyces, U lociumiosimellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucana <IF0 6113) , Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Uloc ladium chartarum, EmbelTisia all-ior Dreschlera ha Iodes »

Other preferred fungi include strains belonging to the sub-division Basidiomycotina, class Basidiomycetes, for example Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus 20 35 1 6 10 15

Mod. 71-20,000 ex. - 90 (08 20 25 30 65555

(IFO 8371), Coprinus macrarhizus, Phanerochaete chrysosporium (for example, NA-12.) &quot; or Coriolus versicolor (e.g., PR428-A). Other fungi. preferred include strains belonging to the Zigomycotina subdivision, class Mycoracsae, for example Rhizopus or Mucor, in particular Mucor hiemalis. Some preferred bacteria include strains of the order Actinomycetales, for example Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12332) or Streptoverticium 11 a verticilUum ssp. verticillium. Other preferred bacteria include Bacillus pumillus (ATCC 12905), Bacillus stearothermophi lus, Rhodobacter sphaeroides, Rhodomonas palustris, Streptococcus lactis, Pseudonomas purrocinia (ATCC 15958) or Pseudomonas fluorescs (NRRL B-11). Other potential sources of useful peroxidases are listed in Et.C.Saunders et al., Quoted. 41-43. Methods for producing enzymes to be used according to the invention are described in the art, eg FEBS Letters 1625, 173 (1). Applied and Environmenta 1 liicrobio.Loav. Feb. 1985, pp. 273-278, Applied Mic: Robot 1. B io tec hno 1.26. 1987, pp. 158-163, Biotechnology Letters 9 (5), 1987, pp. 357-360, Nature 326. 2 April 1987, FEBS Letters 4270, 209 (2), p.321, EP 179 486, EP 200 565, 8B 2 167 421, EP 171 074 and Agr. .Chem 50 (1), 1986, p. 247. Particularly preferred peroxidases are those which are active at the pH typical of the wash liquors, i.e., at a pH of 6.5-10.5, preferably 6.5-9, 5 and more preferably 7.5-9.5 Such enzymes may be isolated by screening for the detection of relevant enzyme production by means of alkalophilic microorganisms.

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Mod. 71 -20,000 ex. Example using an ABTS assay described in R, E, Chers. And W. 8, Bardsley, Biochem. : 1.45. 1975, pp. Other preferred peroxidases are those having good thermostability as well as good stability over commonly used detergent components, such as nonionic, cationic anionic surfactants, detergency enhancers, phosphates, and the like. Another group of useful peroxidases are by the haloperoxidases, such as peroxidases, and bromo. The enzyme-peroxidase &quot;. may further be a kit which can be produced by a method consisting of culturing a host cell transformed with a DNA receptor vector having an ADIM coding sequence of said enzyme as well; as DNA sequences encoding functions that. allow expression of the DNA sequence encoding &quot; enzyme in a medium. culture, under conditions which allow the expression of the enzyme and the recovery of the enzyme from the culture. A DNA fragment encoding the enzyme may, for example, be isolated by establishing a cDNA or genomic library of an enzyme producing microorganism which It is of interest as is one of the above-mentioned organisms and screening for detection of positive clones by conventional methods such as by hybridization with oligonucleotide probes synthesized on the basis of the total or partial amino acid sequence of the enzyme or by selection of clones which express the appropriate enzymatic activity, or by selecting clones producing a protein that is reactive with an antibody against the native enzyme. Once selected, the DNA sequence may be 22 35

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Mod. 71 -20,000 ex. Inserted into a suitable repopulating expression vector comprising suitable promoter-operator and terminal sequences which allow the enzyme to be expressed in a particular host organism as well as an origin of replication, making it impossible for the vector to replicate in the host organism in question. The resulting expression vector can then be transformed into a suitable host cell, such as a fungal cell, of which preferred examples are a species of Aspergillus, most preferably Aspergillus oryzae or Aspergillus niger. Fungal cells can be transformed by a process involving the protoplast formation and transformation of the protoplasts, followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238,023 &lt; da Novo Industri A / 3), the contents of which are hereby incorporated by reference. Alternatively, the host organisms may be a bacterium, in particular strains of Streptomyces and Bacillus, or E. coli. be carried out according to conventional methods, for example as described in T.Man iatis et al., Molecular Clonings A-Laboratory Handbook. Cold Spring Harbor, 1982 ,. Detection of the appropriate DNA sequences and construction of the vectors can be carried out by standard procedures, cf. Maniatis et al., Quoted. The medium used to culture the transformed host cells may be any suitable conventional medium to develop the host cells in question. The expressed enzyme can be conveniently secreted into the culture medium and can be recovered therefrom by well-known procedures. 15

Mod. 71-20,000 e *. - including separation of the cells from the medium by centrifugation or filtration, precipitation of the protein components from the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as chromatography ion exchange, affinity chromatography or the like. Detection of the appropriate DNA sequences and construction of the vectors may also be performed by standard procedures, cf. T.Maniatis et al., quot; The medium used to culture the transformed host cells can be any suitable conventional means to develop host cells. In particular, the expressed virus can be conveniently secreted into the culture medium and can be recovered therefrom by well known procedures which include separation of the cells from the medium by centrifugation or filtration , precipitation of the protein components from the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like. Starting or during the process, HeOa may be added in an amount of for example 0.001-5 mM, particularly 0.01-1 mM. When using Coprinus peroxidase, 0.01-0, 5 mM He0e ® is preferred with 0.1-1 mM B.pumillus peroxidase HB0e. The hydrogen peroxide may be added in the form of hydrogen peroxide or a precursor thereof, preferably a perborate or a percarbonate. The level of hydrogen peroxide precursor which may be used is dependent on the specific properties of the chosen peroxidase, for example, Coprinus peroxidase should be applied in a detergent composition containing less than 4 35 1 5 10 15

Mod. 71-20,000. - 70/03 20 25 30 65555

5% perborate. In the process of the present invention it may be desirable to use an enzymatic process for the formation of hydrogen peroxide. Thus the process according to the invention may also comprise the addition of an enzyme system (i.e., an enzyme and the respective substrate thereof) which is capable of generating hydrogen peroxide at the start "or during the washing and / or enhancer process. Such a category of hydrogen peroxide generating systems comprises enzymes which are able to convert molecular oxygen and a substrate organic or inorganic, respectively in hydrogen peroxide and oxidized substrate. These enzymes produce only low levels of hydrogen peroxide, but may be more advantageously employed in the process of the invention, since the presence of peroxidase ensures efficient use of the hydrogen peroxide produced. Preferred hydrogen peroxide generating enzymes are which act on cheap and easy-to-obtain substrates which may conveniently be included in detergent compositions. An example of such a substrate is glucose which can be used for the production of hydrogen peroxide by means of a glucose oxidase. Suitable oxidases include those which act on aromatic compounds such as phenols and related substances, for example oxidases of catechol, laccase. Other suitable oxidases are urate oxidase, galactose oxidase, alcohol oxidases, amine oxidases, amino acid oxidases, amyloglucosidase and cholesterol oxidase. Preferred enzyme systems are alcohol and aldehyde oxidases. 35 1 5 10 15

Mod. 71-20,000 «-90 / 08 20 25 Ó5555

More preferred systems for application in granular detergents will have solid alcohols, for example glucose whose oxidation is catalyzed by glucose oxidase to lucloronic acid, with the formation of hydrogen peroxide. More preferred systems for application in liquid detergents will involve liquid alcohols which may also act as, for example, solvents. An example of this is ethanol / ethanol oxidase. The amount of oxidase to be employed in the compositions according to the invention should be at least sufficient to provide a constant generation of 0.01 to 10 ppm Avo per minute in the wash bath. With the glucose oxidase this can be achieved, for example, at room temperature and at a pH of 6 to 11, preferably 7 to 9, with 50,000 U / l glucose oxidase, 0.005 to 0.5 The addition of another oxidizable substrate to the peroxidase at the beginning or during the washing and / or rinsing process can enhance the inhibitory effect of the peroxidase dye transfer employed. It is believed that this may be attributed to the formation of short lived radicals or other oxidized states of this substrate which participate in the bleaching or other modification of the colored substance. Examples of such oxidizable substrates are the metal ions, for example Mn + ions of halides, for example chloride or bromide ions, or organic compounds such as phenols, for example β-hydroxyninic acid or Î ±, β-dichlorophenol. Further examples of phenolic compounds which may be used for the present purpose are those provided in M. Kato and S. Shimizu, Plant Ce 11 Physiol. E6 &lt; 7 &gt;, 1985, pp. 1E91-1301 (particularly cf. Table 1) or B.C.Saunders et al., Quoted work, p141 ff.

In the process according to the invention, the peroxidase will typically be added as a component of a compound of formula (I) to a compound of the formula (I): ## STR1 ## in which the oxidizable substrate to be added is suitably between about 1 μΙΊ and 1 mM. and may be added in an amount of 0.01-100 mg of enzyme per liter of washing liquid. As such, it may be included in the detergent composition in the form of a non-sprayable granulate, a liquid, especially a stabilized liquid, or a protected enzyme. Non-sprayable granules may be produced, for example as described in U.S. Patents 4,106,991 and 4,661,451 (both issued to Movo Industri A / S) and may optionally be coated by methods known in the art. Liquid enzyme preparations may, for example, be stabilized by the addition of a palliate such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid, according to established methods. Other enzymatic stabilizers are well known in the art. The protected enzymes may be prepared according to the method described in EP-A-38216. The detergent composition may also consist of a further substrate for the peroxidase. Alternatively, the pH of a solution of the detergent composition according to the invention will preferably be 7-iH, especially from 7.5 to 9.5. The pH of the wash bath is dependent on the peroxidase chosen, for example the Coprinus peroxidase should be applied at a bath pH of less than 9.5. Detergents The composition according to the invention may contain the usual components of such compositions £ 7 35 0 &quot; X 1 δ 10 16 mmv

Mod. 71-20,000 ex. The present invention relates to anionic, nonionic, ampholytic or zwitterionic or, more rarely, cationic and surfactant organic surfactants. Suitable surface active agents are well known in the art and an extensive list of such compounds is disclosed in U.S. Patent 3,717,630 and U.S. Patent Application Serial No. 58,116. Detergent compositions useful in the present invention contain between 1 and 95%, preferably 5% to 40%, of a nonionic, anionic, zwitterionic or mixtures thereof. Detergent enhancers, whether inorganic or organic, phosphatic or non-water soluble or insoluble, and other water soluble salts , may be present, and salts of this type may be employed, organic detergents are present or not. A description of suitable enhancers is set forth in US Pat. U.S. Patent 3,936,537 and U.S. Patent Application Serial No. 589,116. Detergent builders are present from 0 to 50%, preferably from 5 to 40%. Other components used in detergent compositions may be employed, such as foam enhancing agents or foam depressing agents, enzymes and stabilizers or activators, soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, stain inhibitors, dyes and perfumes. These components, particularly the enzymes, optical brighteners, coloring agents and perfumes, should preferably be selected in such a way as to be compatible with the bleaching component of the composition. 88 35 1 1 5 10 15

Mod. 71-20,000 ex. 20 25 30

The detergent compositions according to the invention may have liquid, paste or granular forms, the enzyme may be formulated in any convenient form, for example - in the form of a powder or a liquid. The enzyme may be stabilized in a liquid by inclusion of enzyme stabilizers. Liquid detergents may further include stabilized hydrogen peroxide precursors. The granular compositions according to the present invention may also be in &quot; compact form &quot;, i.e., they may have a relatively higher density than conventional granular detergents, i.e. between 550 and 950 g / l? in such a case, the granular detergent compositions according to the present invention will contain a lower amount of inorganic filler salt, compared to conventional granular detergents, typical filler salts are the alkaline earth metal salts of sulfates and chlorides, typically sodium sulfate; the "compact" detergents &quot; typically comprise not more than 10% filler salt. The present invention also relates to a process for inhibiting the transfer of dyes from one fabric to another of the dissolved and suspended dyes encountered during laundry operations involving dyed fabrics. The process comprises placing the fabrics in contact with a washing solution as described above. The process according to the invention is conveniently carried out in the course of the washing process. The washing process is preferably carried out at from 5Â ° C to 75Â ° C, especially from 20Â ° C to 60Â ° C. The pH of the treatment solution is preferably from 7 and 12, 1 1 10 15

Mod. 71 -20,000 and especially 90 to 90, especially 7 to 9, 5. The process and the compositions according to the invention may also be used as additives during laundry operations.

The following examples illustrate the present invention and the unexpected superior color treatment benefits obtained therefrom.

EXAMPLE I

Importance of the cellulase performance parameter of claim 1

The following tests were carried out Test conditions:

Washing temperature 60 ° C (progressive heating cycle)

Washing time s A-O minutes pH 7.5

Water hardness 4 mmol / L Concentration of detergents 1%

Composition of detergents cf »EPA 350 098 ex.l

Celulasess 1) Celluzyme * produced by Novo Mordisk = reference

S) endoglucanase of 43kD = cellulase according to. the invention Test Results:% Removal of C14-CMC by Cellulase

Detergent without cellulase (-reference) 0

Detergent + Celluzyme &quot; 1.5 mg protein / L (150x10â † '50 12.7 3.0 mg protein / L (300x10â †' 50 17.7 4.5 mg protein / L (450x10â † '%) 21.5 30 35

Detergent + endoglucanase with 43kD 0.3mg protein / L (30x10-6%) 20.3

Mod. 71-20,000 e *. The data presented above clearly shows the importance of the parameter claimed for the ceiulases according to the invention over the Celluzyme on the market. EXAMPLE II Two sets of each of four types are prepared of detergent compositions, all based on a granular compact. Typically a compact granular detergent composition contains the following ingredients: 20 25 30

Sulphate of linear alkyl benzene (SAL) Nonionic alkyl sulphate Citrate of citric acid Zeolite citric acid Po 1, imel Chelated Solubility Sodium silicate 11% Cr (1), (2) and (3)

Perbarate 0.5 phenol sulphanate 0.1 The above detergent composition was supplemented as indicated below

35 / CCTCCT òDjju

A5 without endoglucanase with 43kD nor peroxidase = * reference B) with endoglucanase of 43kD) with peroxide D) with endoglucanase of 43kD and peroxidase

Cells with cellulite and peroxidase B) with cellulose and peroxidase B) with cellulose and peroxidase (C) with peroxidase D) with celluzyme * and peroxidase 15

Mod. 71-20,000 ex. The first type of each set of detergent compositions does not contain any cellulase or peroxidase (composition of references A) In the first set of detergent compositions the endoglucanase of 43kD is added at a level of 2mg of protein of the enzyme / liter of wash solution <55 ceu / liter). In the second set of detergent compositions, the cellulose is added to a level of 76 mg enzyme IV protein / liter of the wash solution (55æCi / liter).

Test conditions s "Miele washer test ~ Cotton program, low water level (181), short cycle. - 4 cycles

· Temperature 40 ° C · Composition of the washer part 35 1) 1 kg of clean load s 32 I: 1 I: 1 10 - 150 g of - 150 g of ··· 00 g of - 300 g of

Mod. 71 -20,000 ex. - 90108 20 25 30 cotton (unvarnished velvet) knitted or crocheted cotton fabric (cotton underwear) 1 1 - 1 g cotton - polyethylene (E) For the grading of 3 white articles, (4 replicates of each), 3) 10 by 5 cm of acid red dye 151 in &quot; nylon &quot; s To create a low dye transfer level. 4) Stain prepared to provide a typical laundry source of dirt. - hard water (15 grs / 3.78 liters), - Detergent concentration = O, ΚΚ.

Test procedure: The intent of this test is to compare the whiteness of the textile articles, washed cumulatively 4 times, between the compositions to be tested and the composition of yield. For this test three dirty articles are used. For each treatment of an article four replicates are used. The articles to be examined are exposed on a flat, graduated, neutral colored surface placed parallel to the source of illumination. As a light source a fluorescent light is used Philips Philips 7 &quot; cold &quot; of color TL 40/57 which produces 1080 WATTS of light, designed to correspond to normal daylight (Dé> 5). The color T ° is 7400 ° K, the reflectivity. of the light is excellent (74) and the luminous emission is 46 LH / W. 33 35 5 1 5 1 10

Mod. 71-20,000 ex. · 90/08 20

Differences are indicated in counting panel units (upc), and are positively better performing than those of the reference treatment *

Graduation scale (graduation UPC5 0 = Same 1 = I think this is better 2 = I know this is a little better 3 = This is much better 4 = This is much better

The UPC graduation data are statistically recounted, an average of the 4 replicates is obtained, the DMS (least significant difference) is mentioned in Tables 1 and 11.

Results of the peroxidase / celluzymeR test B vs. C vs. vs. D vs. A DM5 Average -cic the E 0, EI, 67 s 1.80 s 0.61 Mean - 0, 0, ES E, OE s &gt; E, 86 s 0.50 cycles liédia - 4 0.68 E, 48 s S, 66 s 0.76 c iclos 34 35

65555 TABLE II 8

Test results: 4-3kD peroxidase / -endoglucanase ____________________________B vs. A____C vs. A___D vs. The mean DM5 - S -0.10 1.39 s 1.97 s 0.40 cycles Mean - 3 cycles r S t, 0.46 m, 4 - 0.07 2 , 44 s 2.97 s 0.29 cycles

Conclusion The combination of a better

The above results clearly show that peroKidase / 43kD of the present invention provides statistically significant performance than individual actions of both ingredients. EXAMPLES III TO VIII The following compositions are obtained: a) Compact granular detergent: Examples II to IV;

EXAMPLES III IV

Sodium linear alkyl benzene 11.4 Acetic acid sulphate 1.80 2.40 Sulfate Cue alkyl 3.00 3.10 Ethoxylated Cw alcohol 7 times 4.00 Ethoxylated alcohol 11 times 1.80 1 , 90 Dispersant 0.07 0.1 Liquid silicone O GO O 0.80 19! t / v v-

Mod. 71-20,000 ex. - 90/08 Citric acid 3.00 2.50 Zeo1 Ate OO Er UC η w 32.10 6 Copolymer of maleic acid acrylic acid 5.00 5.00 DETMPA 1.00.00 Cellulase de-endoglucanase 43kD 0.02 A Calcium 0.60 0.60 10 Lipase 0.360.40 Amylase 0.30 0.30 Si sodium α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, Smaller constituents up to 100 20 b) Conventional granular detergent 5 examples V and VI EXAMPLE V IV Sodium sulphonate Cta linear alkyl benzene Ò 1 | 8.0 25 Sodium sulfate 15.0 1S, 0 Zeolite A E6, 0 22.0 Sodium nitrile acetate 5.0 5.0 Endolanase of 43 kD endoglucanase 0.02 0.03 30 PVP0.57 TAED 3.0 3.0 Boric acid 4.0 - Perbarate 0.5 1 Phenoxide 1 0.1 0.2 35 Paroxidase 0.1 0.2 36

Jr and

Components smaller than 100

Mcd. 71 · 20,000 ex. - 90 08 c) Liquid detergent s examples VII and VIII EXAMPLES VII VIII C18-1 acid &lt; k alkenyl succinic acid 3.0 8.0 Citric acid monohydrate 10 s 0 L5.0 Sodium sulfate ClB alkyl 8.0 8.0 10 Alcohol Sodium Sulfate, Ethoxylated 2 times 3.0 Cte_l 5 Ethoxylated Alcohol 7 times S, O Alcohol 5 times ethoxylated 8.0 - Penta diethylene triamine (15-methylene phosphonic acid), ε - Oleic acid 1.8 - Ethanol, · Ι · π 0 4, 0 Propanediol Ξ, 0 2.0 Protease 0, 20 0 20 Cells of endoqlucanase of 43 kD 0, 2 0.05 PVP. f. 0 0.1 Foam suppressor 0.15 0.15 NaOH to tea pl-1 7.5 Perborate 0.15 25 25 1 1 1 0 0 0 0 0 0 0 0 0 id ase 0, 4 · 0, 1 Water and smaller components up to 100 parts 30 INFORMATIO N FOR SEQ ID N ° 1. (i) SEQUENCE CHARACTERISTICS (A) Length s 10% pairs of <B) Nucleic acid types (C) Type of single strands base 35 (D) Topology 1inear 37

1 t κγκγεγιsí · QUvJiJD 5 10 15

Mod. 71-20,000 ex. (A) Name (s) of peptide (c) Type of MOLECULE to cDNA (iii) HYPOTHETICS to IMS O <iv) ORIGINAL SOURCE (A) Organism 5 Humicola insolens (B) Strain DSM 1800 Cix) CHARACTERISTICS . (B) Locus 1: 73. »927 < i) CHARACTERISTICS (A) Name / Key s sig. (B) Location: 10 »= 72 (ix) CHARACTERISTICS (A) Name (s) CDS (B) Location (s) 10. = 957 SEQUENCE DESCRIPTION SEG SEGMENTS IDG CGATCCAAG ATO CGT TCC TCC CCC CTC CTC CCG TCC GCC GTT GTG GCC Hei Arg Ser Ser Pro Leu Leu Pro Ser Ala Goes Ala -EI -ao -15 -10

GCC CTG CCG GTG TTG GCC CTT GCC GCT GAT GGC AGG TCC ACC CGC TAC 96 A Leu Pro Vai Leu Ala Leu • Ala Ala Asp Gly Arg Ser Thr Arg Tyr -5 1 5 TGG GAC TBC TGC AAG CCT TCG TGC GGC TGG GCC AAG AAG GCT CCC GTC 144 Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Go 10 15 EO AAC CAG CCT GTC TTT TCC TGC AAC GCC AAC TTC CAG CGT ATC ACG GAC 192 Asn Gin Pro Will Phe Ser Cys Asn Ala Asn Phe Gin Arg Ile Thr Asp S5 30 35 AO 38 35

TTC GAC GCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC Phe Asp Ala Lys Ser Gly Cys Glu Pro Gly Gly Uai Ala Tyr Ser Cys 50 55 45 GCC GAC CAG ACC CCA TGG GCT GTG AAC GAC GAC TTC GCG CTC GGT TTT Ala Asp Bln Thr Pro Trp Ua Asn Asp Asp Asp As Leu Gly Phe is O 65 70 GCT GCC ACC TCT ATT GCC GGC AGC AAT GAG GCG GGC TGG TGC TGC GCC Ala Ala Thr Ser Ile Ala Gly Ser Asn Glu. Ala Gly Trp Cys Cys Ala 75 80 85

Mod. 71-20,000 ex. - 90 | Q8 20 25 30 TGC TAC GAG CTC ACC TTC ACA TCC GGT CCT GTT GCT GGC AAG AAG ATG Cys * Tyr Glu Leu Thr Phe Thr Ser Gly Pro. Go Ala Gly Lys Lys Het 90 95 100 GTC GTC CAG. TCC ACC AGC ACT GGC GGT GAT CTT GGC AGC ÂAC CAC TTC Go Go Gin Be Thr Be Thr Gly Gly Asp Leu Gly Be Asn His Phe 105 · UO. US 120 GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC. TTC GAC-GGA TGC ACT Asp thy Asn Ile Gly Gly Gly Gly Gly Cly Phe Asp Gly Cys Thr 125 130 _ 135 CCC CAG TTC GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG TCC Pro Gin Phe Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly Ile Ser Ser 140 145 150 CGC AAC GAG TGC GAT CGG TTC CCC GAC GCC CTC AAG CCC GGC TGC TAC Arg Asn Glu Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr 155 160 165 TGG CGC TTC GAC . TGG; TTC AAG AAC GCC GAC AAT 'CCG AGC TTC: AGC TTC Trp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe 170' 175 180 CGT CAG GTC CAG TGC: CCA GCC GAC CTC GTC GCT CGC ACC GGA TGC CGC Arg Gin Go Gin Cys Pro Ala Glu Leu Go Ala Arg Thr Gly Cys Arg 185 190 195 200 CGC AAC GAC GAC GGC AAC TTC CCT GCC GTC CAG ATC CCC TCC AGC AGC Arg Asn Asp Asp Gly Asn Phe Pro Ala Vai Gin Ile Pro Ser Ser Ser 205 210 215 ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG TCC ACC Thr Ser Ser Pro Pro Asn Gin Pro Thr Ser Thr Ser Thr Thr Ser Thr 220 225 230 TCC ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC Ser Thr Thr Ser Ser Pro Pro Go Pro Gin Thr Thr Pro Ser Gly Cys 235 240 245 ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC Thr Ala Glu Arg Trp Ala Gin Cys Gly Gly Asn Gly Trp Ser Gly Cys 250 255 260

39 35 65553 ACC ACC TBC GTC OCT GGC AGC ACT TGC ACG AAG ATT AAT GAC TGG TAC 912 Thr Thr Cys Go Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr 2 270 270 275 280 DAT CAG TGC CTG TAGACGCAGG GCAGCTTGAG GBCCTTACTG GTGGCCGCAA My Gin Cys Lsu 9 is 4 10 15

Mod. 71-20,000 cc. 20 25 30 35 285 CQAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 1024 GATTGTCACA TAAATGCAAT 6AGGAACAAT GAGTAC 1060 INFORMApXQ FOR SEQ.ID.W8 2s (1) SEQUENCE CHARACTERISTICS (A) 305 Amyrioacids Type C (75%) Topology: linear Ui) MOLECULE 5 protein SEQUENCE OF SEQUENCE SEQ.ID.NS 2: Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Will Go To Leu Pro Leu -H -20 -15 -10 Go Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys "5 1 5 10 {> = Lys Pro Ser Cys Gly Trp a Lys Lys Ala Pro Go Asn Gin Pro 15 20 25 Go Phe Ser Cys Asn Ala Asn Phe Gin Arg Ile Thr · Asp Phe Asp Ala 30 35 40 Lys Ser Gly Cys Glu Pro Gly Gly Va Ala Tyr Ser Cys Ala Asp Gin 4. 50

j. Q * l 10 15

Mod. 71 - 20,000 ex.-90 / OS 20 25 30

Thr Pro Tro Ala Go Asn Asp Asp Phe Ala Leu Gly p | -, and Ala Ala Thr ÓO 65 70? 5 Ser Ile Ala Gly Ser Asn Blu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 80 85 90 Leu Thr Phe Thr Ser Gly Pro Go Ala Gly Lys Lys Het Go Go Gin 95 100 105 Be Thr Be Gly Gly Asp Leu Gly Be Asn His Phe Asp Leu Asn 110 115 180 He Pro Gly Gly Gly Go Gly He Phe Asp Gly Cys Thr Pro Gin Phe 125 130 135 Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly He Ser Ser Arg Asn Glu 140 145 150 155 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 160 165 170 Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gin Go 175 180 185 Gin Cys Pro Ala Glu Leu Go Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp 190 195 200 Asp Gly Asn Phr Pro Ala Vai Gin He Pro Ser Ser Ser Ser Thr Ser Ser 805 23.0 2.15 pro Go Asn Gin Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr £ 20 225 230 235 4: 1. 35 a * 1

Ser Ser Pro Pro Go Gin Pro Thr Thr 240

Pro Ser Gly Cys Thr Ala Glu 245 250 15

Mod. 71 20,000 ex. - 90/09 20 25

Arg Trp Ala Gin Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys 255 .., - 260 265

Va'.l. Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gin Cys 270 275 230

(B) TYPES OF BASE TYPE s Nucleic acid (C) of simple CDRDXO s (D) TOPOLOGY s linear (l) TYPE OF MOLECULE (B) TYPES OF MOLECULE (s) HYPOTHETICS s NXG (iv) INVERSE SENSE s NIO (vi) ORIGINAL SOURCE (A) ORGANISM fusarium oxysporum (B) STRAINS s DSM 2672 (i '<) CHARACTERISTICS (A) NAME / KEY * CDS (B ) LOCATION s 97. «1224 30 SEQUENCE DESCRIPTION: SEQ.ID.NS 3:

GAATTCGCGG CCGCTCATTC ACTTCATTCA TTCTTTAGAA TTACATACAC TCTGTTTCAA

AACAGTCACT CTTTAAACAA AACAACTTTT GCAACA ATG CGA TCT TAC.ACT CTT ilet Arg Ser Tyr Thr Leu 1 5 114 35

Mod. 71 · 20,000 ex. -

CTC GCC CTG 1 / GGC CCT CTC GCC GTG L.eu Ala Leu Ala Gly Pro Leu Ala Go 10 15 CAC TCT ACT CG A TAC TGG GAT TGC TGC His Ser Thr Arg Tyr Trp Asp Cys Cys 25 30 GGA AAG GCT GCT GTC AAC GCC CCT GCT 8 and Lys wing Wing Asn Wing Pro Wing 40 45 AAC CCC ATT TCC AAC ACC AAT GCT GTC Asn Pro lie Ser Asn Thr Asn At Vai 55 6Q AGT GCT GCT TCT GGA AGC GGT 162 Be Wing To Be Gly Be Gly To AAG CCT TCT TGC TCT TGG AGC 8.10 Lys Pro Ser Cys 35 Ser Ser Tys ACT TBT GAT AAG AAC GAC 858 Leu Thr Cys Asp Lys Asn Asp 50 AAC GGT TGT GAG GGT GGT GGT 306 Asn Gly Cys Glu Gly Gly Gly 65 70 TCT GCT TAT GCT TGC ACC AAC TAC TCT CCC TGG GCT GTC AAC GAT GAG 354 Ser Wing Tyr Ala Cys Thr Asn Tyr Ser Pro Trp A .La Val Asn Asp Glu 75 80 85 CTT GCC TAC GGT TTC GCT GCT ACC AAG ATC TCC GGT GGC TCC GAG GCC 402 Leu Ala Tyr Gly Phe Ala Ala Thr Lys Ile Ser Gly Gly Ser Glu Ala 90 95 100 AGC TGG TGC TGT GCT TGC TAT GCT TTG ACC TTC ACC ACT GGC CCC GTC 450 Ser Trp Cys Cys Cys Tyr A Leu Thr Phe Thr Thr Gly Pro Va 105 110 1.15 AAG GGC AAG AAG ATG ATC GTC CAG TCC ACC AAC ACT GGA GGT GAT CTC 49S Lys Gly Lys Lys Het 1 le Go Gin Gin Thr Asn Thr Gly Gly Asp Leu 120 125 130 4. * * * 35 1 i 5 10 15

Mod. 71 -20,000 ex. · 92/12 20 25 30 54,555

i GGC GAC AAC CÀC TTC. GAT CTC ATG ATG CCC GGC GGT GGT GTC GGT ATC Gly Asp Asn His Phe Asp leu Het Het: Pro Gly Gly Gly Vly Gly Ile 135 140 145 * 150 TTC GAC GGC TGC. ACC TCT GAG TTC GGC AAC GCT CTC GGC GGT GCC CAG Phe Asp; Gly Cys Thr Ser Glu Phe. Gly Lys Ala read Gly Gly Ala Gin 155 150. 165 TAC GGC GGT ATC TCC TCC CGA AGC GAA TGT GAT AGC TAC CCC GAG CTT Tyr Gly Gly Ile Ser Ser Arg Arg Glu Cys A Ser Ser Tyr Pro Glu leu 170 175 180 CTC AAG GAC GGT TGC CAC. TGG CGA TTC 6ACTGG. TTC GAG AAC GCC-GAC Leu Lys-Asp Gly Cys His Trp Arg Phe Asp. Trp Phe Glu Asn Ala Asp. 185 190 195 I AAC CCT GAC TTC ACC TTT GAG CAG GTT CAG TGC CCC AAG: GCT. CTC CTC I Asn Pro Asp Phe Thr Phe Glu Gin Vai Gin Cys Pro lys Ala Leu Leu ZOO 205 210 GAC ATC AGT GGA TGC AAG CGT GAT GAC GAC TCC AGC TTC CCT GCC TTC, Asp Ile Ser Gly Cys lys Arg Asp Asp Asp Ser Ser Phe Pro Ala Phe 215 220 225 230 AAG GTT GAT ACC TCC GCC AGC AAG 'CCC CAG CCC: TCC. AGC 'TCC GCT AAG lys Go Asp Thr Be Ala Ser Lys Pro Gin Pro Ser Ser Be Ala Lys 235 240 245 AAC ACC ACC TCC. GCT GCT GCT GCC GCT CAG CCC CAG AAG ACC AAG GAT Ivs Thr Thr Ser Ala Ala Ala Ala Ala Gin Pro Gin Lys. Thr Lys Asp 250 255 260 TCC GCT CCT GTT GTC CAG AAG TCC TCC ACC AAG CCT GCC GCT CAG CCC Be Pro Wing Will Be Gin Be Ser Thr Be Pro Wing Ala Gin Pro 255 270 275. c GAG CCT ACT AAG CCC GCC GAC AAG CCC ACC ACC GAC AAG CCT GTC GCC Glu Pro Thr lys Pro Ala.Asp Lys Pro Gin Tljr Asp Lys Pro Vail Ala 280. 285 290 ACC AAG CCT GCT GCT ACC AAG CCC GTC CAA CCT GTC AAC AAG CCC AAG Thrlys Pro Ala Ala Thr lys Pro Vai Gin Pro Go Asn lys Pro Lys 295-300 305- 310 ACA- ACC CAC AAC GIC CGT GGA ACC AAA- ACC CGA GGA AGC TGC CCG GCC Thr Thr Gin. lys is Arg Gly Thr lys Thr Arg Gly Ser Cys. Pro Al 315 320, 325 546 594. i 642; 690 738 786 834 882 930 97 1 1026 1074. AAC ACT GAC GCT ACC GCC AAG GCC TCC GTT GTC CCT GCT TAT TAC CAG 1122 Lys · Thr Asp: Ala Thr Ala lys Ala Ser Vai Vai Pro Ala Tyr Tyr Gin 330 335 340 TGT GGT GGT TCC AAG 'TCC GCT TAT CCC. AAC. GGC AAC CTC GCT TGC GCT '1170 Cys Gly Gly Ser Lys. Be Wing Tyr Pro Asn Gly Asn Leu Wing Cys Wing 345 350 355 ACT GGA AGC AAC TGT GTC AAG CAG AAC. GAG TAC TAC TCC CAG TGT GTC 12181 Thr Gly Ser lys Cys Go Lys Gin Asn Glu Tyr Tyr Ser Gin Cys Go 360 365_ ________370 _! 44 35

1 Î »CC AÎ" TAAA Î "GAT ATCCATCGGT TGT6SAADAG ACTATGCGTC TCAGAAGGGA 1274-Î" Asr &gt; S75 5 TCCTC &lt; CftTS AvCAQGCTTG ΤΟΑΪ'ϊΌΤΑΪΑ GCATG6CATC CTGGACCAAG TB7TCGACCC 1334 T7Q7T8TACA TAGTATATCT TCATTSTATA TATTTABACA CATAQATASC CTCTTGTCAG 1394

CijACAAfjves C «ACAAAA8A CTTGGCAQGC 7T8TTCAATA TTGACACAGT TTCCTCCATA 1454

10 ADDITIONAL INFORMATION FOR SEQ.ID.NO 4

&lt; U CA: &quot; SEQUENCE ACTURES &quot; CDI · SPILLS 376 to »

Mod. / 1 · 20,000 ex. 20 25 (B) Tyr-O-amino acids &lt; C) R0.: OLOGIA s linear

Cii &gt; 7 '(70)> MOLECULAR SEQ ID NO: 4: SEQ ID NO: 4: SEQ ID NO:

Met Arg Ser Tyr Thr Leu Leu Ala Leu Ala Gly Pro Leu Ala Will Be 15 10 15

Ala Ala Ser Gly Ser Gly His Ser Thr Arg Tyr Trp Asp Cys Cys Lys 20 25 30

Pro Ser Cys Ser Trp Ser Gly Lys Ala Ala Go Asn Ala Pro Ala Leu 35 40 45

Thr Cys Asp Lys Asn Asp Asn Pro lys Ser Asn Thr Asn Ala Val Asn SO 55 60

Gly Cys Glu Gly Gly Gly Ser Ala Tyr Ala Cys Thr Asn Tyr Ser Pro 65 70 75 80

Trp Ala Go Asn Asp Glu Leu Ala Tyr Gly Phe Ala Ala Thr Lys Ile 85 90 95

Ser Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr 100 10S 110

Phe Thr Thr Gly Pro Go Lys Gly Lys Lys Het lie Go Gin Gin Ser Thr 115 120 125

Asn Thr Gly. Gly Asp Leu Gly Asp Asn His Phe Asp leu Het Het Pro 130 135 140

Gly Gly Gly Gly Gly Ile Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys 145 ISO 1S5 160 &lt; 15 35

.

Ala Leu Gly Gly Ala Gin Tyr Gly Bly Ile Ser Arg Arg Ser Glu Cye IO 170 175 Asp Ser Tyr Pro Glu Leu Leu Lys Asp Bly Cys His Trp Arg Phe Asp I8 185 185 Trp Phe Glu Asn Ala Asp Asn Pro Asp Phe Thr Phe Glu Gin Vai Gin 195 200 SOS Cys Pro Lys Ala Leu Leu Asp Ile Ser Bly Cys Lys Arg Asp Asp Asp £ 10 215 220

Ser Ser Phe Pro Ala Phe Lys Go Asp Thr Ser Ala Ser Lys Pro Bln S25 230 235 240

Mo d. 71-20,000 ex. - 20/08 20 25 30

Pro Ser Ser To Be Wing Lys Lys Thr Thr Be Wing Wing Wing Wing Wing 245 250 255

Pro Gin Lys Thr Lys Asp Ser Pro Wing Go Go Gin Lys Ser Ser Thr 2 &lt; S0 265 270

Lys Pro Ala Ala Gin Pro Glu Pro Thr Lys Pro Ala Asp Lys Pro Gin 275 280 285 Thr Asp Lys Pro Go Ala Thr Lys Pro Ala Ala Thr Lys Pro Vai Gin 290 295 300

Pro Go Asn Lys Pro Lys Thr Thr Gin Lys Go Arg Gly Thr Lys Thr 305 310 315 320

Arg Gly Ser Cys Pro Ala Lys Thr Asp Ala Thr Ala Lys Ala Ser Va 325 330 335 35 * 11%

Mal Pr d Ala Tyr Tyr Gin Cys Gly Gly Ser Lvs Ser Ala Tyr Pr o Asn * 11% 34Í 345 350

Gly Asn Leu Ala Cys Ala Thr Gly Ser Lys Cys Go Lvs Gin Asn Glu 355 360 3Ã-5

Tyr Tyr Ser Gin Cys Go Pro Asn 370 * 375 10 15

Lisbon 51G.Wffi.1993

By THE PROCTER &amp; GAMBLE C0MPANV

Mod. 71-20,000 ex. - 90/08 20 25 30

VASCO MARQUES UifKt Official Agiote de Propf.Hjade InduMrisl «erlório-Aíce ao Conceição, 3, 1. · -110β UtfMH

Claims (1)

1 5 10 15 DESCRIPTION 90/04 - * '000 - 02 - U' P ° W CLAIMS. A dye transfer inhibiting composition comprising - an enzyme having a peroxidase activity, - a hydrogen peroxide or a hydrogen peroxide precursor, or an enzyme system capable of generating hydrogen peroxide, - a substrate and a cellulase, characterized in that said cellulase provides at least 10% removal of the immobilized carboxymethylcellulose labeled radiolabelled according to the C14CMC method at 85% by weight of the cellulose protein in test solution 21 The dye transfer inhibiting composition according to claim 1, characterized in that the cellulase is essentially constituted by a homogeneous endoglucanase component, which is immunoreactively raised with an antibody raised against a highly purified 43 kD cellulase derived from the Hummingbird _____insolens , SM 1800, or which is homologous to such 43kD endoglucanase "3Î" - Inhibitory Composition The dye transfer agent of claim 2, wherein the endoglucanase component of said cellulase has an isoelectric point of about 5.1%. A detergent composition according to claim E or 3, characterized in that said endoglucanase component is produced by a method which comprises culturing a transformed host cell, 1g • 1 • 15 Mod. 71-20,000 ex. - 90/08 65555 with a recombinant β-DNA vector carrying a DNA sequence encoding said endoglucose component or a precursor of said endoglucanase component, as well as ADM sequences encoding functions, which allow the expression of the sequence of DNA encodes the endoglucanase component, or a precursor thereof, &quot; in a culture medium and under conditions which allow the expression of the endoglucanase component or its precursor and the recovery of the endoglucanase component of the culture. 5. The dye transfer inhibiting composition according to claim 1, characterized in that the ceiulase has the amino acid sequence shown in the Sequence Listing together, or is homologous thereto, exhibiting endoglucanase activity. The dye transfer inhibiting composition according to claim 3, characterized in that said cellulase can be produced by a species of the genus Humicola, for example, Humicola insolens. The detergent composition according to claim 1, characterized in that the ceiulase compound is an endoglucanase enzyme, which has the amino acid sequence shown in the Sequence Listing ID # A, or is an endoglucanase active homologue thereof. Composition, detergent according to claim 7, characterized in that said glucanase enzyme is produced by a species of Fusarium, for example, Fusarium ium Qxysporum 9. A detergent composition according to any one of claims 5 to 8, characterized in that said enzyme is produced by a DNA construct constituting a DNA sequence encoding the enzyme. A detergent composition according to claim 9, characterized in that the DNA sequence is as depicted in the accompanying sequence listings ID # 1 or ID # 2. A detergent composition according to any one of claims 5 to 10, characterized in that said host cell is a strain of a fungus such as Tricloderuca or Aspergillus. preferably Asoer α lus oryzae or As per q i. 1 or a yeast cell belonging to a strain of Hansenula or Saccharomyces, for example a strain of Saccharomyces cerevisae 12. A detergent composition according to any one of claims 5 to 10, characterized in that said host cell is a strain of a bacterium, for example, Bacillus Streptomoves or E.coli. A dye transfer inhibiting composition according to claims 1 to 1, characterized in that said enzyme having peroxidase activity is derived from a strain of Coorinus or Bromeliaceae. Inhibitory transfer composition characterized by a color perborate according to claims 1 to 13, in that the hydrogen peroxidase precursor is O w 65555 or a composition according to claim 14, characterized in that the level of said perhorate is from 1 μl to 10 ml / l of the solution of. The dye transfer inhibiting composition of claim 1 to 15, wherein said further oxytable substrate is selected from a metal ion, a halide ion or an organic compound such as a phenol 1, for example p-hydrazoxic acid or S, 4-dichlorophenol or a phenol sulfonate. The dye transfer inhibiting composition according to any one of claims 1, to 16, characterized in that said enzymatic system capable of generating hydrogen peroxidase is an oxidase selected from the group consisting of glucose oxidase, urate oxidase, galactose oxidase, alcoholic oxidases, amine oxidases, amino acid oxidase and cholesterol oxidase Dye transfer inhibiting composition according to any one of the preceding claims, characterized in that it is a detergent, additive in the form of a non-pulverulent granulate, of a liquid, in particular a stabilized liquid, or of a protected enzyme system. A detergent composition comprising an additive detergent according to any one of claims 4 to 6, wherein the detergent composition comprises a detergent composition according to claim 19, in compact granular form, or in liquid form. A process for inhibiting the transfer of a dye textile fabric from a fabric dyed to another fabric, when said fabrics are washed and / or rinsed together in a washing resolution, wherein said fabrics come into contact with a composition according to claims 1 to 10, characterized in that the peroxidase is used at levels between 0.01 and 100 mg / L of the washing solution, the level of hydrogen peroxide is between 0.001 and 5 μl of the washing solution, the absolute level of additional oxidizable substrate is between 1 μM and ImM and the Cellulose is added at levels between 0.005 and 40 mg of enzyme protein / Per liter of washing solution. GAMBLE C0MPANY 5 VASCO MARQOE * M aggle Office of Industrie Gtrtêtlo-Asiê * ê ConcelÇâe, 3, LMlfi · UKttMI