PT100027B - PROCESS FOR THE PREPARATION OF COMPOSITIONS GRANULAR DETERGENTS COMPACT WITH HIGH-ACTIVITY CELLULASE INTENDED FOR WASHING FABRICS IN A WASHING MACHINE - Google Patents

Description

PROCESS FOR THE PREPARATION OF COMPACT GRANULAR DETERGENT COMPOSITIONS WITH HIGH ACTIVITY CELLULASE, INTENDED FOR WASHING TISSUES IN A WASHING MACHINE The present invention relates to a process for obtaining granular detergent compositions containing cells that have a compact shape, that is, they have a compact form. relatively high density and contain a relatively low amount of inorganic filler salt, compared to conventional detergent compositions. In these tergent compositions, cellulase consists of a high activity cellulase, defined by the C14CM method described here. Preferably cellulase is a specific single component endoglucanase.

Background of the Invention

The need to produce detergent compositions that have not only good cleaning properties, but also a good fabric softening performance and other fabric treatment benefits is well established in the art.

The effectiveness of cellulotic enzymes, that is, cellulases, in terms of cleaning textiles and reducing agents of weight in fabrics has been recognized for some time; GB-A-2,075,028, GB-A-2,095,275 and GB-A-2,094,826, describe detergent compositions with cellulase for better washing performance; GE — A — 1,368,599 describes the use of cellulase to reduce the roughness of fabrics containing cotton; U.S. 4,435,307 describes the use of a cellulotic enzyme derived from Humicola insolens as well as a fraction, called ACXI, as a roughness-reducing detergent additive.

EP-A-0 269 168 describes compositions containing

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optimized cellulase-containing people, which are formulated in a slightly alkaline pH range providing a combined cleaning, softening and tissue treatment performance.

WO 89109259 describes ellase preparations useful for reducing the roughness of fabrics containing cotton, consisting of an endoglucanase component having a high endoase activity and an affinity for cellulase.

The practical exploration of cellulases has, however, been reversed due to the fact that the cellulase preparations described in the decoctions of the prior art mentioned above, if15

Mod. 71 - 20,000 ex. - 90/08 rem complex mixtures, having only a certain fraction effectively in the context of tissue treatment, is therefore difficult.;. ®arálarinaustria. „;. de_-détergeútés. <Impléméiitar an industrial production of cellulase, which is effective as far as costs; and large amounts of such cellulase preparations would be applied to obtain the desired tissue effects.

Improvements in cellulase production have also often not proven to be sufficiently identifiable in terms of applicability to detergents. The definition of a relevant cellulase selection criterion for detergent application was made possible by the C14CMC method described in EP-A-350 098. It was found that a minimum of 10% removal of radioactive, actively labeled immobilized carboxymethylcellulose provided a cellulase of high activity. A preferred group of cellulase, which is by definition agrangido high activity according to the present invention has been described in Patent Dinamarguês ^s co-pending C 1159/90 filed May 5, 1990: It is described a preparation of cellulase essentially constituting a homogeneous endoglucanase component, which is immunoreactive with a monoclonal antibody created against a partially purified 43kD cellulase, derived from Humicola in35

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The discovery that this particular cellulase endoglucanase component is advantageous for the treatment of cellulose-containing materials, now makes it possible to produce cellulase in a cost-effective manner, for example, through the use of recombinant DNA techniques, and allows to apply only a small amount of cellulase preparation is obtained and the desired effects on tissues are achieved.

On the other hand, a new generation of detergent compositions is now being commercialized, which can best be described as compact detergents although they have been given a variety of commercial names, such as Ultra, Supra, Micro ... The particularity of such compositions detergents is their high density compared to conventional detergent compositions and their ability to achieve the same efficiency as conventional detergent compositions, using a considerably lesser amount of compact detergent composition. This particle: density is best reflected, in terms of composition, by a relatively low amount of inorganic filler salt. The efficiency of such compact detergent compositions is best achieved by eliminating the pre-wash cycle and using dispersion and diffusion devices, which are placed directly in the washing machine drum at the beginning of the main wash cycle.

It is an object of the present invention to provide detergent compositions in a compact form, having a relatively high density and containing a low amount of inorganic filler salt, representing an optimum cellulase efficiency.

EP-A-381 397 described the effect of low ionic resistance on the performance of the enzyme, particularly lipase.

However, it was surprisingly found that the effect of the compact matrix on the enzymes selected from the

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the present invention, is much higher than could be expected from the cellulases of the current technique, such as described in EP-A-381 397.

It is another object of the present invention to provide a method for the treatment of fabrics in an alvar machine, consisting of the use of the present detergent compositions at low levels for the main wash cycle.

Summary of the Invention The present invention relates to granular detergent compositions that contain a surfactant, an activator, an enzyme and, if desired, conventional additives, characterized in that the enzyme consists of a cellulase preparation that provides at least 10% removal of radiolabeled immobilized carboxymethylcellulose in accordance with the C14 CMC method, at 25 x 10-4% by weight of cellulase protein in the wash assay solution.

Preferably, the cellulase compound consists essentially of a homogeneous component of endoglucanase that is immunoreactive with a monoclonal antibody created against a partially purified cellulase of about 43kD derived from Humicola insolens, DSM 1800, or that is homologous to the said endoglucanase> ^ 43kD .

Detailed Description of the Invention

The present detergent compositions are in granular form and are characterized by their density, which is higher than the density of conventional detergent compositions. The density of the compositions treated here varies between 550 to 950 g / liter, preferably 650 to 850 g / liter of composition, measured at 202 ° C.

The compact form of the compositions present is better

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reflected, in terms of composition, by the amount of inorganic filler salt; inorganic filler salts are conventional ingredients of detergent compositions in powder form; in conventional detergent compositions, the filling salts are present in substantial quantities, typically in 17-35% of the total weight of the composition

In the present compositions, the filler salt is present in amounts that do not exceed 15% of the total composition, preferably not exceeding 10% and more preferably not exceeding 5% of the weight of the composition.

The inorganic filler salts, as defined in the present compositions, are selected from alkali and alkaline earth metal salts of sulphates and

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A preferred filler salt is sodium sulfate.

TENSIO AGENT — ACTIVE

A wide variety of surfactants can be used in detergent compositions. A typical listing of the anionic, nonionic, ampholytic and switterionic classes and species of these surfactants is provided in US Patent 3,664,961 published in favor of Norris on May 23, 1972.

Mixtures of anionic surfactants are particularly suitable here, especially mixtures of sulfonate surfactants, in a weight ratio of 5: 1 to 1: 2, preferably 3: 1 to 2: 3, more preferably 3 : 1 to 1: 1. Preferred sulfonates include alkylbenzene sulfonates containing 9 to 15, especially 11 to 1Ξcarbon atoms in the alkyl radical and esters of alpha-sulfonated methyl fatty acids in which the fatty acid is derived from a C „-C ₁ fat source, preferably from a goi source of C _ir -C _in . In all cases the cation is an alkali metal

18

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ethoxy sulfates containing between 10 to 20, preferably 10 to 16 carbon atoms in the alkyl radical and an iflédwtdeetõxylation degree of 1 to 6. Examples of preferred alkyl sulfates in this invention are tallow alkyl sulfate, coconut alkyl sulfate and alkyl Em sulfates in each case, the cation is again an alkali metal cation, preferably sodium. A class of nonionic surfactants that are used in the present invention are ethylene oxide condensates with a hydrophobic portion to provide an active surfactant with a hydrophilic-lipophilic balance (EHL) ranging from 8 to 17, preferably between 9.5 and 13.5, more preferably between 10 and 12.5. The hydrophobic (lipophilic) portion can be aliphatic or aromatic in nature and the extent of the polyoxyethylene group that is condensed with any particular hydrophobic can be easily adjusted to provide a water-soluble compound, with the desired degree of balance between hydrophilic and hydrophobic elements.

Especially preferred nonionic surfactants of this type are the primary alcohol ethoxylates Cg which contain 3-8 moles of ethylene oxide per mole of alcohol, particularly the primary C ^^ alcohols which color have 6-8 moles of ethylene oxide per mole of alcohol, and the primary alcohols containing 3-5 moles of ethylene oxide per mole of alcohol.

Another class of agents. nonionic surfactants comprise the polyglycoside alkyl compounds of the general formula

R0 (CnH2n0) tZx where z is a portion derived from glucose; R is a saturated hydrophobic alkyl group that contains between 12 to 18 atoms of

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carbon; t is 0 to 10 and n is 2 to 23, x is 1.3 to 4, the compounds including less than 10% unreacted fatty alcohol and 50% less lower alkyl polyglucosides. Compounds of this type and their use in detergents are described in EP-B-0 070 077, 0 075 996 and 0 094 118.

Equally suitable as nonionic surfactants are polyhydroxy fatty acid starch surfactants of the formula

1

R -CNZ _r where R is H

R ¹

C 4 hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl or a mixture thereof, R ² is C _c , ...,. . ,

5-31 hydrocarbyl and Z and a polyhydroxyhydrocarbyl with a linear hydrocarbyl chain with at least 3 hydroxyls directly attached to the chain, or an alkoxylated derivative thereof. Preferably, R is methyl, R is an 11-15 straight alkyl or alkenyl chain C such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar, such as glucose, fructose, maltose, -lactose, in a reducing reaction amination.

Another class of surfactants is semi-polar surfactants such as amine oxides. Suitable amine oxides are chosen from mono CC _on amine oxides, preferably C C. N -alkyl or alkenyl and propylene-1,3-diamine dioxides where the remaining N positions are replaced by methyl, hydroxyethyl or hydroxypropyl groups.

Another class of surfactants is amphoteric surfactants, such as polyamine-based species. Cationic surfactants can also be used in the present detergent compositions and suitable quaternary ammonium surfactants are selected from mono C _o C _ic ammonium surfactants, preferably N-alkyl or alkenyl, with the remaining N positions are replaced by methyl, hi3 groups

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xethyl or hydroxypropyl.

Mixtures of types of surfactants are preferred, more especially anionic-nonionic mixtures and also anionic-nonionic-cationic mixtures. Particularly preferred mixtures are those described in British Patent No. 2040987 and Published European Patent Application No. 0 087 914. Detergent compositions may comprise between 1% 70% by weight of surfactant, but usually the surfactant is found present in the compositions of the present invention in an amount between 1% and 3%, more preferably 10-25% by weight.

Activator

Activating materials will typically be present between 10% and 60% of the present detergent compositions. The present compositions do not have or have practically no phosphate containing activators (not having practically is defined here as constituting less than 1% of the total detergent activating system),

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This activator consists of water-soluble activators, water-insoluble activators or mixtures thereof.

Water-insoluble activators can be an inorganic ion exchange material, generally an inorganic hydrated aluminum silicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite Α, Χ, Β or HS.

Preferred aluminum silicate ion exchange materials have the unit cell formula

Mz [(A10 ₂ ) z (SiO ₂ )] xH ₂ 0 where M is a calcium exchange cation, z and y are at least 6; the molar ratio of z pagan y is between 1.0 to 0.5 x and at least 5, preferably between 7.5 and 276, more preferably

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between 10 and 264. The aluminum silicate materials are in hydrated form and are preferably crystalline, containing between 10% and 28%, more preferably 18% to 22% water.

The above-mentioned aluminum silicate ion exchange materials are further characterized by a particle size between 0.1 and 10 micrometers, preferably between 0.2 and 4 micrometers. The term particle diameter size here represents the average particle diameter size of a given ion exchange material, determined by conventional analytical techniques such as, for example, useful microscopic determination. using an electronic screening microscope. Aluminum silicate ion exchange materials are also characterized by their calcium ion exchange capacity, which is at least 200 mg of CaC03 / g aluminum silicate water hardener equivalents, calculated on an anhydrous basis. and which generally range from 300 mg eq./g and 352 mg eq./g. The aluminum silicate ion exchange materials listed here are further characterized by their calcium ion exchange ratio, which is described in detail in GB-1,429,143.

Silicate and aluminum ion exchange materials useful in the practice of the present invention are available on the market and may be naturally occurring materials, but are preferably synthetic derivatives. A method for the production of aluminum silicatc ion exchange materials is disclosed in US Patent No. ^s 3,985,669. The preferred synthetic crystalline aluminum silicate ion exchange materials, usable here, are available under the designation Zeolite A, Zeolite B, Zeolite X, Zeolite HS and mixtures thereof. In an especially preferred embodiment, the crystalline aluminum silicate ion exchange material is Zeolite A and has the formula

IO

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^At 12 ^[(A10 2> 12 ^(SÍ0 2> 12] ^XH 2 ° where x is between 20 and 30, especially 27. 0 Zeolite X of formula Νβθθ [((A10 ₂ ) θθ (SiO ₂ ))] - IO.276H2O it is also suitable as well as the Zeolite HS of formula Na _c [(A10 „) _r (SiO„) _r ] 7.5 ϋ Z Ό Z Ό

H ₂ °).

Another suitable activating material, insoluble in water, inorganic, is layered silicate, for example, SKS-6 (Hoechst) .; SKS-6 is crystalline layered silicate consisting of sodium silicate (Na ^^ O ^) .: The high Ca ++ / Mg ++ binding capacity is mainly a cation exchange mechanism. In hot water the material becomes more soluble.

water soluble activator can be a monomeric or oligomeric carboxylate chelating agent.

Suitable carboxylates that contain a carboxy group include lactic acid, glycolic acid and its ether derivatives, as described in Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid (ethylenedioxy) diacetic acid, maleicc acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Patents 2,446 .686 and 2,446,687 and in U.S. Patent 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. ^Q 840,623. Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates, as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No. ^Q 1,379,241, lactoxisuccinates described in Dutch Patent Application 7205873 and the materials oxypolycarboxylate such as 2-oxa-1, 1,3-propane tricarboxylates described in British Patent No. ^Q 1,387,447.

Polycarboxylates containing four carboxy groups include oxisuccinates described in British Patent N ^s

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1,261,829, 1,1,2,2-ethane tetracarboxylates, 1,1,3,3-tetracarboxylates, -propane and 1,1,2,3-pro-tetracarboxylates. Polycarboxylates containing sulfur substituents include the sulfosuccinate derivatives disclosed in British Patent Nos 1,398,421 and 1,398,422 and U.S. Patent No. 3,936,448 and ^second sulfonated pyrolysed citrates described in British Patent No. 1,082,179 ^5, while xilatos that polycarbosilanes containing phosphone substituents are disclosed in British Patent No. ^s 1,439,000.

Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis, cis, cis-tetracarboxylate, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydrofuranocis, 2,5-tetrahydrofurans-cis-dicarboxylates, 2,2,5, 5-tetrahydrofurans-tetracarboxylates, 1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. ^s 1,425,343.

Among those described above, the preferred polycarboxylates are hydrocarboxylates which contain up to three carboxy groups per molecule, more particularly citrates.

Pre-cooled activator systems for use in the present compositions include a mixture of water-insoluble aluminum silicate activator such as Zeolite A and a water-soluble carboxylate chelating agent such as citric acid.

Other activating materials that may form part of the activating system for the purposes of the present invention include inorganic materials such as carbonates, bicarbonates, and alkali metal silicates and organic materials such as organic phosphatates, amino polyalkylene phosphates and amino polycarboxylates.

Other suitable water-soluble organic salts are homo- or co-polymeric acids or their salts, where the

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polycarboxylic acid comprises at least two carboxyl radicals separated from each other by no more than two carbon atoms.

Polymers of this type are described in GB-A-1,596,756. ⁵ Examples of such salts are PM 2000-5000 polyacrylates and their copolymers with maleic anhydride, these copolymers having a molecular weight between 20,000 and 70,000, especially about 40,000.

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CELLULASE

Enzyme activity and particularly cellulase enzyme activity has been defined for various applications by different analytical methods. These methods all try to provide a realistic assessment of expected performance when using or at least a measurement correlated with performance: when using. As detailed, in European Patent Application EP-A-350098, many of the methods, particularly those used frequently by cellulase manufacturers, are not sufficiently correlated with performance when using cellulase in laundry detergent compositions. This is due to the various other conditions of use for these activity measurement methods have been developed.

The method described in EP-A-350098, was developed to be and to have a predictive correlation regarding the hierarchy of cellulase activity in laundry detergent compositions.

Therefore, the present invention uses the method described in EP-A-350098 for selection of cellulases, in order to distinguish cellulases that are useful in the present invention and those that would not provide the objectives of the present invention. The selection method, hereinafter referred to as Method C14CMC, which was adapted from the method described in EP-A-350098, can be described as follows:

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First principle Method C14CMC to select is to measure, at a defined cellulase concentration in a lava solution

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gem, the removal of immobilized carboxy methyl cellulase (CMC) from a tissue substrate. The removal of CMC is measured by means of radioactive marking of some of the CMC, using radioactive carbon C14. The simple counting of the amount of radioactive C14 in the tissue substrate and after treatment with cellulase, allows the evaluation of cellulase activity.

Sample preparation:

Preparation of CMC: The base solution of radioactive CMC is prepared according to Table I. Radioactive CMC can be obtained by methods referred to in EP-A-350098.

Tissue substrates. The substrates of fabric are cotton muslin with dimensions of 5 cm x 5 cm. They are inoculated, in their center, with 0.35 ml of radiolabelled CMC base solution. The cotton muslin samples are then air dried.

CMC immobilization: To immobilize the radioactivated CMC marked on cotton muslin samples, Linitest Original Hanau laundry meter equipment manufactured by Original Hanau, Germany, is used. A metal bottle of the lavage meter is filled with 400 ml of hard water (4 mmol / liter of Ca ** ions). A maximum number of 13 samples per bottle can be used. The flask is then incubated in a heating cycle of 20 ^and C at 60 ² C for 40 minutes in a lavage equipment. After incubation, the samples are rinsed under running tap water for 1 minute. It was squeezed and allowed to air dry for at least 30 minutes.

According to EP-A-350098, specimens of samples with immobilized radioactive CMC can also be measured co64.264

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Sample treatment:

Wash solution test: The wash test solution is prepared according to the composition of Table II. It is balanced until it reaches a pH of 7.5. The wash test solution is the base to which a cellulase test sample is added. Care should be taken not to dilute the wash test solution by adding water to reach a 100% equilibrium, before determining the amount of cellulase to be added. The amount of cellulase that is used in this screening assay should be added to provide 25x 10 ”by weight percentage of cellulase protein in the wash assay solution (equivalent to 0.25 milligram / liter at 14.5 ^ C).

Washing process: Flaps so inoculated with radiolabeled CMC are then treated in a washing simulation process. The washing process is simulated on a Linitest, Original Hanau _# device from Original Hanau, Hanau Germany. An individual flap is placed in a 20cm glass bottle. The bottle is filled with 10 ml of wash test solution and then sealed tightly. up to 5 bottles are placed in each of the lavage meter bottles. The flask is filled with water as a heat transfer medium for simulation of washing. The wash simulation is conducted in the form of a heating cycle between 20 ^and C and 60 ^to C over 40 minutes.

After processing the samples, the vials are submerged in cold water and then each of the flaps is extracted from its bottle, rinsed in a beaker under coherent fresh water, squeezed and allowed to air dry for at least 30 minutes.

Measurement;

In order to measure CMC removal radioactively

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checked, a scintillation counter is used, for example, an LKB 1210 Ultrabeta Scintillation Counter, In order to obtain the most accurate results, the instructions manual for the optimal operation of that particular scintillation counter should be followed. For example, for the LKB 1210 Ultrabeta Scintillation Counter, the following procedure should be followed, the flap to be measured is placed in a plastic bottle filled with 12ml of scintillating liquid (for example, scintillator 299 from Packard). The flap is then allowed to stabilize for at least 30 minutes. The bottle is then placed inside the LKB 1210 Ultrabeta Scintillation Counter and the respective flap radioactivity counts are obtained.

In order to measure the value of CMC removal due to cellulase alone, it is necessary to measure a flap that has been inoculated at the same time but that has been treated in the wash test solution without cellulase. The cellulase activity is then expressed as a percentage of removal of the radioactively labeled CMC. This percentage is calculated using the following formula:

% removal of radioactive CMC = XO xc x 100

X0 where X0 is the scintillation percentage of the redioactivity of a flap treated with the washing test solution without cellulase

X0 is the radioactivity scintillation count of a flap treated with the cellngase containing lavngen test solution, to be evaluated.

Statistical considerations, confirmation of the procedure:

In order to provide statistically valid results, standard statistical analysis should be employed. For the example given, using the LKB 1210 Ultra16

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beta Scintillation Counter, it was found that it could be useful. A sample number of 3 flaps was used for each count. scintillation of radioactivity.

In order to confirm the procedure by means of an internal counter-verification, it is recommended to measure and calculate the blank sample according to EP-A-350098. This will allow you to detect and eliminate errors.

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Interpretation of results.

The described research assay provides a fast, unique and reliable method for identifying cellulases that meet the activity criteria of the present invention in relation to cellulases that are not part of it.

It has been found that a removal of 10% or more of the redioactively labeled CMC immobilized according to the C14CMC method described above, indicates that the respective cellulase meets the requirements of the invention.

It will become apparent to those skilled in the art that removal percentages above 10% indicate a higher activity for the respective cellulase. It is therefore considered that a cellulase that provides more than 25%, or preferably more than 50% removal of radioactive CMC each day, at the protein concentration of the wash test solution according to the C14CMC method, will provide indication even better cellulase performance for use in laundry detergents.

It is also considered that the use of higher cellulase concentrations for the C14CMC method will provide higher removal percentages. However, there is no proven linear correlation between cellulase concentration and the percentage of removal obtained by it.

It is also considered that the use of higher concentrations of cellulase for the C14CMC method, will provide higher removal percentages.

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Table ΙΣ:

(all

CMC solution marked with radioactive C ^ percentages are by weight of the total solution)

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CMC * Total (CMC should be detergent grade CMC with a substitution grade between about 0.47 and 0.7) τη 99.2 x 10% Ethanol 14985.12 x 10 ³ % Deionized water 84015.68 x 10 ^-3 % Total: : 100%

* Total CMC contains both non-radioactive and radioactive CMC, to provide radioactivity that allows sufficiently clear readings on the scintillation counter used. for example, radioactive CMC can have an activity of 0.7 milicuries / g and be mixed.

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TABLE II: Wash test solution (all percentages are by weight of the total solution)

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5 Benzene C ₁₂ linear alkyl sulfonic acid 0.110% * 10 Coconut alkyl sulfate (TEA salt) 0.040% • Ethoxylate alcohol 0.100% ç 12-15 15 Coconut fatty acid 0.100% Oleic acid 0.050% ' Citric acid 0.10% 20 Triethanolamine 0.040% Ethanol 0.060% • Propanadiol 0.015% 25 Sodium hydroxide 0.030% Sodium Formate 0.010% 30 Protease 0.006% Water (2, i5mmol / liter Ca ⁺⁺ ^ 'pH of the adjusting agent balance up to 100% 35 (HCL or NaOH solutions) and cellulase

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In accordance with the present invention, preferred cellulases are those described in Danish Patent Application 1159/90. For example, a cellulase preparation useful in the compositions of the invention, can essentially consist of a homogeneous component of endoglucanase, which is immunereactive with an antibody formed against a highly purified 43kl cellulase, derived from Humicola insolens, DSM 180C or which is homologous to said 43kD endoglucanase.

It should be noted that all cellulase enzymes in accordance with the present invention must meet the criteria of the aforementioned screening assay. However, in Danish Patent Application 1159/90 additional criteria are established to identify preferred cellulase enzymes in combination with the present screening assay,

Cellulase preparations particularly useful in the compositions of the invention are those in which, in addition to the screening assay, the endoglucanase component exhibits a CMC-endoase activity of at least about 50, preferably at least about 60, in particular at least about of 9C units of CMC-endoase per mg of total protein. A particularly preferred component of endoglucanase has a CMC-endoase activity of at least 'IQW CMC-endoase units per mg of total protein.

In the present context, the term CMC-endoase activity refers to the endoglucanase activity of the endoglucanase component, in terms of its ability to degrade cellulose into glucose, cellobiosis and triosis, as determined by a decrease in viscosity of a carboxymethyl cellulose (CMC) solution after incubation with the cellulase preparation of the invention, as described in more detail below.

The activity of CMC-endoase (endoglucanase) can be determined from the decrease in the viscosity of CMC, such as _t ^? scrub: A substrate solution containing 35g / l CMC (Hercules 7 LFD) in 0.1 M tris buffer at pH

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9.0. The enzyme sample to be analyzed is dissolved in the buffer MEJ _L hand, are mixed 10 ml substrate solution and 0.5 & mld enzyme solution and transferred to a viscosimeter (e.g. Haake VT 181, Nyr sensor, 181 rpm ), with the mustate set at 402cC. Viscosity readings are taken as soon as possible after mixing and again 30 minutes later. The amount of enzyme that halves viscosity under these conditions is defined as Ϊ unit of CMC-endoase activity.

The SDS polyacrylamide jelly electropheresis (SDS-PAGE) and isoelectric focusing with marker proteins in a manner known to those in the art, were used to determine the molecular weight and isoelectric point (pl), respectively, of the endoglucanase component in the preparation of cellulase useful in the present context. In this way the molecular weight of a specific component of endoglucanase was determined to be 43kD. The isoelectric point of this endoglucanase was determined to be about 5.1.

Cell-biohydrolase activity can be defined as activity towards p-nitrophenyl cellobiose. Activity is determined as umole of nitrophenyl released per minute at 37 ° C and pH 7.0. the present endoglucanase component was found to have virtually no cell-biohydrolase activity.

The endoglucanase component in the cellulase preparation indicated here was initially isolated by means of extensive purification processes, involving, among others, purification by reverse phase HPLC of a crude mixture of H. insolens cellulase according to U.S. 4,435. 307. This procedure resulted, surprisingly, in the isolation of a 43lgg endoglucanase as a single component with unexpectedly favorable properties, due to surprisingly high endoglucanase activity.

Likewise, in addition to the ras-lellis assay, decellulase enzymes useful in the present compositions can still be

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defined as enzymes that exhibit endogluase activity (hereinafter referred to as the endoglucanase enzyme), enzymes that have the amino acid sequence shown in the Sequence Listing ID # 2 attached, or a homologue that exhibits endoglucanase activity.

In the present context, the term homologous is understood to indicate a polypeptide encoded by DNA that hybridizes to the same probe as the DNA encoding for the enzyme endoglucanase, with this amino acid sequence, under certain specified conditions (such as embedding previous in 5xSSC and pre-hybridization for 1 hour at 40 ^B C in a 20% formamide solution, 5 x Denhardt solution, 50 mM sodium phosphate, pH 6.8 and 50 µg of calf thymus DNA denatured, followed by hybridization in the same solution supplemented with 100 uM ATP for 18 h at 40 C ^B). The term is intended to include derivatives' of the aforementioned sequence, obtained by adding one or more amino acid residues to each or both of the C- and N- terminals of the native sequence, replacing one or more amino acid residues at one or more locations in the native sequence, deletion of one or more amino acid residues at both or at each end of the native amino acid sequence, or insertion of one or more amino acid residues at one or more locations in the native sequence.

endoglucanase enzyme indicated here may be one that can be produced by Humicola species such as Humicola insolens, for example strain DSM 1800, deposited on October 1, 1981 in Deust ^ ch Sammlung von Mikro organismen, Mascheroder Weg 1B, D -3300 Braunschweig, Germany-1. in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure Proceedings (the Treaty of Budapesfce ·· ^ <

In another aspect, the cellulase enzymes used here can also be defined, in addition to the screening assay, as endoglucanase enzymes that have the sequence

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Mod. 71 - 20,000 ex. - 90/08 of amino acids represented in the attached Sequence Listing, or a homologue (cyberform defined above) that has endoglucanase activity. The said glucanase enzyme may be one that can be produced by a species of Fusarium, such as Fusarium oxysporum, for example strain 2672 DSM, deposited on June 6, 1983 in Deustch Sammlung vcn Microorganismen, Mascheroder Weg 1B, D- 3300 Braunschewieg, Germany, according to the provisions of the Budapest Treaty.

In addition, it is contemplated that homologous endoglucanases may be derived from other microorganisms that produce cellulotic enzymes, for example, species of Trichoderma, Myceliophtora, Phanerochaete, Schizophyllum, Penicillium, Asperqillus and Geotricim.

For the industrial production of the present cellulase preparation, however, it is preferred to employ recombinant DNA techniques or other techniques that involve adjustments of fermentations or mutation of the microorganisms involved, to ensure the overproduction of the desired enzymatic activities. Such methods and techniques are known in the art and can be easily practiced by experts in the field.

The endoglucanase component can thus be one that is produced by a method that comprises culturing a host cell transformed with a recombinant DNA vector that carries a DNA sequence encoding the said endoglucanase component, or a precursor of said component. endoglucanase sequences, as well as DNA sequences that encode functions that allow expression of the DNA sequence encoding the endoglucanase component or a precursor thereof, in a culture medium under conditions that allow the expression of the endoglucanase component or its precursor and the recovery of endoglucanase component of the culture.

DNA constructs that comprise a 'DNA sequence encoding an endoglucanase enzyme as described

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above, or a precursor form of the enzyme, include the constructs of 'DNA as a coniform DNA sequence represented in

Listings of sequences ID ^ l or ID | r3 attached, or a modification thereof. Examples of suitable modifications of the DNA sequence are nucleotide substitutions that do not give rise to another amino acid sequence that responds to the use of codon from which the construction of nucleotide DNA that gives rise to the endoglucanase of the host organism denir is introduced or substitutions a different sequence of but which color amino acid and therefore possibly a different protein structure may give rise to an endoglucanase mutant

Mod. 71 - 20,000 ex. - 90/08 with different properties than the native enzyme. Other examples of possible modifications are the insertion of one or more nucleotides at either end of the sequence or in the middle of the sequence, or the removal of one or more nucleotides at either end or in the middle of the sequence.

DNA constructs encoding endoglucanase enzymes usable here can be prepared synthetically by established standard methods, for example, the phosphoamidite method described by S.L. Beaucage and M.H.Caruthers, Tetrahedron Letters 22. 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp, 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, ligated and cloned in suitable vectors.

A DNA construct encoding the endogluca nase enzyme or its precursor can, for example, be isolated by means of (establishment of a cDNA library or genome of a cellulase-producing microorganism, such as Humicola insolens, DSM 1800 and looking for of positive clones by means of conventional processes such as by means of hybridization using probes; õligonucleotides synthesized based on the · complete or partial amino acid sequence of englucanase, according to standard techniques (cf. Sambrool · et al., Molecular Cloning: A Laboratory Manual, 2nd ed.

£ 4

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Cold spring Harbor, 1989), or by selecting clones that express the appropriate enzyme activity (ie CMC endoase activity as defined above), or by selecting clones that produce a protein that is reactive with an antibody against a native cellulase (endoglucanase).

Finally, the DNA construct can be of mixed synthetic and genomic origin, mixed of synthetic and cDNA or mixed genomic and original cDNA, prepared by linking the synthetic fragments, genomics or original cDNA (appropriate co-form) to the fragments corresponding to the various parts of the entire DNA construction, according to standard techniques. The DNA construct can also be prepared by polymerase chain reaction using specific primers, for example as described in the patents of

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U.S.A. 4,683,202 by R.K.Saiki et al., Science 239,1988, pp, 487-491.

The recombinant expression vectors in which they contain them. DNA translations above are inserted, include any vector that can be conveniently subjected to recombinant DNA processes, and the choice of the vector will depend on the host cell into which it is to be introduced. Thus the vector can be an autonomously replicating vector, that is, a vector that exists as an extra-chromosomal entity, whose replication is independent of chromosomal replication, for example a plasmid. Alternatively, the vector can be one that, 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 the endoglucanase should be operably linked to a single suitable promoter and terminator. The promoter can be any DNA sequence that shows transcriptive activity in the selected host cell and can be derived from proteins. genes, both homologous and heterologous, of the host cell. The processes used to link the strings

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Mod. 71 - 20,000 ex. -90/08 DNA encoding endoglucanase, respectively, at pr. motor and terminator, and to introduce them in the appropriate vectors, are well known to those in the field (cf., for example, Sambrook et al., cited work).

The host cells that are transformed with the above DNA constructs or the expression vectors indicated above, may belong, for example, to a species of Aspergillus, more preferably Aspergillus oryzae or Asperqillus niqer. Fungal cells can be transformed by a process that involves the formation of protoplasts 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 (from New Industry A / S), the contents of which are incorporated herein by reference. The host cell can also be a yeast cell, for example a strain of Saccharomyces cerevisiae.

Alternatively, the host microorganism may be a bacterium, in particular strains of Streptomyces and Bacillus and Ecoli. The transformation of bacterial ·-células' can be carried out according to conventional methods such as described in Sambrook et al _T Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982.

The search for appropriate DNA sequences and the construction of vectors can also be performed by means of standardized processes, ef. Sambrook et al., Cited work.

medium used to grow the transformed host cells, can be any of the conventional means, which is suitable for the growth of the host cells in question. The expressed endoglucanase can conveniently be secreted into the culture medium and can be recovered from it by well-known processes, which include separating the cells from the medium by centrifugation or filtration, precipitating the protein components of the medium by means of salt. such as sulfate-deammonium, followed by pro35

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chromatographic processes such as ion exchange chromatography, affinity chromatography or the like.

By employing recombinant cHnfor DNA techniques indicated above, protein purification techniques, fermentation and mutation techniques and other techniques that are well known in the art, it is possible to provide high purity endoglucanases.

The cellulase level in the present composition, described above, should be such that the amount of enzyme protein to be administered in the washing solution is from 0.005 to 40 mg / liter of the washing solution, preferably 0.01 to 10 mg / liter of the washing solution.

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OPTIONAL INGREDIENTS

Typically, the present compositions will include optional ingredients that are normally part of detergent compositions. Anti-redeposition agents and dirt suspenders, optical brighteners, brighteners, bleach activators, foam suppressants, anti-viscous agents, dyes and pigments are examples of such optional ingredients and can be added in various amounts, as desired.

Anti-redeposition agents and soil suspenders suitable for use in the present invention include cellulose derivatives such as methylcellulose, carboxylmethylcellulose and hydroxyethylcellulose and polycarboxylic acids homo-copolymeric or their salts. Polymers of this type include polyacrylates and copolymers of acrylic acid-maleic anhydride previously mentioned as intensifiers, as well as copolymers of maleic anhydride with ethylene, methyl vinyl ether or methacrylic acid, constituting maleic anhydride at least 20 mole percent of the copolymer. . These materials are normally used at levels between 0.5% and 10% by weight, more preferably between 0.75% to 8%, and most preferably.

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between 1% and 6% by weight of the composition.

Preferred optical brighteners are of anionic character, examples of which are 2: 4,4-bis-disulfonate (2-diethanolamino-4-anilino-s-triazin-6-ylamino) disodium, 2: 2 ^ 4,4 -4-bis- (2-morpholino-4-anilino-s-triazin-6-ylaminostilbene disodium 2: disulfonate, 4,4-bis-disulfonate - (2,4-dianilino-s-triazin -6-ylamino) disodium stylbene, 4 ^, 4 ^^ 2-sulfonate - bis- (2,4-dianilino-s-triazin-6-ylamino) stalbene monosodium 2.2 ^ 4.4 ^1- bis disulfonate (2-anilino-4- (N-methyl-N-2-hydroxyethylamino) -s-triazino-6-ylamino) disodium 2,2b-4,4 ^I- bis- (4-phenyl-2, disulfonate, 1,3, -Triazol-2-yl) -stilbene disodium 2,2-4,4-bis (2-anilino-4- (1-methyl-2-hydroxyethylamino) -s-triazin-6-ylamino disulfonate ) disodium stilbene and 2 ¹¹ sulfonate 2 (stylbil-4 ¹ · ^ -naphth-1 ¹ , 2 ^: 4.5) -1,2,3-sodium triazole.

Any inorganic perhydrate bleach can be used, in an amount between 3% and 40% by weight, more preferably between 8% and 25% by weight and most preferably between 12% and 20% by weight of the compositions. Preferred examples of such bleaches are percarbonate ether monohydrate ^: sodium percabonate and mixtures thereof.

Another preferred ingredient, separately mixed, is a peroxy carboxylic acid bleach precursor, generally referred to as a bleach activator, which is preferably added in a crystallized or agglomerated form. Examples of suitable compounds of this type are described in British Patent Nos. 1586769 and 2143231 and a method for their formation in a crystallized form is described in Published European Patent Application No. ^Q 0 062 523.Exen. Preferred examples of such compounds are tetracetyl ethylene diamine and 3,5,5-trimethylhexanoyloxybenzene sodium sulfoanto.

Bleach activators are normally employed at levels between 0.5% and 10% by weight, more often between 1% and 8% and preferably between 2% and 6% of the composition.

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Another optional ingredient is a foam suppressant, exemplified by silicones and silica-silicon mixtures. Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xerellias and hydrophobic silicas of various types. These materials can be incorporated as particulates in which the foam suppressors can be advantageously releasably incorporated into a substantially non-surfactant, water-soluble, water-soluble detergent impermeable vehicle. Alternatively, the foam suppressor can be dissolved or dispersed in a liquid vehicle and applied by spraying over one or more of the other components.

As mentioned above, useful foam controlling silicone agents can comprise a mixture of an alkylated siloxane, of the type referred to hereinafter, and solid silica. Such mixtures are prepared by affixing the silicone to the surface of the solid silica. A preferred foam-controlling silicone agent is represented by a hydrophobic silanadan silica (more preferably trimethyl silane) with a particle size between 10 millimicron and 20 millimicron and a specific weight of up to 50 g / m, infinitely mixed with dimethyl liquid silicone with a molecular weight between about 500 and about 200,000 in a weight ratio of silicone to silane silica between about del: l and about del: 2.

A foam controlling silicone agent is described in Bartollota et al., U.S. Patent 3,931. 672. Other particularly useful foam suppressors are the self-emulsifying suppressing silicone foam, _U des critos in German Patent Application DTOS 2,646,126 published 28 April 1977. ^ An example dean such a compound is DC-544 , marketed by Dow Corning, which is a siloxane / glycol copolymer.

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The foam suppressants described above are normally employed at levels between 0.001% to 2% by weight of the composition, preferably between 0.01% to 1% by weight. The incorporation of foam modifiers is preferably done in the form of separate particulates and this allows the inclusion of other foam controlling materials such as C20-C24 fatty acids, microcrystalline waxes and high PM copolymers. ethylene oxide and propylene oxide, which would otherwise adversely affect the dispersibility of the matrix. Techniques for forming such particulates modificadoresdê-foams are described in US Patent 3,933,672 of ² Bartollota et al, mentioned above.

Other useful polymeric materials are polyethylene glycols, particularly those having a molecular weight of 1000-10000, more particularly 2000 to 8000 and more preferably about-4000. These are used in levels between 0.20% and 5%, more preferable = between 0.25% and 2.5% in weight These polymers and the homo- or copolymeric polycarboxylate salts mentioned above, are valuable for improving maintenance of whiteness, ash deposition on fabrics and cleaning performance on earth, protein and oxidizable stains in the presence of transition metal impurities.

Stain suppressing agents useful in the compositions of the present invention are. conventionally, terephthalic acid copolymers or terpolymers with ethylene glycol and / or propylene glycol units in various arrangements. Examples of such polymers are disclosed in copending Patentes'EUA N ⁹ are 4116885 and 4711730 and European Patent Application No. 0 272 033. A ^second especially preferred polymer in accordance with EP-A-0272033 has the formula (CH ₃ (PEG) ₄₃ ) ₀ . ₇₅ (POH) ₀ _ _25r T-PO) ₂ . ₈ (TP _AND G) _o . ₄ ] T ( _P OH) ₀₂₅ ((PEG) ₄₅ CH ₃ ) ₀ . ₇₅

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where PEG is - (0C ₂ H ₄ ) 0-, P0 is (C0 ₃ H ₆ 0) is T is (pcOC H ₄ C0).

Certain polymeric materials, such as polyvinyl pyrrolidone, typically from PM 5000-20000, preferably 10000-15000, also form useful agents for predicting the transfer of labile dyes between fabrics during the washing process.

Fabric softening agents can also be incorporated into the detergent compositions according to the present invention. These agents can be of an inorganic or organic type. Inorganic softening agents are exemplified by the smectite clays described in GB-A-1,400.89í Organic fabric softening agents include water-insoluble tertiary amines as described in GB-A-1514276 and EP-B-0 oll 340 and their combination with mono salts C12-C14 quaternary ammonium is described in EP-B-0 026 527 and EP-B-0 026 528 and long-chain starches as described in EP-B-0 0242 919. Other useful organic ingredients from systems fabric softeners include high molecular weight polyethylene oxide materials as described in EP-A-0 299 575 and 0 313 146.

The smectite clay levels are normally between 5% and 20%, more preferably between 8% and 15% by weight, with the materials to be added to the rest of the formulation in the form of a dry mixed component. Hi agents. tissue softening agents such as water-insoluble tertiary amines or long-chain starch materials are incorporated at levels between 0.5% and 5% by weight, usually between 1% and 3% by weight, while materials of high molecular weight polyethylene oxide and water-soluble cationic substances are added at levels between 0.1% and 2%, usually between 0.15% and 1.5% by weight. These materials are normally. added to the dry sprayed portion of the composition, although in some cases it may be more convenient to add them in the form of a dry mixed particulate,

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Mod. 71 - 20,000 ex. -90/08 or spray them as a molten liquid over the other solid components of the composition.

Enzymes other than the specific cellulase preparation presented herein, such as proteases, iases and amylases, may be present in this composition.

MANUFACTURING PROCESS

The comoposições according to the present invention can be manufactured by a variety of methods incluemea dry blending _t spray drying, agglomeration and granulation and combinations of any of these ·· Computer Technician cas.

PREFERRED MANUFACTURING PROCESS

A preferred process for the manufacture of the present compositions involves a spray drying combination. agglomerate in a high speed mixer and dry mix.

A first granular component containing a relatively insoluble anionic surfactant is spray-dried and part of the dried product is separated and subjected to a spray of low level anionic surfactant beforehand; to be remixed with the rest. A second component grj. Nular is manufactured by dry neutralization of an anionic surfactant, using sodium carbonate as a neutralizing agent in a continuous high speed mixer, such as a Lodige KM mixer. The first and second components, together with other dry-mix ingredients such as the carboxylate chelating agent, inorganic peroxygen bleach, whitening enhancer, dirt / siliacide suspending agent and enzyme are then introduced into a carrier belt, from which are transferred to a horizontally rotating drum in which

Mod. 71 - 20,000 ex. - 8/90

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are sprayed on the perfume product and silicone foam suppressant. In highly preferred compositions, one more is employed drum mixing step in which a low level (aprox.2%) of di finely crystalline aluminosilicate is introduced to ⁵ vidido aumeistar density and improve granular flow characteristics.

WASHING PROCESS

The present compact detergent compositions have the ability to achieve the same efficiency as conventional detergent compositions, but considerably less of the composition of the present invention is used in the main washing cycle of a washing machine.

Consequently, in an embodiment of the invention ₍ a process for washing fabrics in a washing machine is provided, in which an amount between 15 and 170 g of a detergent composition according to the present invention is used for the main cycle washing.

Typically, under European conditions, the recommended use is between 80 and 140 g of detergent composition for the main wash cycle, without the need for a pre-wash.

The present detergent compositions are preferably. directly into the drum and not indirectly through the outer casing of the machine. This can easily be achieved by incorporating the composition into a bag or container, from which it can be released at the beginning of the washing cycle, in response to agitation, a rise in temperature or immersion in the washing water in the drum. Such a container will be placed inside the drum, together with the fabrics to be washed. Alternatively, the washing machine may itself be adapted for perm. direct drawing of the composition to the drum, for example, by means of a device provided on the access door.

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Products that comprise a detergent composition enclosed in a bag or container are generally designed in such a way that the integrity of the container is maintained in the dry state to prevent the contents from escaping dry, but are adapted to release the contents of the container when exposed to the washing environment, usually by immersion in an aqueous solution.

Generally, the container will be flexible like a bag or pouch. The bag may be of fibrous construction, covered with a water-impermeable protective material in order to retain its contents, as described in Published European Patent Application N ^Q 0 018 687. Alternatively, it may be formed of synthetic polymeric material insoluble in water, provided with an edge seal or seal intended for ron ptíx in an aqueous medium, as described in Published European Patron Orders N ^Q s0 011 500, 0011 501, 0 011 502 and 0 011 968. A convenient way water-permeable closure comprises a water-soluble adhesive, arranged along and closing an edge of the pouch formed by a water-impermeable polymeric film, such as polyethylene or polypropylene.

In a variant in the form of a product bag or container, laminated sheet products can be employed in which a flexible central layer is impregnated and / or coated with a composition, and then one or more outer layers are applied to produce an aesthetic effect. similar and woven. The chambers can be placed together in order to remain connected during use or they can separate when in contact with water to facilitate the release of the coated or impregnated material.

An alternative laminated form comprises an embossed or deformed layer to provide a series of pouch-like containers within each of which the detergent components are deposited in measured quantities, with a second layer overlapping the first and the second.

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it glued, in the areas located between the boj containers. sa, where the two layers are in contact. The components can be deposited in the form of particles, pastes or molten material and the laminated layers must prevent the color from leaving. content of bag-type containers, before being placed in water. The layers can separate or remain connected to each other when in contact with water, the only requirement being that the structure allows a quick release of the contents of the bag-like containers into the solution. The number of bag-shaped containers per unit area of the substrate is a matter of choice, but will normally vary between 500 and 25,000 per square meter.

Materials suitable for use in flexible laminate layers in this aspect of the invention include, but are not limited to, sponges, paper and non-woven fabrics and fabrics.

However, preferred means for carrying out the washing process according to the present invention include the use of a reusable delivery device with walls that are permeable to liquids but impermeable to the solid composition.

Devices of this type are described in European Patent Application Publication Nos. ⁹ 34 0 063 069 and 0 344 070. The latest Application describes a device comprising a flexible bag-shaped sheet that is prolvable from a ring. support that defines an orifice, the orifice being adapted to admit in the bag enough product for a washing cycle, in a washing cycle. A portion of the washing medium flows through the hole into the bag, dissolves the product and the solution then passes out through the hole into the washing medium. The support ring is provided with a cover arrangement to prevent the exit of the wet, undissolved product, comprising this arrangement, typically, walls extending radially from a central projection with the configuration35

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spoked wheel ration, or a similar structure in which the walls have a helical shape.

EXAMPLES

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The following examples illustrate the invention and facilitate its understanding.

Abbreviations for individual ingredients have the following meaning:

LAS: linear salt of dodecyl benzene linear sulfonate

TAS: sodium sulfate sodium tallow alcohol

AS: alkyl sulfate sodium salt (C14 - C15)

A0: C12-C14 Alkyldimethylamine oxide

FA45E7: fatty alcohol (C14-C15) ethoxylated with about 7 moles of methylene oxide

CAT: C12 alkyl trimethyl ammonium chloride

Clay: smectite clay

Zeolite 4A: sodium salt of zeolite 4A with an average particle size between 1-10 micrometers

SKS-6: crystalline silicate layer (Hoechst)

AA / MA copolymer: acrylic acid and maleic acid polymer

PAA: polyacrylic acid PM 1000—10000

CMC: carboxymethylcellulose

Phosphonate: sodium salt of ethylenediamino-tetramethylenc acid

-phosphonic

EDTA: ethylene diamine tetraacetate sodium salt

PB1: NaB02.H202

PB4: NaB02.H202.3H20

TAED: tetra acetyl ethylene diamine

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NOBS: -nonanoyl sulfonate-oxybenzene sodium

P.A .: zinc sulfonated phthalocyanine

Silicate (R = n): SiO2 / Na2O = n

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Amylase: Termamyl 60T

Lipase: Lipolase 100T

Protease: Savinase 4T (Novo-Nordisk) (Novo-Nordisk) (Novo-Nordisk)

SSS: Foam Suppressor System (silica / silica mixture)

EXAMPLE I

Critical points of the cellulase performance parameter of claim 1

The following test was carried out:

Test conditions:

Wash temperature: 603C (heating cycle)

Washing time: 40 min.

pH = 7.5

Water hardness: 4 mmol / L

Detergent concentration: 1%

Detergent composition: acc. EPA 350 098 ex. 1

Cellulases:

1) Celluzyme provided by Novo Nordisk = reference

2) 43kD endoglucanase = cellulase according to the invention

Test results:

% Cellulase C14-CMC removal

Detergent without cellulase (= reference) 0 Detergent + Celluzyme ^R 0.25 mg protein / L below 3 0.9 mg protein / L 10 1.5 mg protein / L 12.7

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3.0 mg protein / L

4.5 mg protein / L

Detergent + 43kD endoglucanese ⁵ 0.3 mg protein / L

0/25 mg Protein / L

17.7

2.1 / 5

20.3

18.5

Discussion of results +

The data above clearly demonstrate the critical points of the claimed parameter for the cellulases of the invention through the commercially available Celluzyme.

EXAMPLE II

The following base compositions were prepared:

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COMPOSITIONS:

(all levels in% of weight)

LAS

TAS

FA45E7

Na citrate / citrus acid

Zeolite 4A

Copolymer AA / MA

Phosphonate

Na carbonate

Silicate (R = 2)

Protease

Sulfate

SSS

Compact Non-Compact Detergent Detergent 9.40 6.27 3.00 2.00 2.65 1.77 18.50 12.33 32, i65 21.77 4.90 3.27 0.19 0.13 3.00 2.00 2.90 1.93 1.62 1.08 4.50 30.00 0.40 0.27

£ 3

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COMPOSITIONS; (continued) (all levels in% of weight)

Minors + Water

Density (g / L at 20 ^and C)

Compact

Detergent

Non-Compact

Detergent rest to 100%

680

415

Recommended product use

Washing

120

180

Color Rejuvenation Essay

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Test conditions:

Washing temperature: 40 ^Q C

Wash time: 3h

Number of washing cycles; 2 pH = non-compact detergent 8.2 compact detergent 8.5

Water hardness: 15gr / 3.96gr / L

Detergent concentration:

0.75% for non-compact detergent

0.66% for compact detergent

Test fabric: blue cotton pajamas (90/10 cotton /; Polyester) p Cellulases: 1) Celluzymz supplied by Novo Nordisk (= ^; reference)

2) 43kD endoglucanase - cellulase according to the present invention

Washing test: Patches of 8g of debauched blue pajama fabric were treated with the different washing solutions. After rolling drying, the fabrics were graded for the purpose of color clarification by means of direct comparison of the two different detergent matrices at equal levels of cellulase. Visual grading by expert evaluators using a scale of 0 to 4 was the pre3 ^

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wound. (Ó means no difference and 4 means a very big difference,)

Test results:

I) Non-Compact Detergent

PSU

No cellulase

Celluzyme

138mg protein / L + 2,; 43kD endoglucanase

18.6 mg protein / L + 2.2 mg protein / PSU

8.5

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II) Compact Detergent

No cellulase

Celluzyme

165 mg protein / L

Endoglucanase 43kD

3.4 mg protein /

PSU mg of protein / PSU

0 + = '- 3.8 43 + 3.4 1.0

LSD (Least Significant Difference) = 0.5 PSU

From the mg protein / PSU result, the following efficiency factors were calculated:

Efficiency factor of 43kD endoglucanase in relation to

Celluzyma:

In Non-compact detergent In Compact detergent

60 / 8.5 = 7 43 / 1.0 = 43

Efficiency factor in Compact Detergent in relation to Detergent do not compact · f: 1 e \ ·: ei

From Celluzyma

60/43 = 1.4

Conclusions:

of endoglucanase 43kD

8.5 / 1 = 8.5

MÔ

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The above results show that a cellulase selected according to the present invention is 43 times more effective than the current technique cellulase in the claimed compact matrix. In addition, the above results show that the performance improvement due to the claimed compact matrix, observed with the cellulases selected, is surprisingly much higher than what can be obtained with the cellulase of the current technology.

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EXAMPLE III

EARTH STAIN REMOVAL TEST cellulase enzymes are also very effective in removing soil stains from tissues. This particular performance characteristic was verified for the 43kD endoglucanase in the two detergent compositions provided in example II.

Conditions;

Tissue testing equipment- Linitest

Dry ^and C 60 ^(UEC cycle ^and q i ^men t °)

Wash time: 4Q- minutes

Water hardness: Brussels city water

Detergent concentrations:

0.66% for compact detergent

1.0% for non-compact detergent

Cellulase concentrations: 1.55; 3.10; 4.65; and 6.2mg of prc>

t.eine7.enzyme / L washing liquor

Washing test

An Arab cotton fabric was stained with land of natural origin from two different locations (USA; GB). The cellulase performance was evaluated by comparing the washed earth stains with an equal level of cellulase in the two different detergent compositions. The visual graduation scale

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Results

Cellulase level 1.55 3.1 4.7 6.2 (mg of prot.enz./L of liquor washing) Compact detergent USA land + 1.50 + 2.50 + 2, D0 + 1.50 GB land +0.50 + 1.00 + 1.50 + 2, .50 Non-compound detergent (= reference) 0 0 0 0 LSD (least significant difference) = 0.42 to 95% of trust

Mod. 71 - 20,000 ex. - 8/8 ça. The performance of removing soil stains from the cellulase selected in accordance with the present invention, in the compact dé-detergent composition of the invention, is significantly superior to its performance.

EXAMPLES IV-XI compact detergent compositions are also prepared:

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1 O O in l 1st O l in Η ’1 cn O i 1st O i mr Η 1 X X - 1 1 - X 1 X 1 cn CD CM 1 1 LO O l cn 1—1 CM

O in O 1 1 1st O i in H in CM CM 1 1 1st O 1 Ν ' N X - x '1 1 1 ' 1 ω co CM | 1 1 CM CD i cn t — 1 i — 1

O LD ΙΓ) | 1 1 O 1 1st O X- CM CD l-H 1 1 i tn i i in in X 'X '1 1 1 - l l X co CM cn i i l cn i CN l cn you

00 co 1 r-1 1 1 1st in O O X LO 1 r4 1 1 1st CD LD σ> H- X X 1'1 1 1 X X X Γ ' CM 1 LO 1 1 1 in ID CM CN 1—4 f — 4

H CO CO 1 T — 1 1 1 1 IO 1st H LD xr 1 T ^- 1 1 1 1 1 00 1 CD H X X 1'1 1 1 1 1 ► r- -CN i lo i i i i cn CN i id

W ffl +>

□ rd O & co s Φ 0 D u O W T Φ +5 0 Φ s &1 φ H Φ m THE • laughs Φ Φ THE !> Ή W ç Φ iO to uo O laughs M co 0 O & TS g 0 0 _P u

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64,264

Case CM 356M

Mod. 71 - 20,000 ex. - 8/90

INFORMATION FOR SEQUENCE OF ID N9 1:

(i) SEQUENCE CHARACTERISTICS (A) Length: 1060 base pairs (B) Type: nucleic acid (C) Cord shape: simple (D) Topology: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ORIGINAL SOURCE (A) organism: Humicola insolens (B) strain: DSM 1800 (ix) CHARACTERISTICS (A) Name / Key: peptide of random structure (B) Location: 73..927 (ix) CHARACTERISTICS (A) Name / Key: peptide sig.

(B) Location: 10..72 (ix) FEATURE (A) Name / Key: CDS (Bj Location: 10..927

INFORMATION FOR SEQUENCE ID Ν ^ 2:

(i) SEQUENCE FEATURES (A) Length: 305 amino acids (B) Type: amino acid (D) Topology: linear (ii) MOLECULE TYPE: Protein

64.264 CM 356M case

Mod. 71 - 20,000 ex. - 8/90

INFORMATION FOR SEQUENCE OF ID N ^and 3:

(i) SEQUENCE CHARACTERISTICS (A) Length: 1473 base pairs (B) Type-e nucleic acid (C) Cord length: simple (D) Topology: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-DIRECTION: No (vi) ORIGINAL SOURCE (A) Organism: fusarius oxysporum (B) Strain: DSM 2672 (ix) FEATURES (A) Name / Code: CDS (B) Location: 97..1224

INFORMATION FOR SEQUENCE OF ID N ² 4:

(i) SEQUENCE CHARACTERISTICS (A) Length: 376 amino acids (B) Type: amino acid (C) Topology: linear (ii) MOLECULE TYPE: Protein.

64,264

Case CM 356M

Sequence Description: Seq.de ID N® 1:

QSITCCAAQ-.ATG 'CGT TCC TCC CCC .CTC. CTC CCG TCC.GCC GTT GTG GCC 48

Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Vai Vai Ala

-21 -20 -15 -10

Mod. 71 - 20,000 ex. - 8/90

GCC CTG CCG GTG TTG GCC CTT GCC GCT GAT GGC AGG CBT ACC CGC TAC 96 Allah Read Pro Vai Read Allah Read Allah Allah Asp Gly Arg To be Thr Arg Tyr -5 1 5 TGG GAC TGC TGC THE AG CCT TCG TGC GGC TGG GCC THE AG THE AG GCT CCC GTG 144 Trp Asp Cys Cys Lys Pro To be Cys Gly Trp Allah Lys Lys Allah Pro Go 10 15 20 AAC CAG CCT GTC TTT CBT TGC AAC GCC AAC TTC CAG CGT ATC ACG GAC 192 Asn Gin Pro Vai Phe To be Cys Asn Allah Asn Phe Gin Arg Ile Thr Asp 25 30 35 40 CBT GAC GCC AAG CBT GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC 240 Phe Asp Lys Ward To be Gly Cys Glu Pro Gly Gly Go Allah Tyr To be Cys 45 50 55 GCC GAC CAG ACC CCA TGG GCT GTG AAC GAC GAC TTC GCG CTC GGT TTT 288 Allah Asp Gin Thr Pro Trp Allah Go Asn Asp Asp Phe Allah Read Gly Phe 60 65 70 GCT GCC ACC TCT ATT GCC GCC AGC AAT GAG GCG GGC TGG TGC TGC GCC 336 Allah Allah Thr Ser Ile Allah Gly To be Asn G1U Allah Gly Trp Cys Cys Allah 75 80 85 TGC TAC GAG CTC ACC CBT A CA CBT GGT CCT GTT GCT GGC THE AG THE AG ATG 384 Cys Tyr Glu Leu Thr Phe Thr To be Gly Pro Go Allah Gly Lys Lys Met 90 95 100 GTC GTC CAG TCC ACC AGC ACT GGC GGT GAT CTT GGC AGC AAC CAC TTC 432 Go Go Gin Ser Thr To be Thr Gly Gly Asp Read Gly To be Asn His Phe 105 110 115 120 GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT 480 Asp Read Asn Ile Pro Gly Gly Gly Go Gly Ile Phe Asp Gly Cys Thr 125 130 135 CCC CAG TTC GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG CBT 528 Pro Gin Phe Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly Ile To be To be

140 145 150

Bad

64,264

Case CM 356M

Mod. 71 - 20,000 ex. - 8/90

CGC AAC GAG TGC GAT CGG TTC CCC GAC GCC CTC THE AG CCC GGC TGC TAC 576 Arg Asn G1U Cys Asp Arg Phe Pro Asp .. · Leu Lys Ward Pro-Gly Cys Tyr 155 160 165 TGG CGC TTC GAC TGG TTC THE AG ACC GCC GAC AAT CCG AGC TTC AGC TTC 624 Trp Arg Phe Asp Trp Phe Lys Asn Allah asp Asn Pro To be Phe To be Phe 170 175 180 CGT CAG GTC CAG TGC CCA GCC GAG CTC GTC GCT CGC ACC GGA TGC CGC 672 Arg Gin Go Gin cys Pro Allah Glu Read Go Allah Arg Thr Gly Cys Arg 185 190 195 200 CGC AAC GAC GAC GGC AAC TTC GCT GCC GTC CAG ATC CCC CBT AGC AGC 720 Arg Asn Asp Asp Gly Asn Phe Pfo Aia Go Gin Ile Pro To be To be To be 205 210 215 ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG CBT ACC 768 Thr To be To be Pro Go Asn Gin Pro Thr To be Thr To be Thr Thr To be Thr 220 225 230 CBT ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC 816 To be Thr Thr To be To be Pro Pro go Gin Pro Thr Thr Pro To be Gly Cys 235 240 245 ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC 864 Thr Allah Glu Arg Trp Allah Gin Cys Gly Gly 'AsH Gly Trp To be Gly Cys 250 255 260 ACC ACC TGC GTC GGC AGC ACT TGC ACG AAG ATT AAT AAT GAC TGG TAC 912 Thr Thr Cys Go Allah Gly To be Thr Cys Thr Lys Ile Asn Asp Trp Tyr 265 270 275 280

CAT CAG TGC CTG TAGACGAGG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA 964

His Gin Cys Leu

285

CGAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 1024

GATTGTCACA TAAATGCAAT GAGGAACAAT GAGTAC

1060

64,264

Case CM 356M

Mod. 71-20,000 ex. - 8/90

Description of Sequence: Seq. ID 2: MetArg Being Ser Pro Leu Leu Pro Ser Ala Vai Go Ala Ala Leu Pro -21 -20 -15 -10 Go read Wing Leu Wing Wing Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys -5 1 5 18 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Vai 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 Vai ala Tyr Ser Cys Ala Asp Gin 45 50 55 Thr Pro Trp Ala Vai Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 60 65 70 75 Ser Ile Wing Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 80 85 90 Leu Thr Phe Thr Ser Gly Pro Go Ala Gly Lys Lys Met Gonna Gin 95 100 105 Ser Thr Ser Thr Gly Gly Asp Leu gly Ser Asn His Phe Asp Leu Asn 110 115 120 Ile Pro Gly Gly Gly Vãl Gly Tle Phe Asp Gly Cys Thr Pro Gin Phe 125 130 135 Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 140 145 150 155 Cys Asp Arg Phe Pro Asp Wing 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 Vai 175 180 185 Gin Cys Pro Glu Leu Wing Go Ala Arg Thr Gly Cys Arg Arg Asn Asp 190 195 200 Asp Gly Asn Phe Pro Wing Go gin Ile Pro Ser Ser Ser Thr Ser Ser 205 210 215 Pro Vai Asn Gin Pro Thr Ser Thr Thr Thr Thr Ser Thr Thr Thr Thr 220 230 235 Ser Ser Pro Pro Go Gin Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 240 245 250

£ 0

64,264

Case CM 356M

Arg Trp Allah Gin Cys Gly Gly Asn Gly Trp To be Gly Cys Thr Thr Cys 255 260 265 Go Allah Gly To be Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gin Cys 270 275 280 Read

Sequence Description: Seq. RS 3 ID:

Mod. 71 - 20,000 ex. - 8/90

GAATTCGCGG CCGCTCATTC ACTTCATTCA TTCTTTAGAA TTACATACAC TCTCTTTCAA

AACAGTCACT CTTTAAACAA AACAACTTTT GCAACA ATG CGA TCT Met Arg Ser TAC ACT CTT Read Tyr Thr 5 1 CTC GCC CTG GCC GGC CCT CTC GCC GTG AGT GCT GCT TCT GGA AGC GGT Read Allah Read Allah Gly Pro Read Allah Go To be Allah Allah To be Gly To be Gly 10 15 20 CAC TCT ACT CGA TAC TGG GAT TGC TGC THE AG CCT TCT TGC TCT TGG AGC His To be Thr Arg Tyr Trp Asp Cys Cys Lys Pro To be Cys To be Trp To be 25 30 35 GGA THE AG GCT GCT GCT AAC GCC CCT GCT TTA ACT TGT GAT THE AG AAC GAC Gly Lys Allah Allah Go Asn Allah Pro Allah Read Thr Cys Asp Lys Asn Asp 40 45 50 AAC CCC ATT CBT AAC ACC AAT GCT GTC AAC GGT TGT GAG GGT GGT GGT Asn Pro Ile To be Asn Thr Asn Allah Go Asn Gly Cys G1M Gly Gly Gly 55 60 65 70 TCT GCT TAT GCT TGC ACC AAC TAC TCT CCC TGG GCT GTC AAC GAT GAG To be Allah Tyr Allah Cys Thr Asn Tyr To be Pro Trp Allah Go Asn Asp G1U 75 80 85 CTT GCC TAC GGT TTC GCT GCT ACC THE AG ATC CBT GGT GGC CBT GAG GCC Read Allah Tyr Gly Phe Allah Allah Thr Lys Ile To be Gly Gly To be Glu Allah 90 95 100 AGC TGG TGC TGC GGT TGC TAT GCT TTG ACC TTC ACC ACT GGC CCC GTC To be Trp Cys Cys Allah Cys Tyr Allah Read Thr Phe Thr Thr Gly Pro Go 105 110 115 THE AG GGC THE AG THE AG ATG ATC GTC CAG CBT ACC AAC ACT GGA GGT GAT CTC Lys Gly Lys Lys Met Ile Go Gin To be Thr Asn Thr Gly Gly Asp Read 120 125 130

:it's the

114

162

210

258

306

354

402

450

S\

498

64,264

Case CM 356M

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

GGC GAC AAC CAC TTC GAT CTC ATG ATG CCC GGC GGT GGT GTC GGT ATC Gly Asp Asn His Phe Asp Read Met Met Pro Gly Gly Gly Go giy Ile 135 140 145 150 TTC GAC GGC TGC ACC TCT GAG TTC GGC THE AG GCT CTC GGC GGT GCC ACG Phe Asp Gly Cys Thr To be G1U Phe Gly Lys Allah Read Gly Gly Allah Gin 155 160 165 TAC GGC GGT ATC CBT CBT CGA AGC GAA TGT GAT AGC TAC CCC GAG CTT Tyr Gly Gly Ile To be To be Arg To be Giu Cys Asp To be Tyr Pro Spin Read 170 175 180 CTC THE AG GAC GGT TGC CAC TGG CGA TTC GAC TGG TTC GAG AAC GCC GAC Read Lys Asp Gly Cys His Trp Arg Phe Asp Trp Phe Glm Asn Allah Asp 185 190 195 AAC CCT GAC TTC ACC TTT GAG CAG GTT CAG TGC CCC THE AG GCT CTC CTC Asn Pro Asp Phe Thr Phe Glu Gin Go Gin Cys Pro Lys Allah Read Read 200 205 210 GAC ATC AGT GGA TGC THE AG CGT GAT GAC GAC CBT AGC TTC CCT GCC TTC Asp Ile To be Gly Cys Lys Arg Asp Asp Asp To be Sgr Phe Pro Allah Phe 215 220 225 230 THE AG GTT GAT ACC TCG GCC AGC THE AG CCC CAG CCC CBT TGC CBT GCT THE AG Lys Go Asp Thr To be Allah To be Lys Pro Gin Pro To be To be To be Allah Lys 235 _: 240 245 THE AG ACC ACC CBT GCT GCT GCT GCC GCT GAG CCC CAG THE AG ACC THE AG GAT Lys Thr Thr To be Allah Allah Allah Allah Allah Gin Pro Gin Lys Thr Lys Asp 250 255 260 CBT GCT CCT GTT GTC CAG THE AG CBT CBT ACC THE AG CCT GCC GCT CAG CCC To be Allah Pro Go Go Gin Lys To be To be Thr Lys Pro Allah Allah Gin Pro 265 270 275 GAG CCT ACT THE AG CCC GCC GAC THE AG CCC CAG ACC GCA THE AG CCT GCT GCC Glu PEO Thr Lys Pro Allah Asp Lys PÊÔ Gin Thr Asp Lys Pro Go Allah 280 285 290 ACC THE AG CCT GCT GCT ACC THE AG CCC GTC CAA CCT GTC AAC THE AG CCC THE AG Thr Lys Pro Allah Allah Thr Lys Pro Go Gin Pro Go Asn Lys Pro Lys 295 300 305 310 A CA ACC CAG. THE AG GTC CGT GGA ACC ΆΑΑ ACC CGA GGA AGC TGC CCG GCC Thr Thr Gin Lys Go Arg Gly Thr Lys Thr Arg Gly To be Cys Pro Allah 315 320 325

546

594

642

690

738

786

834

882

930

978

1026

1074

64,264

Case CM 356M

THE AG ACT GAC GCT ACC GCC THE AG GCC CBT GTT GTC CCT GCT TAT TAC CAG 1122 Lys Thr Asp Allah Thr Allah Lys Allah To be Go Go Pro Allah Tyr Tyr Gin 330 335 340 TGT GGT GGT CBT THE AG CBT GCT TAT CCC AAC GGC AAC CTC GCT TGC GCT 1170 Cys Giy Gly To be Lys To be Allah Tyr Pro Asn Gly Asn Read Allah Cys Allah 345 350 355 ACT GGA AGC THE AG TGT GTC THE AG CAG AAC GAG TAC TAC CBT CAG TGT GTC 1218 Thr Gly To be Lys Cys Go Lys Gin Asn Glu Tyr Tyr To be Gin Cys Go 360 365 370 CCC AAC TAAATGGTAG ATCCATCGGT TGTGGAAGAG ACTATGCGTC TCAGAAGGGA 1274 Pro Asn

375

TCCTCTCATG

TTGTTGTACA

CGACAACTGG

AGCAGGCTTG

TCATTGTATA

GCATGGCATC

CTGGACCAAG

TGTTCGACCC

1334

TAGTATATCT

CTACAAAAGA

TCATTGTATA

TATTTAGZCA

CATAGATAGC

CTCTTGTCAG

1394

CTTGGCAGGC

TTGTTCAATA

TTGACACAGT

TTCCTCCATA

1454

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

AAAAAAAAAA

AAAAAAAAA

Sequence Description: Seq. From ID ΝΘ4:

Wb. · Arg · Ser Tyr Thr Read Read Alan. Read Allah' Gly Pro Leu Ala Val To be 1 5 10 15 Allah Ala Ser Gly To be Gly His To be Thr Arg Tyr Trp Asp Cys Cys Lys 20 25 30 Pro Ser Cys Ser Trp To be Gly Lys Allah Allah Val Asn Allah Pro Allah Read 35 40 45 Thr Cys Asp Lys Asn Asp Asn Pro Ile To be Asn Thr Asn Allah Val Asn 50 55 60 Gly Cys Glu Gly Gly Gly To be Allah Tyr Allah Cys Thr Asn Tyr To be Pro 65 70 75 80 Trp Val Asn Ward Asp G1U Read Allah Tyr Gly Phe Allah Allah Thr Lys Ile 85 90 95 To be Gly Gly Ser Glu Allah To be Trp Cys Cys Allah Cys Tyr Allah Read Thr 100 105 110 Phe Thr Thr Gly Pro Val Lys Gly Lys Lys Met Ile Val Gin To be Thr 115 120 125

64,264

Case CM 356M

Mod. 71 - 20,000 ex. - 8/90

Asn Thr 130 Gly Gly Asp Leu Gly Asp Asn His Phe Asp Leu Met Met Pro 135 140 Gly Gly Gly Vai Gly Ile Phe Asp Gly Cys Thr To be Glu Phe Gly Lys 145 150 155 160 Allah Read Gly Gly Allah Gin Tyr Gly Gly Ile To be To be Arg To be Glu Cys : - 165 - 170 175 Asp To be Tyr Pro Glu Read Read Lys Asp Gly Cys His Trp Arg Phe Asp 180 185 190 Trp Phe Glu Asn Allah Asp Asn Pro Asp Phe Thr Phe Glu Gin Go Gin 195 200 205 Cys Pro Lys Allah Read Read Asp Ile To be Gly Cys Lys Arg Asp Asp Asp 210 215 220 To be To be Phe Pro Allah Phe Lys Go Asp Thr To be Allah To be Lys Pro Gin 225 230 235 240 Pro To be To be To be Allah Lys Lys Thr Thr To be Allah Allah Allah Allah Allah Gin 245 250 255 Pro Gin Lys Thr Lys Asp To be Allah Pro Go Go Gin Lys To be To be Thr 260 265 270 Lys Pro Allah Allah Gin Pro Glu Pro Thr Lys Pro Allah Asp Lys Pro Gin 275 280 285 Thr Asp Lys Pro Go Allah Thr Lys Pro Allah Allah Thr Lys Pro Go 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 To be Cys Pro Allah Lys Thr Asp Allah Thr Allah Lys Allah To be Go 325 330 335 Go Pro Allah Tyr Tyr Gin Cys Gly Gly To be Lys To be Allah Tyr Pro Asn 340 345 350 Gly Asn Read Allah Cys Allah Thr Gly Ser Lys Cys Go Lys Gin Asn Glu 355 360 365 Tyr Tyr To be Gin cys Go Pro Asn

370 375

Lisbon, - 8. APR. 1992

PCIER & CAMBIE COMPANY fâiftáe iWGOÊS ÈOTÇ âjÈSfiie Official liá Property Indusmai «» Gonosi ^ o. 3, l.Mlifc tMWA

Claims (16)

Mod. 71 - 20,000 ex. - 8/90 I ^a · - Process for the preparation of a granular detergent composition for washing fabrics in a washing machine, consisting of surfactant, bulking agent and cellulase, characterized in that said cellulase provides at least 10% removal of immobilized carboxylmethylcellulose , radioactively labeled according to the C14CMC method at 25x10% in. weight of protein: 'cellulase' no: assay solution. .der. wash ^! 'á arefertóapòoi' pbsiçãtr-gíaiailá & -dè- detetgenteo no more than 15% by weight of inorganic filler salt, and said granular detergent composition has a density of 550 to 950g / liter of composition. ^To 2 · - Process for the preparation of a composition according to claim 1, characterized in that. cellulase compound consists essentially of a homogeneous endoglucanase component that is immunoreactive with a monoclonal antibody created by reacting with a paraffinely purified cellulase of about 9 ^ 43kD, derived from Humicolc insolens, DSM 1800, or that is homologous to said 43kD endoglucané . 3. ^The process for preparing a composition according to claim 2, characterized in that the endoglucanase component of said cellulase has an isolation point of about 5.1. 4. ^A process for the preparation of a composition according to claims 2-3, characterized in that said endoglucanase component can be produced by a method consisting of culturing a host cell transformed with a recombinant DNA vector, which carries a DNA sequence encoding said endoglucanase component or a precursor of said component endoglucanase, as well as DNA sequences encoding functions that allow the expression of the sequential DNA sequencing οι the endo-r glucanaseyiou component. a .your precursor, -. in a culture medium under con35 64,264 Case CM 356M Mod. 71 - 20,000 ex. - 90/08 terms that allow the expression of the endoglucanase component or its precursor and recovery of the endoglucanase component of the culture. 5§.- Process for the preparation of a composition according to any one of the preceding claims, characterized in that the cellulase level is such that the amount of enzyme protein to be supplied in the washing solution is between 6, 5 and 40mg / liter of washing solution, preferably 0.01 to 10 mg / liter of washing solution. 6 ^a .- Process for the preparation of a composition according to any one of the preceding claims, characterized in that said inorganic filler salt is chosen from the alkali and alkaline earth metal salts of chloride sulphate. 73. The process for preparing a composition according to any one of the preceding claims, characterized in that the composition does not contain more than 10% by weight of inorganic filler salt. 8. A process for preparing a composition according to claim 7, characterized in that the composition does not contain more than 5% by weight of inorganic filler salt, 9 ^a .- Process for the preparation of a composition according to any one of the preceding claims, characterized in that the composition has a density of 650 to 850g / liter. 10 ^a .- Process for the preparation of a composition according to any one of the preceding claims, characterized in that the composition is practically free of phosphate compounds and said bulking agent is chosen from aluminosilicate ion exchangers, citrates, carbonates and their mixtures. Ll = - 113.- Process for preparing a composition according to claim 1, characterized in that the cellulase compound is an endoglucanase enzyme with the sequence 64,264 Case CM 356M of amino acids represented in the sequence listing ID- ^ 2: Mod. 71 - 20,000 ex. - 8/90 Met Arg To be To be Pro Read Read Pro Be Wing Go Go Allah Allah Read Pro -21 -20 15 10 Go Read Allah Read Allah Allah Asp Gly Arg Ser Thr ARg Tyr Trp Asp Cys -5 1 5 10 Cys Lys Pro To be Cys Gly Trp Allah Lys Lys Allah Pro Go Asn Gin Pro 15 20 25 Go Phe To be Cys Asn Allah Asn Phe Gin Arg Ile Thr Asp Phe Asp Allah 30 35 40 Lys To be Gly Cys Glu Pro Gly Gly Go Allah Tyr To be Cys Allah Asp Gin 45 50 55 Thr Pro Trp Allah Go Asn Asp Asp Phe Allah Read Gly Phe Allah Allah Thr 60 65 70 75 To be Ile Allah Gly to be Asn Glu Allah Gly Trp Cys Cys Allah Cys Tyr Glu 80 85 90 Read Thr Phe Thr To be Gly Pro Go Allah Gly Lys Lys Met Go Go Gin 95 100 105 To be Thr To be Thr Gly Gly Asp Read Gly To be AsnHi’s. Phe Asp Read Asn 110 115 120 Ile Pro Gly Gly Gly Go Gly Ile Phe Asp Gly Cys Thr Pro Gin Phe 125 130 135 Gly Gly Read Pro Gly Gin Arg Tyr Gly Gly Ile To be To be Arg Asn Glu 140 145 150 155 Cys Asp Arg Phe Pro Asp Allah Read Lys Pro Gly Cys Tyr Trp Arg Phe 160 165 170 Asp Trp The Lys Asn Allah Asp Asn Pro To be Phe To be Phe Arg Gin Go 175 180 185 Gin Cys Pro Allah Glu Read Go Allah Arg Thr Gly Cys Arg Arg Asn Asp 190 195 200 Asp Gly Asn Phe Pro Allah Go Gin Ile Pro To be To be To be Thr To be To be 205 210 215 Pro Go Asn Gin Pro Thr To be Thr To be Thr Thr To be Thr To be Thr Thr 220 225 230 235 To be To be Pro Pro Go Gin Pro Thr Thr Pro To be Gly Cys Thr Allah Glu 240 245 250 5} 64,264 Case CM356M 8/0; Arg Trp Allah Gin Cys Gly Gly Asn Gly Trp To be Gly Cys Thr Thr Cys 255 260 265 Go Allah Gly To be Thr Cys Thr Lys Tle Asn Asp Trp Tyr His Gin Cys 270 275 280 Read Mod. 71 -20,000 ex.-90/08 or is its counterpart that has an endoglucanase activity. ^A .-- Process for the preparation of a composition according to claim 11, characterized in that said endoglucanase enzyme is produced by a Humicola species, for example, Humicola insolens. ^A · 13 - Process for the preparation of a composition according to claim 1, characterized in that the gas con be a cellulase endoglucanase enzyme with the amino acid sequence shown in ID ^ E4 sequence listing: Met 1 Arg To be Tyr Thr Leu Leu Ala Leu Ala Gly Pro Leu Ala Vai Sera 5 10 15 Allah Allah To be Gly To be Gly Hys Ser Thr Arg Tyr Trp Asp Cys Cys Lys 20 25 30 Pro To be Cys To be Trp To be Gly Lys Allah Allah Go Asn Allah Pro Allah Read 35 40 45 Thr Cys Asp Lys Asn Asp Asn Pro Ile To be Asn Thr Asn Allah Go Asn 50 55 60 Gly Cys Glu Gly Gly Gly To be Allah Tyr Allah Cys Thr Asn Tyr To be Pro 65 70 75 80 Trp Allah Go Asn Asp Gli Read Allah Tyr Gly Phe Allah Allah Thr Lys Ile 85 90 95 To be Gly Gly To be GlU Allah To be Trp Cys Cys Allah Cys Tyr Allah Read Thr 100 105 110 Phe Thr Thr Gly Pro Go Lys Gly Lys Lys Met Ile Go Gin To be Thr 115 120 125 Asn Thr Gly Gly Asp Read Gly Asp Asn Hys Phe Asp Read Met Met Pro 130 135 140 Gly Gly Gly Vai Gly Ile Phe Asp Gly Cys Thr To be Giu Phe Gly Lys 145 150 155 160 sa 264 Case CM 356M Mod. 71 -20,000 ex.-90/08 Ala Leu Gly Gly Ala Gin Tyr Gly Gly Ile Ser Ser Arg Ser Gli Cys 165 170 175 Asp To be Tyr Pro Glu Read Read Lys Asp Gly Cys Hys Trp Arg Phe Asp 180 185 190 Trp Phe Glu Asn Allah Asp Asn Pro Asp Phe Thr Phe Glu Gin Go Gin 195 200 205 Cys Pro Lys Allah Read Read Asp Ile To be Gly Cys Lys Arg Asp Asp Asp 210 215 220 To be To be Phe Pro Allah Phe Lys Go Asp Thr To be Allah To be Lys Pro Gin 225 230 235 240 Pro To be To be To be Allah Lys Lys Thr Thr To be Allah Allah Allah Allah Allah Gin 245 250 255: Pro Gin Lys Thr Lys Asp To be Allah Pro Go Go Gin Lys To be To be Thr 260 265 270 Lys Pro Allah Allah Gin Pro Gloi Pro Thr Lys Pro Allah Asp Lys Pro Gin 275 280 285 Thr Asp Lys Pro Go Allah Thr Lys Pro Allah Allah Thr Lys Pro Go 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 To be Cys Pro Allah Lys Thr Asp Allah Thr Allah Lys Allah To be Val 325 330 335 Go Pro Allah Tyr Tyr Gin Cys Gly Gly To be Lys To be Allah Tyr Pro Asn 340 345 350 Gly Asn Read Allah Cys Allah Thr Gly Ser Lys Cys Go Lys Gin Asn Glu 355 360 365 Tyr Tyr To be Gin Cys Go Pro Asn 370 375 or is a homologue that has an endoglucanase activity. 14 ^. Process for the preparation of a composition according to claim 11, characterized in that said endoglucanase enzyme is produced by a species of Fusarium, for example, Fusarium oxysporum. 15§. - Process for the preparation of a composition64.264 Case CM 356M Mod. 71 - 20,000 ex. 90/08 according to claims 11-14, characterized in that said enzyme is produced by a DNA construct comprising a DNA sequence encoding the enzyme. 16. - Process for the preparation of a composition according to claim 15, characterized in that the same DNA is as shown in the sequence listing ID 1: GGATCCAAG ATG CGT TCC TCC CCC CTC CTC CCG TCC GCC GTT GTG GCC Met Arg Ser Ser Pro Leu Leu Pro ser Ala Val.Val Ala 48 96 GCC Allah CTG Read -21 -20 CTT Read 15 GCC GCT GAT GGC AGG TCC 10 ACC CGC TAC CCG Pro GTG TTC GCC Go : - 5. Leu Ala Allah Wing Asp Gly Arg Ser Thr Arg Tyr . 1 5 TGG GAC TGC TGC AAG CCT TCG TGC GGC TGG GCC AAG AAG GCT CCC GTG 144 Trp Asp Cys Cys Lys Pro To be Cys Gly Trp Ala Lys Lys Allah Pro Vai 10 15 20 AAC CAG CCT GTC TTT CBT TGC AAC GCC AAC TTC CAG CGT ATC AGC GAC 192 Asn Gin Pro Go Phe Ser Cys Asn Allah Asn Phe Gin Arg Ile Thr Asp 25 30 35 40 TTC GACGCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC 240 Phe Asp Allah Lys Being Gly Cys Glu Pro Gly Gly Go Ala Tyr Be Cys 45 50 55 GCC GAC CAG ACC CCA TGG GCT GTG AAC GAC GAC TTC GCG CTC GGT TTT 288 Allah Asp Gin Thr Pro Trp Allah Go Asn Asp Asp Phe Ala Read Gly Phe 60 65 70 GCT GCC ACC TCT ATT GCC GGC AGC AAT GAG GCG GGC TGG TGC TGC GCC 336 Allah Allah Thr To be Ile Ala Gly To be Asn Glm Ala Gly Trp cys Cys Ala 75 80 85 TGC TAC GAG CTC ACC TTC A CA CBT GGT CCT GTT GCT GGC THE AG AAG ATG 384 Cys Tyr G1U Read Thr phe Thr To be Gly Pro Go Ala Gly Lys lys Met 90 95 100 GTC GTC CAG CBT ACC AGC ACT GGC GGT GAT CTT GGC AGC AAC CAC TTC 432 Go Go Gin To be Thr Ser Thr Gly Gly Asp Read Gly Ser Asn His Phe 105 110 115 120 64,264 Case CM 356M Mod. 71 - 20,000 ex. - 8/90 GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT 480 Asp Read Asn Ile Pro Gly Gly Gly Go Gly Ile Phe Asp Gly Cys Thr 125 130 135 ccc CAG CBT GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG CBT 528 pro Gin Phe Gly Gly Read Pro Gly Gin Arg Tyr Gly Gly Ile To be To be 140 145 150 CGC AAC GAG TGC.GAT CGG TTC CCC GAC GCC CTC THE AG CCC GGC TGC TAC 576 Arg ASn Glu Cys Asp Arg Phe Pro Asp Allah Read Lys Pro Gly Cys Tyr 155 160 165 TGG CGC TTC GAC TGG TTC THE AG AAC GCC GAC AAT CCG AGC TTC AGC TTC 624 Trp Arg Phe Asp Trp Phe Lys Asn Allah Asp Asn Pro To be Phe To be Phe 170 175 180 CGT CAG GTC CAG TGC CCA GCC GAC CTC GTC GCT CGC ACC GGA TGC CGC 672 Arg Gin Go Gin Cys Pro Allah G1U Read Go Allah Arg Thr Gly Cys Arg 185 190 195 200 CGC AAC GAC GAC GCC AAC TTC CCT GCC GTC CAG ATC CCC CBT AGC AGC 720 Arg Asn Asp Asp Gly Asn Phe Pro Allah Go Gin Ile Pro To be To be To be 205 210 215 ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG CBT ACC 768 Thr To be To be Pro Go Asn Gin Pro Thr To be Thr To be Thr Thr To be Thr 220 225 230 CBT ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC 816 To be ^r Thr Thr To be To be Pro Pro Go Gin Pro Thr Thr Pro To be Gly Cys 235 240 245 ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC 864 Thr Allah Gltt Arg Trp Allah Gin Cys Gly Gly Asn Gly Trp TO BE Gly Cys 250 255 260 ACC ACC TGC GTC GCT GGC AGC ACT TGC ACG THE AG ATT AAT GAC TGG TAC 912 Thr Thr Cys Go Allah Gly To be Thr Cys Thr Lys Ile Asn Asp Trp Tyr 265 270 275 280 CAT CAG TGC TAGACGCAGG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA 964 His Gin Cys Leu 285 CGAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 1024 GATTGTCACA TAAATGCAAT GAGGAACAAT GAGTAC 1060 264 CM 356M Case a » Mod. 71 - 20,000 ex. - 90/08 or ID 3: GAATTCGCGG CCGCTCATTC ACTTCATTCA TTCCTTTAGAA TTACATACAC TCTCTTTCAA 60 AACAGTCACT CTTTAAACAA AACAACTTTT GCAACA ATG CGA TCT TAC ACT CTT Met Arg SEr Tyr Thr Leu 114 1 5 CTC GCC CTG GCC GGC CCT CTC GCC GTG AGT GCT GCT TCT GGA AGC GGT 162 Leu Wing Leu Wing Gly Pro Read Wing Will Be Wing Ala Ser Gly Ser Gly 10 15 20 ? A £ í'-TCT ACT CGA TAC TGG GAT TGC TGC AAG CCT TCT TGC TCT TGG AGC 210 lis Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ser Trp Ser 25 30 35 GGA AAG GCT GCT GTC AAC GCC CCT GCT TTA ACT TGT GAT AAG AAC GAC 258 Gly Lys 40 Allah Allah Go Asn Wing Pro Wing 45 Leu Thr Cys Asp Lys Asn Asp 50 ACC CCC ATT CBT AAC ACC AATSGCTAGTCAAAC 1GGT. TGT'-GAG2GGT - GGT “GGT 306 Asn Pro 55 Ile To be Asn Thr 60 Asn Ala Vai Asn Gly Cys Glu Gly Gly Gly 65 70 TCT GCT TAT GCT TGC ACC’-. AAC TCT TAC. CCC TGG -GCT GTC AAC GAT GAG 354 Be Wing Tyr Allah Cys 75 Thr Asn Tyr Ser Pro Trp Ala Vai Asn Asp Glu 80 85 CTT GCC TAC GGT TTC GCT GCT ACC AAG ATC TCC GGT GGC TCC GAG GCC 402 Leu Ala Tyr Gly 90 Phe Allah Allah Thr Lys 95 Ile Ser Gly Gly Ser Glu Ala 100 AGC TGG TGC TGT GCT TGC TAT GCT TTG ACC TTC ACC ACT GGC CCC GTC 450 Ser Trp Cys 105 Cys Allah Cys Tyr Wing Leu 110 Thr Phe Thr Thr Gly Pro Vai 115 AAG GGC THE AG THE AG ATG ATC GTC CAG TCC ACC AAC ACT GGA GGT GAT CTC 498 Lys Gly Lys 120 Lys Met Ile Go 125 Gin Ser Thr Asn Thr Gly Gly Asp Leu 130 GGC GAC AAC CAC TTC GAT CTC ATG ATG CCCGGC GGT GGT GTC GGT ATC 546 Gly Asp 135 Asn His Phe Asp 140 Read Met Met Pro Gly Gly Gly Vai Gly Ile 145 150 TTC GAC GGC TGC ACC TCT GAG TTC GGC AAG GCT TCT GGC GGT GCC CAG 594 Phe Asp Gly Cys Thr To be Glu Phe Gly Lys Ala Leu Gly Gly Ala Gin 155 160 165 64,264 Case CM 356M Mod. 71 - 20,000 eic. - 8/90 TAC GGC ATC CBT CBT CGA AGC GAA TGT GAT AGC AGC TAC CCC GAC CTT 642 Tyr Gly Gly Ile To be To be Arg To be G1U Cys Asp to be Tyr Pro Glu Read 170 175 180 CTC AAG GAC GGT TGC CAC TGG CGA TTC GAC TGG TTC GAG AAC GCC GAC 690 Read Lys Asp Gly Cys His Trp Arg Phe Asp Trp Phe Glu Asn Allah Asp 185 190 195 AAC CCT GAC TTC ACC TTT GAG CAG GTT CAG TGC CCC THE AG GCT CTC CTC 738 Asn Pro Asp Phe Thr Phe Glu Gin go Gin Cys Pro Lys Allah Read Read 200 205 210 GAC ATC AGT GGA TGC THE AG CGT GAT GAC GAC CBT AGC TTC CCT GCC TTC 786 Asp Ile To be Gly Cys Lys Arg Asp Asp Asp To be To be Phe Pro Allah Phe 215 220 225 230 THE AG GTT GAT ACC TCG GCC AGC THE AG CCC CAG CCC CBT AGC CBT GCT THE AG 834 Lys Go Asp Thr To be Allah To be Lys PÍQ Gin Pro To be To be To be Allah Lys 235 240 245 THE AG ACC ACC CBT GCT GCT GCT GCC GCT CAG CCC CAG THE AG ACC THE AG GAT 882 Lys Thr Thr To be Allah Allah Allah Allah Allah Gin Pro Gin Lys Thr Lys Asp 250 255 260 CBT GCT CCT GTT GTC CAG THE AG CBT CBT ACC THE AG CCT GCC GCT CAG CCC 930 To be Allah Pro Go Go Gin Lys To be To be Thr Lys Pro Allah Allah Gin Pro 265 270 275 GAG CCT ACT THE AG CCC GCC GAC THE AG CCC CAG ACC GAC THE AG CCT GTC GCC 978 Glu Pro Thr Lys Pro Allah Asp Lys Pro Gin Thr Asp Lys Pro Go Allah 280 285 290 ACC THE AG CCT GCT GCT ACC THE AG CCC GTC CAA CCT GTC AAC THE AG CCC THE AG 1026 Thr Lys Pro Allah Allah Thr Lys Pro Go Gin Pro Go Asn Lys Pro Lys 295 300 305 310 A CA ACC CAG THE AG GTC CGT GGA ACC AAA ACC CGA GGA AGC TGC CCG GCC 1074 Thr Thr Gin Lys Go Arg Gly Thr Lys Thr Arg Gly To be Cys Pro Allah 315 320 325 THE AG ACT GAC GCT ACC GCC THE AG GCC CBT GTT GTC CCT GCT TAT TAC CAG 1122 Lys Thr Asp Allah Thr Allah Lys Allah To be Go Go Pro Allah Tyr Tyr Gin 330 335 340 TGT GGT GGT CBT THE AG CBT GCT TAT CCC AAC GGC AAC CTC GCT TGC GCT 1170 Cys Gly Gly To be Lys To be Allah Tyr Pro Asn Gly Asn Read Allah Cys Allah 345 350 355 64,264 Case CM 356M Mod. 71 - 20,000 ex. - 8/90 ACT GGA AGC AAG TGT GTC AAG CAG AAC GAG TAC TAC TCC CAG TGT GTC 1218 Thr Gly Ser Lys Cys Vai Lys Gin Asn Glu Tyr Tyr Ser Gin Cys Vai 360 365 370 CCC AAC TAAATGGTAG ATCCATCGGT TGTGGAAGAG ACTATGCGTC TCAGAAGGGA 1274 Pro Asn 375 TCCTCTCATG AGCAGGCTTG TCATTGTATA GCATGGCATC CTGGACCAAG · TGTTCGACCC 1334 TTGTTGTACA TAGTATATCT TCATTGTATA TATTTAGACA CATAGATAGC CTCTTGTCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 17 ^ã . Process for the preparation of a composition according to claims 11-16, characterized in that said host cell is a fungus strain such as Tricloderuca or Aspergillus, preferably Aspergillus oryzae or Aspergillus niger or a yeast cell belonging to a strain Hansenula or Saccharomyces, for example, a strain of Saccharomyces cerevisae. 18 ^â . Process for preparing a composition according to claims 11-17, characterized in that the host cell is a strain of a bacterium, for example, Bacillus, Streptomyces or E. Coli.