MXPA99003713A - Cellulase obtainable from thermomonospora fusca - Google Patents

Abstract

A method for treating cellulosic materials is disclosed which comprises contacting the cellulosic material with a cellulase obtainable from Thermomonospora fusca corresponding to E5 or a derivative thereof. Particularly preferred methods comprise stonewashing and detergent cleaning of cotton fabrics, the production of paper products, as an additive to animal feed and in the production of food, starch, ethanol and sugar.

Description

THERMOMONOSPORA FUSCA OBTAINABLE CELLULOSE FOR USE IN INDUSTRIAL PROCESSES BACKGROUND OF THE INVENTION The present invention is directed to methods comprising the use of cellulases in industrial processes and compositions for these. In particular, the present invention relates to treating textiles, for example, washing and processing, with cellulase derived from Thermomonospora fusca, which is particularly suitable for that purpose. The present invention is also related to the use of cellulase derived from Th ermomon ospora fusca to improve the digestibility of animal feed, in detergents, in the treatment of pulp and paper and in the production of starch and the treatment of secondary products thereof. . Cellulases are enzymes that hydrolyze cellulose (β-1,4-D-glucan bonds) and produce glucose, cellobiose and cellooligosaccharides as primary products. Cellulases are produced by a number of microorganisms and comprise several different classifications of enzymes, including those identified as exo-cellobiohydrolases REF .: 30012 (CBH), endoglucanases (EG) and ß-glucosidases (BG) (M. Schulein, Methods in Enzymology, vol 160, pp. 235-242 (1988)). Current theory holds that enzymes with these classifications can be separated into individual components. For example, the microbial cellulase compositions may consist of one or more components of CBH, one or more components of EG and possibly β-glucosidase. The complete cellulase system comprising components of CBH, EG and BG acts synergistically to convert crystalline cellulose to glucose. Exo-cellobiohydrolases and endoglucanases act together to hydrolyze cellulose to small cello-oligosaccharides. The oligosaccharides (mainly cellobiose) are subsequently hydrolyzed to glucose by a major β-glucosidase. Cellulases and components thereof, used either individually or in combination, are known to be useful in detergent compositions and for treating textiles. In the textile industry, during or shortly after the manufacture of cotton-containing fabrics, it is known to treat such fabrics with cellulase to impart desirable properties to the fabric. One purpose of this treatment is to eliminate lint, that is, unraveled fiber ends protruding from the surface of a yarn or fabric, and balls, ie bunches or tangled spheres of fibers that adhere to the surface of a fabric by a more fibers. Accordingly, in the textile industry, cellulase has been used to improve the feel and / or appearance of cotton-containing fabrics, to remove surface fibers from knitted fabrics containing cotton, and also to impart a stone-washed appearance to denim fabrics that contain cotton. In particular, Japanese Patent Applications Nos. 58-36217 and 58-54032, as well as Ohishi et al., "Reformation of Cotton Fabric by Cellulase" and "hat's New-Weight Loss Treatment to Soften the Touch of Cotton Fabric". Japan Textile News, (December 1988) each describes that the treatment of tissues containing cotton with cellulase results in an improved feeling for the tissue. It is generally believed that this cellulase treatment eliminates the formation of cotton fluff and / or surface fibers, which reduces the weight of the fabric. The combination of these effects imparts an improved feeling to the fabric. Clothing made from cellulase fabric, such as cotton denim fabric, has a rigid texture due to the presence of sizing compositions used to facilitate the manufacture, handling and assembly of garments and typically has A dark and fresh tinted appearance. A desirable feature of indigo-dyed denim clothing is the alteration of strands dyed with white threads, which gives the denim fabric a white or blue appearance. For example, after a period of prolonged use and washing, the garment, particularly the denim fabric, can develop in the panels and in the seam localized areas of variation in the form of a clearance in the depth or density of the color. In addition, a general discoloration of the clothes, some wrinkles in the seams and some wrinkles in the fabric panels may appear frequently. In recent years such deformed or "stone washed" appearance, particularly in denim clothing, has become very desirable for a substantial proportion of the public. Previous methods to produce this deformed appearance include washing stone from an article or articles of clothing in a large vat with pumice that has a particle size from about 2.54 to 25.4 cm (1 to 10 inches) and with particles of smaller pumice stones generated by the abrasive nature of the process. Typically the garment is rotated in a drum with the pumice while it is wet for a sufficient period of time such that the pumice scrapes the fabric to produce on the fabric panels scraped areas of lighter color and areas lighter colored similar at the seams. Additionally, the pumice softens the fabric and produces a fluffed surface similar to that produced by prolonged use and washing of the fabric. The use of pumice has several disadvantages, including damage due to overload to the engines of the machine, mechanical damage to transport mechanisms and washing drums, environmental problems of waste from the sand produced and high labor costs associated with the elimination manual of the stones of the garment bags. In view of the problems associated with pumice stones in stone washing, cellulase solutions are used as a replacement for pumice stones under agitating and cascading conditions, i.e. in a rotating drum washing machine, for imparting a "stone washed" appearance to the denim fabric (U.S. Patent No. 4, 832, 864). A cellulase system derived from the bacterium of the filamentous soil, ter ophilicus Thermomonospora fusca has been detected and the biochemical characteristics of that system and its components have been studied (Wilson, Critical Reviews in Biotechnology, Vol. 12 ^, pp. 45-63 (1992)). The sequence has been determined to a specific component of the endoglucanase of the T. fusca system, E5 (Lao et al., J. Bacter., Vol 173, pp 3397-3407 (1991)), and its disulfide arrangement and functional domains (McGinnis et al., Biochemistry, vol.32, pp. 8157-8161 (1993)). McGinnis discloses that E5 treated with Strept omyces l i vi dans protease results in a cellulose binding domain of 14 kD units and a catalytic active 32 kD fragment, which had lost the ability to bind to cellulose. E5 treated with S protease. l i vi dans catalytically active, pure was shown to have an activity similar to the intact enzyme on CMC. However, mixtures of catalytically active E5 fragments, when combined with intact E3 from T. fusca or intact E3 and CBHI from Tri choderma reesei, showed a decreased efficiency to similar mixtures containing intact E5 instead of the fragment (Publication of PCT No. 96/00281). Despite extensive research related to the use of cellulases in industrial processes, cellulases known and used in the art have shown significant disadvantages. For example, many cellulases have been problematic due to their low activity, poor alkaline or acid stability, poor temperature stability and poor oxidative stability. Surprisingly, applicants herein have discovered that E5 cellulases possess a feature complement that makes their use particularly desirable in certain industrial applications.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, there is provided a method for treating cellulosic material comprising contacting the cellulosic material with a cellulase obtainable from Th ermomon ospora fus ca corresponding to E5, a truncated E5, or a derivative thereof . In one embodiment of the process of the invention, the cellulosic material comprises cellulose-containing tissue and the result of the method is to produce a stonewashing effect or an improvement in the feel and / or appearance of tissue. In an alternative embodiment of the process of the invention, the cellulose-containing fabric is contacted with an aqueous solution containing a detergent composition comprising a cellulase obtainable from T. fusca, which corresponds to E5, a truncated E5, or a derivative thereof. In still another embodiment of the process of the invention, the cellulosic material comprises wood pulp and the addition of cellulase facilitates the production of paper products therefrom. In still another embodiment of the process of the invention, the cellulosic material comprises feed for animals, and the method results in an increase in the digestibility or the value of the feed for animals. In still further embodiments of the invention, the cellulosic material comprises grain or by-product grain used in the production of food, starch, ethanol or sugar. Applicants hereby identify a specific cellulase obtainable from T. fusca, known in the literature as E5, which has a surprising feature set, which are especially beneficial in the processing of textiles (specifically including stone washing of the denim fabric and bio-polishing), cleaning products and detergents, pulp and paper production, food processing and as an additive for animal feed. Specifically, the Applicants have discovered that E5 has an especially broad pH / activity profile on insoluble substrates, being active in the pH range from about pH 5.0 to 10.5 with very little decrease in activity in the alkaline region. On the other hand, E5 has significant activity levels at moderate pH and temperature, is stable for prolonged periods of time and at temperatures above 80 ° C, is essentially insensitive to many compositions and concentrations of buffers, remains active after prolonged proteolytic hydrolysis , and is stable in the presence of oxidants such as perborate and perborate / TAED combination and in detergents. Among the especially surprising aspects of E5 are its exceptional activity at high pH on insoluble substrates such as cotton fabrics compared to its activity on soluble substrates, which decreases significantly after its optimum pH of about 6.0. Thus, E5 is particularly suitable for high pH textile applications, such as simultaneous bleaching and bio-polishing, or laundry and pre-treatment or post-treatment detergents which are formulated for high pH. Another surprising aspect is the ability of T. fusca to remain almost unaltered by incubation in liquid detergent. A further novel aspect of the present invention is that the truncated E5 also possesses many of the same exceptional characteristics as E5. For example, the Applicants discovered that a truncated E5 enzyme can exhibit an almost identical surface fiber removal activity as E5. The considerable advantages of cellulase E5 or truncated E5 cellulase in industrial applications would not have been suggested by the prior art. The invention itself, together with additional accompanying objects and advantages, will be better understood by reference to the following detailed description, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the pH profile of E5 at 70 ° C on soluble substrates. Figure 2 illustrates the activity of E5 from 40 ° C to 80 ° C on soluble substrates. Figure 3 illustrates the stability of E5 at 20 ° C, 20 ppm at pH 8 and 10. Figure 4 illustrates the stability of E5 at 37 ° C, 20 ppm at pH 6, 8 and 10. Figure 5 illustrates the stability from E5 at 50 ° C, 20 ppm at pH 6, 8 and 10. Figure 6 illustrates the stability of E5 in the presence of perborate concentrations of 0 ppm, 90 ppm, 250 ppm, 500 ppm and 900 ppm. Figure 7 illustrates the removal of surface fibers of E5 at pH 6, pH 7.5, pH 8.5 and pH 9.5. Figure 8 illustrates the activity of E5 in the presence of protease at 50 ° C and a pH of 7. Figure 9 illustrates the response to the pH of E5 compared to commercially available Cheer® and heat treated at 38 ° C. Figure 10 illustrates the response to the pH of E5 compared to commercially available Cheer® and heat treated at 60 ° C.

Figure 11 illustrates the stability of E5 in extra strong liquid Wisk® for 35 days compared to a cellulase representative of T. l ongibra chi a t um.

DETAILED DESCRIPTION OF THE INVENTION "Cotton-containing fabric" means sewn or non-sewn fabrics, yarns or fibers made of pure cotton or cotton blends including woven cotton fabrics, cotton knits, cotton denim fabrics, cotton yarns, cotton twine and similar. When cotton blends are used, the amount of cotton in the fabric is preferably at least about 35 weight percent cotton. When used as blends, the accompanying material employed in the fabric may include one or more non-cotton fibers including cellulosic or synthetic fibers such as polyamide fibers (eg, nylon 6 and nylon 66), acrylic fibers (e.g. , polyacrylonitrile fibers), and polyester fibers (eg, polyethylene terephthalate), polyvinyl alcohol fibers (eg, Vinylon), polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers and Aramid fibers. "Cellulose-containing fabric" means any sewn or non-sewn fabric, threads or fibers made of cotton or fiber other than cotton containing cellulose or cotton or non-cotton fiber containing cellulose blends including natural cellulose fibers and artificial cellulosic fibers (such as jute, flax, ramin, rayon and lyocell). Included under the heading of tissues containing artificial cellulose are regenerated tissues that are well known in the art such as rayon. Other fabrics containing artificial cellulose include chemically modified cellulose fibers (for example cellulose derivatized by acetate) and solvent-spun cellulose fibers (for example lyocell). Specifically included within the definition of cellulose-containing fabrics is any yarn or fiber made from such materials. "Stonewashing composition" means a formulation for use in stonewashing tissues containing cellulose. Stonewashing compositions are used to modify cellulose-containing fabrics prior to presentation for sale to the consumer, i.e., during the manufacturing process. In contrast, detergent compositions are proposed for cleaning soiled laundry items. "Stonewashing" means the treatment of tissue containing cellulose with a cellulase solution under agitating and cascading conditions, i.e. in a rotating drum washing machine, to impart a "stone washed" appearance to the denim fabric. The cellulase solution according to the present invention will functionally replace the use of stones in such methods recognized in the art, either completely or partially. Methods for imparting a stone-washed appearance to denim are described in U.S. Patent No. 4,832,864, which is incorporated herein by reference in its entirety. Usually, stonewashing techniques have been applied to denim cotton fabric dyed indigo. "Detergent composition" means a mixture that is proposed to be used in a washing medium for washing soiled cellulose containing fabrics. In the context of the present invention, such compositions may include, in addition to cellulases and surfactants, additional hydrolytic enzymes, filler additives, bleaching agents, bleach activators, coloring agents and fluorescent dyes, cake formation inhibitors, masking agents , cellulase activators, antioxidants and solubilizers. Such compositions are generally used to clean soiled garments and are not used during the manufacturing process, in contrast to stone washing compositions. Detergent compositions comprising cellulase are described in, for example, Clarkson et al., U.S. Patent No. 5,290,474 and EP Publication No. 271,004, incorporated herein by reference. "Derivative" means a protein that is derived from a precursor protein (eg, the native protein) by the addition of one or more amino acids at either or both of the C- and N-ter-inal end, substitution of one or more amino acids in one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends of the protein, or at one or more sites in the amino acid sequence, or insertion of one or more amino acids into one or more sites in the amino acid sequence. The preparation of an enzymatic derivative is preferably performed by modifying a DNA sequence encoding the native protein, transforming this DNA sequence into a suitable host, and expressing the modified DNA sequence to form the derived enzyme. The derivative of the invention includes peptides comprising altered amino acid sequences compared to an amino acid sequence of the precursor enzyme (for example, a wild-type or naturally occurring enzyme), which peptides retain an enzymatic nature characteristic of the enzyme precursor, but that has altered properties in some specific aspect. For example, an altered E5 or altered truncated E5 may have an optimal stability at an increased pH, or an increased stability at temperature or increased oxidative stability, but retain its characteristic cellulolytic activity. Similarly, the derivatives according to the present invention include a cellulose binding domain which has either been entirely removed, or modified in such a manner as to significantly impair its cellulose binding capacity. It is contemplated that derivatives according to the present invention can be derived from a DNA fragment encoding a truncated E5 or E5 (as described below) wherein the functional activity of the expressed truncated E5 or E5 derivative is retained . For example, a DNA fragment encoding a truncated E5 may further include a DNA sequence or portion thereof encoding a linker or linker attached to the E5 DNA sequence truncated at either the 5 'or 3' end, where the functional activity of the encoded truncated E5 domain is retained. The derivative also includes the chemical modification to change the characteristics of the enzyme. The term "truncated E5" refers to a protein comprising a derivative (usually shortened) of an intact E5 enzyme that retains cellulolytic activity. E5 in its intact form is believed to contain a catalytic core and a binding domain. The catalytic core and the cellulose binding domain can act together in a synergistic manner to effect the efficient and often detrimental hydrolysis of the cellulose fibers in a cellulose-containing fabric often resulting in an undesirable loss of strength. A truncated E5 lacking a functional binding domain may include other entities which do not include cellulose binding activity attributable to a cellulose binding domain. For example, the presence of a linker or articulation is specifically contemplated. Similarly, the covalent attachment of another enzyme entity or a cellulose binding domain that is not E5 to a truncated E5 is also specifically contemplated. It is expected that a truncated E5, or derivatives thereof according to the invention will retain at least 10% of the activity. exhibited by E5 when each is tested under similar conditions and dosed based on similar amounts of protein. A truncated E5 can be made by any standard means to produce a truncated enzyme. Particularly effective means include the use of protease or chemical hydrolysis (i.e., cyanogen bromide) to hydrolyze the enzyme, or the use of genetic engineering to directly express a truncated E5 in a microbial host. McGinnis et al., S upra, suggests that S protease. l i vi dans hydrolyzed the 120 amino acids at the N-terminus of the E5 enzyme, leaving the remainder of the enzyme as a catalytically active core. Thus, a preferred embodiment of the present invention contemplates the use of a truncated E5 that differs from E5 in that an N-terminal segment of the enzyme less than 121 amino acids in length is deleted, preferably differing from E5 in that the truncated E5 comprises the sequence of intact E5 starting at threonine at residue 121. In another preferred embodiment of the present invention, a truncated E5 differs from E5 in that the amino acid sequence of E5 in the region of amino acids 1-120 has been altered to reduce or eliminate the cellulose binding activity. According to the present invention, there is provided a method for treating cellulosic material comprising contacting the cellulosic material with a cellulase obtainable from Th ermomonospora fusca, which corresponds to E5, a truncated E5, or a derivative thereof. In one embodiment of the process of the invention, the cellulosic material comprises cellulose-containing fabric, and the result of the method is to produce a stone-washing effect or an improvement in the feel and / or appearance of the fabric. In an alternative embodiment of the process of the invention, the cellulose-containing fabric is contacted with an aqueous solution containing a detergent composition comprising a cellulase obtainable from T fusca corresponding to E5, a truncated E5, or a derivative of the same. In still another embodiment of the process of the invention, the cellulosic material comprises wood pulp and the addition of cellulase facilitates the production of paper products therefrom. In still another embodiment of the process of the invention, the cellulosic material comprises an animal feed, and the method results in an increase in the digestibility or value of the animal feed. E5 as used herein refers to a cellulase having a molecular weight of about 45-47 kD units (deduced from the amino acid sequence and confirmed by SDS-gel) and a pl of about 4.5-4.8 (measured on IEF gel) and which is obtainable from T. fusca. T. fusca is a thermophilic, filamentous actinomycete found in soil and common in decomposing organic material such as decaying wood. The sequence of E5 is described in Lao et al., S upra. The cellulases according to the present invention can be produced by T. fusca by culturing it under conditions that have been described in the literature, to produce a fermentation broth from which E5 can be purified (see for example Walker et al., Biotechnol Bioeng., Vol 40, pp. 1019-1026 (1992)). Alternatively, E5 can be produced by a microorganism that has been genetically modified to produce truncated E5 or E5 as in, for example, McGinnis et al., Supra. As used herein, the cellulase is proposed to include truncated E5, E5, or derivatives thereof. Preferably the truncated E5 comprises a catalytic domain and lacks significant cellulose binding activity. In general, the compositions comprising the cellulase according to the invention can be obtained by purification techniques based on the characteristics and known properties of the cellulase of the invention. Specifically, when the cellulase according to the invention is part of a mixture of cellulases produced by the cultured organism, the complete mixture of cellulase (total cellulase) can be purified into substantially pure components by recognized separation techniques published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography and size exclusion. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the components of the cellulase by eluting with a pH gradient, or a salt gradient, or both of a pH and a salt gradient. After purification, the requisite quantity of the desired components could be recombined. Alternatively, genetic engineering techniques can be used to manipulate the cellulase mixtures produced, for example through the use of suppressed strains in cellulase genes wherein the gene encoding the cellulase according to the invention is transformed and expressed by the host strain deleted otherwise in cellulase. The treatment of textiles according to the present invention contemplates the processing or cleaning of textiles with a composition comprising a cellulase. Such treatment includes, but is not limited to, stone washing, modification of the texture, feel and / or appearance of cellulose-containing fabrics or other techniques used during manufacturing or cleaning / reconditioning of cellulose-containing fabrics. Additionally, treatment within the context of this invention contemplates the removal of "immature" or "dead" cotton from cellulosic fabrics or fibers, i.e. immature cotton that is significantly more amorphous than mature cotton. It is known that dead cotton causes uneven dyeing, and is undesirable. Accordingly, the composition contemplated in the present invention includes a cellulase component proposed for use in washing or weaving containing dirty, manufactured cellulose. For example, the cellulase can be used in a detergent composition for washing with soap and water. Detergent compositions useful in accordance with the present invention include special formulations such as pre-wash, pre-soak and color restoration compositions for use at home. Such treatment compositions, described herein, may be in the form of a concentrate, which requires dilution or in the form of a dilute solution or form that can be applied directly to the cellulose-containing tissue. General treatment techniques known in the art for cellulase treatment of textiles are described in, for example, EP Publication No. 220 016 and Requests GB No. 1,368,599 and 2,095,275.

The treatment of a cellulosic material according to the present invention further contemplates the treatment of animal feed, pulp and / or paper, feed and grains for purposes known in the art. For example, it is known that cellulase increases the value of animal feed, improves the drainability of wood pulp, improves food products and reduces fiber in grains during the wet milling process of grains or the process of dry grinding. According to a preferred embodiment of the present invention, the cellulase compositions described above can be employed as a stone washing composition. Preferably, the stone wash according to the present invention comprises preparing an aqueous solution containing an effective amount of cellulase together with other optional ingredients including, for example, a buffer, a surfactant, and a cleaning agent. An effective amount of cellulase enzyme composition is a concentration of cellulase enzyme sufficient for its intended purpose. Thus, an "effective amount" of cellulase in the stonewashing composition according to the present invention is that amount which will provide the desired treatment, for example, stonewashing. The amount of cellulase used is also dependent on the equipment used, the process parameters employed (the temperature of the cellulase treatment solution, the exposure time to the cellulase solution and the like) and the activity of the cellulase (for example a particular solution will require a lower concentration of cellulase when a more active cellulase composition compared to a less active cellulase composition is used). The exact concentration of cellulase in the aqueous treatment solution to which the fabric to be washed is added to the stone can be easily determined by the skilled artisan based on the above factors, as well as the desired result. Preferably the cellulase is present in a concentration from 1 to 5,000 ppm and more preferably from 10 to 200 ppm of total protein. Optionally, a buffer is employed in the stone washing composition such that the buffer concentration is sufficient to maintain the pH of the solution within the range where the cellulase employed exhibits activity which, in turn, depends on the nature of the cellulase used. The exact concentration of shock absorber used will depend on several factors, which the expert technician can easily take into account. For example, in a preferred embodiment, the buffer as well as the concentration of the buffer is selected to maintain the pH of the final cellulase solution within the pH range required for the optimal activity of the cellulase. Because E5 has a broad pH profile from about 5.0 to about 10.5 on insoluble substrates, the cellulase can be used at any of a slightly acid, neutral or alkaline pH with optimal activity, depending on the specific needs of the textile processor. Suitable buffers at a pH within the range of E5 activity are well known to those skilled in the art in the field. In addition to the cellulase and a buffer, the stone washing composition may optionally contain a surfactant. Suitable surfactants include any surfactant compatible with cellulase and tissue including, for example, anionic, nonionic and ampholytic surfactants. Suitable anionic surfactants for use herein include linear or branched alkylbenzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkane sulfonates and the like. Suitable counterions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; the ammonium ion; and alkanolamines having from 1 to 3 alkanol groups of carbon number 2 or 3. The ampholytic surfactants include sulfonates of quaternary ammonium salts, and ampholytic surfactants of betaine type. Such ampholytic surfactants have both charged groups, the positive and the negative in the same molecule. Nonionic surfactants generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adducts thereof, and glycerin monoesters of fatty acid. Mixtures of surfactants can also be employed in manners known to those skilled in the art. In a preferred embodiment, a concentrated stone wash composition can be prepared for use in the methods described herein. Such concentrates would contain concentrated amounts of the cellulase composition described above, buffer and surfactant, preferably in an aqueous solution. When formulated in this manner, the stone wash concentrate can easily be diluted with water, to quickly and accurately prepare stonewashing compositions according to the present invention and have the requisite concentration of these additives. When formulating aqueous concentrates, these concentrates can be diluted to reach the required concentration of the components in the cellulase solution indicated above. As is readily apparent, such stone wash concentrates will allow easy formulation of the cellulase solutions, as well as allow for viable transportation of the concentration to the site where it will be used. The stone wash concentrate can be in any manner recognized in the art, for example, liquid, emulsion, gel or paste. Such forms are well known to those skilled in the art. When solid stone wash concentrate is employed, the cellulase composition can be a granule, a powder, a bonded material or a solid disk. When granules are used, the granules are preferably formulated to contain a cellulase protective agent. See, for example, U.S. Serial No. 07 / 642,669, filed on January 17, 1991 as Proxy File No. 010055-073 and titled "GRANULOS CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES" (GRANULES CONTAINING BOTH OF AN ENZYME AND AN ENZYME PROTECTIVE AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES), the application is incorporated herein by reference in its entirety. Also, the granules can be formulated to contain materials to reduce the rate of dissolution of the granules in the washing medium. Such materials and granules are described in U.S. Patent No. 5,254,283 which is incorporated herein by reference in its entirety. Other materials may also be used with or placed in the stonewashing composition of the present invention as desired, including stones, pumice, fillers, solvents, enzyme activators, and anti-redeposition agents.

The cellulose-containing fabric is contacted with the stonewashing composition containing an effective amount of the cellulase by mixing the treatment composition with the stonewashing composition, and thereby bringing the cellulase enzyme in proximity with the tissue. . Consequently, the aqueous solution containing the cellulase and the tissue is agitated. If the treatment composition is an aqueous solution, the fabric can be directly soaked in the solution. Similarly, when the stone wash composition is a concentrate, the concentrate is diluted in a water bath with the cellulose-containing fabric. When the stone washing composition is a solid form, for example a pre-wash gel or a solid stick, the stone wash composition can be contacted by directly applying the composition to the fabric or wash liquor. The cellulose-containing fabric is incubated with the stone wash solution under effective conditions to allow the enzymatic action to impart a stone-washed appearance to the cellulose-containing fabric. For example, during stone washing, the pH, the liquor ratio, the temperature and the reaction time can be adjusted to optimize the conditions under which the stone washing composition acts. "Effective conditions" necessarily refer to the pH, liquor ratio, and temperature that allow the cellulase enzyme to react efficiently with the cellulose-containing tissue. The effective reaction conditions for the stone washing compositions of the present invention are substantially similar to the well-known methods used with the corresponding prior art cellulase compositions. Accordingly, it is within the experience of those skilled in the art to maximize the conditions for using the stonewashing compositions according to the present invention. The liquor ratios during stone washing, ie, the weight ratio of the wash composition solution to the stone (ie, wash liquor) to the weight of the fabric, employed herein is generally an amount enough to achieve the desired effect of washing the stone in the denim fabric and is dependent on the process used. Preferably, the liquor ratios are from about 4: 1 to about 50: 1; more preferably from 5: 1 to about 20: 1, and most preferably from about 10: 1 to about 15: 1. The reaction temperatures during the stone washing with the stone washing compositions present are governed by two competitive factors. First, higher temperatures generally correspond to increased reaction kinetics, i.e., faster reactions, which allows reduced reaction times compared to the required reaction times at lower temperatures. Accordingly, the reaction temperatures are generally at least about 10 ° C and higher. Secondly, cellulase is a protein that loses its activity beyond a given reaction temperature, whose temperature is dependent on the nature of the cellulase used. Thus, if the reaction temperature is allowed to be too high, the cellulolytic activity is lost as a result of denaturation of the cellulase. Because E5 shows excellent thermostability, the temperature of the stone wash can be very high, that is, higher than 80 ° C if necessary. However, standard temperatures in the art are generally in the range of 35 ° C to 65 ° C, which will also be suitable for the cellulase of the invention. The reaction times are dependent on the specific conditions under which stone washing occurs. For example, the pH, the temperature and the cellulase concentration will all effect the optimum reaction time. Usually, the reaction times are from about 5 minutes to about 5 hours, and preferably from about 10 minutes to about 3 hours, and, more preferably, from about 20 minutes to about 1 hour. According to yet another preferred embodiment of the present invention, the cellulase compositions described above can be employed in a detergent composition. The detergent compositions according to the present invention are useful as a pre-wash composition, pre-soaking compositions, or for cleaning during the regular washing or rinsing site. Preferably, the detergent composition of the present invention comprises an effective amount of cellulase, a surfactant, and optionally include other ingredients described below.

An effective amount of cellulase employed in the detergent compositions of this invention is an amount sufficient to impart the desirable effects known to be produced by the cellulase on cellulose containing fabrics, for example fluffing, smoothing, lint antiforming, elimination. of surface fiber and cleaning. Preferably, the cellulase in the detergent composition is used in a concentration of about 10 ppm to about 20,000 ppm of detergent. The cellulase enzyme concentration employed in the detergent composition is preferably selected such that with dilution in a washing medium, the concentration of cellulase enzyme is in a range of about 0.01 to about 1000 ppm, preferably from about 0.02 ppm. at about 500 ppm, and more preferably from about 0.5 ppm to about 250 ppm of total protein. The amount of cellulase enzyme used in the detergent composition will depend on the degree to which the detergent is diluted with the addition of water to form a wash solution.

The detergent compositions of the present invention may be in any form recognized in the art, for example, as a liquid diluent, in grains, in emulsions, in gels, or in pastes. Such forms are well known to the skilled artisan. When a solid detergent composition is employed, the cellulase is preferably formulated as granules. Preferably, the granules can be formulated to additionally contain a cellulase protective agent. See, for example, U.S. Serial No. 07 / 642,669 filed on January 17, 1991 as Proxy File No. 010055-073 and entitled "GRANULES CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES" (GRANULES CONTAINING BOTH, ONE ENZYME AND AN ENZYME PROTECTIVE AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES), the application is incorporated herein by reference in its entirety. Also, the granule can be formulated to contain materials to reduce the rate of dissolution of the granule in the washing medium. Such materials and granules are described in U.S. Pat. No. ,254,283, which is incorporated herein by reference in its entirety. The detergent compositions of this invention employ a surface active people, ie, a surfactant, including anionic, nonionic and ampholytic surfactants well known for use in detergent compositions. Suitable anionic surfactants for use in the detergent composition of this invention include linear or branched alkylbenzene sulfonates; alkyl or alkenyl sulfate ether having alkyl or straight or branched alkenyl groups, alkyl or alkenyl sulfates; olefin sulfonates; and alkane sulfonates. Suitable counterions for the anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having from 1 to 3 alkanol groups of carbon number 2 or 3. The ampholytic surfactants include sulfonates of quaternary ammonium salts, and ampholytic surfactants of betaine type. Such ampholytic surfactants have both groups positively and negatively charged in the same molecule. Nonionic surfactants generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adducts thereof, fatty acid glycerin monoesters, and the like. Suitable surfactants for use in this invention are described in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. Mixtures of such surfactants can also be used. The surfactant or a mixture of surfactants is generally employed in the detergent compositions of this invention in an amount of from about 1 percent by weight to about 95 percent by weight of the total detergent composition and preferably from about 5 percent by weight to about 45 percent by weight of the total detergent composition. In addition to the cellulase composition and the surfactant or surfactants, the detergent compositions of this invention may optionally contain one or more of the following components: Hydrolases Except Cellulase Suitable hydrolases include ester carboxylate hydrolase, thioester hydrolase, phosphate monoester hydrolase and phosphate diester hydrolase, which act on the ester linkage; glycoside hydrolase, which acts on glycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds; thioether hydrolase, which acts on the ether bond; and a-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-a-nacre hydrolase, dipeptide hydrolase, and peptidyl-peptide hydrolase, which acts on the peptide bond. Preferred among them are carboxylate hydrolase ester, glycoside hydrolase, and peptidyl peptide hydrolase. Suitable hydrolases include (1) proteases belonging to the peptidyl peptide hydrolases such as pepsin, pepsin B, renin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein, gastrisin, cathepsin D, broelin, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidase A and B, and aminopeptidase; (2) glycoside hydrolases (the cellulase which is an essential ingredient is excluded from this group) α-amylase, β-amylase, glyco amylase, invertase, lysozyme, pectinase, chitinase, and dextranase. Preferred among them are a-amylase and β-amylase. They work in acid-to-neutral systems, but one that is obtained from bacteria exhibits high activity in an alkaline system; (3) carboxylate hydrolase ester including carboxylesterase, lipase, pectin esterase, and chlorophyllase. Especially effective among them is lipase. The hydrolase different from the cellulase is incorporated in the detergent composition as much as required according to the purpose. Preferably it should be incorporated in an amount of 0.001 to 5 percent by weight, and more preferably 0.02 to 3 percent by weight, in terms of purified protein. This enzyme should be used in the form of granules made from crude enzyme alone or in combination with other components in the detergent composition. The crude enzyme granules are used in such an amount that the purified enzyme is 0.001 to 50 percent by weight in the granules. The granules are used in an amount of 0.002 to 20 and preferably of 0.1 to 10 percent by weight. As with cellulases, these granules can be formulated to contain an enzyme protective agent and a dissolution retarding material.

Cationic Surfactants and Large Chain Fatty Acid Salts Such cationic surfactants and large chain fatty acid salts include saturated or unsaturated fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, salts or esters of a-sulfo fatty acid, surfactants of type amino acid, sulfate ester surfactants, quaternary ammonium salts including those having 3-6 alkyl substituents and up to 1 alkyl substituent substituted with phenyl. Suitable cationic surfactants and large chain fatty acid salts are described in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. The composition may contain from about 1 to about 20 percent by weight of such cationic surfactants and large chain fatty acid salts.

Loading additives A. Divalent sequestering agents. The composition may contain from about 0 to about 50 percent by weight of one or more charge additive components selected from the group consisting of alkali metal salts and alkanolamin salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, aminopolyacetates, electrolytes of high molecular weight, polymers that do not dissociate, salts of dicarboxylic acids, and salts of aluminosilicate. Suitable divalent sequestering agents are described in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

B. Alkalis or inorganic electrolytes The composition may contain from about 1 to about 50 percent by weight, preferably from about 5 to about 30 percent by weight, based on the composition of one or more alkali metal salts of the following compounds such as alkalis or inorganic electrolytes: silicates, carbonates and sulphates as well as organic alkalis such as triethanolamine, diethanolamine, monoethanolamine and tri isopropanolamine.

Anti-redeposition Agents The composition may contain from about 0.1 to about 5 percent by weight of one or more of the following compounds as anti-redeposition agents: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. Among them, a combination of carboxymethylcellulose and / or polyethylene glycol with the cellulase composition of the present invention provides for a particularly useful soil-removing composition.

Bleaching Agents The use of the cellulase of the present invention in combination with a bleaching agent such as potassium monopersulfate, sodium percarbonate, sodium perborate, sodium sulfate / hydrogen peroxide adduct and sodium chloride / hydrogen peroxide adduct or / and a photosensitive decolorizing dye such as the aluminum or zinc salt of sulfonated phthalocyanine further improves the detergency effects. Similarly, the bleaching agents and bleach catalysts described in EP 684 304 can be used.

Staining Agents and Fluorescent Stains Various staining agents and fluorescent stains can be incorporated into the composition, if necessary. Suitable anchoring agents and fluorescent dyes are described in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

Inhibitors of Cake Formation The following cake formation inhibitors can be incorporated into the powdered detergent: salts of p-toluenesulfonic acid, salts of xylene sulfonic acid, salts of acetic acid, salts of sulfosuccinic acid, talc, finely powdered silica, amorphous silicas, clay, calcium silicate (such as Micro-Cell of Johns Manville Co.), calcium carbonate and magnesium oxide.

Masking Agents for Factors that Inhibit the Activity of the Cellulase The cellulase composition of this invention is deactivated in some cases in the presence of copper, zinc, chromium, mercury, lead, manganese or silver ions or their compounds. Various metal chelating agents and agents that precipitate metals are effective against these inhibitors. These include, for example, divalent metal ion sequestering agents listed in the above article with reference to optional additives as well as magnesium silicate and magnesium sulfate. Cellobiose, glucose and gluconolactone sometimes act as inhibitors. It is preferred to avoid co-presence of these saccharides with the cellulase as much as possible. In the event that co-presence is unavoidable, it is necessary to avoid direct contact of the saccharides with the cellulase, for example by coating them. Large chain fatty acid salts and cationic surfactants act as inhibitors in some cases. However, the co-presence of these substances with the cellulase is admissible if the direct contact between them is prevented by some means such as tabletting or coating. The masking agents mentioned in the foregoing and the methods may be employed, if necessary, in the present invention.

Cellulase activators Activators vary depending on the cellulase variety. In the presence of proteins, cobalt and its salts, magnesium and its salts, and calcium and its salts, potassium and its salts, sodium and its salts or monosaccharides such as mannose and xylose, the cellulases are activated and their detergent power is improved in a remarkable.

Antioxidants Antioxidants include, for example, tert-buty1-hydroxytoluene, 4,4'-butylidenebis (6-tert-butyl-3-methylphenol), 2,2'-butylidenebis (6-tert-butyl-4-methylphenol), cresol mono-styrene, cresol diestirenadonado, mono-styrene phenol, fenil diestirenado and 1, 1-bis (4-hydroxy-phenyl) ciciohexano.

Solubilizers Solubilizers include, for example, lower alcohols such as ethanol, benzenesulfonate salts, lower alkylbenzene sulfonate salts such as p-toluenesulfonate salts, glycols such as propylene glycol, acetylbenzenesulfonate salts, acetamides, pyridinedicarboxylic acid amides, benzoate salts and urea.

The detergent composition of the present invention can be used over a broad pH range from an acidic to an alkaline pH. In a preferred embodiment, the detergent composition of the present invention can be used in a mildly acidic, neutral or alkaline detergent washing medium having a pH from above of 5 to not more than about 12. In addition to the ingredients above, they can perfumes, buffers, preservatives, dyes and the like are used, if desired, with the detergent compositions of this invention. Such components are conventionally employed in amounts hitherto used in the art. When a detergent base used in the present invention is in the form of a powder, it may be one which is prepared by any known preparation method including a spray-dried method and a granulation method. The detergent base obtained particularly by the spray drying method, the agglomeration method, the dry mixing method and the non-tower route methods are preferred. The detergent base obtained by the spray drying method is not restricted with respect to the preparation conditions. The detergent base obtained by the spray drying method is hollow granules, which are obtained by spraying an aqueous paste of heat resistant ingredients, such as surface active agents and filler additives in a hot space. After spray drying, perfumes, enzymes, bleaching agents, inorganic alkaline fillers can be added. With a highly dense granular detergent base, obtained such as by the granulation method by spray drying or agglomeration, various ingredients can also be added after the preparation of the base. When the base detergent is a liquid, it can be either a homogeneous solution or a non-homogeneous dispersion. In order to eliminate the decomposition of carboxymethylcellulose by the cellulase in the detergent, it is desirable that the carboxymethylcellulose be granulated or coated before incorporation into the composition. The detergent compositions of this invention can be incubated with cellulose-containing fabrics, for example soiled fabrics, in industrial and domestic uses at temperatures, reaction times and liquor ratios conventionally employed in these environments. Incubation conditions, i.e., effective conditions for treating cellulose-containing fabrics with detergent compositions according to the present invention, will be readily determined by those of ordinary skill in the art. Accordingly, the appropriate effective conditions for treatment with the detergents present will correspond to those using similar detergent compositions that include known cellulases. The detergents according to the present invention can additionally be formulated as a pre-wash in the appropriate solution at an intermediate pH where there is sufficient activity to provide desired improvements in softening, lint deformation, lint prevention, elimination of Surface fiber or cleaning. When the detergent composition is a pre-soaking composition (e.g., pre-wash or pre-treatment), either as a liquid, spray, gel or paste composition, the truncated cellulose enzyme is generally employed from about 0.0001 to about 1 percent by weight based on the total weight of the composition of pre-soaking or pre-treatment. In such compositions, a surfactant can optionally be employed and when employed, is generally present in a concentration of about 0.005 to about 20 percent by weight based on the total weight of the pre-soaking composition. The remainder of the composition comprises the conventional components used in the pre-soaking composition, ie, diluent, buffers, other enzymes (proteases), and the like in their conventional concentrations. It is contemplated that the compositions that the compositions comprising truncated cellulase enzymes described herein may be used in home use as a separate composition suitable for restoring color to discolored fabrics (see, for example, U.S. Pat. No. 4,738,682, which is incorporated herein by reference in its entirety) as well as also used in a spot remover and for lint deformation and lint antiforming (prevention of lint formation).

The use of the cellulase according to the invention can be particularly effective in food additives and in the processing of pulp and paper. These additional industrial applications are described, for example, in PCT Publication No. 95/16360 and in the Finnish Granted Patent No. 87372, respectively. To further illustrate the present invention and the advantages thereof, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.

EXAMPLES Example 1 Production and Purification of E5 The prepared clones can be used as in McGinnis et al., Biotechnology, vol. 32, pp. 8157-8161 (1993) to express E5 in S trept omyces l i vi dans and should be cultured under conditions suitable for expression of the plasmid. The E5 enzyme can then be purified from the resulting fermentation supernatant according to the procedure cited in Walker et al., Biotechnol. Bioeng., Vol. 40, p. 1019-1026 (1992) and in Irwin e tal. Biotechnology and Bio engineering ibid. vol. 42. pp. 1002-1013 (1993). Alternatively, the E5 paste can be solubilized in MOPS, pH 7, subjected to centrifugation and the extracted supernatant is prepared (crude E5). A second resolubilization and subjection to centrifugation is done on the loose pellets. The combined recovery is around 75%. The supernatant is precipitated with 0.65 M ammonium sulfate in the cold room with stirring and then subjected to centrifugation (14,000 rpm for 20 minutes in the SS-34 rotor). The pellet is again solubilized in a buffer solution containing 0.25 M ammonium sulfate, 5 mM KP, pH 6.0. To eliminate the antifoam (to make filtration less difficult) the solution is heated to room temperature and subjected to centrifugation (15,000 rpm for 30 minutes in the SS-34 rotor). The white film is removed from the top and the mixture is filtered through a 0.45 micron membrane using a disposable filtering device under vacuum. A phenyl sepharose column is pre-equilibrated with 0.25 M ammonium sulfate, 5 mM KPi, pH 6. The filtered E5 is loaded and then washed with the equilibration buffer until the absorbance of the through flow returns to a low level . The column is then washed with a volume of 0.125 M ammonium sulfate, 5 mM KP ?, pH 6 (little absorbance is observed). The E5 is eluted with 5 mM KVI f pH 6; small fractions are collected. After the absorbance returns to its base, the column is then eluted with water and fractions are collected. Small aliquots of each fraction of possible interest are concentrated in spin columns, washed with water, and concentrated 3-4x. The fractions are then analyzed by IEF gel. The purest fractions are those near the end of the elution with 5 M KPi, and at the beginning of elution with water. On both a small column (25 ml) and a larger one (450 ml), the activity yield of collecting the purest fractions obtained through the previous procedure was 30% total. These fractions were either an IEF band dyed with silver, or additionally contained approximately two small bands. The activity levels were tested as follows: Add 5 to 20 μl of an appropriate enzyme solution in a sufficient concentration to provide a requisite amount of enzyme in the final solution. Add 250 μl of 2 percent by weight RBB-CMC (Brilliant Blue Re azol R-Carboxymethylcellulose - commercially available from Mega Zyme, Nórth Rocks, Australia) in 50mm sodium acetate buffer at pH 5.5. Undergo centrifugation and incubate at 40 ° C for 30 minutes. Cool in an ice bath for 5-10 minutes. Add 1000 μl of 80% ethanol containing 0.3 M sodium acetate and 0.02 M zinc acetate. Undergo centrifugation and let settle for 5-10 minutes. Centrifuge and pour the supernatant into cells or tubes. Measure the optical density of the solution in each cell at 590 nm. The highest levels of optical density correspond to higher levels of enzymatic activity. Protein determinations were made using the BCA method with BSA as the standard according to the distributor's instructions (Pierce, BCA Protein Assay Reagent, Prod. No. 23225). The specific activity of the crude E5 (paste turned to solubilize) was determined to be about 1.6 units / mg of protein. The purified E5 had a specific activity of approximately 11 units / mg.

Example 2 Protease Treatment of E5 to Produce E5 Truncated E5 intact, solubilized and purified as in Example 1, was treated with several proteases to give truncated E5 variants. Five proteases, Alcalase (available from Novo Nordisk, Denmark), Purafect (available from Genencor International, Inc.) and three different mutant bacterial proteases (BA, Bl and B2) each having a different catalytic behavior were used to provide a variety of different hydrolysis patterns in E5. A concentration of either 0.5 or 5 mg / ml E5 was incubated with 2.5 mg / ml protease. Incubations were made at pH 5 (NaOAc buffer), 7 (buffer solution of TES) and 9 (glycine buffer) at 20, 37 and 50 ° C. A final concentration of 15 gpg was added (grains per gallon) of Ca + 2. Aliquots were removed at 1, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours and immediately frozen. The aliquots treated with protease were analyzed by IEF gels (Coomassie staining) and to determine the activity on RBB-CMC as in Example 1.

During the course of all incubations, two new bands with pl's slightly higher than E5 appeared when analyzed with IEF. An overlay using RBB-CMC showed that at least one of these bands always had activity. The Alcalase produced bands of E5 in the fastest time and thus had the greatest effect on E5. For example, given a concentration of 1: 5 E5: Alcalase (measured in mg protein / ml) at 37 ° C and pH 7 and 9, all three bands on an IEF gel were formed after 30 minutes, and the band Higher pl is predominant after 8 hours. The other proteases were less aggressive. Purafect at pH 5 produced only slight activity towards E5 at 37 ° C. At pH 7 and 9, at least 2 hours of incubation was required before any significant development in the high pl band. BA and Bl appeared to affect E5 less than Alcalase and Purafect, and required at least 4 hours of incubation before a significant development of the pl band. BA digests E5 to the other bands but can also degrade E5 (the bands become very light). Digestions with E5: protease 1: 1 (measured in mg protein / ml) were made at 37 ° C and pH 9 with Purafect, B2 and Alcalase. Again, Alcalase digested E5 more quickly, to one band after 8 hours. With Purafect and B2, both of the new bands appeared after 30 hours, showing that these proteases digest E5 more slowly than Alcalase. The stability of E5 with Purafect against time is shown in Figure 8. As shown in Figure 8, E5 retains a significant portion of its original activity on RBB-CMC after more than 25 hours at 50 ° C, pH 7 In addition, each of the E5 mixtures treated with other proteases retained at least 50% of their activity.

Example 3 Temperature Stability, pH Stability, Storage Stability and Oxidative Stability of E5 The pH profile of E5 was determined by incubating separate aliquots of crude E5 with RBB-CMC at 70 ° C for 30 minutes. A buffer solution of citrate-phosphate was used from pH 4-9 and a glycine buffer was used from pH 9-11. The activity was determined by measuring the absorbance at 620 nm and the results are shown in Figure 1. As shown in Figure 1, E5 has an optimum pH on RBB-CMC of about 6. Aliquots of crude E5 were incubated at pH 5.5 in buffer solution of 0.05 M sodium acetate for 30 minutes with RBB-CMC at temperatures ranging between 40 ° C and 80 ° C and the activity was determined by measuring the absorbance at 590 nm. The results are given in Figure 2. As shown in Figure 2, E5 shows an optimum temperature in the range of 70-75 ° C. The stability at the pH of E5 for prolonged periods of time was measured at a diluted concentration (20 ppm) at 20 ° C, 37 ° C and 50 ° C at pH of 6 (buffer solution of 50 mM MOPS), 8 (50 mM MOPS buffer) and 10 (50 mM glycine buffer). During the course of nine days, aliquots were removed and assayed using the RBB-CMC activity assay described in Example 1. The results are shown in Figures 3-5. As shown in Figures 3-5, As shown in Figures 3-5, at 37 ° C and 50 ° C and pH 6, at least 60-70% of the activity remains after nine days. At pH 8, none of the incubations showed more than 20% loss in activity after nine days, and at pH 10, there was no significant loss in activity at 20 ° C and 37 ° C, while incubation at 50 ° C. C lost 50% of his activity after nine days. As can be seen from the results shown in Figures 3-5, E5 is very stable in each of the conditions tested for extended periods of time. The oxidative activity of E5 was tested by preincubating 15 ppm of crude E5 with 90 to 900 ppm of perborate in MOPS buffer solution (pH 7) at 40 ° C. Aliquots were removed for a period of 30 minutes and assayed by RBB-CMC to determine their activity as in Example 1. There was no detectable loss of activity compared to the control of any of the incubations (Figure 6). For comparison, EGIII from Tri choderma l ongibra chi a tum is completely inactivated by approximately 500 ppm perborate + 170 ppm TAED at pH 5.5 and 50 ° C. The activity of E5 in the presence of perborate was also tested by surface fiber removal studies in a washing meter. The test was carried out by contacting the crude cellulase at 60 ° C for one hour at pH 9 (glycine buffer) with cotton samples. The results indicate that there may be a small loss of activity with perborate, but it is not dependent on perborate concentration. Even the incubation with 900 ppm perborate showed good surface fiber removal. Control of 900 ppm perborate without enzyme showed a reduced surface fiber removal.

Example 4 Effect of Various Shock Absorbers on the Activity of E5 E5 was incubated with RBB-CMC with a variety of buffers at varying pH to determine the effect on the activity of each buffer. Each buffer was tested at 20, 100 and 200 mM. At pH 5, citrate, succinate and sodium acetate were used; at pH 7, phosphate, MOPS and TES were used; and at pH 9, borate and CHES were used. The solutions containing E5, buffer and RBB-CMC were incubated for 30 minutes at 40 ° C, and then analyzed for cellulase activity as described in Example 1. The activity was standardized to the buffer concentration. mM under each condition of the shock absorber. As shown in Table 1 below, E5 proved to be relatively insensitive to the buffers or concentrations used.

TABLE 1 Activity at pH 5 Concentration of Sodium Acetate Citrate Succinate Shock absorber, mM 20 100 LOO 100 100 84 87 95 200 78 65 88 Activity at pH 7 MOPS concentration Potassium phosphate TES Shock absorber, mM 20 100 100 100 100 127 118 120 200 129 83 117 Activity at pH 9 Concentration of Borate TES Shock absorber, mM 20 100 100 100 100 113 200 130 129 Example 5 Surface Fiber Removal for E5 and E5 Treated with Protease E5 was tested using 100, 300, 500 and 1000 units of RBB-CMC in 400 ml of buffer at pH 6, 7.5, 8.5 and 9.5 at 60 ° C. A dose-response was observed, with excellent SFR activity observed in either 500 or 1000 units. E5 is optimally active over a wide range, extending at least from about pH 6.0 to pH 9.5. These results are summarized in Figure 7. As shown in Figure 7, the pH profile for E5 with insoluble substrate differs considerably from the pH profile for E5 with RBB-CMC in Figure 1. Two series of studies in the meter of washing were performed with E5, all tests comprised 350 units of cellulase in the washing meter. In the first, E5 was preincubated with protease before adding it to the wash meter. The two representative proteases selected were Purafect and Alcalase. For the pre-incubation, 40 mg of protease (1000 ppm, resulting in 100 ppm after dilution in the washing meter) were mixed with the cellulase and allowed to stand for 20 hours at 37 ° C and pH 9 (buffer solution of glycine) with 15 ppm of calcium. The conditions used had been established by the Applicants as sufficient to hydrolyze E5 to a truncated E5 protein. A control was prepared with E5 that had been preincubated with protease and a second control was prepared with buffer alone (without added enzyme). The study of the washing meter was made with the mixture pre-incubated at 60 ° C for 1 hour at pH 9 (glycine buffer) on cotton samples. The pants were then dried in a clothes dryer and sorted by a panel of 4 assessors. The grading scale for surface fiber removal was in the range from 1 (highest surface fume) to 5 (lowest surface fume). The results are shown in Table 2. As shown in Table 2, E5 samples pre-incubated with any protease did not show a significant loss in surface fiber removal activity compared to E5 incubated without protease. Table 2 In the second series of studies on the washing meter, the E5 samples were not pretreated, but were added to the washing meter with 1, 10 or 50 ppm of either Purafect or Alcalase. The control experiments provided comparative results for E5 without protease, EGII cellulase (Tri ch oderma l ongibra chi a tum) without protease, buffer, and either Alcalase or Purafect alone. The results are shown in Table 3. As shown in Table 3, the panel classification of the samples treated with E5 and protease showed only a small reduction in surface fiber removal compared to E5 without protease.

Table 3 As stated in the above, E5 is digested by proteases to a truncated protein that has excellent activity both on RBB-CMC (see Figure 8) and on cotton surface fibers. This is especially apparent in the wash meter test, which shows that E5 is active even in the presence of a high level of protease during a 1 hour incubation with the sample.

Example 6 Abrasion of the Denim Fabric with Truncated E5 and E5 Raw E5 was tested to determine the abrasion efficiency on denim to determine the effectiveness of E5 in stone washing applications. The stone washing experiments were carried out in a Unimac. The enzyme (13,500 units of truncated E5 or E5 RBB-CMC made by incubation with Alcalase) was added to the solution, which contained 20 mM MOPS, 9.5 g Triton X-100, pH 7.0, and eight trouser pemiles. denim previously desprestados. The temperature was 60 ° C. Four pemiles were removed after 60 minutes and rinsed in cold water. They returned to Unimac 30 minutes later. At this time, a post-wash with detergent at 70 ° C was made using 50 g of detergent. The pemiles were then dried in a clothes dryer and sorted by a panel of assessors. The grading scale is a denim sample panel that varies in abrasion from 1 (non-abrasion) to 10 (highly abraded).

TABLE 4 TABLE 5 As can be seen from Tables 4 and 5, E5 has excellent abrasion levels after both 60 and 90 minutes. More surprisingly, in this example the abrasion is not reduced by the truncation of the enzyme.

Example 7 Depressed and Discolored Simultaneous Twelve squeezed jeans were used to demonstrate the effectiveness of E5 in a simultaneous process of desizing and discoloration. The pants were combined in a solution that had a final buffer concentration of 40 mM MOPS, pH 7 and also contained 10 g of Triton X-100. To these were added 14,000 units of crude E5 and 5 ml of α-amylase derived from Bacil l us li cheniformi s, and the cycle ran at 65 ° C and 36 rpm. Half of the pants were removed after 30 minutes and rinsed in cold water, while the others continued until 60 minutes. There was no post-wash with detergent. After drying, the pants were sorted by a panel of assessors. The classification scale is a panel of denim samples that vary in abrasion from 1 (non-abrasion) to 10 (very scratched).

TABLE 6 Surprisingly, the amount of abrasion and the pattern are similar to those obtained from jeans that have been first desensitized before stone washing with E5 (see, for example, Example 6).

Example 8 E5 Efficiency in Detergents The evaluation of the surface fiber removal of cotton samples with E5 was conducted in a Terg-O-Tometer under conditions that included a temperature of 100 ° C or 140 ° C for one wash cycle. 2.5 hours at a stirring speed of 125 rpm and a water hardness of 150 ppm (CaCO3). 8 samples (4 of knitted fabric, 4 of woven fabric) were loaded per test container. Two types of fabrics were tested: a cross-linked cotton knit material obtained from Burlington Mills in North Carolina, and 400 woven cotton material, from Teslfabrics, Inc. in New Jersey. All tests were performed on commercially available liquid laundry detergents, Cheer® "Fee", purchased at a local supermarket. The liquid detergent was heat treated for 30 minutes at 95 ° C to destroy the activity of the cellulase (HT Cheer®) and E5 was added at a concentration of 5 mg / l. Identical conditions were used in comparative tests with non-heat-treated Cheer® and heat-treated Cheer® without E5. The treated samples were compared to a group of standard classification samples by a panel of assessors who assigned a Surface Appearance Classification score. The samples with standard classifications were in the range of 0 = (a lot of fluff and pilled) to 7 = (no fluff or pills). The results were averaged and are shown in Figures 9 and 10.

Example 9 Stability of E5 in Liquid Detergent E5 was tested to determine the stability in liquid detergent. E5 or a cellulase isolated from Tri choderma l ongibra chi a tum were incubated in Wisk® detergent at a temperature of 38 ° C and a concentration of 1300 mg / l and 695 mg / l, respectively. Samples were taken at various time points and the remaining cellulase activity was tested by the PAHBAH method as described in M. Lever, Anal. Biochem., Vol. 47, 'pp. 273-279 (1972) (The assay conditions included: 12 mg / ml CMC substrate, 12 mM MOPS buffer, 30 minutes incubation at 40 ° C). The results are shown in Figure 11. As shown in Figure 11, the residual activity of E5 was essentially unchanged after incubation for 35 days, while the residual activity of the cellulase derived from T. The ongibra chi a t um fell to almost zero in less than 15 days. Of course, it should be understood that a wide range of changes and modifications can be made to the preferred embodiment described in the foregoing. Therefore, it is proposed that the above detailed description be understood in the context of the following claims, including all equivalents, which are proposed to define the scope of this invention. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (19)

CLAIMS Having described the invention as above, property is claimed as contained in the following:

1. A method for treating cellulosic material characterized in that it comprises contacting the material with a cellulase obtainable from Thermomonospora fusca corresponding to E5, a truncated E5, or a derivative thereof.

2. The method according to claim 1, characterized in that the truncated E5 is obtained through proteolytic hydrolysis.

3. The method according to claim 1, characterized in that the truncated E5 is obtained through genetic engineering techniques.

4. The method according to claim 1, characterized in that the cellulosic material comprises cellulose-containing fabric.

5. The method according to claim 4, characterized in that the cellulose-containing fabric is treated to effect a stone washing effect.

6. The method according to claim 4, characterized in that the cellulose-containing fabric is treated to improve the appearance and / or feel of the fabric.

7. The method according to claim 4, characterized in that the cellulase is incorporated in a detergent composition.

8. The method according to claim 6, characterized in that the cellulose-containing fabric comprises a bleached colored fabric, and the improvement comprises improving the appearance of the colored bleached fabric by rejuvenating the color thereof.

9. The method according to claim 9, characterized in that the improvement comprises a softer or smoother feeling to the fabric.

10. The method according to claim 1, characterized in that the cellulosic material comprises wood pulp.

11. The method according to claim 1, characterized in that the cellulosic material comprises feed for animals or grains.

12 »A stonewashing composition characterized in that it comprises a cellulase selected from the group consisting of truncated E5, E5 or a derivative thereof.

13. The composition according to claim 12, characterized in that it also comprises a surfactant.

14. The composition according to claim 12, characterized in that the composition is a concentrate.

15. A detergent composition characterized in that it comprises a cellulase selected from the group consisting of truncated E5, E5 or a derivative thereof.

16. The detergent composition according to claim 15, characterized in that the cellulase is present in a concentration of at least 10 ppm.

17. An additive for food characterized in that it comprises truncated E5, E5 or a derivative thereof.

18. The composition according to claim 12 or 15, characterized in that the truncated E5 differs from E5 because it has any amino acid residue segment 1-120 of the E5 deleted.

19. The detergent composition according to claim 15, characterized in that the detergent is a liquid.