BACKGROUND OF THE INVENTION The present invention is directe to improved methods for the use of cellulases and compositions incorporating such improvements. In one particular aspect, the present invention is related to improved methods for treating cellulose containing textiles with cellulase, including improved detergents and improved stonewashing compositions. In other aspects, the present invention relates to improving the performance of cellulase in animal feed, treating pulp and paper, biomass reduction and production of starch and treatment of by-products thereof.

Cellulases are enzymes which hydrolyze cellulose (ß-1,4-D-glucan linkages) and produce as primary products glucose, cellobiose and cellooligosaccharides. Cellulases are produced by a number of microorganisms and comprise several different enzyme classifications including those identifie as exo-cellobiohydrolases (CBH), endoglucanases (EG) and p-glucosidases (BG) (M. Schulein, Methods in Enzymology, Vol. 160, pp.

235-242 (1988)). Current theory holds that the enzymes within these classifications can be separated into individual components. For example, microbial cellulase compositions may consist of one or more CBH components, one or more EG components and possibly, B- glucosidase. The complete cellulase system comprising CBH, EG and BG components synergistically act to convert crystalline cellulose to glucose. The exo-cellobiohydrolases and the endoglucanases act together to hydrolyze cellulose to small cello- oligosaccharides. The oligosaccharides (mainly cellobioses) are subsequently hydrolyzed to glucose by a major p-glucosidase.

In addition to the different enzymological classifications which have been developed for cellulase, differences have been noted between the cellulases produced by the various microbial classes. Among the cellulases which have been characterized to date, significant differences have been noted between, e. g., fungal cellulases and bacterial cellulases. For example, it has been noted that cellulose binding domains between various fungal cellulases are highly conserve. However, the binding domains of many bacterial cellulases differ significantly from the fungal domains. Additionally, while many fungal cellulases (e. g., Trichoderma spp., and Humicola spp.) are limited in their ability to act efficiently at high pH, instead exhibiting their characteristic agressive behavior patterns only in the acidic or neural range, bacterial cellulases have been found which act efficiently at a much broader range of pu rand appear particularly well suited for use in high pH environments such as laundry detergents.

Cellulases and components thereof, used either singularly or in combination, are known to be useful in detergent compositions and for treating textiles. In the detergent industry, it is known to include cellulase in laundry detergent compositions for several reasons, including as a cleaning aid and to impart desirable properties to the fabric. One purpose of this treatment is to remove fuzz, i. e., untangled fiber ends that protrude from the surface of a yarn or fabric, and pills, i. e., bunches or balls of tangled fibers that are held to the surface of a fabric by one or more fibers. Accordingly, cellulase has been used to improve the feel and/or appearance of cotton-containing fabrics and to remove surface fibers from cotton-containing knits.

In the textile industry cellulase has additionally been used for imparting a stone washed appearance to cotton-containing denims. In particular, Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al., "Reformation of Cotton Fabric by Cellulase"and"What's New--Weight Loss Treatment to Soften the Touch of Cotton Fabric", Japan Textile News, (December 1988) each disclose that treatment of cotton-containing fabrics with cellulase results in an improved feel for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing and/or surface fibers which reduces the weight of the fabric. The combination of these effects imparts improved feel to the fabric.

Reinikainen et al., Proteins: Structure, Function and Genetics, Vol. 22, pp. 392-403 (1995) disclose that high ionic strength improves the activity of CBHI cellulases from Trichoderma reeseiwhich have been mutated to decrease their ability to bind to cellulose. The authors note that while the activity of the mutant having lessened binding ability increased in the presence of ionic strength, the activities of the wild type CBHI and another mutant were practically insensitive to high salt concentrations.

Despite intensive research related to the use of cellulases in industrial processes, cellulase use, particularly in laundry detergent and other surfactant containing matrices, remains hampered by less than optimal activity in response to the often harsh environment.

SUMMARY OF THE INVENTION It is an object of the invention to provide for a method of altering the activity of cellulase in industrial processes by altering the ionic strength of the solution wherein the cellulase is used.

It is a further object of the invention to provide for a detergent composition which facilitates more effective usage of cellulase therein.

It is yet a further object of the invention to provide for a method of decreasing backstaining in stonewashing compositions comprising cellulase.

According to the present invention, a method of enhancing the activity of a cellulase against a cellulosic substrate is provided wherein said cellulase is utilized in an aqueous solution comprising an ionic strength of 20 mM or greater.

In another embodiment of the invention, the cellulase is utilized in a laundry detergent composition which, when diluted into an aqueous laundry, provides an ionic strength of 20 mM or greater.

In yet another embodiment of the invention, the cellulase is utilized for the treatment of cellulosic fabric to improve the feel and/or appearance of the fabric and the ionic strength of the solution in which the cellulase is contacte with said textile comprises an ionic strength of 20 mM or greater.

In yet another embodiment of the invention, the cellulase is utilized in stonewashing of dyed cellulosic fabrics.

In yet another embodiment, the present invention comprises compound capable of producing an ionic environment which inclue, for example, sodium, calcium, magnesium, potassium, borate, ammonium and phosphorus. Additionally, sodium chloride, calcium chloride, magnesium chloride, sodium dibasic phosphate (SDP), sodium bicarbonate and sodium sulfate are particularly preferred ionic compound for use in conjunction with the present invention.

In a preferred embodiment, the cellulase is from a microbial source, preferably from a fungal or bacterial source. In a most preferred embodiment, the cellulase is from Bacillus spp. or from an Actinomycete, most preferably from spp. or Bacillus spp. Also preferably, the cellulase has not been modifie so as to compromise the ability of the enzyme to find the substrate.

In preferred embodiments according to the present invention, the ionic strength of the composition in which the cellulase is utilized to achieve a specific purpose, i. e., bio- polishing, pill prevention, color maintenance, stonewashing or other such uses, is 40 mM or greater, more preferably 60 mM or greater and most preferably 80 mM or greater.

An avantage of the present invention is to provide methods for easily enhancing the activity of a given cellulase. For example, detergent matrices into which cellulase has been incorporated, while including ionic compound, generally show an ionic strength when diluted into the wash liquor in the micromolar range. Surprisingly, Applicant herein has discovered that certain cellulases respond to an increase in the ionic strength into the millimolar range of a composition comprising the cellulase, e. g., a solution comprising

dissolve detergent or a stonewashing composition, and will exhibit desirable performance and/or activity changes.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 illustrates the effect of ionic strength at pH 7 on T. fusca E5 cellulase performance in surface polishing. Ionic strength is due to dibasic sodium phosphate buffer (5.73 mM), citric acid (0.87 mM), and sodium sulfate (varied from 0 to 100mM). Fabrics used where a Tencel/Cotton blend (O), and 100% cotton (o).

DETAILED DESCRIPTION OF THE INVENTION "lonic strength"is identifie herein as a method of measuring the total charge effect of an environment within which cellulase is used. For the purposes herein, the calculation of ionic strength is as provided in Gould, Mechanism and Structure in Organic Chemistry, John Wiley and Sons N. Y., p. 185 (1959). As provided in Gould, calculation of ionic strength may be according to the formula: Y, [AZ2 + BZ2 + CZ2... wherein A, B, C... represent the molarity of each ion in solution and z is the charge for that species. Applying this principle, 20 mM calcium chloride (CaCl2) would result in a total <BR> <BR> ionic strength of (. 5) x ( (20 x 22) + (40 x 12)) = 60 mM. Calculating ionic strength involving buffered solutions requires consideration of pH and degree of dissociation, however, such calculations are well within the skill in the art. Ionic strength according to the present invention means the concentration of free ion in the solution comprising the cellulase during the relevant application. Thus, the ionic strength of a cellulase in a detergent composition would be calculated according to the actual ionic strength of the detergent in the laundry wash solution. Generally, according to the present invention, the ionic strength is at least 20 mM, preferably at least 40 mM, more preferably at least 60 mM and most preferably at least 100 mM. Ionic strength may be conferred by the addition of any compound which releases free ions, i. e., cations and/or anions, into the aqueous solution in which the cellulase is used. While it is possible to adjust the ionic strength by the addition of any ion which provides charge to the treating solution certain ions are particularly useful due to their ready availabiiity, low cost and established suitability in industrial compound. Thus compound capable of producing ionic strength which inclue, for example, sodium, calcium, magnesium, potassium, borate, ammonium and phosphorus

are particularly useful. Additionally, sodium chloride, calcium chloride, magnesium chloride, sodium dibasic phosphate (SDP), sodium bicarbonate and sodium sulfate are particularly preferred ionic compound.

The ion selected should further be compatible with the enzyme used, i. e., not substantially deleterious to activity or performance in connection with the given substrate.

The identification of such compatible ions may be routinely determined by methods standard in the art. It is desirable to adjust the ionic strength of the aqueous solution in which the cellulase is utilized according to the specific application. For example, in laundry detergents, the ionic strength is 20 mM or greater, preferably between 40 mM and 1000 mM, more preferably between 60 mM and 750 mM and most preferably between 100 mM and 500 mM. However, with respect to stonewashing of cotton denim fabric dyed with indigo dye, ionic strength appears to be linked to backstaining. Thus, for stonewashing it may be preferable to utilize the lower end of the range indicated for laundry detergents if backstaining is undesirable.

"Cotton-containing fabric"means sewn or unsewn fabrics, yarns or fibers made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, raw cotton and the like. When cotton blends are employed, the amount of cotton in the fabric is preferably at least about 35 percent by weight cotton. When employed as blends, the companion material employed in the fabric can include one or more non-coton fibers including cellulosic or synthetic fibers such as polyamide fibers (for example, nylon 6 and nylon 66), acrylic fibers (for example, polyacrylonitrile fibers), and polyester fibers (for example, polyethylene terephthalate), polyvinyl alcohol fibers (for example, Vinylon), polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers and aramid fibers.

"Cellulose containing fabric"means any sewn or unsewn fabrics, yarns or fibers made of cotton or non-coton containing cellulose or cotton or non-coton containing cellulose blends including natural cellulosics and manmade cellulosics (such as jute, flax, ramie, rayon, and lyocell). Included under the heading of manmade cellulose containing fabrics are regenerated fabrics that are well known in the art such as rayon. Other manmade cellulose containing fabrics include chemically modifie cellulose fibers (e. g, cellulose derivatized by acetate) and solvent-spun cellulose fibers (e. g. lyocell).

Specifically included within the definition of cellulose containing fabric is any yarn or fiber made of such materials.

"Stonewashing composition"means a formulation for use in stonewashing cellulose containing fabrics. Stonewashing compositions are used to modify cellulose containing

fabrics prior to presentation for consumer sale, i. e., during the manufacturing process. In contrast, detergent compositions are intended for the cleaning of soiled garments.

"Stonewashing"means the treatment of cellulose containing fabric with a cellulase solution, i. e., in a rotary drum washing machine, to impart a"stonewashed"appearance to the denim. The cellulase solution according to the instant invention will functionally replace the use of stones in such art recognized methods, either completely or partially. Methods for imparting a stonewashed appearance to denim are described in U. S. Patent No.

4,832,864 which is incorporated herein by reference in its entirety. Generally, stonewashing techniques have been applied to indigo dyed cotton denim.

"Detergent composition"means a mixture which is intended for use in a wash medium for the laundering of soiled cellulose containing fabrics. In the context of the present invention, such compositions may inclue, in addition to cellulases and surfactants, additional hydrolytic enzymes, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, cellulase activators, antioxidants, and solubilizers. Such compositions are generally used for cleaning soiled garments and are not used during the manufacturing process, in contrast to stonewashing compositions.

Detergent compositions comprising celiuiase 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.

Treatment of textiles according to the present invention contemplates textile processing or cleaning with a composition comprising a cellulase. Such treating inclues, but is not limited to, stonewashing, modifying the texture, feel and/or appearance of cellulose containing fabrics or other techniques used during manufacturing or cleaning/reconditioning of cellulose containing fabrics. Additionally, treating within the context of this invention contemplates the removal of"immature"or"dead"cotton, from cellulosic fabric or fibers, i. e. immature cotton which is significantly more amorphous than mature cotton. Dead cotton is known to cause uneven dyeing and is undesirable.

Accordingly, the composition contemplated in the present invention inclues a cellulase component intended for use in washing of a soiled manufactured cellulose containing fabric. For example, cellulase may be used in a detergent composition for washing laundry. Detergent compositions useful in accordance with the present invention include special formulations such as pre-wash, pre-soak and home-use color restoration compositions. Such treating compositions, as described herein, may be in the form of a concentrate which requires dilution or in the form of a dilute solution or form which can be applied directly to the cellulose containing fabric. General treatment techniques known in

the art for cellulase treatment of textiles are described in, for example, EP Publication No.

220 016 and GB Application Nos. 1,368,599 and 2,095,275.

Treatment of a cellulosic material according to the present invention further contemplates the treatment of animal feed, pulp and/or paper, food and grain for purposes known in the art. For example, cellulase is known to increase the value of animal feed, improve the drainability of wood pulp, enhance food products and reduce fiber in grain during the grain wet milling process or dry milling process.

According to a preferred embodiment of the present invention, the aqueous composition comprising cellulase according to the present invention comprises a laundry detergent. Where the detergent composition is in concentrated form, it would be formulated so as to provide the requisite ionic strength upon dilution. The detergent compositions according to the present invention are useful as pre-wash compositions, pre- soak compositions, or for cleaning during the regular wash or rinse cycle. Preferably, the detergent composition of the present invention comprises an effective amount of cellulase, a surfactant, and optionally inclues 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 cellulase on cellulose containing fabrics, for example, depilling, softening, anti-pilling, surface fiber removal and cleaning. Preferably, the cellulase in the detergent composition is employed in a concentration of about 10 ppm to about 20,000 ppm of detergent.

The concentration of cellulase enzyme employed in the detergent composition is preferably selected so that upon dilution into a wash medium, the concentration of cellulase enzyme is in a range of about 0.01 to about 1000 ppm, preferably from about 0.02 ppm to about 500 ppm, and most preferably from about 0.5 ppm to about 250 ppm total protein. The amount of cellulase enzyme employed in the detergent composition will depend on the extent to which the detergent will be diluted upon addition to water so as to form a wash solution.

The detergent compositions of the present invention may be in any art recognized form, for example, as a liquid diluent, in granules, in mulsions, 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 so as to additionally contain a cellulase protecting agent. See, for instance, U. S. Serial No. 07/642,669, filed January 17,1991, as Attorney Docket No. 010055-073 and entitled,"GRANULES CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH

GRANULES,"which application is incorporated herein by reference in its entirety.

Likewise, the granule can be formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium. Such materials and granules are disclosed in U. S. Patent No. 5,254,283 which is incorporated herein by reference in its entirety.

According to another preferred embodiment of the present invention, the cellulase compositions described above may be employed as a stonewashing composition comprising the requisite ionic strength upon contact with the cellulosic fabric. Preferably, stonewashing according to the instant invention comprises preparing an aqueous solution which contains an effective amount of cellulase together with other optional ingredients including, for example, a buffer, a surfactant, and a scouring 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, e. g., stonewashing. The amount of cellulase employed is also dependent on the equipment employed, the process parameters employed (the temperature and pH of the cellulase treatment solution, the exposure time to the cellulase solution, and the like), and the cellulase activity (e. g., a particular solution will require a lower concentration of cellulase where a more active cellulase composition is used as compare to a less active cellulase composition). The exact concentration of cellulase in the aqueous treatment solution to which the fabric to be stonewashed is added can be readily determined by the skilled artisan based on the above factors as well as the desired result. Preferably the cellulase is present in a concentration of from 1 to 5,000 ppm and most preferably 10 to 200 ppm total protein.

In a preferred embodiment, a concentrated stonewashing 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 so formulated, the stonewashing concentrate can readily be diluted with water so as to quickly and accurately prepare stonewashing compositions having the requisite concentration of these additives. When aqueous concentrates are formulated, these concentrates can be diluted so as to arrive at the requisite concentration of the components in the cellulase solution as indicated above. As is readily apparent, such stonewashing concentrates will permit facile formulation of the cellulase solutions as well as permit feasible transportation of the concentration to the location where it will be used. The stonewashing concentrate can be in any art recognized

form, for example, liquid, mulsion, gel, or paste. Such forms are well known to those skilled in the art.

When a solid stonewashing concentrate is employed, the cellulase composition may be a granule, a powder, an agglomerate or a solid disk. When granules are used, the granules are preferably formulated so as to contain a cellulase protecting agent. See, for example, U. S. Serial No. 07/642,669, filed January 17,1991, as Attorney Docket No.

010055-073 and entitled,"GRANULES CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES,"which application is incorporated herein by reference in its entirety.

Likewise, the granules can be formulated so as to contain materials to reduce the rate of dissolution of the granules into the wash medium. Such materials and granules are disclosed in U. S. Patent No. 5,254,283 which is incorporated herein by reference in its entirety.

Other materials can also be used with or placed in the stonewashing composition of the present invention as desired, including stones, pumice, filles, solvents, enzyme activators, and anti-redeposition agents.

The cellulose containing fabric is contacte with the stonewashing composition containing an effective amount of the cellulase by intermingling the treating composition with the stonewashing composition, and thus bringing the cellulase enzyme into proximity with the fabric. Subsequently, the aqueous solution containing the cellulase and the fabric is often agitated. If the treating composition is an aqueous solution, the fabric may be directly soaked in the solution. Similarly, where the stonewashing composition is a concentrate, the concentrate is diluted into a water bath with the cellulose containing fabric.

When the stonewashing composition is in a solid form, for example a pre-wash gel or solid stick, the stonewashing composition may be contacte by directly applying the composition to the fabric or to the wash liquor.

The cellulose containing fabric is incubated with the stonewashing solution under conditions effective to allow the enzymatic action to confer a stonewashed appearance to the cellulose containing fabric. For example, during stonewashing, the pH, liquor ratio, temperature and rection time and dosage of enzyme may be adjusted to optimize the conditions under which the stonewashing composition acts."Effective conditions" necessarily refers to the pH, liquor ratio, and temperature which allow the cellulase enzyme to react efficiently with cellulose containing fabric. The rection conditions effective for the stonewashing compositions of the present invention are substantially similar to well known methods used with corresponding prior art cellulase compositions. Accordingly, it is within

the skill of those in the art to maximize conditions for using the stonewashing compositions according to the present invention.

The liquor ratios during stonewashing, i. e., the ratio of weight of stonewashing composition solution (i. e., the wash liquor) to the weight of fabric, employed herein is generally an amount sufficient to achieve the desired stonewashing effect in the denim fabric and is dependent upon 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.

Rection temperatures during stonewashing with the present stonewashing compositions are governed by two competing factors. Firstly, higher temperatures generally correspond to enhanced rection kinetics, i. e., faster rections, which permit reduced rection times as compare to rection times required at lower temperatures.

Accordingly, rection temperatures are generally at least about 10°C and greater.

Secondly, cellulase is a protein which often loses activity beyond a given rection temperature, which temperature is dependent on the nature of the cellulase used. Thus, if the rection temperature is permitted to go too high, the cellulolytic activity is lost as a result of the denaturing of the cellulase.

Rection times are dependent on the specific conditions under which the stonewashing occurs. For example, pH, temperature and concentration of cellulase will all effect the optimal rection time. Generally, rection 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.

It is contemplated that compositions comprising cellulase enzymes formulated as described herein can be used in home use as a stand alone composition suitable for restoring color to faded fabrics (see, for example, U. S. Patent No. 4,738,682, which is incorporated herein by reference in its entirety) as well as used in a spot-remover and for depilling and antipilling (pilling prevention).

The use of the cellulase according to the invention may be particularly effective in feed additives and in the processing of pulp and paper. These additional industrial applications are described in, for example, PCT Publication No. 95/16360 and Finnish Granted Patent No. 87372, respectively.

In order to further illustrate the present invention and avantages 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 The performance of a T. Fusca E5 variant having a mutation at T1401 was tested under varying ionic strength with respect to stonewashing performance on denim. The expriment analyzed the effect of altering the concentration of sodium sulfate and determined the level of bio-polishing of cotton and 60/40 Tencel/cotton blend surface by the following method: Equipment: Unimac (50 Ibs lab scale tumbler) Liquor ratio: 10 to 1 Substrats: 3kg of denim (12 denim legs) 6 legs: sulfur bottom standard legs from American cotton growers 3 legs: sulfur bottom legs from Greenwood Mills 3 legs: bioindigo legs from Cone Mills Treatment pH: pH 7.0 (10mM sodium phosphate/1.7 mM citric acid buffer) Treatment temperature: 60° C Treatment time: 60 minutes Surfactant: 0.25g/L of Triton X-100 (non-ionic surfactant) Clean up: Table 1 Treatment of 100% Cotton Knit and 60/40 Tencel/Cotton Blend With Cellulase Under Varying lonic Strength total SDP (mM) citric sodium tencel/cotton 100% cotton cellulase acid sulfate Panel Score Panel Score mM)(mM 0 5. T3 0.87 0 1.03 2.41 1 6.73 0.87 0 1. 75 2.31 1 5.73 0.87 1 1.94 1.47 1 5.73 0.87 5 3.25 2.72 1 5. 73 0.87 10 3.59 4.22 1 5. 73 0.87 20 4.88 5.16 1 5.73 0.87 100 6.25 6.19 As shown in Table 1, activity as illustrated by abrasion increases with the increase in sodium sulfate concentration.

Table 2 Treatment of Denim With E5 Cellulase

ACG Denim treated w/E5 variant @ 1g protein abrasion (CIE L')'''backstaining (CIE b-) _ : ; : :'..'. : :'... : : :.. : : : : : : : : :. : _ : : : :. :... : : : : : : : : : ; :. _ : : : :.. : : : : : :.. :. : : :. : : ; : : :'... : . :. . .................................. , ............................................................ .,..... ............................. ........ ..........................................,,................ ...... ............. ..........-3 :'r................-........... ........ 5£............., : : : : : : :.. : : :. : : : : :. : : : : : :". : : : : : : : : : :... : : : : : : : : : : :. :...... : : : : : : : : : : : ;... .. .- : :.. : : : : :. .............,............,.........................,.. '................,....... Backstaining .....,................................,.. :.......... y.............,....... :. : :. :., ;. ;, ;. ; :. : :. : :. : :. : :. : :. : :. :. :. : :. :. : :. : :. : :. : :. : :..". : : : :. :. : :. : :. : : ; :. :. : : : : : : :. :.,.,.., Y. ; :. : :. : : _ ...................... ............. .......................................... : : : :.,. : : : : :.. rix\..-....... _......... ........... .... : :...................................... s..... ;.................. : : : : : :. : : : : : :. : : ; : : : : : : : :. : : T : : : : : :' : : : : : : :..............5 : : : :. : :. : :... tOmM SmM tOmM 30mM lOmM SDP + SDP + SDP + SDP + SDP + 1.7mM 0.85mM 1.7mM 5mM CA 1.7mM CA CA CA CA + 40mM NaCI As shown in Table 2, both abrasion and backstaining levels of E5 variant treated denim were increased as the ionic strength is increased. The treated denim look at high buffer concentration resulted in reduced contrast, lighter shade, and blue cast. When 40mM of sodium chloride was added in the 10mM sodium dibasic phosphate + 1.7mM citric acid buffer, both abrasion and backstaining levels of treated denim were increased.

Also, when 40 mM of NaCI was added, the abrasion and backstaining levels were increased as well. This result illustrates that the effects are not caused specifically by sodium sulfate, but by the increase in ionic strength through increased salt concentration.

Table 3 Effect on Activitv of Varvina The lonic Strenath When Treatina With Whole Humicola Insolens Cellulase ACG Denim treated wl Denimax XT @ 45g product abrasion (CIE L*)'-0'backstaining (CIE b*) ........................................................ .. : : : : : : : :........ : :. :............................... :........................ ......................... ....................... .................... >-2 ................................. : : : :. :. : : : : : : : : : : : : : :. : : :. :...... : : : : : : :. : : : : : : : : : : : : :. : :- : : : : : : : : : : : : : : : : : : : : : :. : : : :........................... .. 5 : : : : onrl : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :. : : : : : : : : : : : : : : : : : : : : : : 24 : :... :.. :., : : : : :.. : :.. : : :. : :. : .............. : : : :.. :. ........... 20-8 1 omm 1 omm 30mM SDP + SDP + SDP + 1.7mM 1.7mM 5mM CA CA CA

As shown in Table 3, abrasion level and fabric strength loss using Denimax XT (commercially available from Novo Nordis, Denmark) treated denim was not affecte by the buffer concentration. Backstaining level was slightly improved (lowered) under the high ion concentration.