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Title:
METHOD FOR REDUCING LINT GENERATION DURING TREATMENT OF COTTON-CONTAINING AND NON-COTTON-CONTAINING CELLULOSIC FABRICS
Document Type and Number:
WIPO Patent Application WO/1994/023113
Kind Code:
A1
Abstract:
Disclosed are methods for treating cotton-containing and non-cotton-containing cellulosic fabrics with cellulase during manufacture of the fabric. In particular, the disclosed methods are directed to contacting the fabric with a fungal cellulase composition comprising a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein excess lint production is reduced. Additionally, disclosed are methods of treating cotton-containing and non-cotton-containing fabrics in a continuous process comprising contacting the fabrics with a fungal cellulase composition having a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 at a predetermined place in the manufacturing process for an effective contact time. When cotton-containing fabrics are treated with the cellulase composition of the invention under the conditions of the invention, the fabrics show the benefits of improved feel and appearance and/or stone-washed appearance without producing an excessive amount of lint or producing excessive fabric weight loss as compared to treatment with a complete cellulase composition.

Inventors:
CLARKSON KATHLEEN A (US)
LARENAS EDMUND A (US)
WEISS GEOFFREY L (US)
Application Number:
PCT/US1994/003407
Publication Date:
October 13, 1994
Filing Date:
March 29, 1994
Export Citation:
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Assignee:
GENENCOR INT (US)
CLARKSON KATHLEEN A (US)
LARENAS EDMUND A (US)
WEISS GEOFFREY L (US)
International Classes:
D06M16/00; (IPC1-7): D06M16/00; C11D3/386; C11D11/00
Domestic Patent References:
WO1992006183A11992-04-16
WO1992017574A11992-10-15
WO1992007134A11992-04-30
Other References:
JOËL BAZIN ET AL.: "Cellulasebehandlung von Cellulosewaren mit Hilfe von Enzymen", TEXTIL PRAXIS INTERNATIONAL, vol. 47, no. 10, October 1992 (1992-10-01), LEINFELDEN DE, pages 972 - 974, XP000311561
DEBORAH CLARKE: "Enzyme treatment for removing pills from garment dyed goods", INTERNATIONAL DYER, vol. 178, no. 7, July 1993 (1993-07-01), LONDON GB, pages 20 - 21, XP000382295
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Claims:
WHAT IS CLAIMED IS:
1. A method for treating a cottoncontaining fabric during manufacture of the fabric with a fungal cellulase composition naturally comprising CBH and EG components, which method comprises contacting the fabric with an effective amount of a fungal cellulase composition having a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein excess lint production is reduced.
2. A method according to Claim 1 wherein said fungal cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 10:1.
3. A method according to Claim 2 wherein said fungal cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 20:1.
4. A method according to Claim 1 wherein said fungal cellulase composition comprises at least about 30 weight percent EG type components based on the total weight of protein in the cellulase composition.
5. A method according to Claim 1 comprising treating said fabric with agitation of the cellulase solution under conditions so as to produce a cascading effect of the cellulase solution over the fabric.
6. A method according to Claim 1 wherein said EG type component comprises EG type I.
7. A method according to Claim 1 wherein said EG type component comprises EG type II.
8. A method according to Claim 1 wherein said EG type component comprises EG type III.
9. A method according to Claim 1 comprising treating said fabric in a batch process.
10. A method according to Claim 1 comprising treating said fabric in a continuous process.
11. A method of treating a cottoncontaining fabric in a continuous manufacturing process with a fungal cellulase composition naturally comprising CBH and EG components which method comprises contacting said fabric with an effective amount of a fungal cellulase composition in an aqueous solution having a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 at a predetermined place in said manufacturing process for an effective contact time and under conditions wherein excess lint production is reduced and removing any remaining aqueous solution from the fabric.
12. A method according to Claim 11 wherein said fungal cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 10:1.
13. A method according to Claim 12 wherein the fungal cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 20:1.
14. A method according to Claim 11 wherein the cellulase composition comprises at least about 40 weight percent EG type components based on the total weight of protein in the cellulase composition.
15. A method according to Claim 11 wherein the EG type component is EG I.
16. A method according to Claim 11 wherein the EG type component is EG II.
17. A method according to Claim 11 wherein the EG type component is EG III.
Description:
METHODS FOR REDUCING LINT GENERATION DURING TREATMENT OF COTTON-CONTAINING AND NON-COTTON-CONTAINING CELLULOSIC FABRICS

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The present invention is directed to improved methods for treating cotton-containing and non-cotton-containing cellulosic fabric during manufacture of the fabric without excessive lint generation and excessive fabric weight loss during treatment. In particular, the improved methods of the present invention comprise contacting the fabric with an effective amount of a fungal cellulase composition comprising CBH type components and EG type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein lint production is reduced. When the cotton-containing and non-cotton-containing cellulosic fabric is treated with such solutions under these conditions, the generated lint is reduced as well as the weight loss of the fabric as compared to the fabric treated with a complete cellulase composition.

2. State of the Art.

During or shortly after their manufacture, cotton-containing fabrics can be treated with a complete cellulase composition in order to impart desirable properties to the fabric. For example, in the textile industry, a complete cellulase composition has been used to improve the feel and/or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, and to impart a

stone washed appearance to cotton-containing denims and the like.

In particular, Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al., "Reformation of Cotton Fabric by Cellulase" and JTN December 1988 journal article "What's New — Weight Loss Treatment to Soften the Touch of Cotton Fabric" each disclose that treatment of cotton- containing fabrics with complete cellulase compositions results in an improved feel for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing and/or surface fibers. The combination of these effects imparts improved feel to the fabric, i.e., the fabric feels more like silk.

Additionally, it was known in the art to treat cotton- containing knitted fabrics with an aqueous solution containing a complete cellulase composition under agitation and cascading conditions, for example, by use of a jet, for the purpose of removing broken fibers and threads common to these knitted fabrics. When so treated, buffers are generally not employed because they are believed to adversely affect dye shading with selected dyes.

It was still further known in the art to treat cotton- containing and non-cotton-containing cellulosic fabrics with an aqueous solution containing a complete cellulase composition under agitation and cascading conditions. When so treated, the cotton-containing and non-cotton-containing cellulosic fabric possesses improved feel and appearance as compared to the fabric prior to treatment.

Lastly, it was also known that the treatment of cotton- containing dyed denim with complete cellulase solutions under agitating and cascading conditions, i.e., in a rotary drum washing machine, would impart a "stone-washed" appearance to the denim.

A common problem associated with the treatment of such cotton-containing fabrics with an aqueous solution containing a complete cellulase composition is that such treatment can result in the production of an excessive amount of lint which

can cause problems with further handling of the fabric, such as cutting and sewing of the fabric. Also, excessive lint can result in equipment damage and lint released into the atmosphere of the plant can pose a serious health problem to workers.

In addition, treatment of such cotton-containing fabrics with an aqueous solution containing a complete cellulase composition can result in a reduction in the weight of the fabric. This necessitates the selection of a heavier fabric prior to treatment in order to ensure that a sufficient weight of fabric remains. The selection of a heavier weight of cloth results in an incremental increase in the cost of the fabric.

Further, because of the problems described above, and because the degree of processing could not be adequately controlled so as to achieve a consistent product, previously the fabric had to be treated with an aqueous solution containing a complete cellulase composition in batches rather than in a continuous process.

Accordingly, it would be particularly desirable to modify such cellulase treatment methods so as to provide reduced lint production and reduced fabric weight loss while still achieving the desired enhancements in the treated cotton- containing fabric arising from treatment with cellulase.

Additionally, because fungal sources of cellulase are known to secrete very large quantities of cellulase and further because fermentation procedures for such fungal sources as well as isolation and purification procedures for isolating the cellulase are well known in the art, it would be particularly advantageous to use such fungal cellulases in the treatment of cotton-containing fabric.

SUMMARY OF THE INVENTION

The present invention is directed to the novel and unexpected discovery that known methods for treating cotton- containing and non-cotton-containing cellulosic fabrics with

complete fungal cellulases can be improved to reduce lint production by contacting said fabric with an e fective amount of a fungal cellulase composition comprising a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions which further inhibit lint production.

It has been found that EG type components are capable of imparting enhancements to the treated fabric with regard to feel, appearance, softness, color enhancement, and/or a stone- washed appearance as compared to fabric before treatment with such a cellulase composition. Additionally, it has been found that it is the CBH type components in combination with the EG type components which account for a sizable portion of the production of lint and the loss of fabric weight during treatment of the fabric. Further, it has been found that while use of EG type components reduces lint production, further reductions in lint production can be achieved by using conditions which result in the reduced production of lint. Accordingly, in the present invention, the cellulase composition employed to treat cotton-containing fabrics and non-cotton-containing cellulosic fabrics is tailored so as to be enriched for EG type components and the treatment conditions are tailored to reduce lint production.

In view of the above, in one of its method aspects, the present invention is directed to a method for treating cotton- containing and non-cotton-containing cellulosic fabric during manufacture of the fabric, with a fungal cellulase composition naturally comprising CBH type and EG type components, wherein the improvement comprises contacting the fabric with an effective amount of a fungal cellulase composition having a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein excess lint production is reduced. In a preferred embodiment, the fungal cellulase composition comprises at least 30 weight percent of EG type components based on the total weight of protein in the cellulase composition.

In another of its method aspects, the present invention

is directed to a method for treating cotton-containing and non-cotton-containing cellulosic fabric with a fungal cellulase composition naturally comprising CBH type and EG type components in order to impart enhancement with regard to feel, appearance, softness, color enhancement, and/or a stone- washed appearance to the fabric which method comprises contacting said fabric with an effective amount of a fungal cellulase composition in an aqueous solution in order to effect such enhancement wherein said cellulase composition comprises a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 wherein said contacting is conducted at a predetermined place in a continuous manufacturing process for said cotton-containing fabric and under conditions wherein excess lint production is reduced. In another preferred embodiment, the fungal cellulase composition comprises at least 30 weight percent of EG type components based on the total weight of protein in the cellulase composition.

Cotton-containing and non-cotton-containing cellulosic fabrics treated by the methods of this invention have the desired enhancement(s) of improved softness, feel and appearance, and/or a stone-washed appearance and/or surface polishing, with a reduction in the amount of lint and the amount of fabric weight loss produced during treatment of the fabric as compared to the amount of lint and the amount of fabric weight loss produced when fabrics are treated with fungal cellulase compositions containing greater amounts of CBH type components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outline of the construction of pΔCBHIpyr..

FIG. 2 illustrates deletion of the T. longibrachiatum gene by integration of the larger .EcoRI fragment from pΔCBHIpyr4 at the cJb l locus on one of the T. longibrachiatum chromosomes.

FIG. 3 is an autoradiograph of DNA from T.

longibrachiatum strain GC69 transformed with EcoRI digested pΔCBHIpyr- after Southern blot analysis using a 32 P labelled pΔCBHIpyr- as the probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.

FIG. 4 is an autoradiograph of DNA from a T. longibrachiatum strain GC69 transformed with .EcoRI digested pΔCBHIpy 4 using a P labeled plntCBHI as the probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.

FIG. 5 is an isoelectric focusing gel displaying the proteins secreted by the wild type and by transformed strains of r. longibrachiatum. Specifically, in FIG.5, Lane A of the isoelectric focusing gel employs partially purified CBH I from T. longibrachiatum; Lane B employs a wild type T. longibrachiatum: Lane C employs protein from a T. longibrachiatum strain with the cbhl gene deleted; and Lane D employs protein from a T. longibrachiatum strain with the cbhl and cbh2 genes deleted. In FIG. 5, the right hand side of the figure is marked to indicate the location of the single proteins found in one or more of the secreted proteins. Specifically, BG refers to the 3-glucosidase, El refers to endoglucanase I, E2 refers to endoglucanase II, E3 refers to endoglucanase III, CI refers to exo-cellobiohydrolase I and C2 refers to exo-cellobiohydrolase II.

FIG. 6A is a representation of the T. longibrachiatum cbh2 locus, cloned as a 4.1 kb .EcoRI fragment on genomic DNA and FIG. 6B is a representation of the cbh2 gene deletion vector pPΔCBHII.

FIG. 7 is an autoradiograph of DNA from T. longibrachiatum strain P37PΔCBHIPyr " 26 transformed with EcoRI digested pPΔCBHII after Southern blot analysis using a 32 P labelled pPΔCBHII as the probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.

FIG. 8 is a diagram of the plasmid pEGIpyr..

FIG. 9 is an outline of the construction of plasmid pTEX.

FIG. 10 illustrates the RBB-CMC activity profile of an

acidic EG enriched fungal cellulase composition (CBH I and II deleted) derived from _rric_-θ_.er__.a longibrachiatum over a pH range at 40°C; as well as the activity profile of an enriched EG III cellulase composition derived from Trichoderma longibrachiatum over a pH range at 0°C.

FIG. 11 illustrates the effect of different cellulase compositions on the redeposition of colorant during the stonewashing process.

FIG. 12 illustrates strength loss results after three wash cycles in a laundero eter for cotton-containing fabrics treated with cellulase compositions having varying amounts of CBH components.

FIG. 13 illustrates fiber removal results (based on panel test scores) for cotton-containing fabrics treated with cellulase secreted by a wild type Trichoderma longibrachiatum (whole cellulase) at various pHs.

FIG. 14 illustrates fiber removal results (based on panel test scores) for cotton-containing fabrics treated with varying concentrations (in ppm) of cellulase secreted by a wild type Trichoderma longibrachiatum and for a cotton fabric treated with cellulase secreted by a strain of Trichoderma longibrachiatum genetically engineered so as to be incapable of secreting CBH I and CBH II.

FIG. 15 illustrates the softness panel test results for varying concentrations (in ppm) of an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be incapable of producing CBH I and CBH II.

FIG. 16 illustrates the softness panel test results for a non-cotton-containing cellulosic fabric treated with an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be incapable of producing CBHI&II.

FIG. 17 illustrates an appearance panel test results for a non-cotton-containing cellulosic fabric treated with an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be

incapable of producing CBHI&II.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As noted above, the methods of this invention are directed to treating cotton-containing and non-cotton- containing cellulosic fabrics during manufacture with an effective amount of a cellulase composition comprising CBH type components and EG type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 5:1. The improvement comprises using a specific cellulase composition which imparts the desired enhancement(s) to the fabric and reduces lint production while treating the fabric under conditions which still further reduce the production of lint and the amount of fabric weight loss. Further, the fungal cellulase compositions of this invention can be used in a continuous treatment process as well as a batch treatment process. However, prior to discussing this invention in detail, the following terms will first be defined.

The term "cotton-containing fabric" refers to sewn or unsewn fabrics made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, cotton toweling and the like. When cotton blends are employed, the amount of cotton in the fabric should be at least about 40 percent by weight cotton; preferably, more than about 60 percent by weight cotton; and most preferably, more than about 75 percent by weight cotton. When employed as blends, the companion material employed in the fabric can include one or more non-cotton fibers including 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 ara id fibers. It is contemplated that regenerated cellulose or a

cellulosic fabric, such as rayon, could be used as a substitute for cotton in the methods of this invention.

The term "cellulosic-containing or cellulosic fabric" refers to any non-cotton-containing cellulosic fabric or non- cotton-containing cellulosic blend including natural cellulosics (such as jute, flax, ramie and the like) and manmade cellulosics. Included under the heading of manmade cellulosics are the regenerated cellulosics that are well known in the art such as rayon. Other manmade cellulosics include chemical modification of cellulose fibers (e.g, cellulose derivatized by acetate and the like) and solvent- spun cellulose fibers (e.g.lyocell) .

The above non-cotton-containing cellulosics can also be employed as blends that include lyocell-rayon, lyocell-linen, viscose rayon acetate, rayon-wool, silk-acetate, and the like.

The term "finishing" as employed herein means the application of a sufficient amount of finish to a cotton- containing or non-cotton-containing cellulosic fabric so as to substantially prevent cellulolytic activity of the cellulase on the fabric. Finishes are generally applied at or near the end of the manufacturing process of the fabric for the purpose of enhancing the properties of the fabric, for example, softness, drapability, etc., which additionally protects the fabric from reaction with cellulases. Finishes useful for finishing a cotton-containing fabric are well known in the art and include resinous materials, such as melamine, glyoxal, or ureafor aldehyde, as well as waxes, silicons, fluorochemicals and quaternaries. When so finished, the cotton-containing fabric is substantially less reactive to cellulase.

The term "lint" refers to small loose threads and fibers, typically less than 1 cm in length, which form as a result of treatment of cotton containing fabrics with cellulase. Without being limited to a theory, it is believed that treatment with a complete cellulase composition causes excessive degradation of the fabric and excessive formation of loose fibers. The resulting loose fibers are no longer associated with the fabric and, accordingly, form lint which

remains suspended in the treatment liquor or collects on the surface of the fabric or in machine filters. On the other hand, without being limited to any theory, it is believed that the specific cellulase compositions recited herein when used under the conditions of the present invention do not excessively degrade the fabric and thus do not create excessive amounts of lint. As the amount of lint produced increases there is a corresponding increase in the amount of fabric weight lost. Typically the amount of lint produced by this method will be at least 20% less than the lint produced during treatment with a complete cellulase, more preferably the amount of lint produced will be at least 30% less than the lint produced during treatment with a complete cellulase.

The term "fabric weight loss" refers to the decrease in the weight of a piece of fabric after treatment with a cellulase composition. Typically the amount of fabric weight loss during treatment with a complete cellulase composition can be from 5 to 25% of the weight of the total fabric. In general, a reduction in fabric weight loss means that the amount of fabric weight lost during treatment with the cellulase composition of this invention when used under the conditions of the present invention is at least 20% less than that loss observed during treatment of the same fabric with a complete cellulase composition when used under the same conditions. Typically, the amount of fabric weight loss will be at least 20% less than the fabric weight loss observed during treatment with a complete cellulase composition, more preferably, the amount of fabric weight loss will be at least 30% less than the fabric weight loss observed during treatment with a complete cellulase composition.

The term "feel" (also referred to as "hand") as used herein refers to the physical smoothness of a cotton- containing and non-cotton-containing cellulosic fabric to touch. Fabrics having improved feel are smoother and silkier to the touch than other fabrics and accordingly are viewed as higher quality products. As defined, the term "feel" is distinguished from qualities such as thickness, color, or

other physical characteristics not involved in smoothness of the fabric.

The term "appearance" as used herein refers to the physical appearance of the cotton-containing and non-cotton- containing cellulosic fabric to the eye and is determined in part, by the presence or absence of, fuzz, surface fibers, and the like on the surface of the fabric as well as by the ability or inability to discern the construction (weave) of the fabric. Fabrics, which have little if any fuzz and surface fibers and wherein the construction (weave) is clearly discernable, possess improved appearance as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weave. As defined, the term "improved appearance" may include surface polishing of the fabric.

In general, the improvements in feel and appearance of cotton-containing and non-cotton-containing cellulosic fabrics after treatment by the methods of the present invention are readily ascertained by simple analytical tests which provide a numerical rating to the fabric both before and after treatment by the methods of this invention. The test procedure is conducted as a side-by-side comparison of a fabric sample before treatment by the process of this invention with a sample of that fabric after treatment by the process of this invention. Specific test procedures useful herein for ranking the improvements in feel and appearance are disclosed in U.S. Application Serial No. 07/598,506 filed October 16, 1990 and entitled "Methods for Improving the Appearance and Feel Characteristics of Cotton Woven Fabrics" the disclosure of which is incorporated herein by reference in its entirety.

The term "fungal cellulase" refers to the enzyme composition derived from fungal sources or microorganisms genetically modified so as to incorporate and express all or part of the cellulase genes obtained from a fungal source. Fungal cellulases act on cellulose and its derivatives to hydrolyze cellulose and give as primary products, glucose and cellobiose. Fungal cellulases are

distinguished from cellulases produced from non-fungal sources including microorganisms such as actinomycetes, gliding bacteria (myxobacteria) and true bacteria. Fungi capable of producing cellulases useful in preparing cellulase compositions described herein are disclosed in British Patent No. 2094826A, the disclosure of which is incorporated herein by reference.

Most fungal cellulases generally have their optimum activity in the acidic or neutral pH range although some fungal cellulases are known to possess significant activity under neutral and slightly alkaline conditions, i.e., for example, cellulase derived from Humicola insolens is known to have activity in neutral to slightly alkaline conditions.

Fungal cellulases are known to be comprised of several enzyme classifications having different substrate specificity, enzymatic action patterns, and the like. Additionally, enzyme components within each classification can exhibit different molecular weights, different degrees of glycosylation, different isoelectric points, different substrate specificities, etc. For example, fungal cellulases can contain cellulase classifications which include endoglucanases (EGs) , exo-cellobiohydrolases (CBHs) , 3-glucosidases (BGs) , etc. On the other hand, while bacterial cellulases are reported in the literature as containing little or no CBH components, there are a few cases where CBH-like components derived from bacterial cellulases have been reported to possess exo-cellobiohydrolase activity.

A fungal cellulase composition produced by a naturally occurring fungal source and which comprises one or more CBH and one or more EG components wherein each of these components is found at the ratio produced by the fungal source is sometimes referred to herein as a "complete fungal cellulase system" or a "complete fungal cellulase composition" to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism

genetically modified so as to overproduce, underproduce, or not produce one or more of the CBH and/or EG components of cellulase.

As used herein "cellulase" or "cellulase composition" refers to cellulase expressed by fungal sources or microorganisms genetically modified to incorporate and express all or part of the cellulase genes obtained from fungal sources which composition may also contain non-cellulase proteins.

The fermentation procedures for culturing fungi for production of cellulase are known per βe in the art. For example, cellulase systems can be produced either by solid or submerged culture, including batch, fed-batch and continuous- flow processes. The collection and purification of the cellulase systems from the fermentation broth can also be effected by procedures known per se in the art.

"Endoglucanase ("EG") type components" refer to all of those fungal cellulase components or combination of components which exhibit textile activity properties similar to the endoglucanase components of Trichoderma longibrachiatum (previously classified as T. reesei) . In this regard, the endoglucanase components of Trichoderma longibrachiatum (specifically, EG I, EG II, EG III, and the like either alone or in combination) impart improved feel, improved appearance, softening, color enhancement, and/or a stone-washed appearance to cotton-containing fabrics (as compared to the fabric prior to treatment) when these components are incorporated into a textile treatment medium and the fabric is treated with this medium. Additionally, treatment of cotton-containing fabrics with endoglucanase components of Trichoderma longibrachiatum result in less strength loss as compared to the strength loss arising from treatment with a similar composition but which additionally contains CBH type components in a ratio of EG components to CBH components approaching that found in naturally complete cellulase compositions which contain both CBH and EG components. Further, treatment of cotton- containing fabrics with endoglucanase components of

_T_ric--θ < -7er_-ia longibrachiatum result in less production of excessive lint and less fabric weight loss compared to treatment with a similar composition but which additionally contains CBH type components.

Accordingly, endoglucanase type components are those fungal cellulase components which impart improved feel, improved appearance, softening, color enhancement, and/or a stone-washed appearance to cotton-containing fabrics (as compared to the fabric before treatment) when these components are incorporated into a medium used to treat the fabrics. Additionally, a cellulase composition containing endogluconose type components imparts reduced strength loss to cotton- containing fabrics as compared to the strength loss arising from treatment with a similar cellulase composition but which additionally contains CBH type components.

Such endoglucanase type components may not include components traditionally classified as endoglucanases using activity tests such as the ability of the component (a) to hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (CMC) , thereby reducing the viscosity of CMC containing solutions, (b) to readily hydrolyze hydrated forms of cellulose such as phosphoric acid swollen cellulose (e.g., Walseth cellulose) and hydrolyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.). On the other hand, it is believed that not all endoglucanase components, as defined by such activity tests, will impart one or more of the enhancements to cotton- containing fabrics as well as reduced strength loss to cotton- containing fabrics. Accordingly, it is more accurate for the purposes herein to define endoglucanase type components as those components of fungal cellulase which possess similar textile activity properties as possessed by the endoglucanase components of Trichoderma longibrachiatum.

Fungal cellulases can contain more than one EG type component. The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities,

different enzymatic action patterns, etc. The different isoelectric points of the components allow for their separation via ion exchange chromatography and the like. In fact, the isolation of components from different fungal sources is known in the art. See, for example, Bjork et al., U.S. Patent No. 5,120,463, Schulein et al.. International Application WO 89/09259, Wood et al.. Biochemistry and Genetics of Cellulose Degradation, pp. 31 to 52 (1988) ; Wood et al.. Carbohydrate Research, Vol. 190, pp. 279 to 297 (1989); Schulein, Methods in Enzymology, Vol. 160, pp. 234 to 242 (1988); and the like. The entire disclosure of each of these references is incorporated herein by reference.

The term "enriched EG type components" refers to a cellulase composition containing at least 30 weight percent, preferably at least 70 weight per-cent and most preferably at least 90 weight percent of EG type cellulase components or a particular EG type component specified based on the total weight of the proteins in the cellulase composition.

The term "EG I cellulase" refers to the component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pi) of from about 4.5 to 5.3, and a molecular weight of about 47 to 50 Kdaltons. Preferably, EG I cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride . EG I cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.0, an isoelectric point (pi) of about 4.7 and a molecular weight of about 47 to 49 Kdaltons. EG I cellulase derived from Trichoderma viride has a pH optimum of about 5.0, an isoelectric point (pi) of about 5.3 and a molecular weight of about 50 Kdaltons.

The term "EG I type cellulase" refers to fungal cellulase components exhibiting textile activity properties similar to EG I cellulase.

It is noted that EG II has been previously referred to by the nomenclature "EG III" by some authors but current nomenclature uses the term EG II. In any event the EG II protein is substantially different from the EG III protein

defined below in its molecular weight, pi and pH optimum. Accordingly, the term "EG II cellulase" refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pl) of from about 5.4 to 6.0 and a molecular weight of about 35 to 50 Kdaltons. Preferably, EG

II cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride.

The term "EG II type cellulase" refers to fungal cellulase components exhibiting textile activity properties similar to EG II cellulase.

The term "EG III cellulase" refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 5.5 to 6.0, an isoelectric point (pi) of from about 7.2 to 8.0, and a molecular weight of about 23 to 28 Kdaltons. Preferably, EG III cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride . EG

III cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.5 to 6.0, an isoelectric point (pl) of about 7.4, and a molecular weight of about 25 to 28 Kdaltons. EG III cellulase derived from Trichoderma viride has a pH optimum of about 5.5, an isoelectric point (pi) of about 7.7, and a molecular weight of about 23.5 Kdaltons.

The term "EG III type cellulase" refers to fungal cellulase components exhibiting textile activity properties similar to EG III cellulase.

It is contemplated that EG type components can be derived from bacterially derived cellulases.

In general, it is contemplated that combinations of EG type components may give a εynergistic response in reducing lint production and fabric weight loss. On the other hand, a single EG type component may be more stable or have a broader spectrum of activity over a range of pHs. In this regard, it has been found that enriched EG I type cellulase retards lint formation and reduces fabric weight loss. Accordingly, the EG type components employed in this invention can be either a single EG type component or a combination of two or more EG

type components. When a combination of components is employed, the EG type component may be derived from the same or different fungal sources.

It is possible that proteins other than CBH type cellulase components present in the whole cellulase composition may produce lint or cause fabric weight loss alone or in combination with EG , ε or other proteins during the treatment of cotton fabric with cellulases. Therefore, it is contemplated that the use of enriched EG I type, EG II type or EG III type components may result in a reduction of some or all of these proteins present in the whole cellulase composition and may result in a further reduction in lint.

"Exo-cellobiohydrolase type ("CBH type") components" refer to those fungal cellulase components which exhibit textile activity properties similar to CBH I and/or CBH II cellulase components of Trichoderma longibrachiatum. In this regard, when used in the absence of EG type cellulase components (as defined above) , the CBH I and CBH II components of Trichoderma longibrachiatum alone do not impart any significant enhancements in feel, appearance, color enhancement and/or stone washed appearance to the so treated cotton-containing fabrics. Additionally, when used in combination with EG type components in a ratio approaching that found in natural complete cellulase compositions which contain both CBH and EG components, the CBH I components of Trichoderma longibrachiatum impart enhanced strength loss and enhanced weight loss to the cotton-containing fabrics and production of excess lint.

Accordingly, CBH I type components and CBH II type components refer to those fungal cellulase components which exhibit textile activity properties similar to CBH I and CBH II components of Trichoderma longibrachiatum, respectively. As noted above, for CBH type components, this includes the property of enhancing strength loss of cotton-containing fabrics when used in the presence of EG type components. Additionally, it is contemplated that the CBH components of Trichoderma longibrachiatum, when used alone or in combination

with EG type components, can impart an incremental softening benefit.

Such exo-cellobiohydrolase type components could possibly not include components traditionally classed as exo- cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II from Trichoderma longibrachiatum. For example, such components (a) are competitively inhibited by cellobiose (K f approximately lmM) ; (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., and (c) hydrolyze phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose. On the other hand, it is believed that some fungal cellulase components which are characterized as CBH components by such activity tests, will impart improved feel, appearance, softening, color enhancement, and/or a stone-washed appearance to cotton- containing fabrics with minimal strength loss when used alone in the cellulase composition. Accordingly, it is believed to be more accurate for the purposes herein to define such exo- cellobiohydrolases as EG type components because these components possess similar functional properties in textile uses as possessed by the endoglucanase components of Trichoderma longibrachiatum.

Fungal cellulase compositions having enriched EG type components can be obtained by purification techniques. Specifically, the complete cellulase system can be purified into enriched components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography) , it is possible to separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient. After purification, the requisite amount of the desired components could be recombined.

It is also contemplated that mixtures of cellulase components enriched for EG type components could be prepared

by means other than isolation and recombination of the components. In this regard, it may be possible to modify the fermentation conditions for a natural microorganism in order to give relatively high ratios of EG type to CBH type components. Likewise, recombinant techniques can alter the relative ratio of EG type components to CBH type components so as to produce a mixture of cellulase components having a relatively high ratio of EG type components to CBH type components.

In regard to the above modification, a preferred method for the preparation of cellulase compositions described herein is to genetically modify a microorganism so as to overproduce one or more EG type components. Likewise, it is also possible to genetically modify a microorganism so as to be incapable of producing one or more CBH type components which methods do not produce any heterologous protein.

In regard to the above, U.S. Serial No. 07/954,113 filed September 30, 1992 which is a continuation-in-part of U.S. Serial No. 07/770,049, filed October 4, 1991 which is a continuation-in-part of U.S. Serial No. 07/593,919, filed October 5, 1990, all of which are incorporated herein by reference in their entirety, disclose methods for genetically engineering Trichoderma longibrachiatum so as to be incapable of producing one or more CBH components and/or overproducing one or more EG components. Likewise, Miller et al., "Direct and Indirect Gene Replacement in Aspergillus nidulans n , Molecular and Cellular Biology, p. 1714-1721 (1985) discloses methods for deleting genes in Aspergillus nidulans by DNA mediated transformation using a linear fragment of homologous DNA. The methods of Miller et al., would achieve gene deletion without producing any heterologous proteins.

In view of the above, the deletion of the genes responsible for producing CBH I type and/or CBH II type cellulase components would have the effect of enriching the amount of EG components present in the cellulase composition. An increase in the number of genes responsible for producing EG type components would also have the effect of enriching the

amount of EG components in the cellulase composition.

It is still further contemplated that fungal cellulase compositions used herein can be derived from fungal sources which produce low concentrations of CBH type components.

Additionally, a requisite amount of one or more CBH type components purified by conventional procedures can be added to a cellulase composition produced from a microorganism genetically engineered so as to be incapable of producing CBH type components so as to achieve a specified ratio of EG type components to CBH type components, i.e., a cellulase composition free of all CBH type components so as to be enriched in EG type components can be formulated to contain 2 weight percent of a CBH type component merely by adding this amount of a purified CBH type component to the cellulase composition.

"β-Glucosidase (BG) components" refer to those components of cellulase which exhibit BG activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides ("cellobiose") and give glucose as the sole product. BG components do not adsorb onto or react with cellulose polymers. Furthermore, such BG components are competitively inhibited by glucose (K,- approximately ImM) . While in a strict sense, BG components are not literally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the combined action of CBH components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur. BG components are often characterized on aryl substrates such as p-nitfophenol 0-D- glucoside (PNPG) and thus are often called aryl-glucosidases. It should be noted that not all aryl glucosidases are BG components, in that some do not hydrolyze cellobiose.

It is contemplated that the presence or absence of BG

components in the cellulase composition can be used to regulate the activity of any CBH components in the composition. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG components in the cellulase composition will

"turn-off" CBH activity when the concentration of cellobiose reaches inhibitory levels. It is also contemplated that one or more additives (e.g., cellobiose, glucose, etc.) can be added to the cellulase composition to effectively "turn-off", directly or indirectly, some or all of the CBH I type activity as well as other CBH activity. When such additives are employed, the resulting composition is considered to be a composition suitable for use in this invention if the amount of additive employed is sufficient to lower the CBH type activity to levels equal to or less than the CBH type activity levels achieved by using the cellulase compositions described herein.

On the other hand, a cellulase composition containing added amounts of BG components may increase overall hydrolysis of cellulose if the level of cellobiose generated by the CBH components becomes restrictive of such overall hydrolysis in the absence of added BG components.

Methods to either increase or decrease the amount of BG components in the cellulase composition are disclosed in U.S. Serial No 07/807,028 filed December 10, 1991 which is a continuation-in-part of U.S. Serial No. 07/625,140, filed December 10, 1990, both of which are incorporated herein by reference in their entirety.

Fungal cellulases can contain more than one BG component. The different components generally have different isoelectric points which allow for their separation via ion exchange chromatography and the like. Either a single BG component or a combination of BG components can be employed.

When employed in textile treatment solutions, the BG

component is generally added in an amount sufficient to prevent inhibition by cellobiose of any CBH and EG components found in the cellulase composition. The amount of BG component added depends upon the amount of cellobiose produced in the textile composition which can be readily determined by the skilled artisan.

Preferred fungal cellulases for use in preparing the fungal cellulase compositions used in this invention are those obtained from Trichoderma longibrachiatum, Trichoderma koningii, Pencillum sp., Humicola insolens, and the like. Certain fungal cellulases are commercially available, i.e.,

CELLUCAST TM (available from Novo Industry, Copenhagen,

TM

Denmark), RAPIDASE (available from Gist Brocades, N.V. ,

TM

Delft, Holland), CYTOLASE 123 (available from Genencor International, South San Francisco, California) and the like. Other fungal cellulases can be readily isolated by art recognized fermentation and isolation procedures.

The term "buffer" refers to art recognized acid/base reagents which stabilize the cellulase solution against undesired pH shifts during the cellulase treatment of the cotton-containing fabric. In this regard, it is art recognized that cellulase activity is pH dependent. That is to say that a specific cellulase composition will exhibit cellulolytic activity within a defined pH range with optimal cellulolytic activity generally being found within a small portion of this defined range. The specific pH range for cellulolytic activity will vary with each cellulase composition. As noted above, while most cellulases will exhibit cellulolytic activity within an acidic to neutral pH profile, there are some cellulase compositions which exhibit cellulolytic activity in an alkaline pH profile.

During cellulase treatment of the cotton-containing and non-cotton-containing cellulosic fabric, it is possible that the pH of the initial cellulase solution could be outside the range required for cellulase activity. It is further possible for the pH to change during treatment of the cotton-containing fabric, for example, by the generation of a reaction product

which alters the pH of the solution. In either event, the pH of an unbuffered cellulase solution could be outside the range required for cellulolytic activity. When this occurs, undesired reduction or cessation of cellulolytic activity in the cellulase solution occurs. For example, if a cellulase having an acidic activity profile is employed in a neutral unbuffered aqueous solution, then the pH of the solution will result in lower cellulolytic activity and possibly in the cessation of cellulolytic activity. On the other hand, the use of a cellulase having a neutral or alkaline pH profile in a neutral unbuffered aqueous solution should initially provide significant cellulolytic activity.

In view of the above, the pH of the cellulase solution should be maintained within the range required for cellulolytic activity. One means of accomplishing this is by simply monitoring the pH of the system and adjusting the pH as required by the addition of either an acid or a base. However, in a preferred embodiment, the pH of the system is preferably maintained within the desired pH range by the use of a buffer in the cellulase solution. In general, a sufficient amount of buffer is employed so as to maintain the pH of the solution within the range wherein the employed cellulase exhibits activity. Insofar as different cellulase compositions have different pH ranges for exhibiting cellulase activity, the specific buffer employed is selected in relationship to the specific cellulase composition employed. The buffer(s) selected for use with the cellulase composition employed can be readily determined by the skilled artisan taking into account the pH range and optimum for the cellulase composition employed as well as the pH of the cellulase solution. Preferably, the buffer employed is one which is compatible with the cellulase composition and which will maintain the pH of the cellulase solution within the pH range required for optimal activity. Suitable buffers include sodium citrate, ammonium acetate, sodium acetate, disodium phosphate, and any other art recognized buffers.

Enhancements to the cotton-containing and non-cotton-

containing cellulosic fabric are achieved by those fabric treatment methods heretofore used with the improvements of the invention being incorporated into such methods. For example, cotton-containing fabrics having improved feel can be achieved as per Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al., "Reformation of Cotton Fabric by Cellulase" and JTN December 1988 journal article "What's New - - Weight Loss Treatment to Soften the Touch of Cotton Fabric". The teachings of each of these references is incorporated herein by reference.

Similarly, methods for improving both the feel and appearance of cotton-containing and non-cotton-containing cellulosic fabrics include contacting the fabric with an aqueous solution containing cellulase under conditions so that the solution is agitated and so that a cascading effect of the cellulase solution over the cotton-containing fabric is achieved. Such methods result in improved feel and appearance of the so treated cotton-containing fabric and are described in U.S. Serial No. 07/598,506, filed October 16, 1990 and which is incorporated herein by reference in its entirety.

Methods for the enhancement of cotton-containing knits are described in International Textile Bulletin, Dyeing/Printing/Finishing, pages 5 et seq., 2 Quarter, 1990, which is incorporated herein by reference.

Likewise, methods for imparting a stone-washed appearance to cotton-containing denims are described in U.S. Patent No. 4,832,864, and U.S. Serial No. 07/954,113 filed September 30, 1992, both of which are incorporated herein by reference in their entirety.

Other methods for enhancing cotton-containing fabrics by treatment with a complete cellulase composition are known in the art. Preferably, in such methods, the treatment of the cotton-containing fabric with the complete cellulase . is conducted prior to finishing the cotton-containing fabric, i.e., during manufacture of the cotton-containing fabric.

The term "agitation" as used herein means any mechanical and/or physical force which agitates the cellulase solution so

as to result in agitation of the cotton-containing or non- cotton-containing cellulosic fabric. Without being limited to any theory, it is believed that such agitation facilitates the removal (clipping) of loose fibers, surface fibers (fuzz) and the like from the cotton-containing or non-cotton-containing cellulosic fabric. As is apparent, the agitation required in the methods described herein defines a vigorous action of the cellulase solution against the fabric surface which is substantially greater than mere mechanical stirring of the cellulase solution in order to achieve uniform cellulase concentration throughout the cellulase solution. Agitation may also include fabric to fabric contact.

Agitation suitable for use in the methods described herein can be achieved, for example, by employing a laundrometer, a jig, a jet, a mercerizer, a beck, a paddle machine, continuous bleach range, continuous wash range and the like.

The agitation employed herein is either repetitive (e.g., intermittent) or continuous agitation. For example, the cellulase solution can be continuously agitated by employing a laundrometer, a jet and the like. In a laundrometer, the cotton-containing or non-cotton-containing cellulosic fabric is loaded into stainless steel water-tight canisters. Continuous agitation is achieved by rotation of the fixed canisters on a frame within a temperature adjustable water bath. The degree of agitation is defined by the speed at which the canisters rotate. In a preferred embodiment, canisters rotated at a speed of at least about 40 revolutions per minute (rpms) achieve the agitation effect required in the herein described methods. Laundrometers are well known in the textile art and are generally employed as laboratory equipment. Suitable laundrometers are commercially available from, for example. Custom Scientific Instruments, Inc., Cedar Knolls, N.J.

In a jet, the cotton-containing or non-cotton-containing cellulosic fabric, in a rope form, continuously rotates through and with the cellulase solution. Specifically, jets

are based on a venturi tube in which the circular movement of liquor carries the fabric with it in a totally enclosed tubular chamber, annular in shape. The tubular chamber is filled in part with solution and the fabric is rotated through the chamber via a lifter roller so that at any given time a portion of the fabric is being lifted upward. The venturi tube is a constriction in the annular passage through which the speed of the flow of the liquor must be increased, thus causing suction which imparts movement to the fabric. The primary flow is given by a centrifugal pump, but it is usual to incorporate also a few inclined steam jets to boost the movement of both the fabric and the liquor. The movement of the fabric through the jet, preferably at a rate of at least about 6 ft/sec, provides the agitation required in the herein described methods. A jet is a well known apparatus found in mills and are generally used for the purpose of dyeing and after treating fabrics.

Repetitive agitation can be achieved by employing a jig, a mercerizer, a beck, and the like. A jig is a well known apparatus found in mills manufacturing cotton-containing and non-cotton-containing cellulosic fabrics and is generally used for the purpose of scouring fabrics prior to dyeing. In a jig, a defined length of cotton-containing or non-cotton- containing cellulosic fabric, in its open width position, is maintained on and between two rollers wherein the fabric is passing from one roller which is in the unwinding stage to a second roller which is in the winding stage. Once the unwinding/winding process is completed, the process is reversed so that the previous unwinding roll becomes the winding roll and the previous winding roll becomes the unwinding roll. This process is continuously conducted during the entire cellulase treatment time. A trough containing the cellulase solution is placed between the two rollers and the rollers are adjusted so that the cotton-containing or non- cotton-containing cellulosic fabric becomes immersed in the cellulase solution as it passes from one roller to the other.

Repetitive agitation is achieved in the jig by

continuously rolling and unrolling the cotton-containing and non-cotton-containing cellulosic fabric from the rolls, preferably at a rate of speed of at least about 1 yd/sec and more preferably at least about 1.5 yd/sec so that at any given time, part of the length of the fabric is moving through the cellulase solution at this defined rate of speed. The net result of such rolling and unrolling is that at any given time a portion of the cotton-containing or non-cotton- containing cellulosic fabric found on the rolls is immersed in the cellulase solution and over a given period of time, all of the fabric (except for the very terminal portions found at either end of the fabric—these terminal ends are often composed of leader fabric, i.e., fabric sewn to the terminal portions of the treated fabric and which is not intended to be treated) has been immersed into the cellulase solution. Moving the fabric, preferably at a rate of speed of at least about 1 yd/sec, through the cellulase solution provides the agitation required in the herein described methods.

A mercerizer unit is similar to a jig in that the cotton- containing or non-cotton-containing cellulosic fabric, in its open width position, is passed through a trough of solution, e.g., cellulase solution, at a set speed. Passing the cotton- containing or non-cotton-containing cellulosic fabric through the trough, preferably at a speed of at least 1 yd/sec, and more preferably at a rate of at least 1.5 yd/sec, provides the agitation required in the herein described methods. The mercerizer unit operates in only one direction and the length of time the fabric is exposed to the cellulase solution can be varied by modifying the mercerizer so as to contain more than one trough. In this embodiment, the length of time the fabric is exposed in such a modified mercerizer depends on the number of troughs and the speed the fabric is moving through the troughs.

When repetitive agitation is employed, each portion of the cotton-containing or non-cotton-containing cellulosic fabric is preferably exposed to the cellulase solution under agitating conditions at least once every minute on average.

and more preferably at least 1.5 times every minute on average. For example, when a jig is employed, this required degree of repetitive agitation can be achieved by limiting the length of the fabric so that when conducted at the requisite speed, each portion of the cotton-containing or non-cotton- containing cellulosic fabric is exposed to the cellulase solution under agitating conditions at least once every minute on average. When a modified mercerizer is employed, the desired degree of repetitive agitation can be achieved by adding a sufficient number of troughs appropriately spaced so that the fabric repetitively passes through different troughs.

As used herein, the term "cascading" means the rapid flow of cellulase solution across and eventually away from the surface of the cotton-containing or non-cotton-containing cellulosic fabric. That is to say that cascading occurs when a stream of cellulase solution (liquid) is moving on and relative to at least part of the surface of the cotton- containing or non-cotton-containing cellulosic fabric and this stream eventually moves away from this part of the surface of the fabric. A cascading effect can be achieved, for example, by use of a laundrometer, a jig, a jet, a mercerizer and the like. For example, when a laundrometer is employed, rotation of a partially filled canister will result in movement of the cellulase solution relative to the surface of the cotton- containing or non-cotton-containing cellulosic fabric thereby creating a flow of cellulase solution across and eventually off part of the surface of the cotton-containing or non- cotton-containing cellulosic fabric thereby resulting in a cascading effect. When the canister is rotated at the requisite rpms needed to achieve agitation, the flow of cellulase solution will be sufficiently rapid so as to cause agitation and additionally create a cascading effect of the cellulase solution. When such a cascading effect is desired, the canister should be filled to no greater than about 75 percent of capacity, and preferably no greater than about 50 percent capacity.

Cascading can also be accomplished with the use of a jig.

For example, when a jig is employed to achieve the requisite agitation described above, the cotton-containing fabric rapidly departs from the trough containing the cellulase solution and is lifted somewhat upward in order to be wound onto the winding roller. When this occurs, any cellulase solution remaining on the surface of the cotton-containing or non-cotton-containing cellulosic fabric as it exits from the cellulase solution rapidly flows across and eventually off this part of the fabric surface. Specifically, cascading in a jig is achieved by the passage of the cotton-containing and non-cotton-containing cellulosic fabric through the cellulase solution, preferably at a speed of at least 1 yd/sec and more preferably at a speed of at least 1.5 yd/sec, coupled with the gravitational effect of the upward lift of the fabric as it is being rolled which results in the rapid flow of the cellulase solution across and eventually away from the surface of the cotton-containing and non-cotton-containing cellulosic fabric theretofore covered with the cellulase solution.

Cascading can also be accomplished by use of a jet. Specifically, movement of the fabric relative to the cellulase solution provides agitation whereas rotation of the fabric upward and downward during rotation in the circular jet results in solution cascading over and from the fabric. When the fabric is so moved, preferably at a rate of at least about 6 ft/sec, through the jet, cascading of the cellulase solution on the fabric is achieved.

As used herein, the term "a predetermined place in said manufacturing process" means the place in the process where the fabric is treated with cellulase. In general, the fabric is treated with cellulase at a point in manufacturing which will not interfere with the other manufacturing processes but will allow the cellulase to act on the fabric. The particular place in the manufacturing process is not critical, but in general the cellulase treatment will occur after scouring of the fabric and before finishing of the fabric. It may occur either before or after the bleaching or dying of the fabric. In the case of stonewashing, the treatment may occur after the

denim clothes have been finished and sewn so as to achieve the stonewashed "stressed" look.

As noted above, the present invention is an improvement over previously disclosed methods for treating cotton- containing non-cotton-containing cellulosic fabrics insofar as the present invention employs an effective amount of a specific cellulase composition used under conditions which minimize the production of lint and fabric weight loss during treatment. The cellulase composition described herein reduces lint formation. Further lint formation can be achieved by using reaction conditions which reduce lint production. Suitable conditions for further reducing lint production are set forth below and include using one or more of a specific EG type component (e.g. EG I cellulase) and/or a specific concentration of cellulase; modifying the reaction time, reaction temperatures and liquor ratios, etc., as compared to methods set forth in U.S. Serial No. 07/677,385, filed March 29, 1991 and entitled "Methods For Treating Cotton-containing Fabrics with Cellulase", which disclosure is incorporated herein by reference in its entirety. The exact conditions employed will depend on the type of machine used and the type of cellulase employed. The skilled artisan can readily take into account these factors and modify the conditions of temperature, time, liquor ratio and cellulase concentration so as to achieve reduced lint production and fabric weight loss based on the teachings of this invention.

The cellulase composition employed herein is a fungal cellulase composition which comprises a protein weight ratio of all EG type components to all CBH type components of greater than 5:1.

The use of the cellulase compositions described herein may result in fabric/color enhancement of stressed cotton- containing fabrics. Specifically, during the manufacture of cotton-containing and non-cotton-containing cellulosic fabrics, the fabric can become stressed and when so stressed, it will contain broken and disordered fibers. Such fibers detrimentally impart a worn and dull appearance to the fabric.

However, when treated by the method of this invention, the so stressed fabric is subject to fabric/color enhancement. This is believed to arise because some of the broken and disordered fibers are removed which has the effect of restoring the appearance of the fabric to its appearance prior to becoming stressed and yet, not excessively degrade the fabrics which typically results in the production of excessive lint. Additionally, the use of the cellulase compositions described herein also result in reduced strength loss compared to treatment of the fabric with a complete fungal cellulase. See U.S. Serial No. 07/678,865 filed March 29, 1991 and U.S. Serial No. 07/677,385 filed March 29, 1991 both of which are incorporated herein by reference in their entirety.

Additionally, the use of the cellulase composition described herein with pigment type dyed fabrics (e.g., denims) , may cause a stone-washed appearance with less redeposition of dye. These anti-redeposition properties can be enhanced for one or more specific EG type component(s) as compared to other components. See U.S. Serial No. 07/954,113 filed September 30, 1992 and entitled "Stonewashing of Denim Garments using Endoglucanase I and III" which is incorporated herein by reference.

An "effective amount" of the cellulase composition is that amount which will provide one or more of the desired enhancements of improved feel, softness, hand, color enhancement and/or a stone-washed appearance with reduced lint formation and reduced fabric weight loss. In this regard, it has been found that it is the amount of cellulase, and not the relative rate of hydrolysis of the specific enzymatic components to produce reducing sugars from cellulose, which imparts the desired enhancement(s) to cotton-containing and non-cotton-containing cellulosic fabrics, e.g., one or more of improved color restoration, improved softening, improved stonewashing, etc.

The fungal cellulase compositions described above are employed in an aqueous solution which contains cellulase and other optional ingredients including, for example, a buffer.

a surfactant, a scouring agent, and the like. The concentration of the cellulase composition employed in this solution is generally a concentration sufficient for its intended purpose. That is to say that an amount of the cellulase composition is employed to provide the desired enhancement(s) to the cotton-containing fabric. The amount of the cellulase composition employed is also dependent on the equipment employed, the process parameters employed (the temperature of the cellulase solution, the exposure time to the cellulase solution, and the like) , the cellulase activity in imparting the desired enhancement (e.g., a lower concentration of a more active cellulase composition may be required as compared to the concentration required for a less active cellulase composition) , and the like. The exact concentration of the cellulase composition can be readily determined by the skilled artisan based on the above factors as well as the desired effect. Preferably, the concentration of the cellulase composition in the cellulase solution employed herein is from about 0.005 grams of cellulase composition per liter; to about 5.0 grams/liter; and more preferably, from about 0.01 grams/liter to about 2 grams/liter. (The cellulase concentration recited above refers to the weight of total protein) .

When a buffer is employed in the cellulase solution, the concentration of buffer in the aqueous cellulase solution is that which is sufficient to maintain the pH of the solution within the range wherein the employed cellulase exhibits activity which, in turn, depends on the nature of the cellulase employed. The exact concentration of buffer employed will depend on several factors which the skilled artisan can readily take into account. For example, in a preferred embodiment, the buffer as well as the buffer concentration are selected so as to maintain the pH of the cellulase solution within the pH range required for optimal cellulase activity. In general, buffer concentration in the cellulase solution is about 0.005 N and greater. Preferably, the concentration of the buffer in the cellulase solution is

from about 0.01 to about 0.5 N, and more preferably, from about 0.05 to about 0.15 N.

In addition to cellulase and a buffer, the cellulase solution can optionally contain a small amount of a surfactant, i.e., less than about 2 weight percent, and preferably from about 0.01 to about 2 weight percent. Suitable surfactants include any surfactant compatible with the cellulase and the fabric including, for example, anionic, non-ionic and a pholytic surfactants.

Suitable anionic surfactants for use herein include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates and the like. Suitable counter ions for 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 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.

Nonionic surfactants generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.

Mixtures of surfactants can also be employed.

The liquor ratios, i.e., the ratio of weight of liquid to the weight of fabric, employed herein is generally an amount sufficient to achieve the desired enhancement in the cotton- containing fabric and is dependent upon the process used and the enhancement to be achieved. Preferably, the liquor ratios are generally from about 3:1 to 40:1, and more preferably from about 5:1 to 20:1. Use of liquor ratios of greater than about 50:1 are usually not preferred.

Reaction temperatures for cellulase treatment are governed by two competing factors. Firstly, higher

temperatures generally correspond to enhanced reaction kinetics, i.e., faster reactions, which permit reduced reaction times as compared to reaction times required at lower temperatures. Accordingly, reaction temperatures are generally at least about 30°C and greater. Secondly, cellulase is a protein which loses activity beyond a given reaction temperature which temperature is dependent on the nature of the cellulase used. Thus, if the reaction temperature is permitted to go too high, then the cellulolytic activity is lost as a result of the denaturing of the cellulase. As a result, the maximum reaction temperatures employed herein are generally about 70°C. In view of the above, reaction temperatures are generally from about 30 β C to about 70 β C; preferably, from about 35°C to about 60 β C; and more preferably, from about 35°C to about 55°C.

Reaction times are generally from about 0.1 hours to about 24 hours and, preferably, from about 0.25 hours to about 5 hours.

In a preferred embodiment, a concentrate 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 concentrate can readily be diluted with water so as to quickly and accurately prepare cellulase solutions having the requisite concentration of these additives. Preferably, such concentrates will comprise from about 0.1 to about 20 weight percent of a cellulase composition described above (protein) ; from greater than about 0 to about 50 weight percent buffer; from greater than 0 to about 50 weight percent surfactant; and from greater than 0 to 80 weight percent water. When aqueous concentrates are formulated, these concentrates can be diluted by factors of from about 2 to about 40,000 so as to arrive at the requisite concentration of the components in the cellulase solution. As is readily apparent, such 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 cellulase composition as described above can be added to the concentrate either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the like. Such forms are well known to the skilled artisan.

When a solid cellulase concentrate is employed, the cellulase composition is generally a granule, a powder, an agglomerate and the like. When granules are used, the granules are preferably formulated so as to contain a cellulase protecting agent such as ammonium sulfate. Typically, the ammonium sulfate employed as part of the granule is from about 0.1 to 40 percent based on the weight of the granule, the cellulase is from about 0.001 to 50 percent based on the weight of the granule and the balance comprises inert materials and the like. See, for instance, U.S. Serial No. 07/642,669, and PCT Application Publication No. PCT/US91/03044 both of which are incorporated herein by reference in their 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. Serial No. 07/642,596 filed on January 17, 1991 as Attorney Docket No. GCS-171-US1 and entitled "GRANULAR COMPOSITIONS" which application is incorporated herein by reference in its entirety.

The following examples are offered to illustrate the present invention and should not be construed in any way as limiting its scope.

EXAMPLES

Examples 1 - 14 demonstrate the preparation of Trichoderma longibrachiatum genetically engineered so as to be incapable of producing one or more cellulase components ϋr so as to overproduce specific cellulase components.

EXAMPLE 1

Selection for pyr4 ~ derivatives of Trichoderma longibrachiatum

The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. The toxic inhibitor 5-fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells. However, cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for pyr4 derivative strains using FOA. In practice, spores of T. longibrachiatum (previously classified as T. reesei ) strain RL-P37 (Sheir- Neiss, G. and Montenecourt, B.S., Apol. Microbiol. Biotechnol. 20, p. 46-53 (1984)) were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resiεtant colonies appeared within three to four days and it was possible to subsequently identify those FOA-resistant derivatives which required uridine for growth. In order to identify those derivatives which specifically had a defective pyr4 gene, protoplasts were generated and transformed with a plasmid containing a wild- type pyr4 gene (see Examples 3 and 4) . Following transformation, protoplasts were plated on medium lacking uridine. Subsequent growth of transformed colonies demonstrated complementation of a defective pyr4 gene by the plasmid-borne pyr4 gene. In this way, strain GC69 was identified as a pyr4 ~ derivative of strain RL-P37.

EXAMPLE 2 Preparation of CBH I Deletion Vector

A cbhl gene encoding the CBH I protein was cloned from the genomic DNA of T. longibrachiatum strain RL-P37 by hybridization with an oligonucleotide probe designed on the basis of the published sequence for this gene using known

probe synthesis methods (Shoemaker et al., 1983b). The cbhl gene resides on a 6.5 kb PstI fragment and was inserted into PstI cut pUC4K (purchased from Pharmacia Inc., Piscataway, New Jersey) replacing the Kan r gene of this vector using techniques known in the art, which techniques are set forth in Sambrook et al. (1989) MOLECULAR CLONING A LABORATORY MANUAL, Cold Spring Harbor Press, and incorporated herein by reference. The resulting plasmid, pUC4K::cbhl was then cut with ffindlll and the larger fragment of about 6 kb was isolated and religated to give pUC4K: :cbhlΔH/H (see FIG. 1). This procedure removes the entire cbhl coding sequence and approximately 1.2 kb upstream and 1.5 kb downstream of flanking sequences. Approximately, 1 kb of flanking DNA from either end of the original PstI fragment remains.

The T. longibrachiatum pyr4 gene was cloned as a 6.5 kb Hindi11 fragment of genomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991) following the methods of Sambrook et al., supra . The plasmid pUC4K: :cbhlΔH/H was cut with Hindi! I and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was ligated with the 6.5 kb ifindlll fragment containing the T. longibrachiatum pyr4 gene to give pΔCBHIpyr.. FIG. 1 illustrates the construction of this plasmid.

Digestion of pΔCBHIpyr4 with .EcoRI liberated a larger fragment which consisted of flanking regions of the c_ -2 locus at either end with the pyr4 gene replacing the cbhl coding sequence in the center. The only DNA on this fragment which was not derived from T. longibrachiatum was a 21 bp fragment derived from the multiple cloning site of pUC4K.

EXAMPLE 3 Isolation of Protoplasts

Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2% glucose) in a 500 ml flask with about 5 x IO 7 T. longibrachiatum GC69 spores (the pyr4 ~ derivative

strain) . The flask was then incubated at 37 β C with shaking for about 16 hours. The mycelium was harvested by centrifugation at 2,750 x g. The harvested mycelium was further washed in a 1.2 M sorbitol solution and resuspended in 40 ml of a solution containing 5 mg/ml Novozym" 234 solution (which is the trade name for a multicomponent enzyme system containing 1,3-alpha-glucanase, 1,3-beta-glucanase, la inari- nase, xylanase, chitinase and protease from Novo Biolabs, Danbury, CT) ; 5 mg/ml MgS0 4 .7H 2 0; 0.5 mg/ml bovine serum albumin; 1.2 M sorbitol. The protoplasts were removed from the cellular debris by filtration through Miracloth (Calbioche Corp, La Jolla, California) and collected by centrifugation at 2,000 x g. The protoplasts were washed three times in 1.2 M sorbitol and once in 1.2 M sorbitol, 50 mM CaCl 2 , centrifuged and resuspended at a density of aapppprrooxxiimmately 2 x 10 protoplasts per ml of 1.2 M sorbitol, 50 mM CaCl 2 .

EXAMPLE 4 Transformation of Fungal Protoplasts with pΔCBHIpyr.

200 μl of the protoplast suspension prepared in Example 3 was added to 20 μl of EcoRI digested pΔCBHIpyr4 (prepared in Example 2) in TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) and 50 μl of a polyethylene glycol (PEG) solution containing 25% PEG 4000, 0.6 M KCl and 50 mM CaCl 2 . This mixture was incubated on ice for 20 minutes. After this incubation period 2.0 ml of the above-identified PEG solution was added thereto, the solution was further mixed and incubated at room temperature for 5 minutes. After this second incubation, 4.0 ml of a solution containing 1.2 M sorbitol and 50 mM CaCl 2 was added thereto and this solution was further mixed. The protoplast solution was then immediately added to molten aliquots of Vogel's Medium N (3 grams sodium citrate, 5 grams KH 2 P0 4 , 2 grams NH 4 N0 3 , 0.2 grams MgS0 4 "7H 2 0, 0.1 gram CaCl 2 "2H 2 0, 5 μg α-biotin, 5 mg citric acid, 5 mg ZnS0 4 * 7H 2 0, 1 mg Fe(NH 4 ) 2 "6H 2 0, 0.25 mg CuS0 4 "5H 2 0, 50 μg MnS0 4 '4H 2 0 per liter) containing an

additional 1% glucose, 1.2 M sorbitol and 1% agarose. The protoplast/medium mixture was then poured onto a solid medium containing the same Vogel's medium as stated above. No uridine was present in the medium and therefore only transformed colonies were able to grow as a result of complementation of the pyr4 mutation of strain GC69 by the wild type pyr4 gene inserted in pΔCBHIpyr.. These colonies were subsequently transferred and purified on a solid Vogel's medium N containing as an additive, 1% glucose and stable transformants were chosen for further analysis.

At this stage stable transformants were distinguished from unstable transformants by their faster growth rate and formation of circular colonies with a smooth, rather than ragged outline on solid culture medium lacking uridine. In some cases a further test of stability was made by growing the transformants on solid non-selective medium (i.e. containing uridine) , harvesting spores from this medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium lacking uridine.

EXAMPLE 5 Analysis of the Transformants

DNA was isolated from the transformants obtained in Example 4 after they were grown in liquid Vogel's medium N containing 1% glucose. These transformant DNA samples were further cut with a PstI restriction enzyme and subjected to agarose gel electrophoresis. The gel was then blotted onto a Nytran membrane filter and hybridized with a 32 P labelled pΔCBHIpyr. probe. The probe was selected to identify the native cbhl gene as a 6.5 kb PstI fragment, the native pyr4 gene and any DNA sequences derived from the transforming DNA fragment.

The radioactive bands from the hybridization were visualized by autoradiography. The autoradiograph is seen in FIG. 3. Five samples were run as described above, hence samples A, B, C, D, and E. Lane E is the untransformed strain

GC69 and was used as a control in the present analysis. Lanes A-D represent transformants obtained by the methods described above. The numbers on the side of the autoradiograph represent the sizes of molecular weight markers. As can be seen from this autoradiograph, lane D does not contain the 6.5 kb CBH I band, indicating that this gene has been totally deleted in the transformant by integration of the DNA fragment at the cjbj-l gene. The cbhl deleted strain is called P37PΔCBHI. Figure 2 outlines the deletion of the T. longibrachiatum cbhl gene by integration through a double cross-over event of the larger EcoRI fragment from pΔCBHIpyr4 at the cJbhl locus on one of the T. longibrachiatum chromosomes. The other transformants analyzed appear identical to the untransformed control strain.

EXAMPLE 6 Analysis of the Transformants with plntCBHI

The same procedure was used in this example as in Example 5, except that the probe used was changed to a 32 P labelled plntCBHI probe. This probe is a pUC-type plasmid containing a 2 kb Bgrlll fragment from the cJbhl locus within the region that was deleted in pUC4K: :cbhlΔH/H. Two samples were run in this example including a control, sample A, which is the untransformed strain GC69 and the transformant P37PΔCBHI, sample B. As can be seen in FIG. 4, sample A contained the cJbhl gene, as indicated by the band at 6.5 kb; however the transformant, sample B, does not contain this 6.5 kb band and therefore does not contain the cbhl gene and does not contain any sequences derived from the pUC plasmid.

EXAMPLE 7 Protein Secretion by strain P37PΔCBHI

Spores from the produced P37PΔCBHI strain were inoculated into 50 ml of a Trichoderma basal medium containing 1% glucose, 0.14% (NH 4 ) 2 S0 4 , 0.2% KH 2 P0 4 , 0.03% MgS0 4 , 0.03% urea,

0.75% bactotryptone, 0.05% Tween 80, 0.000016% CuS0 4 * 5H 2 0, 0.001% FeS0 4 '7H 2 0, 0.000128% ZnS0_ 7H 2 0, 0.0000054% Na^oO^^O, 0.0000007% MnCl'4H 2 0. The medium was incubated with shaking in a 250 ml flask at 37°C for about 48 hours. The resulting mycelium was collected by filtering through Miracloth (Calbiochem Corp., La Jolla, CA) and washed two or three times with 17 mM potassium phosphate. The mycelium was finally suspended in 17 mM potassium phosphate with 1 mM sophorose and further incubated for 24 hours at 30°C with shaking. The supernatant was then collected from these cultures and the mycelium was discarded. Samples of the culture supernatant were analyzed by isoelectric focusing using a Pharmacia Phastgel system and pH 3-9 precast gels according to the manufacturer's instructions. The gel was stained with silver stain to visualize the protein bands. The band corresponding to the cbhl protein was absent from the sample derived from the strain P37PΔCBHI, as shown in FIG. 5. This isoelectric focusing gel shows various proteins in different supernatant cultures of T. longibrachiatum. Lane A is partially purified CBH I; Lane B is the supernatant from an untransformed T. longibrachiatum culture; Lane C is the supernatant from strain P37PΔCBHI produced according to the methods of the present invention. The position of various cellulase components are labelled CBH I, CBH II, EG I, EG II, and EG III. Since CBH I constitutes about 50% of the total extracellular protein, it is the major secreted protein and hence is the darkest band on the gel. This isoelectric focusing gel clearly shows depletion of the CBH I protein in the P37PΔCBHI strain.

EXAMPLE 8 Preparation of pPΔCBHII

The cbh2 gene of T. longibrachiatum, encoding the CBH II protein, has been cloned as a 4.1 kb EcoRI fragment of genomic DNA which is shown diagrammatically in FIG. 6A (Chen et al., 1987, Biotechnology. 5:274-278). This 4.1 kb fragment was inserted between the .EcoRI sites of pUC4XL. The latter plasmid

is a pUC derivative (constructed by R.M. Berka, Genencor International, Inc.) which contains a multiple cloning site with a symmetrical pattern of restriction endonuclease sites arranged in the order shown here: EcoRI, BamHI, SacI , Smal , Hindlll , Xhol , Bglll , Clal , Bglll , Xhol , Hindlll , Smal , SacI , BamHI , EcoRI . Using methods known in the art, a plasmid, pPΔCBHII (FIG. 6B) , has been constructed in which a 1.7 kb central region of this gene between a Hindlll site (at 74 bp 3' of the CBH II translation initiation site) and a Clal site (at 265 bp 3' of the last codon of CBH II) has been removed and replaced by a 1.6 kb Hindlll- Clal DNA fragment containing the T. longibrachiatum pyr4 gene.

The T. longibrachiatum pyr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb Nhel-SphI fragment and inserted between the Sphi and Xbal sites of pUC219 to create p219M (Smith et al., 1991, Curr. Genet 19 p. 27-33). The vector pUC219 is derived from pUC119 (described by Wilson et al. (1984) Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for Bglll, Clall and Xhol. The pyr4 gene was removed from p219M as a Hindlll-CIal fragment having seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pPΔCBHII (see FIG. 6B) .

Digestion of this plasmid with EcoRI will liberate a fragment having 0.7 kb of flanking DNA from the cbh2 locus at one end, 1.7 kb of flanking DNA from the cbh2 locus at the other end and the T. longibrachiatum pyr4 gene in the middle.

EXAMPLE 9 Generation of a pyr4 ~ Derivative of P37PΔCBHI

Spores of the transformant (P37PΔCBHI) which was deleted for the cbhl gene were spread onto medium containing FOA. A pyr4~ derivative of this transformant was subsequently

obtained using the methods of Example 1. This pyr4 ' strain was designated P37PΔCBHIPyr * 26. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PΔCBHIPyr26 was selected. This deletion completely removed the pyr4 gene which had integrated at the cbhl locus beyond the extent of the 6.5 kb PstI fragment of genomic DNA which was originally cloned.

EXAMPLE 10

Deletion of the cbh2 gene in a strain previously deleted for cbhl

Protoplasts of strain P37PΔCBHIPyr * 26 were generated and transformed with EcoRI digested pPΔCBHII according to the methods outlined in Examples 3 and 4.

Purified stable transformants were cultured in shaker flasks as in Example 7 and the protein in the culture supernatants was examined by isoelectric focusing. One transformant (designated P37PΔΔCBH67) was identified which did not produce any CBH II (nor CBH I) protein. Lane D of FIG. 5 shows the supernatant from a transformant deleted for both the cJbhl and cJ 2 genes produced according to the methods of the present invention.

DNA was extracted from strain P37PΔΔCBH67, digested with EcoRI and Asp718, and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with 32 P labelled pPΔCBHII (FIG. 7) . Lane A of FIG. 7 shows the hybridization pattern observed for DNA from an untransformed T. longibrachiatum strain. The 4.1 kb _EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization pattern observed for strain P37PΔΔCBH67. The single 4.1 kb band has been eliminated and replaced by two bands of approximately 0.9 and 3.1 kb. This is the expected pattern if a single copy of the EcoRI fragment from pPΔCBHII had integrated precisely at the cbh2 locus.

The same DNA samples were also digested with EcoRI and Southern blot analysis was performed as above. In this

Example, the probe was P labelled plntCBHII. This plasmid contains a portion of the cbh2 gene coding sequence from within that segment of the cbh2 gene which was deleted in plasmid pPΔCBHII. No hybridization was seen with DNA from strain P37PΔΔCBH67 showing that the bh2 gene was deleted and that no sequences derived from the pUC plasmid were present in this strain.

EXAMPLE 11 Selection of a pyr4 null mutant of strain P37PΔΔCBH67 Spores of the transformant (P37PΔΔCBH67) which was deleted for both the cJbhl and cbh2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in Example 1. This pyr4 deficient strain was designated P37PΔΔCBH67Pyr * l. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PΔΔCBH67Pyr * l was selected. This deletion completely removed the pyr4 gene which had integrated at the cbh2 locus beyond the extent of the 4.1 kb EcoRI fragment of genomic DNA which was originally cloned. The short (6 bp and 7 bp) fragments of DNA derived from the pUC219 multiple cloning site which were present at either end of the pyr4 gene would also have been removed from the genome by this deletion.

EXAMPLE 12 Construction of pEGIpyr4 The T. longibrachiatum egll gene, which encodes EG I, has been cloned as a 4.2 kb Hindlll fragment of genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthesized according to the published sequence (Penttila et al., 1986, Gene 45:253-263; van Arsdell et al., 1987, Bio/Technology 5:60-64). A 3.6 kb Hindlll-BamHI fragment was taken from this clone and ligated with a 1.6 kb Hi_.dIII-Ba.__HI fragment containing the T. longibrachiatum pyr4 gene obtained

from pTpyr2 (see Example 2) and pUC218 (identical to pUC219, see Example 8, but with the multiple cloning site in the opposite orientation) cut with Hindlll to give the plasmid pEGIpyr. by standard molecular techniques as outlined in Sambrook et al. (1989), supra (FIG. 8). Digestion of pEGIpyr. with Hindlll would liberate a fragment of DNA containing only T. longibrachiatum genomic DNA (the egll and pyr4 genes) except for 24 bp of sequenced, synthetic DNA between the two genes and 6 bp of sequenced, synthetic DNA at one end (see FIG. 8) . Both these pieces of synthetic DNA were obtained from the multiple cloning site of pUC-type vectors.

EXAMPLE 13 Construction of the EG I expression vector pTEX-EGl

The plasmid, pTEX was constructed following the methods of Sambrook et al. (1989), supra, and is illustrated in FIG. 9. This plasmid has been designed as a multi-purpose expression vector for use in the filamentous fungus Trichoderma longibrachiatum. The expression cassette has several unique features that make it useful for this function. Transcription is regulated using the strong CBH I gene promoter and terminator sequences for T. longibrachiatum. Between the CBH I promoter and terminator there are unique Pmel and Sst I restriction sites that are used to insert the gene to be expressed. The T. longibrachiatum pyr4 selectable marker gene has been inserted into the CBH I terminator and the whole expression cassette (CBH I promoter-insertion sites- CBH I terminator-pyr4 gene-CBH I terminator) can be excised utilizing the unique NotI restriction site or the unique NotI and Nhel restriction sites.

This vector is based on the bacterial vector, pSLllδO (Pharmacia Inc., Piscataway, New Jersey) , which is a pUC-type vector with an extended multiple cloning site. pTEX was digested with Sstll and Pmel and then ligated with a synthetic DNA linker designed to join the cJbhl promoter with the egll coding sequence and with an approximately 2 kb Sfil - Seal

fragment of T. longibrachiatum DNA containing most of the egll coding sequence and the terminator region. This ligation produced the vector pTEX-EGI. This vector was digested with WotI and Whel to release the expression cassette which comprised the following components: a) 11 bp of linker DNA derived from the multiple cloning site of pSLllδO. b) An approximately 2.2 kb Pstl-Sstll fragment of T. longibrachiatum DNA from the promoter region of the cJb l gene. The Sstll site is at a position 15 bp 5' of the translation initiation codon (ATG) . c) A synthetic DNA linker used to join the cbhl promoter with the egll coding sequence, having single-stranded overhanging ends compatible with Ss til and Sfil digested DNA, and having the following sequence:

***

5' GGACTGGCATCATGGCGCCCTCAGTTACACTGCCGTTGACCACGGCCATCC 3' 3' CGCCTGACCGTAGTACCGCGGGAGTCAATGTGACGGCAACTGGTGCCGGT 5' The asterisks mark the translation initiation codon (ATG) of the egll gene coding region. The DNA sequence 5' to the ATG codon is exactly the same as that found in this region of the cbhl gene. The DNA sequence 3' of the ATG codon is exactly the same as that found in this region of the egll gene. d) An approximately 2 kb Sfil-Scal fragment of T. longibrachiatum DNA containing the egll coding sequence, starting at an Sfil site 30 bp after the first ATG codon, and approximately 300 bp of 3' flanking DNA containing the transcription termination and polyadenylation signals. e) An approximately 1 kb Smal-Bglll fragment of T. longibrachiatum DNA from the 3' flanking region of the cJbhl gene with a Pmel restriction site added adjacent to the Smal site using the synthetic linker DNA shown in FIG. 9. f) The T. longibrachiatum pyr4 gene on a 1.6 kb Bglll fragment which has 13 bp on one end and 17 bp on the other end of synthetic linker DNA. Both linkers were derived from the

multiple cloning site of the pUC219 vector. The latter synthetic linker was additionally modified by insertion of a Bglll site at the Hindlll site using the linker shown in FIG. 9. Oligonucleotide directed mutagenesis (Sambrook et al. (1989) Molecular Cloning a Laboratory Manual, Cold Spring Harbor Press) was used to change a single nucleotide within the pyr4 coding region (FIG. 9) . The third nucleotide of the codon coding for the arginine residue at amino acid position 251 of the protein was changed from a C nucleotide to an A nucleotide by this method. This alteration would not change the amino acid sequence of the orotidine 5' monophosphate decarboxylase produced but destroyed an Sstll site in the DNA sequence and thus facilitated construction of the plasmid. g) An approximately 0.5 kb Bglll-Nhel fragment of T. longibrachiatum DNA from the 3' flanking region of the cJbhl gene.

It would be possible to construct plasmids similar to pTEX-EGI but with any other T. longibrachiatum gene replacing the egll gene. In this way, over-expression of other genes and simultaneous deletion of the cbhl gene could be achieved.

EXAMPLE 14

Construction of EG I over-expression strains The linear fragment of DNA containing the egll and pyr4 genes released from pEGIpy 4 by digestion with Hindlll, was purified from an agarose gel. Similarly, the linear fragment of DNA containing the egll , pyr4 genes and flanking regions of the cJbhl gene was purified from pTEX-EGI after digestion with lVotl and Nhel . These fragments of DNA were used in separate experiments to transform T. longibrachiatum strain P37PΔΔCBH67Pyr " l by the method of Examples 3 and 4. Several transformants were obtained with each DNA fragment which transformants produced elevated levels of EG I compared to the parent strain. Total DNA was isolated from these transformants, digested with PstI , subjected to agarose gel

electrophoresis and blotted to a membrane filter. Southern blot analysis using radiolabeled pUCEGI (a pUC plasmid containing the egll gene on a 4.2 kb Hindlll fragment of genomic DNA) showed that each transformant contained multiple copies of the egll gene integrated at sites within the genome which could not be determined.

Similar Southern analysis was also performed using a pUC vector as a probe. This analysis revealed that the pUC plasmid fragment of either pEGIpyr. or pTEX-EGI had not been incorporated by any of these strains.

Transformants of strain P37PΔΔCBH67Pyr " l obtained with either pEGIpyr. or pTEX-EGI as described above were inoculated into 50 ml shake flask cultures in order to determine the amount of secreted endoglucanase produced. The liquid medium used for these experiments had the following composition: alpha-lactose, 30 g/1; (NH 4 ) 2 S0 4 , 6.5 g/1; KH 2 P0 4 , 2.0 g/1; MgS0 4 '7H 2 0, 0.3 g/1; CaCl 2 , 0.2 g/1; lOOOx trace salt solution, 1.0 ml/1; 10 % Tween 80, 2.0 ml/1; Proflo, 22.5 g/1; CaC0 3 , 0.72g/l. Source for Tween 80 and Proflo. The lOOOx trace salt solution had the following composition: FeS0 4 "7H 2 0, 5.0 g/1; MnS0_ H 2 0, 1.6 g/1; ZnS0 4 , 1.4 g/1. These shake flask cultures were incubated with shaking for seven days at 30°C. Samples of the supernatant were taken from these cultures and assays designed to measure the endoglucanase activity were performed as described below.

The endoglucanase assay relied on the release of soluble, dyed oligosaccharides from Remazol Brilliant Blue- carboxymethylcellulose (RBB-CMC, obtained from MegaZyme, North Rocks, NSW, Australia) . The substrate was prepared by adding 2 g of dry RBB-CMC to 80 ml of just boiled deionized water with vigorous stirring. When cooled to room temperature, 5 ml of 2 M sodium acetate buffer (pH 4.8) was added and the pH adjusted to 4.5. The volume was finally adjusted to 100 ml with deionized water and sodium azide added to a final concentration of 0.02%. Aliquots of T. longibrachiatum control culture, pEGIpyr. or pTEX-EGI transformant culture supernatants or 0.1 M sodium acetate as a blank (10-20 μl)

were placed in tubes, 250 μl of substrate was added and the tubes were incubated for 30 minutes at 37°C. The tubes were placed on ice for 10 minutes and 1 ml of cold precipitant (3.3% sodium acetate, 0.4% zinc acetate, pH 5 with HC1, 76% ethanol) was then added. The tubes were vortexed and allowed to sit for five minutes before centrifuging for three minutes at approximately 13,000 x g. The optical density was measured spectrophotometrically at a wavelength of 590-600 nm.

The results of the endoglucanase assay performed on triplicate cultures of 5 different transformants obtained with pEGIpyr. DNA and on cultures of the parental strain P37PΔΔCBH67 are given in Table 1 below. It is apparent that the transformants produced significantly more secreted endoglucanase activity compared to the parental strain described above, are shown in Table 1.

TABLE 1 Secreted Endoglucanase Activity of T. longibrachiatum Transformants

TRANSFORMANT RBB-CMC UNITS/ML

FLASK 1 FLASK2 FLASK3 FLASK4 AVERAGE

P37 EP1 5.7 14.3 14.1 11.4

P37 EP8 11.0 3.8 6.1 7.0

P37 EP9 3.4 11.5 14.9 9.9

P37 EP10 7.0 9.1 5.0 7.0

P37 EP11 5.0 5.5 10.0 6.9 parental

P37PΔΔCBH67 3.0 2.8 5.5 1.1 3.1

The above results are presented for the purpose of demonstrating the overproduction of the EG I component and not for the purpose of demonstrating the extent of overproduction. In this regard, the extent of overproduction is expected to vary with each experiment.

Similar shake flask cultures and endoglucanase assays were performed with transformants obtained with pTEX-EGI and those transformants which over-produced endoglucanase activity were identified.

The methods of this example could be used to produce T.

aZongiJbraσhiatU--- strains which would over-produce any of the other EG components.

It would also be possible to transform pyr4 derivative strains of T. longibrachiatum which had previously been deleted for other genes in addition to CBH I and CBH II, eg. for EG II, with pEGIpyr. or pTEX-EGI to construct transformants which would, for example, produce no exocellobiohydrolases or EG II and overexpress EG I.

EXAMPLE 15

Purification of CYTOLASE 123 Cellulase into Cellulase Components

CYTOLASE 123 cellulase was fractionated in the following manner. The normal distribution of cellulase components in this cellulase system is as follows:

CBH I 45-55 weight percent

CBH II 13-15 weight percent

EG I 11-13 weight percent

EG II 8-10 weight percent

EG III 1-4 weight percent

BG 0.5-1 weight percent.

The fractionation was done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Mo) , QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Maryland) . CYTOLASE 123 cellulase, 0.5g, was desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, was then loaded onto a column of 20 ml of QA Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I. These components were separated by gradient elution using an aqueous gradient containing from 0 to about 500 mM sodium chloride. The fraction not bound on this column contained CBH II and EG II. These fractions were desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 3.3. This solution, 200 ml, was then loaded onto a column of 20 ml of SP Trisacryl M cation exchange resin.

CBH II and EG II were eluted separately using an aqueous gradient containing from 0 to about 200 mM sodium chloride.

Following procedures similar to this Example, other cellulase systems which can be separated into their components include CELLUCAST (available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist Brocades, N.V., Delft, Holland) , and cellulase systems derived from Trichoderma koningii, Penicillium sp. and the like.

EXAMPLE 16 Purification of EG III from CYTOLASE 123 Cellulase Example 15 above demonstrated the isolation of several components from CYTOLASE 123 Cellulase. However, because EG III is present in very small quantities in CYTOLASE 123 Cellulase, the following procedures were employed to isolate this component. See also U.S. Serial No. 07/862,846 filed April 3, 1992 and entitled "METHODS FOR PRODUCING SUBSTANTIALLY PURE EG III CELLULASE USING POLYETHYLENE GLYCOL", which is incorporated herein in its entirety by reference.

λ. Large Scale Extraction of EG III Cellulase Enzyme

One hundred liters of cell free cellulase filtrate were heated to about 30°C. The heated material was made about 4% wt/vol PEG 8000 (polyethylene glycol, MW of about 8000) and about 10% wt/vol anhydrous sodium sulfate. The mixture formed a two phase liquid mixture. The phases were separated using an SA-1 disk stack centrifuge. The phases were analyzed using silver staining isoelectric focusing gels. Separation was obtained for EG III and xylanase. The recovered composition contained about 20 to 50 weight percent of EG III.

Regarding the above procedure, use of a polyethylene glycol having a molecular weight of less than about 8000 gave inadequate separation; whereas, use of polyethylene glycol having a molecular weight of greater than about 8000 resulted in the exclusion of desired enzymes in the recovered

composition. With regard to the amount of sodium sulfate, sodium sulfate levels greater than about 10% wt/vol caused precipitation problems; whereas, sodium sulfate levels less than about 10% wt/vol gave poor separation or the solution remained in a single phase.

Alternatively, EG III cellulase may be extracted by the method described in U.S. Serial No. 07/862,641 filed April 3, 1992 which is entitled "METHODS FOR PRODUCING SUBSTANTIALLY PURE EG III CELLULASE USING ALCOHOL," which reference is incorporated herein in its entirety.

B. Purification of EG III Via Fractionation

The purification of EG III is conducted by fractionation from a complete fungal cellulase composition (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, California) which is produced by wild type Trichoderma longibrachiatum. Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Missouri) , QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Maryland) . CYTOLASE 123 cellulase, 0.5g, is desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, is then loaded onto a column of 20 ml of QA Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I. The fraction not bound on this column contains CBH II, EG II and EG III. These fractions are desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto a column of 20 ml of SP Trisacryl M cation exchange resin. The EG III was eluted with 100 mL of an aqueous solution of 200 mM sodium chloride.

In order to enhance the efficiency of the isolation of EG III, it may be desirable to employ Trichoderma longibrachiatum genetically modified so as to be incapable of producing one or

more of EG I, EG II, CBH I and/or CBH II. The absence of one or more of such components will necessarily lead to more efficient isolation of EG III.

Likewise, it may be desirable for the EG III compositions described above to be further purified to provide for enriched EG III compositions, i.e., compositions containing EG III at greater than about 80 weight percent of protein. For example, such an enriched EG III protein can be obtained by utilizing material obtained from procedure A in procedure B or vice versa. One particular method for further purifying EG III is by further fractionation of an EG III sample obtained in part b) of this Example 16. The further fraction was done on a FPLC system using a Mono-S-HR 5/5 column (available from Pharmacia LKB Biotechnology, Piscataway, NJ) . The FPLC system consists of a liquid chromatography controller, 2 pumps, a dual path monitor, a fraction collector and a chart recorder (all of which are available from Pharmacia LKB Biotechnology, Piscataway, NJ) . The fractionation was conducted by desalting 5 ml of the EG III sample prepared in part b) of this Example 16 with a 20 ml Sephadex G-25 column which had been previously equilibrated with 10 mM sodium citrate pH 4. The column was then eluted with 0-200 mM aqueous gradient of NaCl at a rate of 0.5 ml/minute with samples collected in 1 ml fractions. EG III was recovered in fractions 10 and 11 and was determined to be greater than 90% pure by SDS gel electrophoresis. EG III of this purity is suitable for determining the N-terminal amino acid sequence by known techniques.

Enriched EG III as well as EG I and EG II components purified in Example 15 above can be used singularly or in mixtures in the methods of this invention. These EG components have the following characteristics:

MW Dl PH optimum

EG I -47-49 kD 4 .7 -5

EG II -35 kD 5. 5 -5

EG III -25-28 kD 7.4 -5.5-6. 0

1. pH optimum determined by RBB-CMC activity as per Example 17 below.

As can be seen from the above table, EG III has both a higher pH optimum and a higher pi as compared to the other endoglucanase components of Trichoderma longibrachiatum. In Example 17 below, it is seen that EG III also retains significant RBB-CMC activity under alkaline pHs.

Likewise, EG III cellulase from other strains of Trichoderma spp. can be purified. For example, EG III cellulase derived from Trichoderma viridae has been described by Voragen et al.. Methods in Enzymology, 160:243-251, 1988. This reference describes the EG III cellulase as having a molecular weight of about 23.5 kdaltons, a pH optimum of 5.5 and a pi of 7.7.

EXAMPLE 17

Activity of Cellulase compositions

Over a pH Range

The following procedure was employed to determine the pH profiles of two different cellulase compositions. The first cellulase composition was a CBH I and II deleted cellulase composition prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be unable to produce CBH I and CBH II components. Insofar as this cellulase composition does not contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase composition derived from Trichoderma longibrachiatum, this cellulase composition is necessarily free of CBH I type and CBH II type cellulase components and accordingly, is enriched in EG components, i.e., EG I, EG II, EG III, and the like.

The second cellulase composition was an approximately 20 to 40% pure fraction of EG III isolated from a cellulase composition derived from Trichoderma longibrachiatum via purification methods similar to part b) of Example 16.

The activity of these cellulase compositions was determined at 40°C and the determinations were made using the following procedures.

Add 5 to 20 μl of an appropriate enzyme solution at a concentration sufficient to provide for normalized enzyme activity at pH 5.5 in the final solution. Add 250 μl of 2 weight percent RBB-CMC (Remazol Brilliant Blue R- Carboxymethylcellulose — commercially available from MegaZyme, 6 Altona Place, North Rocks, N.S.W. 2151, Australia) in 0.05M citrate/phosphate buffer at pH 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8.

Vortex and incubate at 40 β C for 30 minutes. Chill in an ice bath f r 5 to 10 minutes. Add 1000 μl of methyl cellosolve containing 0.3M sodium acetate and 0.02M zinc acetate. Vortex and let sit for 5-10 minutes. Centrifuge and pour supernatant into cuvets. Measure the optical density (OD) of the solution in each cuvet at 590 nm. Higher levels of optical density correspond to higher levels of enzyme activity.

The results of this analysis are set forth in FIG. 10 which illustrates the relative activity of the CBH I and II deleted cellulase composition compared to the EG III cellulase composition. From this figure, it is seen that the cellulase composition deleted in CBH I and CBH II possesses optimum cellulolytic activity against RBB-CMC at near pH 5.5 and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8. On the other hand, the cellulase composition enriched in EG III possesses optimum cellulolytic activity at pH 5.5 - 6 and possesses significant activity at alkaline pHs.

From the above example, one skilled in the art would merely need to adjust and maintain the pH of the aqueous textile composition so that the cellulase composition is active and preferably, possesses optimum activity. As noted

above, such adjustments and maintenance may involve the use of a suitable buffer.

EXAMPLE 18 Stonewashed Appearance

This example demonstrates that the presence of CBH type components is not essential for imparting a stonewashed appearance to denim fabrics. Specifically, this example employs a cellulase composition derived from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and II components) as well as a complete cellulase composition derived from Trichoderma longibrachiatum and which is available as Cytolase 123 cellulase from Genencor International, South San Francisco, California.

These cellulase compositions were tested for their ability to impart a stonewashed appearance to dyed denim pants. Specifically, the samples were prepared using an industrial washer and dryer under the following conditions:

10 mM citrate/phosphate buffer pH 5

40 L total volume

110°F

Four pair of denim pants

1 hour run time

50 ppm CBH I and II deleted cellulase or 100 ppm whole cellulase (i.e., at approximately equal EG concentrations)

Samples were evaluated for their stonewashed appearance, but not the level of redeposition of dye, by 8 panelists. All eight panelists choose 100 ppm whole cellulase over non-enzyme treated pants as having the better stonewashed look. Four of the 8 panelists choose the CBH I and II deleted cellulase treated pants over whole cellulase as having the better stonewashed look; whereas the other four panelists choose the whole cellulase treated pants as having the better stonewashed look. These results indicate that the CBH I and II deleted cellulase treated pants were indistinguishable from whole cellulase treated pants and that CBH I and/or CBH II are not

essential for imparting a stonewashed appearance to denim fabrics.

EXAMPLE 19 Reduced Redeposition of dye This example demonstrates that the use of EG type components substantially free of CBH type components results in a reduced redeposition of dye onto the fabric during stonewashing. Specifically, this example employs the following cellulase compositions: a) a complete cellulase composition derived from Trichoderma longibrachiatum and which is available as CYTOLASE 123 cellulase from Genencor International, South San Francisco, California. b) a cellulase composition from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and II components) ; c) CBH I purified by the method of Example 15; d) EG I purified by the method of Example 15; e) EG II purified by the method of Example 15; and f) EG III purified by the method of Example 16. These cellulase compositions were tested for their ability to impart a stonewashed appearance to dyed denim pants and their ability to prevent the redeposition of dye onto the fabric.

Specifically, the samples were prepared using an industrial washer and dryer under the following conditions:

20 mM citrate/phosphate buffer pH 4.9 40 L total volume 55°C 3.8 kg of desized indigo dyed denim pants 1 hour run time at 36 rpms 35 ppm CBH I and II deleted cellulase or 70 ppm whole cellulase or 15-30 ppm of purified EG I, EG II or EG III cellulase

The garments were rinsed according to a standardized

protocol in three consecutive cycles of clean liquor. Rinse ≠l — 24 gallons hot water, approximately 50°C, plus -100 grams standard detergent WOR (from American Association of Textile Chemists and Colorists [AATCC], WOB - without brighteners) . Agitation was for 12 minutes at 36 rpms. The bath was dropped. Rinse 02 — 24 gallons warm water, -40°C, with no additional detergents, agitated for 5 minutes. The bath was dropped. Rinse #3 — 24 gallons cold water, -30°C, with no additional detergents, agitated for 5 minutes. The bath was dropped. Garments were extracted and dried in a standard electric clothes dryer.

FIG. 11 shows the results obtained with the different cellulase compositions.

The results indicate that EG III gave the least redeposition of dye on the fabric. However, purified EG I, EG II and CBH I/II deleted cellulase also gave a satisfactory stonewashed appearance with less redeposition as compared to that obtained with whole cellulase. Although CBH I alone did not produce redeposition, it also did not generate a stonewashed effect.

The presence of substantial amounts of CBH type components in the cellulase composition appears to result in an increase in the redeposition of dye onto the fabric.

Example 20. Laundero eter Strength Loss Assay Cellulase Compositions This example examines the ability of different cellulase compositions to reduce the strength of cotton-containing fabrics. This example employs an aqueous cellulase solution maintained at pH 5 because the activity of most of the cellulase components derived from Trichoderma longibrachiatum is greatest at or near pH 5 and accordingly, strength loss results will be most evident when the assay is conducted at about this pH.

Specifically, in this example, the first cellulase composition analyzed was a complete fungal cellulase system

(CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma longibrachiatum and is identified as GC010.

The second cellulase composition analyzed was a CBH II deleted cellulase composition prepared from Trichoderma longibrachiatum genetically modified in a manner similar to Examples 1 to 14 above so as to be incapable of expressing CBH II and is identified as CBH lid. Insofar as CBH II comprises up to about 15 percent of the cellulase composition, deletion of this component results in enriched levels of CBH I, and all of the EG components.

The third cellulase composition analyzed was a CBH I and CBH II deleted cellulase composition prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II and is identified as CBH I/IId. Insofar as CBH I and CBH II are not produced by this modified microorganism, the cellulase is necessarily free of all CBH I type components as well as all CBH components.

The last cellulase composition analyzed was a CBH I deleted cellulase composition prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and is identified as CBH Id. Insofar as the modified microorganism is incapable of expressing CBH I, this cellulase composition is necessarily free of all CBH I type cellulase components.

The cellulase compositions described above were tested for their effect on cotton-containing fabric strength loss in a launderometer. The compositions were first normalized so that equal amounts of EG components were used. Each cellulase composition was then added to separate solutions of 400 ml of a.20 mM citrate/phosphate buffer, titrated to pH 5, and which contains 0.5 ml of a non-ionic surfactant. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate strength loss as well as a 16 inch x 20

inch cotton fabric (100% woven cotton, available as Style No. 467 from Test Fabrics, Inc., 200 Blackford Ave. , Middlesex, NJ 08846) . The canister was then closed and the canister lowered into the launderometer bath which was maintained at 43°C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

In order to maximize strength loss results, the above procedure was repeated twice more and after the third treatment, the cotton fabrics were removed and analyzed for strength loss. Strength loss was measured by determining the tensile strength in the fill direction ("FTS") using a Instron Tester and the results compared to the FTS of the fabric treated with the same solution with the exception that no cellulase was added. The results of this analysis are reported as percent strength loss which is determined as follows:

% Strength Loss = 100 x 1 - FTS with cellulase

FTS without cellulase

The results of this analysis are set forth in FIG. 12 which shows that compositions containing CBH I, i.e., whole cellulase (GC010) and CBH II deleted cellulase, possessed the most strength loss whereas, the compositions containing no CBH I possessed significantly reduced strength loss as compared to whole cellulase and CBH II deleted cellulase. From these results, it is seen that the presence of CBH I type components in a cellulase composition imparts increased strength loss to the composition as compared to a similar composition not containing CBH I type components.

Likewise, these results show that CBH II plays some role in strength loss.

Accordingly, in view of these results, strength loss resistant cellulase compositions are those compositions free of all CBH I type cellulase components and preferably, all CBH type cellulase components. In this regard, it is contemplated

that such cellulase compositions will result in even lower strength loss at pH _> 7 than those results observed at pH 5 shown in FIG 12.

In the following Examples 21 and 22 worn cotton T-shirts (knits) as well as new cotton knits are employed. The faded appearance of the worn cotton-containing fabric arises from the accumulation on the fabric of loose and broken surface fibers over a period of time. These fibers give rise to a faded and matted appearance for the fabric and accordingly, the removal of these fibers is a necessary prerequisite to restoring the original sharp color to the fabric. Additionally, the accumulation of broken surface fibers on new cotton knits imparts a dull appearance to such fabrics.

Example 21 Color Enhancement The ability of EG components to enhance color in cotton- containing fabrics was analyzed in the following experiments. Specifically, the first experiment measures the ability of a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type

Trichoderma longibrachiatum to remove surface fibers from a cotton-containing fabric at various pHs. This cellulase was tested for its ability to remove surface fibers in a launderometer. An appropriate amount of cellulase to provide for either 25 ppm or 100 ppm cellulase in the final composition was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated so as to provide for samples at pH 5, pH 6, pH 7 and pH 7.5. Each of the resulting solution was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100% woven cotton, available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) . The canister was then closed and the

canister lowered into the launderometer bath which was maintained at 43°C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

The so treated fabrics were then analyzed for fiber removal by evaluation in a panel test. In particular, the fabrics (unmarked) were rated for levels of fiber by 6 individuals. The fabrics were visually evaluated for surface fibers and rated on a 0 to 6 scale. The scale has six standards to allow meaningful comparisons. The standards are:

Rating Standard"

0 Fabric not treated with cellulase

1 Fabric treated with 8 ppm cellulase

2 Fabric treated with 16 ppm cellulase

3 Fabric treated with 20 ppm cellulase

4 Fabric treated with 40 ppm cellulase

5 Fabric treated with 50 ppm cellulase

6 Fabric treated with 100 ppm cellulase

a In all of the standards, the fabric was a 100% cotton sheeting standardized test fabric (Style No. 439W) available from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846 b All samples were treated with the same cellulase composition. Cellulase concentrations are in total protein. The launderometer treatment conditions are the same as set forth in Example 16 above.

The fabric to be rated was provided a rating which most closely matched one of the standards. After complete analysis of the fabrics, the values assigned to each fabric by all of the individuals were added and an average value generated.

The results of this analysis are set forth in FIG. 13. Specifically, FIG. 13 illustrates that at the same pH, a dose dependent response is seen in the amount of fibers removed.

That is to say that at the same pH, the fabrics treated with more cellulase stowed for higher levels of fiber removal as compared to fabrics treated with less cellulase. Moreover, the results of this figure demonstrate that at higher pHs, fiber removal can still be effected merely by using higher concentrations of cellulase.

In a second experiment, two different cellulase compositions were compared for their ability to remove fiber. Specifically, the first cellulase composition analyzed was a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma longibrachiatum and is identified as GC010.

The second cellulase composition analyzed was a cellulase composition substantially free of all

CBH type components (including CBH I type components) which composition was prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II and is identified as CBH I/II deleted. Insofar as CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG components.

These compositions were tested for their ability to remove surface fibers in a launderometer. An appropriate amount of cellulase to provide for the requisite concentrations of EG components in the final compositions was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100% woven cotton, available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) . The canister was then closed and the canister lowered into the launderometer bath which was

maintained at 43°C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

The so treated fabrics were then analyzed for fiber removal by evaluation in the panel test described above. The results of this analysis are set forth in FIG. 14 which is plotted on estimated EG concentrations. Specifically, FIG. 14 illustrates that both GC010 and CBH I/II deleted cellulase compositions gave substantially identical fiber removal results at substantially equal endoglucanase concentrations. The results of this figure suggest that it is the EG components which provide for fiber removal. These results coupled with the results of FIG. 13 demonstrate that EG components remove surface fibers.

Example 22 Tergotometer Color Enhancement

This example is further to Example 21 and substantiates that CBH type components are not necessary for color enhancement and the purpose of this example is to examine the ability of cellulase compositions deficient in CBH type components to enhance color to cotton-containing fabrics.

Specifically, the cellulase composition employed in this example was substantially free of all CBH type components (including CBH I type components) insofar as this composition was prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II. Insofar as CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG components.

The assay was conducted by adding a sufficient concentration of this cellulase composition to a 50 mM citrate/phosphate buffer to provide 500 ppm of cellulase. The solution was titrated to pH 5 and contained 0.1 weight percent of nonionic surfactant (Grescoterg GL100 — commercially

available from Gresco Mfg., Thomasvilie, N.C. 27360). A 10 inch x 10 inch faded cotton-containing fabric as well as a 10 inch x 10 inch new knitted fabric having loose and broken surface fibers were then placed into 1 liter of this buffer and allowed to sit at 110°F for 30 minutes and then agitated for 30 minutes at 100 rotations per minute. The fabrics were then removed from the buffer, washed and dried. The resulting fabrics were then compared to the fabric prior to treatment. The results of this analysis are as follows:

Cotton-Containing Material Result

Worn Cotton T-Shirt benefit seen Cotton Knit benefit seen

The term "benefit seen" means that the treated fabric exhibits color restoration (i.e., is less faded) as compared to the non-treated fabric which includes removal of broken surface fibers including surface fibers generated as a result of using the tergotometer. These results substantiate the results of Example 21 that the presence of CBH type components is not necessary for effecting color restoration of faded cotton-containing fabrics.

It is contemplated that the use of such cellulase compositions would be beneficial during fabric processing because such compositions would remove broken/loose fibers generated during processing without detrimental strength loss to the fabric.

Example 23 Softness This example demonstrates that the presence of CBH type components are not essential for imparting improved softness to cotton-containing fabrics. Specifically, this example employs a cellulase composition free of all CBH type components which composition is derived from Trichoderma longibrachiatum genetically engineered in the manner described

above so as to be incapable of producing CBH I and II components.

This cellulase composition was tested for its ability to soften terry wash cloth. Specifically, unsoftened 8.5 ounce cotton terry cloths, 14 inches by 15 inches (available as Style No. 420NS from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846), were cut into 7 inch by 7.5 inch swatches.

The cellulase composition described above was tested for its ability to soften these swatches in a launderometer. Specifically, an appropriate amount of CBH I and II deleted cellulase to provide for 500 ppm, 250 ppm, 100 ppm, 50 ppm, and 10 ppm cellulase in the final cellulase solution was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.025 weight percent of a non-ionic surfactant (Triton X114). Additionally, a blank was run containing the same solution but with no added cellulase. Samples so prepared were titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate softness as well as cotton swatches described above. All conditions were run in triplicate with two swatches per canister. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at 37°C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the swatches were removed, rinsed well and dried in a standard drier.

The swatches were then analyzed for softness by evaluation in a preference test. Specifically, six panelists were given their own set of swatches and asked to rate them with respect to softness based on the softness criteria such as the pliability of the whole fabric. Swatches obtained from treatment with the five different enzyme concentrations and the blank were placed behind a screen and the panelists were asked to order them from least soft to most soft. Scores were assigned to each swatch based on its order relative to the

other swatches; 5 being most soft and 0 being least soft. The scores from each panelists were cumulated and then averaged.

The results of this averaging are set forth in FIG. 15. Specifically, these results demonstrate that at higher concentrations, improved softening is obtained. It is noted that this improved softening is achieved without the presence of either CBH I or II in the cellulase composition.

Example 24 Feel and Appearance

This example demonstrates that the presence of CBH type components are not essential for imparting improved feel and appearance to cotton-containing fabrics. Specifically, this example employs a cellulase composition derived from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and II components) .

This cellulase composition was tested for its ability to improve the appearance of cotton-containing fabrics. Specifically, appropriately sized 100% cotton sheeting (available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the appearance aspects of this example.

The cellulase composition described above was tested for its ability to improve the appearance of these samples in a launderometer. Specifically, an appropriate amount of CBH I and II deleted cellulase to provide for 25 ppm, 50 ppm, and 100 ppm cellulase in the final cellulase solution was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.025 weight percent of a non-ionic surfactant (Triton X114) . Additionally, a blank was run containing the same solution but with no added cellulase. Samples so prepared were titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer

canister. Into these canisters were added a quantity of marbles to facilitate improvements in appearance as well as cotton samples described above. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at about 0 β C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the samples were removed, rinsed well and dried in a standard drier.

The samples were then analyzed for improved appearance by evaluation in a preference test. Specifically, 6 panelists were given the 4 samples (not identified) and asked to rate them with respect to appearance. The panelists were instructed that the term "appearance" refers to the physical appearance of the cotton-containing fabric to the eye and is determined in part, by the presence or absence of, fuzz, surface fibers, and the like on the surface of the fabric as well as by the ability or inability to discern the construction (weave) of the fabric. Fabrics which have little if any fuzz and surface fibers and wherein the construction (weave) is clearly discernable possess improved appearance as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weave.

The panelists then assigned scores were assigned to each sample based on its order relative to the other samples; 4 having the best appearance and 1 having the worst appearance. The scores from each panelists were cumulated and then averaged. The results of this test are as follows: Arot Cellulase Average Appearance None 1

25 ppm 2

50 ppm 3

100 ppm 4

The CBH I and II deleted cellulase composition was then tested for its ability to improve the feel of cotton- containing fabrics. Specifically, appropriately sized 100%

cotton sheeting (available as Style No. 39W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the feel aspects of this example.

The cellulase composition described above was tested for its ability to improve the feel of these samples in a launderometer. Specifically, an appropriate amount of cellulase to provide for 500 ppm, 1000 ppm, and 2000 ppm cellulase in the final cellulase solution was added to separate solutions of 24 L of a 20 mM citrate/phosphate buffer. Additionally, a blank was run containing the same solution but with no added cellulase. All tests were conducted at pH 5.8 and run in an industrial washer. The washer was operated at 50°C, a total volume of 24 L, a liquor to cloth ratio of 50:1 (weight to weight) and the washer was run for 30 minutes. Afterwards, the samples were removed and dried in an industrial dryer.

The samples were then analyzed for improved feel by evaluation in a preference test. Specifically, 5 panelists were given the 4 samples (not identified) and asked to rate them with respect to feel. The panelists were instructed that fabrics having improved feel are smoother and silkier to the touch than other fabrics and that feel is distinguished from qualities such as softness (which refers to the pliability of the fabric rather than its feel) , thickness, color, or other physical characteristics not involved in smoothness of the fabric.

The panelists then assigned scores to each sample based on its order relative to the other samples; 4 having the best feel and 1 having the worst feel. The scores from each panelists were cumulated and then averaged. The results of this test are as follows:

Amt Cellulase Average Feel None 1.5 ± 0.5 500 ppm 1.7 ± 0.4 1000 ppm 3.2 ± 0.4 2000 ppm 3.8 ± 0.4

The above results demonstrate that improvements in feel and appearance can be achieved with cellulase compositions free of all CBH type components.

Example 25

Reduction in Lint Production in Treatment of Denim pants

Desized denim pants (3.8 kg) were washed under the following conditions:

Unimac Washer

20 mM citrate/phosphate buffer pH 4.9

40 L total volume

55 β C

3.8 kg of desized indigo dyed denim pants

1 hour run time at 36 rpms

19 ml of 130 g/1 whole cellulase [Indiage 44L,

Genencor International, Inc.] or 12 ml of a 100 g/1 of EGI cellulase expressed by the pEGIpyr4 transformant prepared by the method of

Example 14.

In this regard, each of the cellulase compositions employed were dosed to give equal stone-washing performance and, in the case of the EGI expressed by pEGIpyr4 transformants, this cellulase actually contained more EG components than whole cellulase.

The garments were rinsed according to a standardized protocol in three consecutive cycles of clean liquor. Rinse ≠l — 24 gallons hot water, approximately 50 β C, plus -100 grams standard detergent WOR (from American Association of Textile Chemists and Colorists [AATCC] , WOB - without brighteners) . Agitation was for 12 minutes at 36 rpms. The

bath was dropped. Rinse §2 -- 24 gallons warm water, -40 β C, with no additional detergents, agitated for 5 minutes. The bath was dropped. Rinse #3 — 24 gallons cold water, -30 β C, with no additional detergents, agitated for 5 minutes. The bath was dropped. Garments were extracted and dried in a standard electric clothes dryer for 70 minutes.

The lint screen of the dryer was thoroughly cleaned prior to drying the denim pants. After the pants were dried, the lint was removed from the lint screen and weighed on a tared weigh boat.

This was repeated four times and the results of four tests are set forth below.

Weight of Lint (gms) _I II III IV

Whole Cellulase 0.35 0.41 0.41 0.36 EG I 0.35 0.27 0.24 0.24

The results indicate that, in this test, the use of enriched EG I cellulase produces on average about 27% less lint during treatment of denim pants than does the use of whole cellulase.

Example 26

Reduction in Weight Loss in Treatment of Cotton Knits

Three different types of cotton tubular knit fabric were treated with different cellulase compositions in an industrial setting to determine the effect the cellulase compositions employed had on weight loss. Specifically, the fabrics were treated under the following conditions:

50-55 β C

20 mM citrate/phosphate buffer pH 4.9

Machine: Jet

Load Size: 40 kg

Liquor Ratio: 20:1

Whole T. longibrachiatum cellulase (PRIMAFAST

100, Genencor International, Inc.): 1.5 g/1 of treatment solution Enriched EG I cellulase: 3 g/1 of treatment solution

Enriched EG I is the supernatant from a pEGIpyr. transformant prepared by the method disclosed in Example 14 and contains substantially more EG components than whole cellulase. The following results were obtained:

Article Weight loss

Whole Cellulase EG I

1001 6-8% 2-4%

700 8-10% 2-4%

1510 5-7% 1-3%

The feel and appearance of the whole cellulase treated and EG I treated fabric were compared. It was determined that EG I enriched cellulase provided as good an appearance as could be achieved with an equivalent amount of whole cellulase. The EGI treated fabric had a softer feel as compared to treatment with whole cellulase. However, the amount of fabric weight loss was less with EGI enriched cellulase.

A second test to determine weight loss was conducted at another time under the following conditions:

Fabrics: Burlington Interlock, scoured or bleached Brazoli Jet 50-55°C 30 minutes

20 mM citrate/phosphate buffer pH 4.9 2 g/1 whole T. longibrachiatum Cellulase

(PRIMAFAST 100, Genencor International, Inc.) 2 g/1 enriched EG I cellulase

The results are presented in the following table:

Scoured Fabric Bleached Fabric

Weight % Loss Weight % Loss (g/sq. yd.) (g/sq. yd.)

Before 6.9 6.1

Complete 6.5 6% 5.8 4.9% cellulase

Enriched 6.9 0% 6.0 1.6% EG I Cellulase

The results indicate that treatment of cotton knit fabric with enriched EG I cellulase results in reduced weight loss as compared to treatment with whole cellulase.

Preferably, upon completing treatment of the cotton- containing fabric with cellulase, the fabric is then treated in a manner to remove and/or inactivate the cellulase. One method of removing the cellulase is by thoroughly rinsing the so treated fabric with a cellulase free aqueous solution (i.e., an aqueous solution containing no cellulase). In such an embodiment, the cotton-containing fabric is then dried at elevated temperatures to inactivate any cellulase remaining. Alternatively, the cotton-containing fabric is first treated to inactivate the cellulase by heating to sufficiently high temperatures (i.e., at temperatures from about 75°C or more, e.g., 75° to 125 β C) for a sufficiently long period of time to inactivate the enzyme. In this embodiment, after inactivation, the cotton-containing fabric can subsequently be thoroughly rinsed and dried. Additionally, subsequent treatment steps for the cotton-containing fabric treated by the methods of this invention can further inactivate any active cellulase remaining either by the use of treatment conditions and/or reagents which deactivate cellulase.

Example 27 Enhanced properties of non-cotton-containing cellulosic fabrics This example demonstrates the ability of EG cellulase composition to enhance appearance, softness and surface polishing of non-cotton-containing cellulosic fabrics. A 200 kg Jet Dyer machine was used to evaluate the enhanced properties of the non-cotton-containing cellulosic fabric TENCEL™. Approximately 10 kg of 100% TENCEL™ mid-weight woven fabric was loaded into the machine in rope form and sewn end- to-end. This process may be performed on greige or dyed fabric. The jet machine was filled with 150 - 200 liters of water (which represents approximately 15-20:1 liquor to fabric ratio) and heated to 120 - 140OF (50O - 60OC) . The pH was adjusted to 4.5 - 5.0 by the addition of 3.6 g/1 (56%) acetic acid and 1.9 g/1 (50%) sodium hydroxide. The sodium hydroxide was added slowly to a dilute acetic acid solution before putting into the machine. Next, 0.25 - 0.5 ml/1 of a nonionic wetting agent (Triton X-100) was added to the liquor. The pH and temperature was checked to ensure that the pH was between 4.5 and 5.0, and the temperature was between 500 - βooc. Next, 3 - 4 g/1 of an enriched EG cellulase composition was added. The enriched EG cellulase composition comprised a cellulase composition free of all CBH type components, which composition is derived from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing CBH I and II components and over¬ produces EG I. After adding the enriched EG cellulase composition, the jet was run for 30 - 60 minutes. At the end of the cycle, 0.25 g/1 soda ash was added to the liquor and run for 10 minutes. The liquor was dropped from the jet, then the jet was filled again with water and the fabric rinsed one more time. The fabric was removed from the jet and dried. Finally, a silicone-based finish was exhausted onto the fabric.

Swatches were analyzed for softness and surface appearance by evaluation in a preference test. Specifically,

four panelists were given their own set of swatches and asked to rate them with respect to softness and surface appearance. Softness was based on the softness criteria such as pliability of the whole fabric. Surface appearance was based on the amount of loose fibers or fuzz present on the fabric. Swatches were compared to a non-enzyme treated fabric control and in the measurement of softness, an additional control was included i.e. a fabric treated with a complete fungal cellulase composition. Scores were assigned to each swatch and the average score was tabulated from the four panalists. The highest score for softness and surface appearance was assigned the value 5.0. The lowest score for least soft and most fuzz was assigned the value 0. The results of this averaging are set forth in FIG. 16 and 17. Specifically, these results demonstrate that softness and surface appearance were both improved following EG cellulase treatment. Additionally, the surface appearance of the TENCEL™ fabric was maintained following 10 home launderings whereas the control fabrics' surface appearance declined substantially.

An additional comparison of the EG enriched cellulase composition treated TENCEL™ fabric was compared to whole cellulase treated TENCEL™ fabric (FIG. 16). In this example, swatches were analyzed for softness by evaluation in a preference test. Four panelists were given their own set of swatches and asked to rate them with respect to softness. Softness was based on the above-mentioned criteria and panel score scale. Swatches were compared to a whole cellulase treated fabric control. Scores were assigned to each swatch and an average score was tabulated from the four panelists. The results of this averaging are set forth in FIG. 16. Specifically, these results demonstrate that EG enriched cellulase treated TENCEL™ fabric was on average softer than the whole cellulase treated fabric control.

Example 28 on-cotton-containing cellulosic fabric showing less lint

The example demonstrates the ability of an enriched EG cellulase composition to enhance appearance and softness while generating less lint. A 45 kilogram rotary drum washing machine was used to generate the non-cotton-containing cellulosic fabrics which were evaluated for enhanced properties with accompanying lower lint generation following EG enriched cellulase treatment. In this example, approximately 20 kilograms of a light weight (4.5 ounce per sq. yd) , 100% TENCEL™ woven fabric was loaded into the machine. The rotary drum machine was filled with 300 liters of water representing a 15:1 liquor to fabric ratio and maintained at a temperature of 50 C. The fabric was run in the machine for 45 minutes. Then, the pH was adjusted to 5.0 by the addition of 3.6 grams per liter of (56%) acetic acid and 1.9 grams per liter (50%) sodium hydroxide. 0.5 milliliters per liter of a nonionic wetting agent was added to the liquor. 2 grams per liter of an enriched EG cellulase composition was added to the machine. The EG enriched enzyme is comprised of a cellulase composition free of all CBH type components, and is derived from Trichoderma longibrachiatum genetically engineered in a manner described above so as to be incapable of producing CBH I and II components and overproduces EG I (See Example 14). After adding the enriched EG cellulase composition, the rotary drum machine was run for 45 minutes. At the end of the cycle, 0.25 grams per liter of soda ash was added to the liquor and the machine was run an additional 10 minutes. The liquor was drained from the machine and the fabric was rinsed twice before being dried. A whole cellulase treated fabric control was run under identical conditions with an identical dose (2 grams per liter whole Trichoderma longibrachiatum cellulase from Genencor International Inc. called PRIMAFAST™ 100) .

Swatches were analyzed for softness and appearance by evaluation in a preference test. The enriched EG I cellulase

composition provided equal or improved softness and satisfactory appearance compared to the whole cellulase by this evaluation. An additional panel rating the amount of lint present on the dried fabric was completed. Three swatches were analyzed in this panel. In the panel score for lint a rating of 5 indicated the least amount of visible lint and a rating of 0 indicated the highest level of visible lint. The results are set forth in the following table. Specifically, these results demonstrate that EG enriched cellulase treatment imparted enhanced fabric properties while generating less lint versus a whole cellulase treated fabric control.

Panel

1 2 3

Whole Cellulase 0.5 0.5 0.5

EG I Enriched 1.5 1.5 2

Example 29 Non cotton-containing cellulosic fabric showing less strength loss A 100 kg jet dyer machine may be used to generate the non- cotton-containing cellulosic fabrics to evaluate for enhanced appearance, softness and surface-polishing properties of non- cotton-containing cellulosic fabrics along with corresponding fabric strength following EG enriched cellulase treatment. In this example, a non-cotton-containing cellulosic fabric such

TM as mid-weight, 100% TENCEL woven fabric maybe loaded into the machine in rope form and sewn end-to-end. The jet machine maybe filled with approximately 600 liters of water representing a liquor to fabric ratio of approximately 15:1. The liquor maybe heated to 50 - 55 C and the pH was adjusted to 5.0 by the addition of 3.6 grams per liter of (56%) acetic acid and 1.9 grams per liter of (50%) sodium hydroxide. Next, 0.5 % weight to volume of a nonionic wetting agent may be was added to the liquor. 3 - 4 grams per liter of an enriched EG

cellulase composition is added to the machine. The enriched EG cellulase comprise a cellulase composition free of all CBH type components and derived from Trichoderma longibrachiatum genetically engineered in a manner described above so as to incapable of producing CBH I and II components and overproduces EG I (See Example 14) . After adding the enriched EG cellulase composition, the jet is run for 60 minutes. At the end of the cycle, 0.25 grams per liter of soda is added to the liquor and the machine run an additional 10 minutes. The liquor is drained from the jet and refilled to rinse the fabric. The fabric is rinsed a second time before being removed from the jet and dried. Lastly, a silicone-based finish is applied to the fabric. A whole cellulase treated fabric control is run under identical conditions with an identical dose of enzyme.

Swatches are analyzed for softness and appearance by evaluation in a preference test (See Example 27) . Corresponding fabric tensile strengths are measured in accordance with the AATCC procedure to measure the breaking force and elongation of textile fabrics (Grab test- ASTM D 5034) . It is expected that treatment of the non-cotton cellulosic fabric with an EG I enriched cellulase composition will provide the enhancing properties (e.g., appearance, softness and/or surface polishing) as previously found in Example 27 with the additional property of lowered fabric strength loss versus whole cellulase treatment of non-cotton- containing cellulosic fabrics.