CA2107206A1 - Methods for treating cotton-containing fabrics with cellulase - Google Patents

Abstract

Disclosed are improved methods for treating cotton-containing fabrics as well as the fabrics produced from these methods. In particular, the disclosed methods are directed to contacting cotton-containing fabrics with an aqueous solution containing a fungal cellulase composition which comprises one or more EG type components and one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH I type components of greater than 5:1. Cotton-containing fabrics so treated possess decreased strength loss as compared to fabrics treated with a cellulase composition containing greater amounts of CBH I type components.

Description

D 92/17574 2 :1 ~ 7 2 U 6 Pcr/usg2/o263l M15T~OD8 FOR TREATING COTTON-CONTA~NING
FAB~IC8 ~IT~ CEI.LlJI~a8E

BACRG}I,O~JND OF ~E IN~ENTION

1. Field of the Invention.
The present invention is directed to improved methods for treating cotton-containing fabrics with cellulase as well as to the fabrics produced from these methods. In particular, the improved methods of the present invention are directed to contacting cotton-containing fabrics with an aqueous solution containing a fungal cellulase compositio~ which comprises one or more EG type components and which contains low concentrations of CBH I type components. When the cotton-containing fabric is treated with such solutions, the resulting fabric possesses the expected enhancements in, for example, feel, appearance, and/or softening, etc., as compared to the fabric prior to treatment and the fabric also possesses decr~ased strength loss as compared to the fa~ric treated with a cellulase composition containing higher concentrations of CBH I type components.

2. State of the Art. -- -D~ring or shortly after their manufacture, cotton-containing fabrics can be treated with callulase in order to impart desirable properties to the fabric. For exa~ple, in the textile industry, cellulase has been used to improve the feel and~or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, for 21 0 7? Ofi - 2 -WOg2/17574 PCT/US92/02631 -~

imparting 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 cellulase results in an improved feel for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing and/or surface fibers which reduces the weight of the f~bric. The combination of these effects imparts improved feel to the fabric, i.e., the fabric feels -more like silk.

Additionally, it was here~ofore known in the art to treat cotton-containing knitted fabrics with a cellulase solution under agitation and cascading conditions, for example, by use of a jet, for the purpo~e of removing broken fibers ~nd threads common to the~e 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 heretofore known in the 2~ art to treat cotton-containing woven fabrics with a cellulase solution under agitation and cascading conditions. When so treated, the cotton-containing woven fabric possesses improved feel and appearance as compared to the fabric prior to treatment.

~ .
, ~ ~

- 3 - ~lO~S
~92/17S74 PCTiUS92/02631 Lastly, it was also heretofore known that the treatment of cotton-containing dyed denim with 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 a cellulase solution is that the treated fabrics exhibit significant strength loss as compared to the untreated fabric. Strength loss arises because the cellulase hydrolyzes cellulose (B-l,4-glucan linkages) which, in turn, can result in a breakdown of z portion of the cotton polymer. As more and more cotton polymers are disrupted (brokendown), the tensile strength of the fabric is reduced.

Because methods involving agitation and cascading of cellulase solutions over cotton woven fabrics require shorter rea~tion times, these methods are believed to provide cotton-containing woven fabrics of reduced strength loss as compared to cellulas~ treatment methods not involving agitation and cascading. In any event, such methods still nevertheless result in significant strength .25 105s.

Accordingly, it would be particularly d~sirable to ~odify such cellulase treatment methods so as to pro~ide reduced strength loss while still achieving the desired enhancements in the treated cotton-containing fabri~ arising from treatment with 21 !37 '~ 4 ~

cellulase as comp~red to the fabric prior to treatment.

Additionally, because fungal sources of cellulase are known to secrete very large quantities of cellulase and further because fermentation procedures far such fungal sources as well as isolation and purification prooedures for isolating the cellulase are well known in the art, it would be particularly advantageous to use such fungal cellulases in the methods for improving feel and/or appearance.

~MNARY OF T~E INVENTSON

The present invention is directed to the discovery that heretofore known methods for treating cotton-containing fabrics with fungal cellulases can :
be improved by employing a fungal cellula~e composition which comprises one or more EG type components and which contains sufficiently low concentrations of CBH I. Surprisingly, it has been found that EG type components ara capable of imparting enhancements to the treated fabric with regard to feel, ppearance, softness, color enhancement, snd/or a stone washed appearance as ~
cGmpared to fabric before treatment wi~h such a cellulase composition. Additionally, it has been found that it is the CBH I type components in combination with the EG type components which account for a sizable portion of the strength loss 3Q in the treated fabric. Accordingly, in the present invention, the cellulase composition employed to treat cotton-containing fabrics is tailored so as to - s - 2~072 06 :~92/17574 PCT/US92/02631 contain sufficiently low concentrations of CBH I
type components so as to be strength loss resistant.

In view of the above, in one of its method aspects, the present invention is directed to an improved method for the treatment of cotton-containing fabrics with a fungal cellulase composition wherein said improvement comprises employing a fungal cellulase composition which comprises one or more EG type components and one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all EG
type components to all CBH I type components of gre~ter than 5:l. In a preferred embodiment, the fungal cellulase composition employed herein comprises one or more EG type components and one or more CBH type components wherein said cellulase : composition has a protein weight ratio of a~l EG
type components to all CBH type components of greater than 5:l. In still another preferred embodiment, the fungal cellulase composition comprises at least about lO weight percent and preferably at l~ast about 20 weight percent of EG
components baaed on the total weight of protein in the cellulase composition.

I~ another of its method aspects, the present invention is direct~d to an improved method for the treatment of cotton-containing fabrics with an aqu~ous fungal cellulase solution wherein said method is conducted with agitation of the cellulase solution under conditions so as to produce a cascading effect of the cellulase solution over the fabric w~erein said improvement comprises employing ~ .

21 ~720~ - 6 - ,~, W092/l7s7~ PCT/USg2/02631 a fungal cellulase composition which comprises one or more EG type components and one or more CBH I
type components wherein said cellulase composition has a protein weight ratio of all EG type components s to all CBH I type components of greater than 5~
In a preferred embodiment, the fungal cellulase composition employed herein comprises one or more EG
type components and one or more CBH 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. In still another preferred embodiment, the fungal cellulase composition comprises at least about 10 weight percent and preferably at least about 20 weight percent of EG components based on the total weight of protein in the cellulase composition.

Cotton-containing fabrics treated by the ;~ ~ethods of this invention have the expected ~' enhancement(s) as compared to the fabric prior to treatment while exhibiting reduced strength loss as compared to the fabric treated with a fungal cellulase composition containing greater amounts of CBH I type components. The reduced strength loss evidences that t,he methods of this invention are strength loss resistant. '~

In its composition aspects, the present invention is directed to a cotton-containing fabric treated in the methods of this invention as defined above.

_ 7 _ 2107206 ? ` ~) 92~17574 PCl`/US92/02631 B~IEF DE8CRIPTION OF l~E DRAWING~;

FIG. 1 is an outline of the construction of p~CB~I~r4.

FIG. 2 illustrates deletion of the T. reesei gene by integration of the larger EcoRI fr~gment from p~CBHIEyE~ at the Çkh~ locus on one of the T.
reesei chromosomes.

FIG. 3 is an autoradiograph of DNA from 1~
reesei strain GC69 transformed with EcoRI digested p~CBHIEyE~ after Southern blot analysis using a 32p labelled pACBHIEyE~ as the probe. The sizes of molecular wei~ht markers are shown in kilobase pairs to the left of the Figure.
o ~ FIG. 4 is an autoradiograph of DNA from a T.
reesei strain GC69 transformed with EcoRI digested p~CBHIpYr4 using a ~P labelled pIntC~HI as the probe~ The sizas 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 tran formed strains of T. Eeesei.
Spe~i~ically, in FIG.5, Lane A of the isoelectric focusing gel employs partially purified CBHI from T.
reesei; Lane B employs a wild type T. eesei : Lane C employs protein from a ~ reesei strain with the cbhl gene deleted; and Lane D employs protein from a reesei strain with the Ç~hl and b~2 genes deleted. In FIG. 5, the right hand side of the figure is marked to indicate the location of the ~ ~ 072 0 ~ - 8 -4 PC~/US92/02631 single proteins. found in one or more of the secreted .:
proteins. Specifically, BG refers to the ~- -glucosidase, El refers to endoglucanase I, E2 refers to endoglucanase II, E3 refers to endoglucanase III, Cl refers to exo-cellobiohydrolase I and C2 refers .
to exo-cellobiohydrolase II.

FIG. 6A is a representation of the T. ~Ç~Çi çbh2 locus, cloned as a 4.l kb EcoRI fragment on genomic DNA and FIG. 6B is a representation of the c~h2 gene deletion vector pP~CBHII.

FIG. 7 is an autoradiograph of DNA from T.
reesei strain P37P~CBHIPyr~26 transformed with EcoRI
digested pP~CBHII after Southern blot analysis using a 32p labelled pP~CBHII 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 pEGIEyE~.

FIG. 9 illustratas ~he RBB-CNC ~ctivity profile of ~n acidic EG enriched fungal cellul~se composition (CBH I and II deleted) derived from Trichode~ma reesei over a pH range at 40C; as well as the activity profile of an ~nriched EG III ---cellulnse co~position derived from Trichoderma rees~i over a pH range at 40C.

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

n72o6 F~G. ll ill~strates fiber removal results (based on panel test scores) for cotton-containing fabrics treated with cellulase secreted by a wild type Trichoderma reesei (whole cellulase) at various pHs.

FIG. 12 illustrates fiber removal results (based on panel teit scores) for cotton-containing fabrics treated with varying concentrations (in ppm~
of cellulase secreted by a wild type Trichoderma reesei and for a cotton fabric treated with cellulase secreted by a str~in of Trichoder~a reese:L
genetically engineered so as to be incapable of se~reting CBH I and CBH II.

FIG. 13 illustrates the softness panel test re~ul~s for varying concentration~ ~in ppm) of an EG
enriched cellulase composition derived from a strain of Tricho~crma reesei genetically modified so as to be incapable of producinq CBHI&II.

FIG. 14 is a diagram of the site specific alterations made in ~he eqll and c~kl genes to create convenient restriction endonuclease cleavage sites~ Tn each c~se, the upper line shows the original DNA sequence, the changes introduced are shown in the ~iddle line, and the new sequence is shown in the lower line.

FIG. l5 is a diagram of the larger EcoRI
fragment which can be obtained from pCEPCl.

FIG. 16 is an autoradiograph of DNA, from an untransformed strain of T. ree$ei RutC30 and from 2107206 - lO-WO92/17~74 PCT/US92/02631 two transformants obt~ined by transforming ~ re~esei with EcoRI digested pCEPCl. The DNA was digested with ~stI, a Southern blot was o~tained and hybridized with 32p ~abelled pUC4K::cbhl. The sizes of marker DNA fragments are shown in kilobase pairs to the left of the Figure.

FIG. 17 is a diagram of the plasmid pEGlI::P-l.

FIG 18. is an autoradiograph of DNA from T.
reesei strain P37P~67P-l transformed with ~i~dIII
and BamHI digested pEGII::P-l. A Southern blot was prepared and the DNA was hybridized with an approximately 4kb PstI fragment of radiolabelled T.reesei DNA containing the ~gi3 gene. Lanes A, c and E contain DNA from the untransformed strain whereas, Lanes B, D ~nd F contain DNA from the untransformed T. reesei strain. The T.reesei DNA
was digested witb ~glII in Lanes A and B, with ~coRV
in Lanes C and D and with PstI in Lanes E and F.
The size of marker DNA fragments are shown in kilobase pairs to the left of the Figure.

FIG. l9 is a diagram of the plasmid pP~EGI-l.

FIG. 20 is an autoradiograph of a Southern-blot of DNA i~olated from trnnsformants of strain GC69 obtPined with ~i~dIII digested p~EGIpyr-3. ~he pattern of hybridisation with the probe, radiolabelled p~EGIpyr-3, expected`for an untransformed strain is shown in Lane C. Lane A
shows the pattern expected for a transformant in which the çgll gene has been disrupted and Lane B
shows a ~ransformant in which pAEGlpyr-3 DN~ has ~ -11 2l0~2n6 ~`)92/17574 PCT/US92/02631 integrated into the genome but without disrupting the Çgll gene. Lane D contains p~EGIpyr-3 digested with ~indIII to provide appropriate size markers.
The sizes of marker DNA fragments are shown in kilobase pairs to the right of the figure.

DBTAILED DB8CRIPTION OF ~E PREFERRED EMBODIMENT8 As noted above, the methods of this invention are improvements in prior art methods $or treating cotton-containing fabrics with cellulase. The improvement comprises using a specific cellulase composition which imparts the desired enhancement(s) to the fa~ric while minimi~ing strength loss in the fabric. 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 and the like. When cotton blends are employed, the amount of cotton in the fabric should be ~t least about 40 percent by weight cotton; prPf erably ~ more than about 60 percent ~y 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 ~nd nylon 66), acrylic fibers (for example, polyacrylonitrile fibers), and polyester fibers (for example, polyethylene terephthalate), polyvinyl alcohol fibers (for ~1072~ - 12 -example, Vinylon), polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers and aramid fibers. It is contemplated that regenerated cellulose, such as rayon, could be used as a substitute for cotton in the methods of this invention.

The term "finishing" as employed herein means the application of a sufficient amount of finish to a cotton-containing 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, lS etc~, which additionally protects the fabric from reaction with cellulases. Finishes useful for finishing a cotton-containing fabric are well known in the art ~nd include resinous materials, such as melamine, glyoxal, or ureaformaldehyde, as well as waxes, silicons, fluorochemicals and quaternaries.
When so finished, the cotton-containing fabric is su~stantially less reactive to cellulase.

The term "fungal cellulase" refers to the enzyme c~mposition derived from fungal sources or microorganisms genetically modified so as to inc~rporate and express all or part of the cellulase genes obtained from a fungal Rource. Fungal cellulases act on cellulose and its derivatives to hydrolyze cellulose and give primary products, glucose and cellobiose. ~ungal cellulases are distinguished from cellulases produced from - 13 _ 21 072 05 ~092/17~74 PCT/VS92/02631 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. 2 094 826A, 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 ~he l~ke. Addltionally, enzyme ~omponents within ea~h classification can exhibit different molecular weights, different degrees of glycosylation, different isoelectric points, different substrate æpecificity etc. For example, fungal cellulases can contain cellulase classificationæ which include endoglucanases (EG~), exo-cellobiohydrolaæes (CBHs), ~-glucosidases (BGs), etc. on ~he other hand, while bacteri~l cellul~es 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 bean reported to possess exo-cellobiohydrolase activity.

2 0 ~ 4 -W092/17S74 PCT/VS92/02631 ~`

A fungal cellulase composition produced ~y a naturally occurring fungal source and which comprises one or more CBH and 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 ~ome 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 CB~
~5 and/or EG components of cellulase.

The fermentation procedures for culturing fungi for production of cellulase are known EÇE se in the art. For example, cellulase systems can be produced either by solid or ~ubmerged 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 er se in the art.

NEndoglucana~e (nEG~) type components" refer to 2~ all of those fungal cellulase components or combination of compcnents which exhibit textile activity properties similar to the endoglucanase components of ~ichode~a reesei. In this reqard, the endoglucanase components of TTichoderma reesei (specifically, EG I, EG II, EG III, and the like either alone or in combination~ impart improved feel, improved appearance, softening, color - 1S 2t~7206 ~'092/17~74 PCT/US92/02631 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 ~sçi results in less strength loss as compared to the strength loss arising from treatment with a similar composition but which additionally contains CBH I
type components.

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

Such endoglucana~e type components may not~-include components traditionally cl~ssified as endoglucanases using activity te~ts such as the ability of the component (a) to hydrolyze soluble cellulose derivatives æuch 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 21~7206 - 16 ~

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 S activity tests, will impart one or more of the enhancements ~o cotton-containing fabrics as well as r~duced ~trength 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 reesei.

Fungal cellulases can cont~in more than one EG
type component. The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificity, different enzymatic action patterns, etc. The d~fferent isoelectric points of the components allow for their ~eparation via ion exchange chromatography ~nd 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. Serial No.
07/422,814, Schulein et al., International 2~ Application WO 89/09259, Wood et al., Biochemistry and Genetics of Cellulose Degradation, pp. 31 to 52 tlg88); Wood et al., Carbohydrate Research, Vol.
l90, 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.

;~092/17574 - 17 ~ 2 ~ O ~ 2 0 6 PCT/US92/02631 In general, it is contemplated that combinations of EG type components may give a synergistic response in imparting enhancements to the cotton-containing fabrics as well as imparting reduced strength loss as compared to a sinqle EG
component. 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.
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 contemplated that EG type components can be derived from bacterially derived cellulases.

"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 reesei. In this regard, when used in the absence of EG type cellulase components (as defined above), the CBH I and CBH II components of Trichode~ reesçi alone do not impart zny ~~
significant enhancements in feel, appearance, color enhancs~ent and/or ~to~e washed appearance to t~e so treated cotton-containing fabrics. Additionally, when used in combination with EG type components, the CBH I co~ponent of Trichoderma reesei imparts enhanced strength loss to the cotton-containing fabrics.

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 S Trichoderma reesei, respectively. As noted above, for CBH I type components, this includes the property of enhancing strength loss of cotton-containing fabrics when used in the presence of EG
type components. In a preferred embodiment and when ..
used in combination with EG type components, the CBH I type components of Trichoderma reesei can impart an incremental cleaning benefit.
Additionally, it i8 contemplated that the CBH I
components of Trichoderma Feesei, when u~ed alone in or in co~bination with EG type components, can impart an incremental softening benefit.

Such ex~-cellobiohydrolase type components could possibly not include components traditionally classed as exo-cellobiohydrol~ses using activity tests such as those used to characterize CBH I and CBH II from Trichoderma reesei. For example, such components (a) are compet~tively inh~bited by cellobiose ~ 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 - 19 - ~107205 `"092/17~74 PCT/US92/02631 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 posses~ed by the endoglucanase components of Tric~oderma ~qçsei.

In regard to the d~tergent compositions containing cellulase compostions which are CBHI
deficient, CBHI enriched or EGIII enriched, 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 detergent properties to cotton-containing fabrics, eg., one or more of improved color restoration, improved softening and improved cleaning to the detergent composition.

Fungal cellulase compositions having one or more EG type components and one or more CBH I type components wherein ~aid cellulase composition has a protein weight ratio of all EG type components to all CBH I type components of greater than 5:l can be obtained by puri~ication techniques. Speci~icaliy, the co~plete cellulase ~ystem can be purified into substantially pure componen~s by recognized separation techniques well published in the literature, including ion exchange chromatography at a ~uitable pH,-a~finity chromatography, size ~0 exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the ~ t n7295 W092/17~74 PCT/US92/02631 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 o~
cellulase components having the requisite ratio of EG type components to CBH I type cellulafie components could be prepared by means other than i601ation 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 to CBH
components. Likewise, recombinant techniques can alter the relative ratio of EG type components to ~5 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, a preferred method for the preparation of cellulase compositions described herein is by genetically modifying a microorganism So ~8 to overproduce one or more acidic EG type components. Likewise, it is also possible to genetically modlfy a microorganism so as to be incapable of producing one or more C~H type --components which methods do not produce any heterologous protein. In such a ca~e, a requisite amount of the cellulase produced by such modified microorganism could be combined with the cellulase produced by the natural microorganism (i.e., containing CBH I type components) so as to provide for a cellulase composition containing one or more EG type components and one or more CBH I type - 21 ~ ~ Q7~ 06 -`
- ~92/17574 PCT/US92/02631 components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH I ty~e components of greater than 5:1.

In regard to the above, U.S. Serial No.
s 07/593,919, filed October 5, 1990 and which is incorporated herein by reference in its entirety, discloses methods for genetically engineering Trichoderma reesei so as to be incapable of producing one or more CBR components and/or overproducing one or more EG components. Moreover, the methods of t~at application create T~ichoderma reesei strains which do not produce any heterologous proteins. Likewise, Miller et al., "Direct and Indirect Gene Replacement in Asper~illus nidulans", Molecular and Cellular Biology, p. 1714-1721 (1985) discloses methods for deleting genes in AsDeraillus nidulans by DNA mediat~d 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 andtor C8H II
type cellulase components would have the effect of enriching the ~mount of EG components present in~the ~
cellulase composition.

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

2~7~0~ - 22 -WO92/17574 PCT/USg2/02631 Additionally, a requisite amount of one or more CBH I 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 I type components so as to achieve a specified ratio of EG
type components to CBH I 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 I type component (or CBH II type component) merely by adding this amount of a purified CBH I type component (or CBH II type component) to the cellulase composition.

"B-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 t"cellobio~e") 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 (~ approximately lmM). While in a ~trict sense, BG c~ponents are not li~erally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because th~se enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products tparticularly cellobiose) produced by the combined action of C8H
components and EG components. Without the presence Or BG components, moderate or 11ttle hydrolysis Or .

- 23 - 2~
'092/17~74 PCT/US92/02631 crystalline cellulose will occur. 8G components are often characterized on aryl substrates such as p-nitrophenol B-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 thst the presence or absence of BG components in the cellulase composition can be uæed 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 activi~y, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG
components in the cellulase composition will Hturn-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 Nturn-off", directly or indirectly, some or all of the CBH I
type activity as well as other CBH activity. When 6uch additives are employed, the resulting co~position is considered to be a composition --suitable for use in this in~ention if the amount of additiYe employed is kufficient to lower the CBH I
type activity to levels equal to or less than the CBH I type activity levels achieved by using ~he cellulase compositions describad herein.

On the other hand, a cellulase composition containing added amounts of BG components may W092/17s7~ 10 7 ?. ~ ~ - 24 - PCT/US92/02631 '`~

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 disclo~ed in U.S. Serial No. 07/625,140, filed December l0, l990, as attorney docket no. 010055-056 and entitled ~SACCHARIF~CATION OF CELLUL0SE BY
CLONING AND AMPLIF~CATION OF THE ~-GLUCOSTDASE GENE
OF TRICHODERMA REESEI", which application is incorporated herein by reference in its entirety.

Fungal cellulases can contain more than one BG
component. The different components generally have different isoelectric points which allow for their ~eparation 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 8G 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 dep~nds upon the amount of cellobiose produced in the textile composition which can be readily determined by the skilled artisan. However, when employed, the weight percent of BG component relative to any CBH type components present in the cellulase composition is preferably from about 0.2 to about l0 weight percent and more preferably, from about 0.5 to about 5 weight percent.

- 25 _ 7~072~6 ~092/17574 PCT/US92/02631 Preferred fungal cellulases for use in preparing the fungal cellulase compositions used in this invention are those obtained from Trichoderma reesei, Trichoderma koninqii, Pencillum 8p., Humicola nsolens, and the like. Certain fungal cellulases are commercially available, i.e., CELLUCAST (available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist ~rocades, N.V., 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 lS acid/base reagents which stabilize the cellulase solution ~gainst undesired pH shifts during the cellulase treatment of the cotton-containing fabric.
In this regard, it is ~rt recognized that cellulase activity is pH dependent. That is to say tha~ a specific cellulase composition will exhibit cellulolytic activity within a defined pH r~nge with : optim~l cellulolytic activity generally being found within a small portion of this defined range. The specific pH range for cellulolytic acti~ity will vary with each cellulase composition. As noted - -above, while ~ost cellulases will exhibit cellulolytic activity within an acidic to neutral pH
profile, there are some cellulase compositions which exhibit cellulolytic actiYity in an alkaline pH
pro~ile.

During cellulase treatment of the cotton-containing fabric, it is possible that the pH of the 2107?,0~ - 26 -W092/l7574 PCT/US92/02631 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 reguired for cellulolytic activity. When this occurs, undesired reduction or ce~ation of cellulolytic activity in tbe cellulase ~olution occurs. For example, if a cellulase having an acidic activity profile i~ 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 ~queous solution should initially provide ~ignificant cellulolytic activity.

In view of the above, the pH of the cellulase solution should be maintained within the range r~quired for cellulolytic activity. One mea~s of accomplishing this is by simply monitoring the pH of the #ystem and adjusting the pH as reguired by the addition of either an acid or a base. However, m 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 èmployed cellulase exhibits activity. Insofar as different cellulase compositions have different pH ranges for exhibiting ,: .

- 27 - 21 ~ 72 0 6 ,'092/17574 PCT/US92/02631 cellulase activity, the specific buffer employed is selected in relationship to the specific cellulase composition employed. The buffer( 8 ) selected for use with the cellulase compo~ition employed can be S readily determined by the skilled artisan taking into account the pH range and optimum for the cellula~e composition employed as well as the pH of the cellul~e solution. Preferably, the buffer employed i8 OJIQ wbich is compatible with the cellulase composition and which will maintain the pH
of the cellula~e solution within the pH range ;~
required for optimal activity. Suitable buffers ~`
include ~odium citrate, ammonium acetate, sodium acetate, di~odium phosphate, and ~ny other art recognized buffers.

The ten~ile strength of cotton-containing rabric~ can be measured in a warp and fill direction which ~re at right angles to each other.
Accordingly, the term ~warp tensile strength" as u~ed herein refers to the tensile strength of the cotton-containing fabric as mea~ured along the length of the cotton-containing fabric wher~as the term ~fill tensile strength" refers to the tensile strength of the cotton-containing fabric as measured across the width of the cotton-containing fabric_ -The tensi~e strength of the resulting cotton-containing fabric treated with a cellulase solution is compared to its tensile strength prior to tr-atment with the cellulase solution so as to determine the strength reducing effect of the treatment. If the tensile strength is reduced too much, the resulting cotton-containing fabric will easily tear and/or form holes. Accordingly, it is 2 In7 2~ - 28 -W092/17574 PCT~USg2/02631 desirable to maintain a tensile strength (both warp and fill) after treatment which is at least about so% of the tensile strength before treatment.

The tensile strength of cotton-containing fabrics is readily conducted following ASTM D1682 test methodology. Equipment suitable for testing the tensile strength of ~uch f~brics include a Scott tester or an Instron tester, both of which are commercially available. In testing the tensile strength of cotton-containing fabrics which have been treated with cellulase solutions, care should be taken to prevent fabric shrinkage after treatment and before testing. Such shrinkage would result in erroneous tensile strength data.

Enhancements to the cotton-containing fabric i5 achieved by those methods heretofore used. 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 ar~icle ~What's New -- Weiqht Loss Treatment to Soften the Touch of Cotton Fabric". The teachings of each of these references is incorporated herein by re~erence.

Similarly, methods for improving both the feel and appearance of cotton-containing 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 - 29 ~ 720~

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, l990 and which is incorporated herein by reference in its entirety.

Methods for the enhancement of cotton-containing knits ~re described in International Textile Bulletin, Dyeing/Printing/Finishing, pages 5 et seq., 2~ Quarter, l990, 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, which is incorporated herein by reference in its entirety.

Other methods for enhancing cotton-containing fabrics by treatment with a cellulase composition are known in the art. Preferably, in such methods, the treatment of the cotton-containing ~abric with cellulase is ~onducted prior to finishing the cotton-containing fabric.

As noted above, the present invention is an i~provement over prior art methods for treating -cotton-containing fabrics insofar as the present invention employs a specific cellulase composition which minimizes strength loss in the treated fabric.
The cellulase composition employed herein is a fungal cellulase composition which comprises one or more EG type components and one or more CBH type components wherein the cellulase composition has a 2i n~'~06 - 30 -WO92/17s74 PCT~U~92/02631 If weight ratio of all EG type components to all CBH
type components of greater than 5:l.

Additionally, the use of the cellulase compositions described herein also result in fabric/color enhancement of stressed cotton-containing fabrics. Specifically, during the manufacture of cotton-containing fabrics, the fabric can become stressed and when so str~ssed, it will contain broken and disordered fibers. Such fibers detrimentally impart a worn and dull appearance to the fabric. However, when treated in the method of this invention, the so stressed fabric is subject to fabric/color enhancement. This is believed to arise by removal of some of the broken and disordered fibers which has the effect of restoring the appearance of the fabric prior to becoming stressed.

Additionally, it is cont~mplated that by employing the cellulase composition described herein with pigment type dyed fabrics (e.g., denims), these cellulase compositions will cause less redeposition of dye. It is also contemplated that these ' anti-redeposition properties can be enhanced for one or more specific EG type component(s) as compared to other components.

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 i5 generally a concentration sufficient for its - 31 - 2 1 ~205 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 cellul~se solution, and the like), the cellulase activity (e.g., a cellulase solution will require a lower concentration of a more active cellulase composition as compared to 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 0.01 gram/liter of cellulase ~olution to about 10.0 grams/liter of cellulase ~olution; and more preferably, from about 0.05 grams/liter of cellulase solution to about 2 gram/liter of cellulase solution. (The cellulase concentration recited above refers to the weight of total protein).

When a buffer is employed in t~e cellulase--solution, the concentration of buffer in the a~ueous cellulase solution is that which is sufficient to malntain the pH of the solution within the range wherein the employed cellulase exhibits activity which, in turn, depends on the nature of tbe cellulase employed. The exact concentration of buffer employed will depend on several factors which the skilled artisan can readily take into account.

2~1~7?.06 - 32 -W092/l7S74 PCT/US92/02631 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 O.15 N. It is possible that increased buffer concentrations in the cellulase solution may enhance the rate of tensile strenqth loss of the treated fabric.

In addition to cellulase and a buffer, the cellulase solution can optionally contain a small ~ount of a surfactant, i.e., less than about 2 weight percent, and preferahly from about o.ol to ~bout 2 weight percent. Suitable surfactants include any surfactant compatible with the cellulase and the fabric including, for example, anionic, non-ionic and ampholytic surfactants.

Suitable anionic surfactants for use herein include linear or branched alkylbenzenesulfonates;
alkyl or alkenyl ether sulfates having lin~ar or branched alkyl groups or alkenyl groups; alkyl or alk~nyl sulfates; olefinsulfonates; alkanesulfonates and the like. Suitable counter ions f or anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and maqnèsium; ammonium ion; and alkanola~ines having 1 to 3 alkanol groups of carbon number 2 or 3.

~ ~92/17574 - 33 ~ ~ ~ 7 2 0 6 pCT/US92~02631 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.

lo Mixtures of surfactants can al50 be employed~

The liquor ratios, i.e., the ratio of w~ight of cellulase solution to the weight of fabric, employed herein is generally an amount sufficient to achieve the desired enhancement in tha cotton-containing fabric and is dependent upon tha process used and the enhancement to be achieve. Preferably, the liquor ratios are generally from about 0.1:1 and greater, and more preferably greater than about 1:1 and even more preferably greater than about 10:1.
U~e of liquor ratios of greater than about Sb:1 are u~ually not preferred from an economic viewpoint.

Reaction temperatures for cellulase treatme~t 2re governed by two competîng factors. Firstly, higher temperatures generally correspond to enhanced reaction kinetics~ i.e., faster reactions, which permit reduced reaction ti~es as compared to reaction times required at lower temperatures.
Accordingly, reaction temperatures are ~enerally at least about 30C and greater. Secondly, cellulase is a protein which loses activity bayond a given 2 l~"~206 WO92/17~74 PCT/US92/02631 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 65C. In view of the above, r~ction temperatures are generally from about 30C
to about 65C; preferably, from about 35C to about 60C; and more preferably, from about 35C to about SOC .

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

The cotton-containing fabrics treated in the : methods described above using such cellulase compositions possess reduced strength loss a~
: compared to the same cotton-containing fabric treated in the same manner with a complete fungal cellulase composition~

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 curfactant, 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 additivesO Preferably, such concentrates will comprise from about O.l to about 20 weight percent of a cellulase composition .

_ 35 _ ~1 07~ o~
~92/17~74 PCT/US92/02631 described above (protein); from about 10 to about 50 weight percent buffer; from about 10 to about 50 weight percent surfactant; and from about 0 to 80 weight percent water. When aqueous concentrates are formulated, these concentrates can be diluted by factors of from about 2 to about 200 so as to arrive at the requisite concentration of the components in the cellul~se solution. As is readily apparQnt, 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 ~5 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 cellula~e composition is generally a granule, a powder, an agglomerate and the like. When gr~nules are used, the granules are preferably formulated so as to contain a ce~lulase protecting agent. See, for instance, U.S. Serial No. 07/642,669, filed January 17, 1991 as Attorney Docket No. 010055-073 nd entitled "GRANULES CONTAINING BOTH AN ENZYME AND
AN ENZYME PROTECTING AGENT AND DETERGENT
CONPOSITIONS CONTAINING SUCH GRANULES" which ~pplication is incorporated herein by reference in its entirety. Likewise, the granule can be formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium. Such materials and granules are disclosed in U.S. Serial No. 07/642,596 filed on January 17, 1991 as Attorney Docket No. GCS-171-USl and entitled 5 1 ~ 7 2 ~) 6 36 wo92/l7s74 PCT/US92/02631 "GRANULAR COMPOSITIONS" which application is incorporated herein by reference in its entirety.

It is contemplated that the cellulase compositions described herein can additionally be used in a pre-wash and as a pre-soak either as a liquid or a spray. ~t is still further contemplated that the cellulase compositions described herein can al~o be used in home use a3 a stand alone composition suitable for enhancing color and appearance of fabrics. See, for example, U.S.
Patent No. 4,738,682, which 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.

E~AMP~8 ~xamples 1-12 and 22-30 demonstrate the preparation of Trichoderma reçsei genetically engineered so as to be incapable of producin~ one or more cellulase components or so as to overproduce specific cellulase components.

Exam~le 1 Selection for ~yr4 derivatives of Trichoderma reesei The EYE~ gene encodes orotidine-S'-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.

?1 ~7206 ' `~g2/17574 PCT/US92/02631 However, cells defective in the EYE~ gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for pyr4 derivati~e strains using FOA. In practice, spores of T. reesei strain RL-P37 (Sheir-Neiss, G.
and Nontenecourt, B.S., Appl. ~icrobiol. Biotechnol.
20, p. 46-53 (1984)) were spread on the surface of a solidified medium oontaining 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resistant colonies appeared within three to four days and it was po~sible to subsequently identify those FOA-resistant deriv~tives which required uridine for growth. In order to identify those derivatives which specifically had a defective E~ gene, protoplasts were generated and transformed with a plasmid containing a wild-type EYE~ gene (see Examples 3 and 4). Following transformation, protoplasts were ~lated on medium ~acking uridine.
Subsequent growth of transformed colonies demonstrated complementation of a defective Eyr~
gene by the plasmid-borne EYE~ gene. In this way, strain GC69 was identified as a yr4 derivative of strain RL-P37.

Example 2 P~eParation of CBHI Deletion Vector A çbhl gene encoding the CBHI protein was cloned from the genomic DNA of T. reesei 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 - 3~ -W092/17S74 PCT/VS92~02631 a 6.5 kb PstI fragment and was inserted into cut pUC4K (purchased from Pharmacia Inc., Piscataway, NJ) replacing the Kanr gene of this vector using technigues known in the art, which s techniques are set forth in Maniatis et al., (1989) and incorporated herein by reference. The resulting plasmid, pUC4X::cbhl was then cut with ~ind~II and the larger fragment of about 6 kb was isolated and religated to give pUC4K::cbhl~H/H (see FIG. 1).
This procedure removes the entire cb~l coding sequence and approximately 1.2 kb upstream and 1.5 kb downstream of flanking sequences. Approximately, 1 kb of fl~nking DNA from either end of the original PstI fraqment remains.

The ~. reesei EYE~ gene was cloned as a 6.5 kb ~i~dIII fragment of genomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991) following the methods of Maniatis et al., sgpra. The plasmid pUC4K::cbhI~H/H
was cut with nindIII and the ends were dephosphorylated with calf intestinal alkaline phosphata~e. This end dephosphorylated DNA was ligated with the 6.5 kb ni~dIII fragment co~taining the T. reesei ~yr4 qene to give p~CBHIEyE~. M G. 1 illustrates the construction of this plasmid.

Exam~le 3 Isolation of Protoplasts Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2% qlucose) in a 500 ml flask with about 5 x 107 T. ~9Q~Ç~ GC69 spores (the ~vr4~ derivative strain). The flask was then _ 39 _ 2lO72~6 ~92/17~74 PCT/US92/02631 incubated at 37OC with shakin~ for about l6 hours.
The mycelium was harvested by centrifugation at 2,750 x g. The harveste~ my~elium was further washed in a l.2 M sorbitol solution and resuspended in 40 ml of a solution containing s mg/ml NovozymR
234 solution (which is the tradename for a multicomponent enzyme system containing 1,3-alpha-glucan~se, l,3-beta-gluaanase, laminarinase, xylanase, chitinase and protease from Novo Biolabs, Danbury, CT); 5 mg/ml MgS04.7H20; 0.5 mg/ml bovine SQrUm albumin; l.2 M sorbitol. The protoplaæts were removed from the cellular debris by filtration through Niracloth (Calbiochem Corp, La Jolla, CA) and col~ected by centrifu~ation ~t 2,000 x g. The protoplasts were washed three times in l.2 M
sorbitol and once in 1.2 M sorbitol, 50 mM CaCl2, centrifuged and resuspended at a density of approximately 2 x lO~ protoplasts per ml of l.2 M
sorbitol, 50 mM CaCl2.

Exam~le 4 al_~rotoplasts with ~CBHIpYr4 200 ~l of the protoplast suspension prepared in Example 3 was added to 20 ~l of EcoRI digested pACBHImYr4 (prepared in Example 2) in TE buffer (lO
mM Tris, pH 7~4; 1 mM EDTA) and 50 ~l of a polyethylena glycol ~PEG) solution containing 2S%
PEG 4000, 0.6 M KCl and 50 mM CaCl2. This mixture was incubated on ice for 20 minutes. After this incubation period 2.0 ml of the above-identified PEG
solution was added thareto, the solution was further mixed and incubated at room temperature for 5 21 07?JO~ ~ - 40 -minutes. After this second incubation, 4.0 ml of a solution containing l.2 M sorbitol and 50 mM CaCl2 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 KH2PO~, 2 grams NH4NO3, O.2 gr~ms MgS04.7H2O, O.1 gram CaCl2.2H20, 5 ~g ~-biotin, 5 mg citric ~cid, 5 mg ZnSO4.7H2O, l mg Fe(NHb)2.6H20, 0.25 mg CuSO4.5H2O, 50 ~g MnSO4.4H20 per liter) containing an additional 1% glucose, l.2 M
sorbitol and 1% agarose. The protoplast/medium mixture was then poured onto a solid medium cont~ining 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 c~mplementation of the pyr4 mutation of strain GC69 by the wild type EY~ g~ne insert in p~CBHIEy~. These colonies were s~bsequently tr~nsferred and purified on a solid Vogel's medium N
- 20 cont~ining as an additive, 1% glucose and stable tr~nsformants were chosen for further analysis.

At this stage stable transformants were distinquished 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 ~urther test of st~bility was made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores from this medium and determining the percen~age of these spores which will subsequently geFminate and grow on selective medium lacking uridine.

2t~72~6 ~92/17574 PCT/US92/02631 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 ~ubjected to agarose gel electrophoresis. The gel was then blotted onto a Nytran membrane filter and hybridized with a 32p labelled pACBHIEy~ probe. The probe was selected to identify the native çbhl gene as a 6.5 kb PstI
fragment, the native EYE~ gene and any DNA sequences derived from the transforming DNA fragment.

The radioactive bands from the hybridization were visualized by autoradiography. The autor~diograph 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.
Lane~ A-D repreæent transform~nts obtained by the methods described above. The numbers on the side of the autoradiograph repre~ent the sizes of molecular weight markers. As can be seen from this autoradiograph, lane D does not cont~in the 6.5 k~
~5 CBHI b~nd, indicating that this gene has been totally deleted in the transformant by integration of the DNA fragment at the c~hl gene. The cbhl deleted strain is called P37P~CBHI. Figure 2 outlines the deletion of the T. reesei ~bhl gene by integration through a double cross-over event of the larger EcoRI fragment from p~CBHIDyr4 at the 5khl 21~72~G - 42 - ..
W092/17574 PCT/US92/02631 t locus on one of the T. reesei chromosomes. The other transforman~s analyzed appear identical to the untransformed control strain.

Example 6 Analvsis of the Trans~ormants wit~_pIn~CB~I
The s~me procedure was used in this ex~mple as in Exa~ple 5, except that the probe used was changed :~
to a 32p labelled p~ntCBHI probe. This probe is a pUC-type plasmid containing a 2 kb ~g~II fragment -~
from the cbhl locus within the region that was deleted in pUC4K::cbhl~HIH. Two samples were run :in thiæ example including a control, sample A, which .is the untransformed strain GC69 and the transformant P37P~CBHI, sample B. A~ can be seen in FIG. 4, snmple A contained the cbhl gene, as indicated by th2 band at 6.5 kb, how~ver the transformant, sample B, does not contain this 6.5 kb b~nd and therefore ~oes not contain the 5khl gene and does not contain any saquences derived from the pUC plas~id.

Exam~le 7 Protein SecretiQ~_~y_~SD~}~ P37~CB~
5pores from the produced P37P~CBHI strain ware inoculated into 50 ml of a Trichoderma basal medium containing l~ glucose, 0.~4% (NH~)2S04- 0-2% KH2P04~
0.03% MgSO4, O.03% urea, 0.75% bactotryptone, 0.05%
Tw~en 80, 0.0000l6% CuS04.5H~0, 0.001% FeS04.7H20, 0.000l28% ZnS04.7H20, 0.0000054% Na~oO4.2H20, 0.0000007% MnCl.4H20). Th~ medium was incubated w~th shaking in a 250 ml flask at 37C for about 48 hours. The resulting mycelium was collected by 92/17574 - 43 - ?I n 72~ ~cT~usg2/o2631 filtering through Miracloth (Calbiochem Corp.) 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 30C 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 P37PACBHI, as shown in FIG. 5. This isoelectric focusing gel shows various proteins in different supernatant cultures of T. reesei. Lane A is partially purified CBHI; Lane B is the supernatant from an untransformed T. reeæei culture; Lane C is the supernatant from strain P37P~CBHI produced according to the methods of the present invention. The position of various cellulase components are labelled CBHI, CBHII, EGI, EGII, and EGII~. Since CBHI constitutes 50% of the total extracellular protein, it is the major secreted protein and hence is the darkest band on the gel. This isoelectriG
focusing gel clesrly sh~ws depletion of the CBHI
protein in the P37PACBHI strain.

W O 92/17574 P(~r/US92/02631 Examp~e 8 Pre~aration of DP~CBH~I
The cbh2 gene of T. ,reesei, encoding the CBRII -protein, has been cloned as a 4.1 kb EcoRI fragment of genomic DNA which is shown diagramatically in FIG. 6A (Chen et al., 1987, Biotechnoloa~, 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 symetrical pattern of restriction endonuclease siteS arranged in the order shown here: EcoRI, ~HI, SacI, SmaI, ~i~dIII, XhoI, ~g~II, ClaI, BqlII, XhoI, HindIII, SmaI, SacI, ~HI, ~çQRI. Using methods known in the art, a pla~mid, pP~CBHII (FIG. 6B3, has been constructed in which a 1.7 kb central region of this gene between a BiadIII site (at 74 bp 3' of the CBHII translation in~tiation site) and a .ClaI site (at 265 bp 3' of the last codon of CBHII) has been removed and replaced by a 1.6 kb ,HindIII- Çl~I DNA fragment containing the $. reesei EY~ gene.

The T. reesei Pvr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb ~h~ hI fragment and -incerted between the ~hI and,X~aI sites of pUC219 (see Example 25) to create p219M ~Smith et al., 1991, curr. Genet 19 p. 27-33). The 'vr4 gene was than removed as a HindIII-ClaI fragment having seven bp of DNA at one end and six ~p of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the ~iadIII and ClaI sites of the _ 45 _ 2 1 ~ 72 0 6 cb~2 gene to form the plasmid pP~CBHII (~ee FIG.
~B).

Digestion of this plasmid with EcoRI will liberate a fraqment having 0.7 kb of flanking DNA
from the cbh~ locus at one end, 1.7 kb o~ flanking DNA from the çbh2 locus at the other end and the T.
reesei EYE~ gene in the middle.

Exam~le 9 Deletion of the cbh2 aene in T. reesei ~ain G~Ç9 .- Protoplasts of strain GC6g will be generated .
and transformed with EcoRI digested pP~CBHII
according to the methods outlined in Examples 3 and 4. DNA from the transformants will be digested with ~co~I and ~e718, and subjected to agarose gel lS electrophoresis. The DNA from the gel will be blotted to a membrane filter and hybridized with 32p labelled pP~CBHII according to the methods in Exampie l~. Transformants will be idsntified which have a singl~ copy of the ECQRI fragment frvm pP~CBHII integrated precisely at the bh~ locus.
The transformants will also be ~rown in shaker flasks as in Example 7 and the protain in the culture super~atants examined by isoelec~ric focusing. In this manner T. ~$ei GC69 transformants which do not produce the CBHII protein will be generated.

2~ 0~ 20 6 - ~6 -WO92/17~74 PCT/US92/02631 Exa~ple l0 Generation f ~ py~ Lyl~ive of ~ ~ÇBHI
Spores of the transformant (P37P CBHI) which was deleted for the cbhl gene were spread onto medium containing FOA. A pvr4~ derivative of this transformant was subsequently obtained using the methods of Example l. This Pvr4- strain was designated P37P~CBHIPyr26.

E~Q~ .:

- peletion of the cbh~ gene ~n a strain ~- ~revious~y~ d~leted for cb~l Protoplasts of strain P37P~CBHIPyr26 were generated and transformed with EcoRI digested pP~CBHII according to the methods outlined in Ex~mples 3 and 4.

Puri~ied 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 tdesignated P37P~CBH67) was identified which did not produce any CBHII
protein. Lane D of FIG~ 5 shows the supernatant from a transformant del~ted for both the cbhl and gene produced according to the methods o~ the pr~sent invention.

DNA was extracted from strain P37P~CBH67, digested with E~RI and As~718, and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with ~92/17574 ~ I 7 '~J O~PCT/VS92/02631 3~ labelled pP~CBHII tFIG. 7). Lane A of FIG. 7 shows the hybridization pattern observed for DNA
from an untransformed T. reesei strain. The 4.l kb EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization pattern observed for strain P37P~aCBH67. The single 4.1 kb band has been eliminated and replaced by two bands of approximately 0.9 and 3.l kb. This is the expected pattern if a single copy of the ~ç_RI
fragment from pP~CBHII 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 32p labelled pIntCBHII. This plasmid contains a portion of the cbh2 gene~ coding seguence from within that segment of the cbh2 gene which was deleted in plasmid pP~CBHII. No hybridization was seen with DNA from strain P37P~CBH67 showing that the ~ gene was deleted and that no sequences derived from the pUC
plasmid were present in this strain.

Exam~le 12 Cons~c~9n~9~ eDeC~
The T. ree~ei e~ll gene, which encodes EGI, has been cloned as a 4.2 kb ~ind II fragment of genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthesized according to the published sequence (Penttila et al., 1986, Gene 4s:253-263; van Arsdell et al., 1987, BiotTechno~oqy 5:60-64). A 3.6 kb ~i~dIII-~HI fragment was taken 2 1 ~7 ~ S - 48 -WO92~17574 PCT/US92/02631 from this clone a~d ligated with a 1.6 kb ~iadIII-BamHI fragment containing the T. reesei ~YE~ gene obtained from pTpyr2 (see Example 2) and pUC218 (identical to pUC219, see Example 25, but with the multiple cloning site in the opposite orien~ation) cut with ~indIII to give the plasmid pEGI~y~
(FIG. 8). Digestion of pEGIpvr4 with ~i~dIII would liberate a fragment of DNA containing only ~. ~eesei genomic DNA (the e~l~ and ~Yr4 genes) exc~pt for 24 bp of sequenced, synthetic DNA between the two genes and 6 bp of sequenced, synthetic DNA at one end (see FIG. 8).

. Ex~mple 1~
~urification of Cytolase 1~3 Cellulase 15 ' into Cellulase Components CYTOLASE 123 cellulase was fractionated in the following manner~ The nor~al distribution of cellulase components in this cellulase system is as follow~:
CB~ I45-55 weight percent CBH II13-15 weight percent EG I11-13 weight percent - EG II8-10 weight percent EG III1-4 weight percent BG `O . S-l weight percent.

The fractionation was done using columns containing the fo~lowing 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, Md). CYTOLASE 123 cellulase, O.5g, was desalted using a column of 3 liters of 5ephadex G 25 gel filtration resin with 10 mM sodi~m phosphate buffer at pH 6.8. The desalted solution, _ 49 ~ ~ 0 7 ~ ~ 6 was t~en 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. Th~se fractions were desalted using a column of Sephadex G-25 gel filtration resin equilibrated with lO 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 u~ing an aqueous gradient containing from O to about 200 mM sodium chloride.

lS Following procedures similar to t~at of Example 13 above, other cellulase systems which can be ssparated into their components include OEr,rUCAST
(available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist Brocades, N.V., Delft, Holland), and cellulase systems derived from Trichoderma koninqii, Penicillum æ. and the like.

Example 14 Purifica~ion of EG II~ ro~
Cvtolase l23 Cellul~se Example 13 above demonstrated the isolation of saveral components from Cytol~se 123 Cellulase.
However, because EG III is present in very small quantities in Cytolase 123 Cellulase, the following procedures ware employed to isolate this component.

A. Lar~e Scale Extracti~n of EG_III Cellulase Enzyme ~J t~ .v.~

WOg2/17574 PCT/US92/02631 One hundred liters of c~ll free cellulase filtrate were heated to about 30C. The heated material was made about 4~ wt/vol PEG 8000 (polyethylene glycol, MW of about 8000) and about 10% wt/vol anhydrous sodium ~ulfate. The mixture formed a two phase liquid mixture~ The phases were separated using an SA-l 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 polyethylen~ 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 lev~ls greater than about 10% wt/vol caused precipitation problems; where~s, sodium sulfate levels less than about l0~ wt/vol gave poor separation or the solution remained in a single phase.

B. Purificatio~o~ G III Via Fr~c~ionatiQn The purification of ~G III is conducted by fractionation from a complete fungal cellulase composition (CYTOL~SE 123 cellulase, commercially availabls from Genencor International, South San Francisco, CA~ which is produced by wild type ~ri~hoderm~ ree$çi~ Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from ~ 6, : 392/17574 PC~/USg2/02631 Sigma Chemical Company (St. Louis, Mo), QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Md).
CYTOLASE 123 cellulase, 0.5g, is desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with lO 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 ~BH II, EG II and EG ~II. These fractions are de~alted using a column of Sephadex G-25 gel filtration re~in eguilibrated with lO mM sodium citr~te, 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 o~ an aqueous solution of 200 mM sodium chloride.

In order to enhance th~ efficiency of the isolation of EG III, it may be desirable to employ richode~ma reesei 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 ~he EG III
compositions described above to be further purified to provide for substantially pure EG III
compositions, i.e~, compositions containing EG ~II
at greater than about 80 weight percent of protein.
For example, such a substantially pure EG III
protein can be obtained by utilizing material 2 1 q ~ 52 -W O 92/17574 PC~r/US92/02631 obtained from procedure A in procedure B or vica 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 14. The further fraction was done on a FPLC ~ystem using a Nono-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 ch~rt recorder (all of which are available from Pharmacia LXB Biotechnology, Piscataway, NJ). The fr~ctionation was conducted by de~alting 5 ml o~ the EG III sample prepared in part b) of this Example 14 - wlth-a 20 ml Sephadex G-25 column which had been 15 , 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 techniqu~s.

Substantially pure EG III ae well as EG I and EG II components purified in Example 13 above can be used singularly or in mi~tures in the methods of this invention. The~e EG components have the following characteristics:

_ 53 - 2~.q7~ 0 6 'Og2/17~74 PCT/US92/02631 MW pI ~H o~timum' EG I -47-49 kD 4.7 -5 EG II -35 kD 5.5 -5 EG III -25-28 kD 7.4 -5.S-6~0 ~ 5 1. pH optimum determined by RBB-CMC activity as per Example 15 below.

The use of a mixture of the~e components in the practice of this invention may give a synergistic response in improving softenin~, feel, appearanc~3, etc., as co~pared to a single component. On the other hand, the us~ of a single component in the practice of this invention may be more stable or have a broader spectrum of activity over a range of pHs. For instance, Example l5 below shows that EG
III has considerable activity against RBB-CMC under alkaline conditions.

Examp~ç l~
Activi~y of ~ellulase 5Q~pQsitions over a pH Ra~e T~e following procPdure was employed to d~texmine the pH profiles of two different cellulase composition~. The first cellulase composition was a CB~I I and II deleted cellulase composition prepared from Tric:hod~rma reesei genetically modif ied in a manner similar to that described above so as to be un~ble to produce CBH I and CBH II components~
In~ofar a5 this cellulase composition does not contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase ~ J~ 54 -.
composition derived from Trichoderma reesei, this cellulase composition is necessarily substantially free of CBH I type and CBH II type cellulase components and accordingly, is enriched in EG
S components, i.e., EG I, EG II, EG III and the like.

The second cellula~e composition was an approximately 20 to 40% pure fraction of EG III
isolated from a cellu~ase c~mposition derived fr~m Trichoderma reesei via purification methods similar to part b) of Example 14.

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

Add~5 to 20 ~1 of an appropriate enzyme solution at a concen~ration ~ufficient to provide the requisite amount of enzyme in the final solution. Add 250 ~1 of 2 w~ight percent RBB-CMC
(Remazol Brilliant Blue R-Carboxymethylcellulose --co~mercially available from MegaZyme, 6 Altona Place, North Rocks, N.S.W. 21S1, 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 40C for 30 minutes.
Chill i~ an ice bath for 5 to 10 minutes. Add 1000 ~1 o~ mathyl 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 S90 nm. Higher lavels - s5 - 2~07,?0~ :
~92/17~74 PCT/US92/02631 of optical density correspond to higher levels of enzyme activity.

The results of this analysis are set forth in FIG. g 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 sQen 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 80 that the cellulase composition is active and preferably, posses~es optimum activity. As noted above, such adjustments and maintenance may involve the use of a suitable buffer.

Example ~
Launderometer Strength Loss ~ss~y Çç~lulase Composi~ions 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 th~ most of the cellulase components derived from Trichoderma reesei is ~ 1n 7 ~ 56 -WO92~17574 PCT/US92/02631 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 Genensor International, South San Francisco, CA) producad by wild type Irichoderma reeQei and is identified as GCOlO.

The second cellulase composition analyzed was a C8H II deleted cellulase composition prepared from - Tri~hoderma reesei genetically modified in a manner similar to Ex~mples 1 to 12 above and 2~-30 below so as to be incapable of expressing CBH II and is identifi~ed as CBHIId. Insofar as CBH II compii~ss up to about 15 percent of the cellulase composition, deletion of this component results in snriched levels of CBH I, and all of the EG components.

The third cellulase composition analyzed was a CBH I and C8H II del~ted cellula~e composition prepared from Trichoderma rees~ ~enetically modified in a manner similar to ~hat described above so a~ to be incapable of expres~ing CBH I ~nd CBH II
and is ide~ti~ied as CBHI/IId. Inso~ar as CBH I and CBH II are not produced by this modified microorganism, the cellulase is necessarily free of all C8H I type components as well as all CBH
components.

The last cellulase composition analyzed was a CBH I deleted cellulase composition prepared from _ 57 _ ~ L ~7~06 092~17574 PCT/VS92/02631 Trichoderma reesei genetically modified in a manner similar to t~at described above so as to be incapable of expressing CBH I and is identified as CBHId. 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 citratetphosphate buffer, titrated to pH 5, and wh~ch contains 0.5 ml of a non-ionic surfactant.
Each of ~he resulting solutions was then added to a separate launderometer canister. Into th~se 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 430C.
The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about l hour. Afterwards, the cloth is removed, rin ed well and dried in a standard drier.

In order to maximize strength 105s resul~s, the above procedure was repeated twice more and after the third treatment, t~e cotton fabrics were removed and analyzed for strength loss. Strength loss was wos2/l7s74 PCT/~S92~02631 measured by determining the tensile strength in the fill direction t"FTS") using a Instron Tester and the results compared to the FTS of the fabric treated with the same solution with the exaeption that no cellula~e wa~ added. The results o$ this analysis are reported as percent strength loss which is determined as follows:

% Strength Loss = lO0 x ~ - FTS with cellulase L FTS without cellula~

The results o~ this analysis ~re set forth in FIG. lO which shows that compositions containing C~H I, i.e., whole cellulase (GCOlO) and CBH II
deleted cellulase~ possessed the most strength lo~;s whereas, the compositions containing no CBH I
possesse* significantly reduced strength loss as compared to whole cellulase and CBH II deleted cellulase. From th~se results, it i~ seen that the presence of CBH I type components in a cellulase composition imparts in~reased strength loss to the composition ~s compared to a similar composition not containing CBH I type components.

Likewise, these results show that C~H 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 _ 59 _ 2.~.0 7~J~ 6 ~9~/17574 PCT/US92/02631 lower strength loss at pH 2 7 than those results observed at pH 5 shown in FIG lO.

During the manufacture of cotton-containing 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, it has been found that the methods of this invention will result in fabrictcolor enhancement. This is believed to arise by removal of some of the broken and disordered fibers which has the effect of r~storing the appearance of the fabric prior to becoming -stre~sed.

The following Examples 17 and 18 illustrate this bene~fit of the present invention. It is noted that these examples employed worn cotton T-shirs (knits) as well as new cotton knits. The faded appearance o~ thQ worn cotton-containing ~abric ari~es from the accumulation on the ~abric of loose and broken surface ~bers over a period of time.
These ~ibers give rise to a faded and matted appearance for the fabric and accordingly, the removal of these fibers is a necessary prerequisite to re~toring 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. Accordingly, these experiments are necessarily applicable to color enhancement of stressed cotton-containing fabrics because both involve removal of surface fibers from the fabric.

21072Q~ - 60 - .
WO92/17~74 PCT/US92/02631 Exam~le 17 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 Genéncor International, South San Francisco, CA) produced by wild type Trichoderma reesei to remove surface fibers from a cotton-containing fabric over various pHs. This cellulase was tested for its ability to re~ove s~rface f~bers in a laundQrometQr. An appropriate amount of cellulase to provide for either 25 ppm or 15 ' lO0 ppm cellulase in the final composition was added to ~eparate solutions of 400 ml of a 20 mM
citrate/phosphate buffer containinq 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 ~.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 remo~al 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., Middlasex, NJ 08846~. The canister was then closed and the canister lowered into the launderometer bath which was maintained at 43C. The canister was then rotated in the bath at a speed of at least about 4D revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

7 ~ 0 6 The so treated fabrics were then analyzed for fiber removal by evaluation in a panel test. In particular, the fabrics (unmarked) were rated ~or levels of fiber by 6 individuals. The fabrics were visually evaluated for surface fibers and rated on a o to 6 scale. The scale has six standards to allow meaningful comparisons. The standards are:
Rating Standard-0 Fabric not treated with cellulase l Fabric treatedb witb 8 ppm cellula~e 2 Fabric treated with 16 ppm cellulase 3 Fabric treated with 20 ppm cellulase 4 Fabric treAted with 40 ppm cellulase Fabric treated with 50 ppm cellulase 6 ' Fabric treated with lO0 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., Niddlesex, NJ 08846 b All samples were treated with the same cellulase composition. Cellulase concentrations are in tota} protein. The launderometer treatment conditions are the same as set forth in Example l6 above.
The fabr~c to be rated was provided a rating which most closely matched one o~ the standards.
A~ter complete analysis of the f abrics, the values :
assigned to each fabric by all of the individuals were added and an a~erage value generated.

The results of this analysis are set forth in FIG. ll. Specifically, FIG. ll illustrates that at .

~0720~ - 62 -g2/17574 PCT/US92/02631 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 tre~ted with more cellulase provided for higher levels of fiber removal as compared to fabrics treated with le~s cellulase. Moreover, the results of this figure demonstrate that at higher pHs, fiber rsmoval can still be effected merely by using higher concentration~ of cellulase.

In a second experiment, two different cellulase compositions were compared for the ability to remove fiber. Specifîcally, the first cellulase composition - analyzed was a complete cellulase sy3tem (CYTOLASE
, 123 cellulAse, commercially available from Genencor International, South San Francisco, CA) produced by :~
wild type Trichoderma reesei ~nd is identified as GCOlO.

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 ~rom Trichoderma reesel genetically modified in a manner similar to that des~ribed above so as to be incapable of expressing CBH I and CBH II and is identified as CB~IIII dele'ed. Insofar as CBH I and CBH II comprises up to about 70 percent of the cellul~se 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.

- 63 ~ 7~.~t~S
~92/17574 PCT/US92/02631 An appropriate amount of cellulase to provide for the requisite concentrations of EG components in the final compositions were 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 cani~ter lowered into the launderometer bath which , was maintained at 43C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about l 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 dQscribed above. The results of this analysis are Qet forth in FIG. 12 which is plotted on estimated EG concentrations. Specifically, FIG. 12 illustrates that both GCOlO and CBH I/II Deleted cellulase compositions gave substantially identical fiber removal results at substantially equal endoglucanass concentrations. The results of this figure suggest that it is the EG components which provide for fiber removal. Thsse results coupled with the results of FIG. ll demonstrate that EG
components remove surface fibers.

2107~6 - 64 -W092/l7574 PCT~US92/02631 Example 18 Teraotometer Color Enhancement T~is example is further to Example 17 and substantiates that CBH type components are not necessary for color enhancément 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 Iriçhoderma reçsei 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 abo~t 70 percent of the cellulase composition, deletion of this component results in enri~hed levels of all of the EG components.

The assay was conducted by adding a sufficient concentrat~on of this cellulase composition to a 50 mM citrate/phosphate buffer to prcvide S00 ppm of cellulase. The solution was titrated to pH 5 and contained 0.1 weight percent of nonionic surfactant (~rescoterg GL100 -- commercially available from Gresco MfgO, Thomasville, 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 bu~fer and allowed to sit at 11.0F for 30 minutes and then a~itated for 30 minutes at 100 J 1 l~ 7 ~ ~ 6 ~92/17574 PCT/US92/02631 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-Containinq Material RÇsult _ Worn Cotton T-Shirt benefit seen Cotton Knit benefit seen The term "benefit seen'1 ~ans that the treated fabric exhibits color restoration (i.e., is 1QSS
faded) as compared to the non-treated fabric which i~cludes removal of broken ~urface fibers includinq s~rface fibers generated as a result of using the tergotometer. These results substantiate the results of Example 17 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 ~uch cellulase compositions would be beneficial during fabric processing because such compositions would remove broken/loose fibers generated during processing wlthout detrim ntal strength loss to the fabric.

Exam~
Softness This ~xample demonstrates that the presence of CBH type components are not es~ential for imparting improved softness to cotton-containing ~abrics.
Specifically, this example employs a cellulase compoæition free of all CBH type componénts which ~iO~20~i - 66 -composition is derived from Trichoderma reesei genetically engineered in the manner described above so as to be incapable of producing C~H I and II
components.

This cellulase composition was tested for its ability to coften terry wash c}oth. Specifically, unsoftened 8.S ounce cotton terry cloths, 14 inches by 15 inches (available as Style No. 420NS from Tect Fabrics, Inc., 200 Bla~kford Ave., Middlesex, lo 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, ~nd 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 sur~actant (Triton X114).
Additionally, a blank was run containing the same solution but with no addad cellulase. Samples so prepared were titrated to pH 5. Each of the resultin~ solution was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to f~cilitate softness as well as cotton swatches described a~ove. All conditions were run in triplicate with two swatches per canister. Each canister was then closed and the canister low~red into the launderometer bath which was maintained at 37C. The canister was then rotated in the bath at a speed of at least about 40 - 67 _ ? l 9 7~? ~ 5 `~92/17S74 PCT/US92/02631 revolutions per minute (rpms) for about l 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 th~ir own ~et of swatches and ask to rate them with respect to softness based on the softness criteria such as the pliability of the whole fabric. 5watches obtained from treatment with the five different enzyme concentrations and the blank were placed behind a screen and the panelists wore ask~d to orde~ them from least so~t to ~o~t - 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 ~hen averaged.

The results of this averaging are set forth in FIG. 13. Specifically, these resultæ 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 c~llulase composition.

E~mDle 2Q
Feçl a~d-~pRe~ ~
This example demonstrates that the presence of CBH type romponents are not essential for imparting improved feel and appearance to cotton-containing fabric~ Specifically, this example employs a cellulase composition derived from Trichoderma reesei genetically engineered in the manner ~107~'~5 - 68 -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 81ackford 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 thQ appsarance of thes~ samples in a launderometer. Specifically, an appropriate amount of CBH I and II deleted cellulase to provide for 25 ppm, 50 ppm, and lO0 ppm cellulase in ths final cellulase solution was added to - -s~parate solutions of 400 ml of a 20 mM
citrate/phosphate buffer containing 0.025 w~ight percent of a non-ionic surfactant (Triton X114).
Additionally, a blank was run containing the same solution but with no added cellulase. Samples 80 prepared were titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters w2re added a ~uantity of marbl~s to facilitate improvements in appearance a-~ well as cotton samples described ~bo~e. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at about 400C. The canister was then rotated in the bat~ at a speed of at least about 40 revolutions per minute (rpms) for about l ' ~92/17~74 - 69 - 2~ ~7206 PCT/US92/02631 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 instructQd that the term "appearance" r~fers 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 ~nabil$ty to diQcern 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 appearanc~ as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weave.

The panelists then assigned ~cores wer~
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 ~re as follows:

25Amt Ce~ ~lase_ Averaqe Appearance None 25 ppm 2 50 ppm 3 100 ppm 4 21Q72~ 70 _ 7~74 PCT/US92/02631 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 ~00% cotton sheeting (available as Style No. 439W from Test F~brics, Inc~, 200 Blackford Ave., Middlesex, NJ 08846) were employed in the feel aspects of this Qxample.

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, lO00 ppm, and 2000 ppm cellulase in the final - cell~lase ~olution was added to Beparate 501utions , 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 industial washer. The washer was operated at 50C, a total volume of 24 L, a liquor to cloth ratio of 50:l ~weight to weight) and the washer wa~ 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 ~he 4 sample~
(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 distinquished from qualities such as so~tness (which refers to the pliability of the fabric rather thAn its feel), thickness, color, or other physical - 71 - ~1 n 7'~06 ~92/17574 PCT/US92/02631 characteristics not involved in smoothness of the fabric.

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

Amt Cellulase Avera~e Feel None 1.5 ~ 0.5 500 ppm 1.7 + 0.4 - ~~lO00 ppm 3.2 + 0.4 2000 ppm 3.8 + 0.4 The~above results demonstrate that improvements in feel and appearance can be achiev~d with cellulase ~ompositions free of all CBH type components.

Exam~le 2l S~one Was~eq Appç~nce This axample demonstrates that the presence of CBH type c~mponents are not essential for imparting a stone washed appearance to cotton-containin~
fabrics. Specifically, this example employs a cellulase composition derived from Trichoderma reesei genetically engin~ered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and XI components) as well as a complete cellulase composition derived from T~içhoderma reesei and which is available as 2 1 0 7 2 a 6 - 72 -WO92/17574 PCT/US92/0~631 Cytolase 123 cellulase from Genencor International, South San Francisco, California.

These cellulase compositions were tested for their ability to impart a stone washed appearance to dyed cotton-containin~ denims pants. Specifically, the ~amples were prepared using an industrial washer and dryer under the following;~onditions:

10 mM citrate/phosphate buffer pH S
40 L total volume ~our pair of denim pants 1 hour run time 50 ppm CBH I and II deleted cellulase or lO0 ppm whole cellulase (i.e., at approximately equal EG concentrations) '~
Samples were evaluated for their stonewashed appearance by 8 panelists. All eig~t panelists choose lO0 ppm whole cellulase over non-enzyme treated pants as having the better stone washed look. Four of the 8 panelists choose the CBH I and II deleted cellulase ~reated pants over whole cellulase as having the better stone washed look;
whereas the other four panelists choose the whole cellulase treated pants as having the better stone washed 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 not es~ential for imparting a stone washed appearance to cotton-containing fabrics.

With regard to Examples 16 to 21, cellulase compositions free of CBH I type components and ~ ~ 0 ~ 6 ~92/17574 PCT/US~2/02631 derived from microorganisms other than Trichoderma reesei could be used in place of the cellulase compositions described in these examples. In particular, the source of the cellulase composition containing the EG type components is not important to this invention and any fungal cellulase comp~sition containing one or more EG type components and sub~tantially free of all CBH I type components can be used herein. For example, funqal cellula~es for use in preparing the fungal cellulase compositions used in this invention can be obtained from Trichoderma koningii, Pencillum SD., and the 11kQ or co o ercially availablQ cellulases can be - u~ed~ i.e., CELLUCAST (available from Novo Industry, , Copenhagen, Denmark), R~PIDASE (available from Gist Brocades, N.V., Delft, Holland), and the like.

Exam~le 22 Transformants of Trichoderma reesei Containina the ~lasmid ~EGI~vr4 A ~r4 defective derivative of T. reesei strain RutC30 ~Sheir-Neiss and Montenecourt, (1984), ~
~ic~obiol. Bioteçhnol. 20:46-53) was obtained by the method outlined in Example l~ Protoplasts of this strain were transformed with undigested pEGImY~4 and stable transformants were purified.

Five of these transformants ~designated EP2, EP4, EPS, EP6, EPll), as well as untransformed RutC30 were inoculated into 50 ml of YEG medium tyeast extract, 5 g/l, glucose~ 20 g/l) in 250 ml shake flasks and cultured with shaking for two days at 28C. The resulting mycelium was washed with 21072~i) 74 _ sterile water and added to 50 ml of TSF medium (0.05M citrate-phosphate buffex, pH 5.0; Avicel microcrystalline cellulose, ~o g/l; KH2P04, 2~0 g/l;
(NEb)2SO~, 1.4 g/l; proteose peptone, 1.0 gll; Urea, 0.3 g/l; Mgsa~.7H2o~ 0.3 g/l; CaCl2, 0.3 g/l;
FeS0~.7H20, 5.0 mg/l; MnS04 .H20, 1.6 mg/l; zns04 ~ 1 4 mgll; CoCl2, 2.0 mg/l; 0.1% Tween 80). These cultures were incubated with Ehaking for a further four days at 28C. Samples of the supernatant were taken from these cultures and assays designed to measure the total amount of protein and of endoglucanase activity were performed as described below.

, The endoglucanase assay relied on the relea~e of soluble, dyed oligosaccharides from Remazol Bri'lian~ Blue-carboxymethylcellulose (RBB-CMC, obtained from MegaZyme, North Rock~, 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, S ml of 2 M s~dium acetate buffer (pH
4.8) was added and the p~ ad~u~ted to 4.5. ThQ
volume was finally adjusted to 100 ml with deionized water and sodium azide added to a final concentration of 0.02%. Aliquots of ~. reesei control culture, pEGI~yE~ transformant culture supernatant or 0.1 M sodium acetate as a blank (lO-20 ~l) wer~ placed in tubes, 250 ~l of substrate wa~
added and the tub~s were incubatQd for 30 minutes at 37~C. The ~ubec were pl~ced on ice for lO minutes and l ml of cold precipitant (3.3% ~odium acet~te, 0.4~ zinc acetate, pH 5 with HCl, 76% ethanol) was then added. The tube were vortexed and allowed to _ 75 _ 2~.~ 7? n6 ~92/17574 PCT/US92/02631 sit for five minutes before centrifuging for t~ree minutes at approximately 13,000 x g~ The optical density was measured spectrophotometrically at a wavelength of 590-600 nm.

The protein assay used was the BCA
(bicinchoninic acid) assay using reagents obtained from Pierce, Rockford, Illinois, USA. The standard was bovine serum albumin (BSA). B~A reayent wa-C
made by mixing 1 part of reagent B with 50 parts of r~agent A. One ml of the BC~ reagent was mixed with 50 ~l of appropriately diluted BS~ or test culture supernatant. Incubation was for 30 minutes at 37C
and the optical density was finally measured spectrophotometrically at a wavelength of 562 nm.

Th~ results of the assays described above are shown in Table l. Tt is clear that some of the transformants produced increased amounts of endoglucanase activity compared to un~ransformed strain RutC30. It is thought t~at the en~oglucanases and exo-cellobiohydrolases produced by untransformed T. ~Ç~Çi constitute approximately 20 and 70 perc~nt respectively of the total amount of protein ~ecreked. Th~refore a transformant such as EP5, which produces approximately four-fold more endoglucanase than strain RutC30, would be expected to ~ecrete approximately equal amounts of endoglucanase~type and exo-cellobiohydrolase-type pxoteins.

The transformants described in this ExamplE
were obtained using intact pEGIEy~ and will contain DNA sequences integrated in the genoms which were 21 07~'~6 W092~17S74 PCT/US92/02631 derived from the pUC plasmid. Prior to transformation it would be possible to digest pEGIEyE~ with HindIII and isolate the larger DNA
fragment containing only T. reesei DNA.
Transformation of T. reesei with this isolated fragment of DNA would allow isolation of transformants which o~erproduced EGI and contained no heterologous DNA sequences except for the two short pieces of synthetic D~A shown in FIG. 8. It would also be possible to use pE~IEy~ to transform a strain which was deleted for either the Çkhl gene, or the cbh2 gQne, or for both gon~s. In this way a strain could be constructed which would over-produce EGI and produce either a limited range of, or no, exo-cellobiohydrolases.

The-~ethods of Example 22 could be used to produce ~. reesei strains which would over-produce any of the other cellulase components, xylanase components or other proteins normally prod~ced by T.
reesei.

Secreted Endoqlucanase Acti~itv of T. reesei Transformants A B
ENDOGLUCANASE
ACTIVITY PROTEIN
STRAINlO.D. AT 59~_nm) ~ma/ml~ ALB
~utC30 0.32 4.1 0.078 EP2 0.70 3.7 0.18g EP4 0.76 3.65 0.208 ~P5 1.24 4.1 0.302 EP6 0.52 2.93 0.177 `EPll 0.99 4.ll 0.241 ~92/17574 ? I ~ 7 2 ~ /US92/02631 The above results are presented for the purpose of demonstrating the overproduction of the EGI
component relative to total protein 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.

Exam~le 23 Construction of pCEPCl A plasmid, pCEPCl, was constructed in which tbe - - cod~ng sequence for EGI was functionally fused to the promoter from the cbhl gene. This was achieved using in vitro, site-specific mutaqenesis to alter the DNA sequence of the cbhl and ea~l genes in order to create convenient restriction endonuclease cleavage sites just 5' (upstre~m) of their respectiYe translation initiation sites. DNA
sequence analysis was performed to verify the expected sequence at the junction between the two DNA segm~nts. The specific a~terations made are shown in FIG. 14.

The DNA fr~gment~ which were combined to form pCEPC1 were inserted between the ~s_RI sites of pU~4K and were as follows (see ~IG. 15):
A) A 2.1 kb fragment from the 5' fl~nking region of ~he Çkh~ locus. This includes the promotex region and extends to the engineered ~lI site and so contains no cbhl coding sequence.
B) A 1.9 kb fragment of genomic DNA from the eqll locus starting at the S' end with the engineered wo~211~774~ 78 - ' PCT/US92/02631 '' BamHI sit~ and extending through the coding reqion and including approximately o.5 kb beyond the translation stop codon~ At the 3' end of the fragment is 18 bp derived from the pUC2l8 multiple cloning site and a 15 bp synthetic oligonucleotide used to link this fragment with the fragment below.
C) A fragment of DNA from the 3'-flanking region of the Çkhl locus, extending from a position approximately l kb downstream to approximately 2.5 kb downstream of the Çkhl translation stop codon.
D) Inserted into an NheI site in fragment (C) was a 3.1 kb ~h~ hI fragment of DNA containing the ,reesei Yr4 gene obtained from pTpyr2 (Example ~) -and having 24 bp of DNA at one end derived from'the , pUCl8 multiple cloning site.

The plasmid, pCEPCl was designed so that the EGI coding sequence would ba integrated at the cbhl locus, replacing the coding sequence for CBHI
without introducing any foreign DNA into the host strain. Digestion of this plasmid with ~çQRI
liberates a fragment which includes the Çkhl promoter region, the egll coding sequence and transcription termination region, the ~ reesei ~y~
gene and a segment of DNA from the 3' (downstream) ~5 flanking region of the Çkh~ locus ~see Fig. 15).

Ex~m~le ,~4 ~r;ansformants con~ainina pCE~Cl DNA

A ~Yr4 defective strain of T. reesei RutC30 (Sheir-Neiss, supra) was obtained by the method outlined in Example l. This strain was transformed with pCEPCl which had been digested with ~_RI.

_ 79 ~tQ7.~
~92/17574 PCT/US92/02631 Stable tr~nsformants were selected and subsequently cultured in shaker flasks for cellulase production as described in Example 22. In order to visualize the cellulase proteins, isoelectric focusing gel electrophoresis was performed on samples from these cultures using the method described in Example 7.
Of a total of 23 transformants analysed in this manner 12 were f~und to produce no CBHI protein, which is the expected result Or integration of the CEPCl DNA at the cbhl locus. Southern blot analysis w~s used to confirm that integration had indeed occurred at the cbhl locus in some of tho~e transformants and that no sequences derived from the bacterial plasmid vector (pUC4K) were present (see lS , Fig. 16). For this analysis the DNA from the transformants was digested with ~I before being subjected to electrophoresis and blotting to a :~ m~mbrane filter. The resulting Southern blot was probed with radiolabelled plasmid pUC4X::¢bhl (see Ex~mple 2). The probe hybridi~ed to the ~khl gene on a 6.5 kb fragment of DNA from the untransformed control culture (F~G. 16, lane A). Integration of the CEPCl fragment of DNA at the Çkh~ locus would be expected to result in the loss of this 6.5 kb band and the appearance of three other bands correæponding to approximately l.0 kb, 2.0 kb and 3.5 kb DNA fragments. This is exactly the pattern observed for the transformant shown in FIG. 16, lane C. Also shown in FIG. 16, lane B is an example of a transformant in which multiple copies of pCEPCl have integrated at sites in the genome other than the cbhl locus~

21~7206 80 -Endoglucanase activity assays were performed on samples of culture supernatant from the untransformed culture and the transformants exactly as described in Example 22 except that the samples were diluted 50 fold prior to the assay so that the protein concentration in the samples was between approximately 0.03 and 0.07 mgtml. The results of as~ays performed with the untransformed control culture and four different transformants (designated CEPCl-101, CEPC1-103, CEPCl-105 and CEPCl-112) are shown in Table 2. Transformants CEPCl-103 and CEPCl-112 are ex~mples in which integration of the CEPCl fragment had led to los~ of CBHI production.

, Table 2 Secreted endoalucanase activitv of T. reesei transformants A B A/B
ENDOGLUCANASE
ACTIVITY PROTEIN
STRAIN~O.D. at 590 nm) (mq/ml~

RutC300 0.037 2.38 0.016 CEPCl-101 0.082 2.72 0.030 CEPCl-103 0.0~9 1.93 0.051 CEPCl 1050.033 2.07 0.016 CEPCl-112 0.093 1.72 0.054 The above results are presented for the purpose of demonstrating the overproduction of the EGI
component relative to total protein and not for the purpose of demonstrating the extent of overproduction. In this regard, tha extent of overproduction is expected to vary with each experiment.

- 8l ~lO7~0~
~92/17~74 PCT/US92/02~31 It would be possible to construct plasmids similar to pCEPCl but with any other T. reesei gene replacing the eall gene. In this way, over-expression of other genes and simultaneous deletion of the cbhl gene could be achieved.

It would also be possible to transform EY~
derivative strains of ~ reesei which had previously been deleted for other genes, e~. for cbh2, w~th pCEPCl to construct transformants which would, for example, produce no exo-cellobiohydrolases and -~
overexpress endoglucana~es.

Using constructions similar to pCEPC1, but in ~ which DNA from another locus of T~ reesei was substituted for the DNA from the cbhl locus, it would be,possible to insert genes under the control of another promoter at another locus in the T.
reesei genome.

~xam~le 25 Construction of pEGII::P-l The e~l3 gene, encoding EGII tpreviously referred to as EGIII by others), has been cloned from T reesei and the DNA ssquence published (Saloheimo et al., 1988, Gene 63~ 21). We have obtained the gane from strain ~L-P37 as an approximately 4 kb PstI- ~h~I fragment of genomic DNA inserted between the PstI and XhoI sites of pUC219. The latter vector, pUC219, is derived from pUCll9 (described in Wilson et al., 1989, Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for B~lII, ClaI and XhoI.

~:107'''~)G - 82 -WO92tl7574 PCT/US92/02631 Using methods known in the art the T. reesei EYE~
gene, present on a 2.7 kb SalI fragment of genomic DNA, was inserted into a SalI site within the EGII
coding seyuence to create plasmid pEGII::P-l (FIG.
17). This resulted in disruption of the EGII coding sequence but without deletion of any sequences. The plasmid, pEGII::P-l can b~ digested with ~i~dIII and ~HI to yield a linear fragm~nt of DNA derived exclusively from T reesei except for 5 bp on one end and l~ bp on the other end, both of which re derived from the multiple cloning site of pUC219.

-- ~xamDle 26 Transformation of T. re~sei GC69 with~ EGII::P-l to crea~e a strain ~nable to produce EGII
T. reesei strain GC69 will be transformed with . .
pEGII::P-l which had been previously digested wit~
~indIII and ~mHI and stable transformants will be selected. Total DNA will be isolated from the transformants and Southern blot analysis used to identify those transformants in which the fragment of DNA conta~`ning the EYE~ and ~gl~ genes had integrated at the ~91~ locus and consequently disrupted the EGII coding sequence. The transformants will be unable to produce EGII. It would also be possible to use pEGII::P-l to tr~nsform a strain which was deleted for either or all of the çbhl, cbh2, or çgll gen~s. In this way a strain could be constructed which would only produce certain cellulase components and no ~ÇI~ comp~nent.

- 83 - ~ 7 ~ ~ fi `~92/17574 PCT/US92/02631 Exam~le 27 Transformation of T. ~eesei with ~EGII::P-l to create a strain unable t.o ~roduce ~BHI
CBHII and ~II
A EYE~ deficient derivative of strain P37P~aCBH67 (fr~m Example ll) was obtained by the method outlined in Example l. This strain P37P~67P
l was transformed with pEGII::P-l which had bQen previously digested with ~i~dIII and ~EHI and stable transformants were selected. Total DNA was isolated from transformants and Southern blot analysis used to identify strains in which the fragme~t of DNA containing the ~vr4 and eal3 gen~s had integrated at the eal3 locus and consequently disrupted the EGII coding sequence. The Southern blot illustrated in FIG. 18 was probed with an approximately 4 kb PstI fragment of ~ reesei DNA
containing the eal3 gene which had been cloned into the PstI site of pUCl8 and subsequently r~-isolated.
When the DNA isolated from strain P37P~67~1 was digested with PstI for Southern blot analysis the eal3 locus was subsequently visualized as a single 4 kb band on the autoradiogr~ph (FIG. 18, lane E).
However, for a transformant disrupted for the e~l3 gene this band was lost and was replaced by two new bands as expected ~FIG. l8, Lane F). I~ the DNA was digested with Eç~oRV or ~glII the size of the band corresponding to the ~g~ gene increased in size by approximately 2.7 kb (the size of the inserted Ey fragment) between the untransformed P37P~67P-l strain (Lanes A and C) and the transformant disrupted for ~5l~ ~FIG. 18, Lanes B and D). The transformant containing the disrupted egl~ gene illustrated in FIG. 18 (Lanes B, D and F) was named 7'~ G
. _ ~4 _ W~92tl7574 PCT/US92/02~31 A22. The transformant identified in FIG. 18 is unable to produce CBHI, CB~II or EGII.

Example 28 Construction of ~P~EGI-l The ~911 gene of T~ re~sei strain RL-P37 was obtained, as described in Example 12, as a 4.2 kb ~i~dIII fragment of genomic DNA. This fragment was inserted at the ~indIII site of pUC100 (a derivative of pUCl8; Yanisch-Perron et al., 1985, Gene 33:103-ll9, with an oligonucleotide inserted into the multiple cloning site adding restriction sites for ~g~ I and ~hQI). Usin~ methodology known in the art an approximately 1 kb EcoRV fragment extending from a position close to the middle of the EGI coding sequence to a position b~yond the 3' end of the coding sequence was removed and replaced by a 3.5 kb ScaI fragment of T. reesei DNA containing the Pvr4 g~ne. The resulting plasmid was callPd pP~EGI-l (see Fig. l9).

The plasmid pP~EGI-l c2n be digested with ~i~dIII to release a ~NA fragment comprising only T.
reesei genomic DNA ha~ing a segment of the eqll gene at either end and the ~ gene replacing part of the EGI coding se~lence, in the cerlter.

~5 Tr~n~formation of 8 suitable T. reesei EY~ deficient strain with the pP~EGI-1 dige~ted with HindIII will lead to integration of this DNA
frag~znt at the ~91l locus in some proportion of the transformants. In this manner a strain unable to produce EGI will be obtained.

- 85 _ ~1 ~7~ ~ 6 ; ~92/17~74 PCT/US92/02631 ~xam~le 29 Construction of ~ EGIpyr-3 and Transfor~ation of a Pvr4 deficient strain o~ T. r~esei The expectation that the EGI gene could be inactivated using the method outlined in Example 28 is strengthened by this experiment. In this case a plasmid, p~EGIpyr 3, was constructed which was similar to pPAEGI-l except that the Asperaillus ~niaer ~vr4 gene replaced the T. reesei EY~ gene as selectable marker. In this c~se the ~gll gene was again present as a 4.2 kb ~iadIII fragment inserted at the HindIII site of pUClO0. The same int~rnal l kb EcoRV fragment was removed ~s during the - construction of pP~EGI-l (see Example 28) but in 15 , this case it was replaced by a 2.2 kb fragment containing the cloned A. niaer vrG gene tWilson et al., 1988, ucl. Acids Res~. l p.2339).
Transformation of a vr4 deficient strain o~ T.
~eesei (strain GC69) with p~EG~pyr-3, after it had been digested with ~i~dIII to release the fragment containing the EYE~ gene with flanking regions from the eqll locus at either end, led to transformants in whi~h the ~gll gene was disrupted. These transformants were recognized by Southern blot analysis of transformant DNA digested with ~i~dIII
and probed with radiolabelled p EGIpyr-3. In the untransformed strain of ~ reesei the ell gene was present on a 4~2 kb ~i~dIII ~ragment of DNA and this pattern of hybridization is represented by Fig. 2~, lane C. However~ following deletion of the ~gll gene by integration of the desired fragment from p~EGIpyr-3 this 4.2 kb fragment disappeared and was replaced by a fragment approximately l.2 kb larger in size, FIG. 20, lane A. Also shown in FIG. 20, ~ ln'72 ~ ~ - 86 -lane B is an example of a transformant in which integration of a single copy of pP~EGIpyr-3 has occurred at a site in the genome other than the Ç9ll locus.

Example 30 Transformation_Qf T.reesei wi~h pP~EGI-l tQ create a strain unabl~ tQL~rodu~e CBHI CBHII~ EGI ~nd EGII

A EY~ deficient derivative of strain A22 (from Exampl~ 27) will be obtained by the method outlined in Example l. This strain will be transformed with pP~EGI-l which had been previously dige~ted with ~indIII to release a DNA fragment comprising only ?.
ree~ei genomic DNA having a segment of the ~gl~ ~ene at either end with part of the EGI coding sequ~nce replaced by the E~E~ gene.

Stable EYE~ transformants will be selected and total DNA isolated from the transformants. The DNA
will be probed with 32p labelled pP~EGI-l after Southern blot analysis in order to identify transformants in which the ~ragment of DNA
containing the ~vr4 gene and_~gl~ ~aquences h~s integrated at the eqll locus and con~e~uently disrupted the EGI coding sequence. The tran~formants identified will be unable to produce CBHI, CBHII, EGI and EG~I~

Claims (10)

WHAT IS CLAIMED IS:

1. An improved method for the treatment of cotton-containing fabrics with a fungal cellulase composition wherein said improvement comprises employing a fungal cellulase composition comprising one or more EG type components and one or more CBH I
type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH I type components of greater than 5:1.

2. The method according to Claim 1 wherein said fungal cellulase composition comprises one or more EG type components and one or more CBH 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.

3. The method according to Claim 2 wherein said fungal cellulase composition has a protein weight ratio of all EG type component to all CBH
type components of greater than 10:1.

4. The method according to Claim 1 wherein said fungal cellulase composition comprises at least about 20 weight percent EG type components based on the total weight of protein in the cellulase composition.

5. An improved method for the treatment of cotton-containing fabrics with an aqueous fungal cellulase solution wherein said method is conducted with agitation of the cellulase solution under conditions so as to produce a cascading effect of the cellulase solution over the fabric wherein said improvement comprises employing a fungal cellulase composition comprising one or more EG type components and one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH I
type components of greater than 5:1.

6. A method according to Claim 5 wherein said fungal cellulase composition comprises one or more EG type components and one or more CBH 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.

7. A method according to Claim 6 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.

8. A method according to Claim 5 wherein said fungal cellulase composition comprises at least about 20 weight percent of EG components based on the total weight of protein in the cellulase composition.

9. A cotton-containing fabric prepared by the method defined in Claim 1.

10. A cotton-containing fabric prepared by the method defined in Claim 5.