CA2107208A1 - 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 a cellulase solution containing a fungal cellulase composition which is substantially free of all CBH I type cellulase components. Cotton-containing fabrics so treated possess decreased strength loss as compared to fabrics treated with a cellulase solution containing a complete cellulase composition.

Description

i~/392/17572 $3 !~ n '7 ~ ~ ~ PCT/US~2/02629 ~3~OD3 ~3~. ~ ~C3~3~-C~3~T~ING
C~ !3 C~

"~ .
~3~ D ~ ~3 ~-3-J~3 ,1 1. Fio'.d of the ~n~ent~on.
T~a pr-s~n. inY2n.ian is diract~d to improved methods f~3r ~r~3ating cotton-containing fabrics with cellul~e ~s wel1 ~s the ra~ s p_odusod 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 composition substantially free of all CBH I type ce}lulase components. When the cotton-containing fabric is treated with 8uch 801utions, the resulting fabric possesses the expected enhancements in, for example, feel, appeaxanc-9, andlor softsning, etc., as ~ ~ compared to the fabric prior to treatment and the I fabric also possesses decreased strength loss as ~ 20 compared to the fabric treated with a cellulase `~ composition containing CB~ ~ ~ype cellulase components.
.

2. State of the Art.
~ , During or shortly after their manufacture, cotton-containing fabrics can be treated with cellulase in order to impart desirable properties to 3 the fabric. For example, in the textile industry, 7~ cellulase has been used to improve the feel and/or 3~

. .

, .':- ~' . ' ~1~7~8 WO92/~7s72 PCT/US92/02629 appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, for 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 ~
"Reformation of Cotton Fabric by Cellulase' and JTN
December 1988 journal article "What's New -- Weight -~;
Loss Treat~ent to Soften the Touch of Cotton ~abric~' each disclose that treatment of cotton-containing -fabrics with cellulase results in an improved feol for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing andlor surface fibers which reduces the weight of the fabric. The combination of these effects imparts ; improved feel to the fabric, i.e., the fabric feels more like siLk.
. .
M ditionally, it was heretofore 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 purpose of removing broken fibers and threads common to these knitted fabrics. When so treated, ~uffers ~ ;
are generally not employed because they are believed ; 25 to adversely affect dye shading with selected dyes.

It was still further heretofore known-in the , ~
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.

~ ' - ' , .

~` ~
~ ~ ~76,~
:~ 92/17572 P(~r/US92/02629 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 com~on problem associated with the treatment of such cotton-containing fabrics with a cellulasa solution is that the treated fabrics exhibit significant strength loss as compared to the untreated fabric. Strength loss arises because th~
cellulase hydrolyzes cellulose (B-1,4-glucan linkages) which, in turn, can result in a breakdown of a portion of the cotton polymer. As more and more cotton polymers are disrupted (broken down), the tensile strength of the fabric is reduced.
, BQcause methods involving agitation and cascading of cellulase solutions over cotton woven fabrics require shorter reaction times, these ` methods are believed to provide cotton-containing woven fabrics of reduced strength loss as compared to celIulase treatment methods not involving agitation and cascading. In any event, such methods 1 25 still nevertheless result in significant strength Ioss.

Accordingly, it would be particularly desirable - to modify such cellulase treatment methods so as to provide reduced stréngth loss while still achieving -~
~; 30 the desired enhancements in the treated cotton-` containing fabric arising from treatment with ;

~: '' ' :
~-, ;~ 2 1 ~ 7 ! ` ~) 8 WO92/17572 PCT~US92/02629 !;~

~' ' .

cellulase as compared to the fabric prior to treatment.
' ~'' ~dditionally, b~cau~2 fungal sources of cellulase are Xnown to secrete very large ~uantities of cPll~ se ar.d fu.~h.e. ~ecaus2 ,e~mentation -procedures for such fungal sources 2S well as isolation _nd rU~ ation prcc2dures .or isolating the c~llulas~ a.2 ~211 ~no~n in tne arc, it would be particul-_ly advan_ag20us to U52 such fungal csllulases in the m2.h3ds for improving reel and/or . appearanca. ~':

s~a~Y OF T~E I~VENTION

. ~ .
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 cellulase composition which is substantially free of all CBH I
type components. Surprisingly, it has been found that EG type components are capable of imparting enhancements to the treated fabric with regard to feel, appearance, softness, color enhancement, -and/or stone washed appearance as compared to the fabric be~ore treatment with 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 in the treated fabric.

!3 ~ In view of the above, in one of its method aspects, the present invention is directed to an 3i~ improved method for the treatment of cotton-,: .
,~.

~ n7~
;~92/17S72 PCT/US92/02629 containing fabrics with a fungal cellulase composition wher~in said improvement comprises employing a fungal cellulase composition which is substantially Lr e of all C~ ype components. In a preferred embodiment, the fungal cellulase composition is free or all CB~ I type and all CBH II
i type components. In still another preferr~d embodiment, th~ Eu~gal cellulas2 composition compri~es at laast about 10 weight percent and lo pre~erably ~ leas. a~out ~0 ~eight percsnt of EG
type components based on the total weight of protein in the cellulas~ composition.

, In another of its method aspects, the present invention is directed to an improved method for the lS treatment of cotton-containing fabrics with an aqueous fungal cellulase solution wherein said method is conducted with agitation under conditions o as to produce a cascading effect of the cellulase solution over the fabric wherein said improvement ~-~ 20 comprises employing a fungal cellulase composition which is su~stantial,y free of all CBH I type ~ components. In a preferred embodiment, the fungal ¦ cellulase composition is free of all CBH I type ~ components and all CBH II type components. In still ,~ 25 another preferred embodiment, the fungal cellulase -composition comprises at least about 10 weight , percent and preferably at least about 20 weight~`
percent of EG type components based on the total weight of protein in the cellulase composition.
~ . :
,~
Cotton-containing fabrics treated by the methods of this invention have the expected enhancement(s) as compared to the fabric prior to 1 :: :"
:, ' J"''.".'',`'','"''.',. "''''';.'',' '~,.',"', . 0 8 wos2~l7s72 PCT/US92/02629 ~i ' ' ' -: ~
'; ' ". -treatment while exhibiting reduced strength loss as compared to the fabric treated with a fungal cellulase composition which contains CBH I type cellulase components. The reduced strength loss evidences that 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 inYention as defined above.
.;!: ' E~ DBSC~PTI9~ OF T~ D~a~I~&8 -~

FIG. 1 is an outline of the construction of p~,CBHIEy~4.

FIG. 2 illustrates dèletion of the ~ reesei , gene by integration of the larger EcoRI fragment `
from~p CBHIEy~ at the .cbhl locus on one of the T.
re-sei chromosomes.

FIG. 3 is an autoradiograph of DNA from T.
reesei strain GC69 transformed with ~ç_RI digected p~CBHI~vr4 after Southern blot analysis using a 32p labelled p~CBHIEyL~ as the probe. The sizes of molecular weight markers are shown in kilobase pairs 3~ ~ ~ to the left of the Figure.
iy ~
FIG. 4 is an autoradiograph of DNA from a T.
reesei strain GC69 transformed with EcoRI digested p~CBHIEyE~ using a 32p labelled pIntC~HI as the ; probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.

';-"~92/17572 ~ t Q7 ,',JO 8 PCT/US92/02629 FIG. 5 is an isoelectric focusing gel displaying the proteins secreted by the wild type and by transformed strains of T. reesei.
Specifically, in FIG.5, Lane A of the isoelectric s focusing gel employs partially purified CBHI from T .
reesei; Lane B employs a wild type T. rei_si_i : Lane C employs protein from a T. reesei strain with the cbhl gene deleted; and Lane D employs protein from a T. reesei strain wi~h the cbhl and cbh2 genes deleted. In FIG. 5, the right hand side of the figure is marXed to indicate the location of the 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. reesei cbh2 locus, cloned as a 4.1 kb EcoRI fragment on genomic DNA and FIG. 6B is a representation of the cbh2 gene deletion vector pP-~CB~
,:
FIG. 7 is an autoradiograph of DNA from T.
reesei strain P37P~CBHIPyr26 transformed with EcoRI
digested pP CB~II after Southern blot analysis using a 32p labelled pPA Q HII 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 illustrates the RBB-CMC activity profile of an acidic EG enriched fungal cellulase ~ ' . .

'i''' ' ' .. '". -" . . ' : . - , '' ' ' ' ' ' : .; '' ' . ,, . ,, ' .. .

~ i . ' ''.' ~ - ': ' " . ' ,, ! , . ' . . , ? ~ ~
WO92/17572 : PCT/US92/02629 "~

composition (CB~ I and II deleted) derived from Trichoderma Leesei over a pH range at 40C; as well as the activity profile of an enriched EG III
cellulase composi-ion derived from Trichoderma reesei over a pH ,ange at 40C.

FIG. lo illustrates strength loss results after ~-three T~ash ~ysles in a launderometer for cot~on-c~.._-.in-ng fa~ics '~e2t~d wi.h cellulase compo~itlons h2ving -~rarylng amounts of CBH
lo components.

FIG. 11 illustrates 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 test 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 strain of Trichoderma reesei ~:
genetically engineered so as to be incapable of :
secreting CBH I and CBH II.

FIG. 13 illustrates the softness panel test ~ :
results for varying concentrations (in ppm) of an EG
enriched cellulase composition derived from a strain of Trichoderma reesei genetically modified so as to be incapable of producing CBHI & II.

: ~. . ' ' . ' ' ::: . ': , ' ' ' ' ,., ' ' .: ' " . . . : ' : .: ' . , . . ' . ' :

:~ . . . ' : . ' '. I :

~` ~ 92/17572 ? ~ ~ 7 ~ ~ 8 PC~r/US92/02629 _ g FIG. 14 is a diagram of the site specific alterations made in the ~511 and cbhl genes to create convenient restriction endonuclease cleavage sites. In ~ach case, thP upper line shows the original DNA sequence, the changes introduced are -~ shown in the middle line, and .he new sequence is ~ shown in the lower lino.

~ I~. Ij is a diagram OL rhe larger EcoRI
frag~en~ -~ihich can ~ o~tained from pCEPC1.

EIG. lo i3 an ~utoradiograph of DNA, from an lntrarsfo~rQd st in of ~ ~oesei RutC30 and from . . .
two transformants obtained by transfor~ing T. reesei with EcoRI digest~d pCE2C1. The DNA was digested with PstI, a Southern blo~ was obtained and hybridized with ~P labelled pUC4X::cbhl. The sizes ~' of marker DNA fragments are shown in kilobase pairs to the left of the Figure.

I FIG. 17 is a diagram of the plasmid pEGII::P~
: . .
~ .
FIG 18. is an autoradiograph of DNA from T.
reesei strain P37P~67Pl transformed with HindIII
and ~3~HI digested pEGII::P-l. A Southern blot was prepared and the DNA was hybridized with an ; approximately 4kb E~I fragment of radiolabelled T.reesei DNA containing the ~9~3 gene. Lanes A, C
25~ and E contain DNA from the untransformed strain ~ whereas, Lanes B, D and F contain DNA from the 4~ untransformed T. reesei strain. The T.reesei DNA
was digested with BalII in Lanes A and B, with EcoRV
in Lanes C and D and with PstI in Lanes E and F.

,~ . ',,' 7 2 -~ 8 WO92/1~572 PCTt~S92/02629 ' `

.
. -- 10 --.. .
The size of marker DNA fragments are shown in kilobase pairs to the left of the Figure.

, FIG. 19 is a diagram of the plasmid pP~GI-1.
.. . .
~, FIG. 20 is an autoradiograph of a Southern blo-c ,'~ 5 of DNA isolated from transformants of strain GC6 ,i obtained with HindIII digested p~GIpy~-3. The ~, 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 eall gene has been dlsrupted and ~2r.e ~
, shows a transformant in which pAEGIpyr-3 DNA has -, integrated into the genome but without disrupting '~, the eall gene. Lane D contains p~EGIpyr-3 digested ~,l 15 with,HindIII to provide appropriate size markers. ' ,, The sizes of marker DNA fragments are shown in ' ~ kilobase pairs to the right of the figure.
~ .
DETAILED DESCRIPTION OF THE PREFERRED EMfiODlM2NTS

As noted above, the methods of this invention ~ 20 are improvements in prior art methods for treating ,,, cotton-containing fabrics with cellulase. The r~'7 improvement comprises using a specific cellulase , , composition which imparts the desired enhancement(s) ' to the fabric while minimizing strength loss in the ', 25 fabric. However, prior to discussing this invention , '! ~ in detail, the following terms will first be -~ defined.

,l The term "cotton-containing fabric~ refers to ~ sewn or unsewn fabrics made of pure cotton or cotton :~ .
.. .
l~

. .

s. ~ . , ~ '' . ; '' : . : :. ' .' 7~8 ~ 92/1757~ PCT/US92/02629 .
., -- 11 --, 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 at least about ~o percent by - -S weight cotton; preferably, more than about 60 percent by weight cotton; and most prefera~ly, more 't~ than about 75 percsnt by weight cotton. When ,j employed as blends, the companion material employed in the fabric can include one or more non-cotton fibers including synthetic fibers such as polyamid~
il fibers (for example, nylon 6 and nylon 66), acrylic ~
f fibers (for example, polyacrylonitrile fibers), and - ~-polyester fibers (for example, poly~thyl~n ,~ terephthalate), polyvinyl alcohol fibers (for 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.
'i;-l ': ' ., , :
The term "finishing" as employed herein means bi;~ , the application of a sufficient amount of-finish to ~ ~ a cotton-containing fabric so as to substantially f prevent cellulolytic activity of the cellulase on ~r~ ~ 25 the fabric. Finishes are qenerally applied at or near the end of the manufacturing process of the ~' fabric for the purpose of enhancing the properties of the fabric,- for example, softness, drapability, etc., which additionally protects the fabric from reaction with cellulases. Finishes useful for finishing a cotton-containing fabric are well known in the art and include resinous materials, such as melamine, glyoxal, or ureaformaldehyde, as well as - , . .

",j wo s2/f~s~ ~ ~
2 PCT/US92/0262s ~i -~; waxes, silicons, fluorochemicals and quaternaries.
When so finished, th~ cotton-containing fabric is substantially less reactive to cellulase.
~ . .
.;1 .
''t~''' The ter~ "~ungal cellulase" refers to the enzyme composition derive~ rrom fungal sources or microorgeni~ms genetically ~odified so as to incorpo~a.e and Pcpre-s all or part of the cellulase genes obta1ned Lrom a ungal source. Fungal callulases act ^r. cellulos2 and its derivatives to hydrolyze cellulose and give primary products, glucose and celloblose. Fun~21 cel 1 ~lases are ~; distingulshed from c21~ ulases produc2d from non-fungal sources including microorganisms such as actinomycetes, gliding bacteria (myxobacteria) and true bacteria. Fungi capable of producing cellulases useful in preparing cellulase compositions described herein are disclosed in ~ritish Patent No. 2 094 826A, the disclosure of which is -;~ incorporated herein by reference.
, . . .
,~, 20 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.
~"' .
",i~ ' ~: Fungal cellulases are known to be comprised of several enzyme classifications having different ~ substrate specificity, enzymatic action patterns, ;'`~ 30 and the liXe. Additionally, enzvme components within each cl~ssification can e~hibit different ij. ~

, . . .

`~ `392/17572 ~ n 7 ? ~ `~ PCT/US92/02629 molecular woights, different degrees of glyco-sylation, differPnt isoelectric points, different substratP specificity, etc. For example, fungal cellulases can contain cellulase classirications S which include endoglucanases (EGs), exo-c~llobiohydro~ases ~CBHs), ~-glucosidases (BGs?, etc. On ~hQ other ~and, whil2 bacterial cellulases are rQpor~ad in the iireratur~ as containing little or no C3H comDcnents, th2re are a few cases where CBH-ii.c2 compon~n;s de_iv2d -from ~acterial cellulases have been reported to possess e~o-~ -Jh~d~-ol~S Q ~.c ~ y . . .
; ' ' A fungal cellulase composition produced by a naturally occurring fungal source and which comprises one or more CBH and EG components wherein each of these co~ponents is found at the ratio produced by the fungal source is sometimes referred ; to herein as a "complete fungal cellulase system" or s a "complete fungal cellulase composition" to distinguish it from the classifications and components Oî cellulase isolat~d therefrom, from , - incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism genetically modified so as to overproduce, `I underproduce, or not produce one or more of the CBH
and/or EG components of cellulase.
.
., .
i The fermentation procedures for culturing fungi for production of cellulase are known per se in the art. For example, cellulase systems can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. The .~ - .

;' '.:"

. L ~. ~ ; ' ' . . ' . ' ' . . ' ' 2~Q7~t~
W 0 92/17572 PC~r/U~92/02629 ;

collection and purification of the cellulase systems from the fermentation broth can also be effect&d by procedures known Der se in the art.

"Endoglucanase (~EG") type components" refer to all of those fungal cellulase components or combination of components which exhibit textila activity properties similar-to the endoglucanase components of Trichode~ma reesei. In this regard, the endoglucanase components of Trichod~rma r~esei ; 10 (specifically, EG I, EG II, EG III, and the like either alone or in combination) impart impro~d feel, improved appearance, softening, color enhancement, and/or a stone washed appearance to cotton-containing fabrics (as compared to the fabric prior to treatment) when these components are incorporated into a textile treatment medium and the ;~ ~ fabric is treated with this medium. Additionally, treatment of cotton-containing fabrics with ~ endoglucanase~components of Trichoderma reesei t' ', ¦ ~ 20 results in }ess strength loss as compared to the strenyth loss arising from treatment with a similar composition but which additionally contains CBH I
type components.

Accordingly, endoglucanase type components are those fungal cellu}ase components which impart improved feel, improved appearance, softening, color enhancement, and/or a stone washed appearance to cotton-containing fabrics (as compared to the fabric before treatment) when these components are incorporated into a medium used to treat the fabrics and which impart reduced strength loss to cotton-containing fabrics as compared to the strength loss ~ . .
~:~
.'. . .

?.'. n7~,98 '~`)92~17572 PC~r/US92/02629 , :

arising from treatment with a similar cellulase composition but which additionally contains CBH I
type components.

Such endoglucanase type components may not include components traditionally classiried as endoglucanases using activity tPsts such as the ability of the component (a) to hydrolyze soluble -cellulose derivatives such as carboxymethylcellulose (CMC), thereby reducing the viscosity of CMC
~ 10 containing solutions, (~) to readily hydrolyze ;~ hydrated forms of cellulose such as phos~horic acid swollen cellulose (e.g., Walseth c~llulos~) and hydrolyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.).
On the other hand, it is believed that not all endoglucanase components, as defined by such activity tests, will impart one or more of the enhancements to cotton-containing fabrics as well as reduced strength loss to cotton-containing fabrics.
Accordingly, it is more accurate for the purposes herein to de.ine endoglucanase~type components as those components of fungal cellulase which possess :~ similar textile activity properties as possessed by `
the endoglucanase components of Trichoderma reesei.

25~ Fungal cellulases can contain more than one EG
type component. The different components generally ~ ~<~
have different isoe}ectric points, different molecuIar weights, different degrees of glyco- -sylation, different substrate specificity, different enzymatic action patterns, etc. The different isoelectric points of the components allow for their separation via ion exchange chromatography and the , . .

2i?7.'..'~
wos2/~7s72 PCT/US92/02629 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.
07/422,81~, Schulein et al., International Application WO 89/09259, ~ood et al., Biochemistry and G~netics o~ Cellulose Degradation, pp. 31 to 52 (19~38)~ '~ood ~t al., Carbohydrate Research, Vol.
~90, pp. 279 o ~97 (1989); Scnulein, Methods in Enzymology, Vol. loO, pp. 234 to 242 (1988); and the lo liX2. Thz en-i 2 disclosure of each of these refer~nces is incorporated herein by reference. ~ -~ y~ a'~ e~pl2~Pd tha~
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 single 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 ~e 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 ~ 392/17572 ~1 n 7 . 9 8 PCT/US92/02629 of Trichoderma reesei. In this regard, when used in the absence or EG type cellulase components (as defined above), the CBH I and CBH II components of Trichoderma reesei alone do not impart any significant onh~n~monts in feel, appearance, color enhancement and/or stone washed appearance to the so treat-od cotton-cor.tainir.g f2brics. Additionally, when used in com3ina-tion ~ith EG type components, the CB~ I component of Trichoderma reesei imparts enhanced s-.-eng.h loss to .he cotton-containing fabrics.

~ccordingly, C3i I ~ype components and CBH II
-type components refer to those fungal cellulase components which exhibit textile activity properties similar to CBH I and CBH II components of Trichoderma 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 ?richoderma reesei can impart an incremental cleaning benefit.
Additionally, it is contemplated that the CBH I
components of Trichoderma reesei, when used alone or in combination with EG type components, can impart an incremental softening benefit.
..
Such exo-cellobiohydrolase type components could possibly not include components traditionally classed as exo-cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II rom Trichoderma reesei. For example, such .' ' .

q~ 7 ~ i ~ f . J ~

.
components (a) are competitively inhibited by cellobiose (~ approximately lmM); (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., S and (c~ hydrolyze phosphoric acid swollen C21lUlG32 and to a lesser degree highly crystalline c~llulose.
On the other hand, it is believ~d that so~a funsa cellulase components which are characterlzed as CBH
components by such activity tests, will im~a t improved feel, appearance, softening, color enhancement, and/or a stone washed appearance to cotton-containing fabrics with minimal atr~n~ ' 0 when used alone in the cellulas2 composition.
Accordingly, it is believed to be more accurate for the purposes herein to define such exo-cellobio-hydrolases as EG type components because these components possess similar functional properties in textile uses as possessed by the endoglucanase components of Trichoderma reesei.

In regard to the detergent compositions ;~ containing cellulase compostions which are CBHI -~
deficient, CBHI enriched or EGIII enriched, it has been found tha~ 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 deter~ent properties to cotton-containing fabrics, eg., one or more of improved color restoration, improved softening and improved cleaning to the detergent composition.
~ .
Fungal cellulases substantially free of all CBH I type components can be obtained by ~: : .
.

: . j : , :. . , . ~ ; ,. , . : ,. ,, . . :.

~ 92/17S72 7~ 7 ?9~ PCl~US92/02629 ~ .

purification techniques. Specifically, the complete cellulase system can be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possibls to separate the cellulase components by eluting with a 1 10 pH gradient, or a salt gradient, or ~oth a pH and a ,~ salt gradient. As used herein, the term ~'cellulase composition substantially r_e of all CB~ I type cellulase components" means that tne cellulase composition, based on the weight of protein, will contain less than 1 weight percent CBH I type ~ cellulase components. ' I ~t is also contemplated that cellulase ~¦~ i compositions substantially free of all CBH I type ,~; components can be prepared by means other than isolation and recombination of the components. For example, recombinant techniques can be used to prèpare microorganisms which are incapable-of producing any CBH I type components or which are incapable of producing any CBH type components.
t; ~ :
In regard to the above, a preferred method for ~- the preparation of cellulase compositions ~1 substantially free of CBH I type components is by ; genetically modifying a microorganism so as to be incapable of expressing CBH I type components which methods do not express any heterologous protein.
Likewise, it is also possible to genetically modify a microorganism so as to additionally overexpress ,:
~ .
. ,. :

WO92/17~-7~ ~' J ~ PCT~US92/02629 ' ~
one or more ~G type components. For example, U.S. ~-Serial No. 07/593,919, filed October 5, 1990 and i which is incorporated herein by reference in its ', entirety, discloses methods for genetically S engine~.ing TL ic~oderma aesei so as to be incapable of expressing on~ or more C8H components and/or OV~LP`~_35Sing One Or mO_9 ~G components. Moreover, the me~ods OL that application cr~ate Trichoderma ~ roesei s -ain.s which do not -xpr~ss any heterologous j 10 proteins. ~ikewise, Miller et al., "Direct and Indir~ct Geno Replacement in As~erqi~lus nidulans", ~012c~1ar a..d CellulaL ~ioiGg~, p. 1714-1721 (1985) SC10g2S 1~2t~0dS L-Or deleting genes in Asperqillus nidulans by DNA mediated transformation using a 1 15 linear fragment of homologous DNA. The methods of ¦ Miller et al., would achieve gene deletion without I producing any heterologous proteins.

l In view of the above, the deletion of the genes i responsible for producing CBH I type and/or CBH II
type cellulase components would also have the effect ¦ of enriching the amount of EG type components present in the cellulase composition. Likewise, the deletion of those genes responsible for producing CB~ I and II type components would result in a cellulase composition free of CBH type components.

It is still further contemplated that fungal cellulase compositions can be used herein from fungal sources which produce an incomplete fungal cellulase composition. For example, it is known that certain fungi produce cellulase compositions free of CBH components. See, for example, Coughlan et al., Biochemistry and Genetics of Cellulose ',~.'' ' ' ' ' . ' , ' , ' ' ',, '; ' ' ` ' '. . . ' ' "''` .' :`,~ ' ~ . ', ' `: ' -~ 92/17572 PCT/US92/02629 : .

.:
.. , : .
~ Degradation, Aubert et al. Editors, pp. 11-30 -i (Academic Press, 1988), disclose that brown rot ~ fungi do not apparently produce CBH components, but ;,~ it may be possible that one or more of these S comFonPn-i, are CB~ I type components.

S-Glurosidase (BG) components" refer to those ~3 componen's of cellulase which exhibit BG activity;
j that is to s2~ that such co~ponants will act from ¦i~ the non-reducing end o~ celIobiose and other soluble cellooligosac~harides ~"cellobiose~ and give glucos7i as tae sol~ producit. BG components do not ~ adsorb onto or rii~act with c~llulose polymers.
!~: Furthermore, such BG components are competitively ;
inhibited by glucose (~ approximately lmM). While 3~: 15 in a strict sense, BG components are not literally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the celIulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory ` `
cellulose degradation products (particularly cellobiose) produced by the combined action of CBH
components and EG components. Without the presence ` of BG components! moderate or little hydrolysis of crystalline cellulose will occur. BG components are often characterized on aryl substrates such as p-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 hOt hydrolyze cellobiose.
{ ~ ~
It is contemplated that the presence or absence of BG components in the cellulase composition can be ' ~ ' ' ' ' 7 7. ~ i~
; W O 92/17572 PC~r/US92/02629 ., .
used to regulate the activity of any C~3H components . in the composition (i.e., non-CBH I type components). Specifically, because cellobiose is produced during cellulose degradation by CBH
components, and because high concentr~tions of cellobiose are known to inhibit CBH actiYity, and further because such cellobiose is hyd.olyzed t~
glucose by BG components, the abssncP of BG
components in the cellulase composition will "turn-off" CBH activity when the concentration or cellobiose reaches inhibitory levels. It is also cont_mplated that one or mor~ additives ~e.g., cellobiose, glucose, etc.) can ~e addiQd to ~ne cellulase composition to effectively "turn-off", ;;~ 15 directly or indirectly, CBH I type activity as well jl~ as other CBH activity. When such additives are employed, the resulting composition is considered to ~;1 be a composition free of all CBH I type components if the amount of additive is sufficient to result in effectively no CBH I type activity.

on the other hand, a cellulase composition containinq added a~ounts of BG components may increase overall hydrolysis of cellulose if the level of cellobiose generated by thé CBH components becomes restrictive of such overall hydrolysis in 3~ the absence of added~BG co~ponents.

' Methods to either increase or decrease the -amount of BG components in the cellulase composition 3 are disclosed in U.S. Serial No. 07/625,140, filed j~ 30 December lO, l990, as attorney docket no. 010055-056 and entitled "SACCHARIFICATION OF CELLULOSE BY
,~ CLONING AND AMPLIFICATION OF THE 3~-GLUCOSIDASE GENE
;3`~

3~

92/1~572 ~ ~ 7 ~ PCT/US92/02629 8Y TRICHODERMA REESEI", which application is incorporated herein by reference in its entirety.

Fungal cellulases can contain more than on~ BG ~-component. The different components generally have different isoelectric points which allow for ~heir - separation via ion exchange chromatography and the like. Either a single BG component or a combination of BG components can be employed.
~ . . When employed in textile treatment solutions, the BG component is generally added in an amount sufficient to prevent inhibition by cellobios~ of any CBH and EG components found in the cellulase composition. The amount of BG component added ~ depends upon the amount of cellobiose produced in ; 15 the textile composition which can be readily ,;
determined by the skilled artisan. However, when employed, the weiaht percent of BG component relative to any C8H type components present in the -cellulase composition i5 preferably from about 0.2 to àbout 10 weight percent and mor2 pref2rably, from ; about 0.5 to about 5 weight percent.
.~ ~ . ...
Preferred fungal cellulases for use in preparing the fungal cellulase compositions used in this invention are those obtained from Trichoderma reesei, Trichoderma koninaii, Pencillum sp., Humicola insolens, and the like. Certain fungal cellu}ases are commercially available, i.e., CELLUCAST (available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist Brocades, - N.V., Delft, Holland), CYTOLASE 123 (available from Genencor International, South San Francisco, , .
~ ' ' . .

W092/l7~72 PCT/US92/02629 California) and the li~e. Other fungal cellulases can be rPadily isolated by art recognized fermentation and isolation procedures.

The term "buffer" refers to art recognized acid/,ase reagents which stabilize the cellulase solution egains~ undesirPd pH shifts during the I cellulase traa-,rmen~ of ,~he cotton-containing fabric.
In this regard, li is art recognized that cellulase ,~ activiLy is p;T dr pQndPnt. That is ~o say that a specific cellulase composition will exhibit cellulolytic acti~rity -~ithin i~ def~ned pH range ~ith optimal c=l'ulslytlc activlty genarally being found ~ within a s~all portion of this defined range. The j specific pH range for cellulolytic activity will vary with each cellulase composition. As noted above, while most cellulases will exhibit cellulolytic activity within an acidic to neutral p~ -profile, there are some cellulase compositions which exhibit cellulolytic activity in an alkaline pH
profile.

During cellulase treatment of the cotton-containing fabric, it is possible that the pH of the initial cellulase solution could be outside the range required for cellulase activity. It is further possible for the pH to change during treatment of the cotton-containing fabric, for example, by the generation of a reaction product which alters the pH of the solution. In either event, the pH of an unbuffered cellulase solution could be outside the range required for cellulolytic activity. When this occurs, undesired reduction or cessation of cellulolytic activity in the cellulase .

~92/17572 2 ~ ~ 7 ~ ~ 8 PCT/USg2/02629 .~ . .; . .

.. . .
,. . ..
solution occurs. For example, if a cellulase having i, an acidic activity profile is employed in a neutral " unbuffer~d aqueous solution, then the pH of the j solu~ion will r~i,ult in lower cellulolytic activity 5 and possibly in the cessation of cellulolytic activi~y. On the otAer hand, the use of a cellulase having a neutral or alkaline pH profile in a neutral unbuf-l_r2d aqueous solution should initially provide significant ceiluiolytic activity.

l ~ - 10 In view or tne above, the pH or the cellulase ! solu~ion should oe maint~ined within the range req~ ed fo~ S~ll ulol~tic acti-~ity. One means of accomplishing this is by simply monitoring the pH of the syste~ and adjusting the pH as required by the addition of either an acid or a base. However, in a ~-preferred embodiment, the pH of the system is preferably maintained within the desired pH range by the use of a buffer in the cellulase solution. -In general, a sufficient amount of buffer is employed so as to maintain the pH of the solution within the ~-~ range wherein the employed callulas2 exhibits ?~ - activity. Insofar as different cellulase compositions have different pH ranges for exhlbiting cellulase activity, the specific buffer employed is selected in~reIationship to the specific cellulase composition employed. The buffer(s) selected for use with the cellulase composition employed can be `~ readily determined by the skilled artisan taking into account the pH range and optimum for the 3~ 30 cellulase composition employed as well as the pH of the cellulase solution. Preferably, the buffer employed is one which is compatible with the cellulase composition and which will maintain the pH

~ .

" n ~ ' ` W092/17572 Pi~T/US92/02629 ;~

. .
of the cellulase solution within the pH range required for optimal activity. Suitable buffers include sodium citrate, ammonium acetate, sodium - acetate, disodium phosphate, and any otAer art recognized buffers.
, .
The tensile strength of cotton-containing fabrics can be measured in a warp and fill direction which are at right angles to each otner.
Accordingly, the term "warp tensile strengt~i~ as used herein refers to the tensile strengih of the cotton-containing fabric as measured along the length of the cotton-containing fabric ~hereas 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 tensile strength of the resulting cotton-!~j containing fabric treated with a cellulase solution is compared to its tensile strength prior to ?j ~ treatment with the cellulase solution so as to determine the strength reducing effect of the treatment. If the tensile strength is red~lc2d too much, the resulting cotton-containing fabric will easily tear and/or form holes. Accordingly, it is desirable to maintain a tensile strength (both warp and fitil) after treatment which is at least about 50% of the tensile strength before treatment.
~?
; The tensile strength of cotton-containing fabrics is readiIy conducted following ASTM D1682 ~ test methodology. Equipment suitable for testing `3,~ 30 - the tensile strength of such fabrics include a Scott tester or an Instron tester, both of which are ; commercially available. In testing the tensile , s.l .. .. .

~ ~, ~ . . . . - . - . . .. .. . ... . . . .. .. . ..

3 92/17572 ~ J- n 7 ~ PC~r/US92~02629 '~

~ 27 ~
.;
strength of cotton-containing fabrics which have - been treated with cellulase solutions, care should be taken to prevent fabric shrinkage after treatment an~ before testing. such shrinkage would resul. in erroneous tensile strength data. -.~ .
~i Enhancaments to the cotton-containing fabric is --,~ achieved by those methods heretofore used. For example, cotton-containing rabrics 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 Decsmber 1988 journal artislo ~ "What's New -- Weight Loss Treatment to Soften the 3~ Touch of Cotton Fabric". The teachings of each of these references is incorporated herein by reference.

Similarly, methods for improving both the feel ; and appearance of cotton-containing fabrics include contacting the fabric with an aqueous solution containing cellulase under conditions so that the solution is agitated and so that a cascading effect of the cellulase solution over the cotton-containing ~ fabric is achieved. Such methods result in improved J`~ feel and appearance of the so treated cotton-25~ ~ containing fabric and are described in U.S. Serial No. 07/598rS06~ filed October 16, 1990 and which is incorporated herein by reference in its entirety.

Methods for the enhancement of cotton-containing knits are described in International Textile Bulletin, Dyeing/Printing/Finishing, pages 5 wo~2~1~s7~ ~ PCT/US92/02629 et seq., 2nd Quarter, l990, which is incorporated herein by rererenc2.
:. ' i Likewis~, mathods for imparting a stone washed appearance to cotton-con~aining denims are described in u.s. ~t~nt ~o. ~,Q32,~54, which is incorporated herein by r~.~renc~ in its en~irQty.
'~ .
O..la~ ;lods for 2nhancing coLton-con~aining fabrics by cr~ment ~ h a csllulasa composition ar~ kncwn in tha ar.... Pr2f-rably, in such methods, the trPatmant of th- cotton-containing fabric with cellulase is conductPd prior to finishing the cotton-containing fabric.

As noted above, the present invention is an improvement 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 cellul2se composition substantially free of CBH I type components and preferably, substantially free of all CBH type components.

Additionally, the use of the cellulase f ~ 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 stressed, it will j ~ contain broken and disordered fibers. Such fibers detrimentally impart a worn and dull appearance to the fabric. ~owever, when treated in the method of 5:~ ~

1 ~ ;

`i~92/17572 71 ~ 7 ? ~ ,~ PCT/US92/02629 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 afrect o- restoring the appearance of the fa~ric prior to becoming stressed.

Additionally, it is contemplatod that by employing `th~a cellula52 ~omposi.ion described herein wiih pigmenc ;_ypa dyed ra:~rics ~e.g., denims), these cellulase co~?csi=ions will caus2 less redeposition of dye. I~ is also contemplated that these anti-redepssition properties can ~e enhanced for one or mor_ 5p~5~ r^i C ~5 t'~o compor.~nt(s) as comparad to other components.

The fungal cellulase compositions described lS 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 ~ 20 is sQnerally a concentration suffici~nt .or its 3 ~ 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 3~ 25 employed is also dependent on the equipment employed, the process parameters employed (the temperature of the cellulase solution, the exposure time to the cellulase solution, and the like), the cellulase activity (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 i1~

1:
1:
.

:: . . , , ~ ' ."~ ~ ' . ! , " ,, 21ni~?~
; wos2~l7s72 PC~/US92/02629 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 solution to about 10.0 grams/liter of cellulase solution; and more preferably, from about 0.05 grams/liter or cellulase solution to about 2 gram/lit~r of callulase solution. (The cellulase concentration recited above refers to the weight of total protein).

When a buffer is employed in the cellulase solution, the concentration of buffer in the aqueous cellulase solution is that which is sufficient to maintain the pH of the solution within the range wherein the employed cellulase exhibits activity ¦ which, in turn, depends on the nature of the cellulase employed. The exact concentration of buffer employed will depend on several factors which the skilled artisan can readily taXe into account.
1 For example, in a preferred embodiment, the buffer 1 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, buffèr concentration in the cellulase solution is about 0.005 N and greater.
Preferably, the concentration of the buffer in the cellulase solution is from about 0.01 to about 0.5 N, and more preferably, from about 0.05 to about ~; 0.15 N. It is possible that increased buffer ! ~ concentrations in the cellulase solution may enhance ' . ' :

'..

? I ~ ~7 ~
392/17572 PCT/US92/02~29 the rate of tensile strength loss of the treated fa~ric.
: :
In addition to cellulase and a buffer, the cellulase solution can optionally contain a small ; 5 amount of a surfactant, i.e., lass than about 2 weight~percent, and preferably from about O.Ol to about Z 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 sulfatès having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates ~ and the like. Suitable counter ions for anionic ; surfactants include alkali metal ions æuch as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alXanolamines having l to 3 alkanol groups of carbon number 2 or 3.

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

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

? ~ ~
WO92~17572 PCT/US92/02629 ~ixturos of such surfactants can also be used.

The liquor ratios, i.e., the ratio of weight of c_llulase solution to the weight of fabric, employed herein is generally an amount sufficient to achieve S th~ d 2s . red e~hanc2m~nt in the cotton-containing fabric and is d pendent upon thP ~rocPss used and the enhanc_ment to b a.-,lieve. PrPferably, the liquo~ raL~ os are gen2rally rrom a~out O.l:l and great_ , al.d more ~L e l- ~rably greater than about l:l and even more preferably greater than about lO:l.
Use of liquor ra~ios of groater than about 50:l are usuall~ no~ pre^o__e~ flem ~ economic viewpoint.

~eaction temperatures for cellulase treatment are governed by two competing factors. Firstly, higher temperatures generally correspond to enhanced reaction kinetics, i.e., faster reactions, which permit reduced reaction times as compared to reaction times reguired at lower temperatures.
Accordingly, reaction temperatures are generally at least about 30C and greatar. Secondly, cellulase is a protein which loses activity beyond a given reaction temperature which temperature is dependent on the nature of the cellulase used. Thus, if the reaction temperature is permitted to go too high, then the cellulolytic activity is lost as a result of the denaturing of the cellulase. As a result, ~ -the maximum reaction temperatures employed herein are generally about 65C. In view of the above, reaction temperatures are generally from about 30OC
to about 65C; preferably, from about 350C to about ~;
60C; and more preferably, from about 35C to about SOC.
.

~ g2/17572 ~ 1 ~ 7 ? ~ 8 PCTtUS92/02629 .:

Reactlon times are generally from about 0.1 hours ~o about 24 hours and, preferably, from about 0.25 hours to about 5 hours.
, ~he cotton-containing fabrics treated in the methods d~scr bed a~o~e using such cellulase compositions ~ossess reduced strength loss as compared to th~ ~2~ cotton-containing fabric , trea.ed in iha ~a~2 ~anner ~ith a comple~e fungal ¦~ cellulas2 c~m~osition.

In a ~rererr~od em~odiment, a concentrate can be preparsd fo~ use in ~h_ m~thods described hereln.
~ Such concentrates would contain concentrated amounts ¦ ~ of the cellulase composition described above, buffer 7; and surfactant, prefer~biy in an aqueous solution.
When so formulated, the concentrate can readily be 1 ¦~ d$1uted with water so as to guickly and accurately prepare cellulase solutions having the requisite concentration of these additives. Preferably, such concentrates will comprise from about 0.1 to about 20 weight percent of a cellulase composition described above (protein); from about 10 to about S0 weight percent buffer; from about 10 to about 50 ;-weight per¢ent surfactant; and from about 0 to 80 weight percent water. When aqueous concentrates are -~
1 .
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 cellulase solution. As is readily apparent, such concentrates will permit facile formulation of the cellulase solutions as well as permit feasible transportation of the concentration to the location where it will be used. The cellulase composition as . ~ . , 1 ~ .
,,~ ' .
: ~ .
' , .

described above can be added to the concentrate either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the likP. Such forms are well known to the sXilled artisan.

.
When a solid cellulase concentrate is ~-mployed, the cellulase compcsition is generally a sranul3, a powder, an agglom~ration, and the like. -~2n granules are used, t~e granules can preI2.-aDly ~e formulated so as to contain a cellulas2 prot~c-ting agent. See, for instance, U.S. Serial ~o.
07/642,669, filed January 17, 1991 25 A~torne~
Docket No. 010055-073 and entitled "GP~TL~S
; CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING
AGENT AND DETERGENT COMPOSITIONS CONTAINING SUC~
¦~ 15 G~ANULES" which application is incorporated herein l~ by reference in its entirety. Likewise, the ~ -1~ granules can be formulated so as to contain ~ materials to reduce the rate of dissolution of the ~ ;
1~ granules into the wash medium. Such materials and granules are disclosed in U.S. Serial No. 07/642,596 filed on January 17, 1991 as~Attorn2y Doc~et No.
GCS-171-US1 and entitled "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. It is still further contemplated that the cellulase compositions described herein can also be used in home use as a stand alone composition suitable for enhancing color and appearance of fabrics. See, for example, U.S.

92/17572 ~ ~ PCT/US92/02629 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.

MP~8 Exampl~s 1-12 and 22-30 demonstrat2 the - preparation of Trichoderma reesei qenetically engineered so as to ~e incapable of prodl~cing onP or more csllulas2 components or so as to overproduce speci~ic cellulase components.
.
Example 1 Selection for pyr4 derivatives of Trichoderma reesei .
The EY~ gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. The toxic inhibitor 5-fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells.
However, cells defective in the ~yr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for EY~ derivative strains using FOA. In practice, spores of T. reesei strain RL-P37 (Sheir-Neiss, G.
and Montenecourt, B.S., A~pl. ~icrobiol. Biotechnol.
20, p. 46-53 (1984)) were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resistant colonies appeared within three to four days and it was possible to subsequently identify those FOA-~ ~ ' r - .
~ ::

~ ? ' ?.~

resistant derivatives which required uridine for growth. In order to identify those derivatives which speci~ically had a defective Dyr4 gene, ~ protoplasts ~r~ genQra-~ed and transform~d with a ; 5 plasmid containing a wild-type Dyr4 gene (see 1 E~ampl~s 3 ~nd 4). ~ollowing transforma~ion, ;~ protoplasts wera platad on medium lacking uridine.
Subsequont sros~h ef t-ansformed colonies demon~tra~d cemplem2n-a-~ion o. a de~ective Dvr4 :-. .
lo gene by '_n~ ~13smid-~orne ~r4 sene. In this way, s~r~in Gcss ~a~ id~ntifi2d as a Dvr4 derivative of strain RL-P37. -;, . . ~4 Example 2 Preparati~nLQf C~H} DeletiQn_Vector ..
.. ..
A cbhl gene encoding the CBHI protein was ~-cloned from the genomic DNA of ~. reesei strain RL-P37 by hybridization with an oligonucleotide probe designed on the b~asis of the published sequence for ~
this gene using known probe synthesis methods ~-(ShoemaXer et al., 1983b). The cbhl gene resides on a 6.5 kb PstI fragment and was inserted into PstI
cut pUC4K ~purchased from Pharmacia Inc., Piscataway, NJ) replacing the Xan' qene of this vector using techniques known in the art, which techniques are set forth in Maniatis et al., (1989) and incorporated herein by reference. The resulting plasmid, pUC4X::cbhl was then cut with HindIII and the }arger fragment of about 6 kb was isolated and religated to give pUC4K::cbhl~ (see FIG. 1).
This procedure removes the entire cbhl coding sequence and approximately 1.2 kb upstream and 1.5 ~; ~
~ , ~ 92/17572 7 1 Q 7 ~ ~ ~ PCT~US92/02629 ' -,,~
kb downstream of flanking sequences. Approximately, 1 kb of flanking DNA from either end of the original PstI fragment remains.

The T. reesei pvr4 gene was cloned as a 6.5 kb S ~dIII ~2as~en. of genomic DNA in pUCl8 to form ~, pTpyr2 ~Smith et al., 1991) following the methods of --Maniatiis 2~ al., su~ra. The plasmid pUC4K::cbhl~H/H
was cut wi-t~ ~indIII and tne ends were dephos~ils~ d -~ith cal. int2stinal alkaline phosphatas2. This end dephosphorylated DNA was ligat~d ~ith the 6.5 k~ HindIII fragment containing the ~. r~Q5~ 4 g~n~ Lo giv~ p~CBHI~vr4. FIG. 1 illustrates the construction of this plasmid. ~;

Example 3 n~ s Isolation Q~_protQRlasts !`
Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2% glucose) in a 500 ml fiask with about S x 107 T. rees~l GC69 spores ~the Y5~L derivative strain). The flask was then ~ incubated at 37~C with shaXing for about 16 hours.
The mycelium was harvested by centrifugation at 2,i50 x g. The harvested mycelium was further washed in a 1.2 M sorbitol solution and resuspended "-in 40 ml of a solution containing 5 mg/ml NovozymR ` `
234 solution (which is the tradename for a multicomponent enzyme system containing 1,3-alpha-glucanase, 1,3-beta-glucanase, laminarinase, :~ :
xylanase, chitinase and protease from Novo ~iolabs, Danbury, Ct.); 5 mg/ml MgS04.7H20; 0.5 mg/ml bovine .~
.~

~ .. 5~i r.~ r~

2 i ~
W092/1~572 PCT~US92/026~9 s;

' serum albumin; 1. 2 M sorbitol. The protoplasts were removed from the cellular debris by filtration through Miracloth (Calbiochem Corp, La Jolla, CA) and collected by centrifugation at 2,000 x g. Th2 protoplasts were washed three times in 1. 2 M
sorbitol and onc~ in i.2 ~ sorbitol, 50 ~ CaCl2, centrifuged and resuspended at a density or approximat~ly 2 x 10' protoplasts per ml o~ 1.2 sorbitol, ~0 ~ Ca 12.
Exammle 4 :'.' Transformation of Funaal Proto~lasts ~ith ~AC~HI~vr~! ~
.~ . .
200 ~1 of the protoplast suspension prepared in Example 3 was added to 20 ~1 of EcoRI digested ',~ p~CBHIEy~ (prepared in Example 2) in TE buffer`(10 ! 15 mM Tris, pH 7.4; 1 mM EDTA) and 50 ~1 of a polyethylene glycol (PEG) solution containing 25%
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 P~G
solution was added thereto, the solution was further mixed and incubated at room temperature for 5 minutes. After this second incubation, 4.0 ml of a , . .
solution containing 1.2 M sorbitol and 50 mM CaCl2 was added thereto and this solution was further ~; 25 mixed. The protoplast solution was then immediately added to molten aliquots of Vogel's Medium N (3 grams sodium citrate, 5 grams KH2P04, 2 grams NH4N03, - 0.2 grams MgS04.7H20, 0.1 gram CaCl2.2H.0, 5 ~g ~-biotin, 5 mg citric acid, 5 mg ZnSo4.782o, 1 mg Fe(NH4)2.6H20, 0.25 mg CuS0~.5H20, 50 ~g MnS04.4H20 per liter) containing an additional 1% glucose, 1.2 M

: .
~ , .~ . .. . : . :..... . . . . .

, sorbitol and 1% agarose. The protoplast/medium mixture was then poured onto a solid medium containing the same Vogel's medium as stated above.
No uridine was present in the medium and thQr~ifor2 only transformed colonies were able to grow as a result of complementation of th~ pYr4 mu~tion of strain GC69 by the wild type ~YE~ gene insert in p~CBHIPvr4. These colonies were subsi~quently transf~rred and purifi d on a solid Vogel's m2dium ~ ;
containing as an additive, 1~ glucose and stabl~
transformants werQ chossin for further analysis.
: .
At this stage stable transformants -i~r 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 further test of stability was made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores ~; 20 from this medium and determining the percentage of ~ -these spores which will subsequently ger~inate and grow on selective medium lac~ing uridine.
, ' '~ '' .: Example ~

~i Analvsis of the ~ransformants .
DNA was isolated from the transformants obtained in Example 4 after they were grown in liquid Yogel's medium N containing 1% glucose.
These transformant DNA samples were further cut with a PstI restriction enzyme and subjected to agarose gel electrophoresis. The gel was then blotted onto :
~: .

2 3 ~
W O 92/17572 PC~r/US92/02629 '' - 40 - :

a Nytran membrane filter and hybridized with a 32p labellPd p~CBHI~y~ probe. The probe was ~elected to identify the nati~e ~k~ gene as a 6.5 ~b PstI
fragment, ~h_ natiYe ~y~ gene and any DNA sequences deri~ed from the transforming DNA fragment.
` :
The radioactive bands from the hybridization wero vi,~l21i7ed by autoradiography. The autoradiograph is seen in EIG. 3. Five samplos wer_ run as desc-~ed zbove, hence samples A, B, C, D, and ~. T an2 E is the untransformed strain GC69 and ~as us~d as a cont.ol in the present analysis.
Lanes A-D reprosent transformants obtained by the methods dPscribed above. The numbers on the side of the autoradiograph represent the sizes of molecular weight markers. As can be seen from this autoradiograph, lane D does not contain the 6.5 kb CBHI band, indicatinq that this gene has been totally deleted in the transformant by integration of the DNA fragment at the cbhl gene. The ç~kl deleted strain is called P37P~CBHI. FIG. 2 outlines the delotion of T he T. reesei cbhl gene by integration through a double cross-over event of the larger EcoRI fragment from pACBHIEyE~ at the cbhl locus on one of the T. reesei chromosomes. The ~- 25 other transformants analyzed appear identical to the untransformed control strain. .

Example 6 Analysis of the Transformants with pIntCBHI
The same procedure was used in this example as in Example 5, except that the probe used was changed to a 32p labelled pIntCBHI probe. This probe is a '.:

~92/17572 ~ ~ 7 ~ ~ 8 PCT/US92,02629 pUC-type plasmid containing a 2 kb aglII fragment ;
from the cbhl locus within the region that was deleted in pUC4~::cbhl~8/H. Two samples were run in this -xamp'e including a control, sample A, which is the untrans.ormed strain GC69 and the transformant P37PACBHI, samDle 3. As can be seen in FIG. 4, ~ sample A contained the çbhl gene, as indicated by `~ the band at ~ . 5 X~; however the transformant, sample B, does not conta~n thi~ ~.5 ~b band and therefor~ -does no~ c~a,n the cbhl gene and does not contain any s2quanc2s deriv2d ~rom ~he pUC plasmid.
,, ~ . .
Examsle 7 Protein Secretion by Strain P37P~CBHI
,:;: ' : ., ~;~ Spores from the produced P37P~CBHI strain were inoculated into 50 ml of a T~ derma basal medium containing 1% glucose, 0.14% (NE~)2SO" 0.2% KH2PO4, 0.03% MgSO4, 0.03% urea, 0.75% bactotryptone, 0.05%
Tween 80, 0.000016% CuS0,.5H20, 0.001% FeSO,.7H20, 0.000128% ZnS0,.7~20, 0.0000054% Na~MoO,.2~20, ¦~ 20 0.0000007% MnCl.4X20). The medium was incubated ; - with shaking in a 250 ml flask at 37C for about 48 hours. The resulting mycelium was collected by fi}tering 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 cuiture supernatant were analyzed by isoelectric focusing using a Pharmacia Phastgel system and pH 3-ç.

~ .

2 1 n~2~ -WO92/17s72 PCT/US92/02629 9 precast gels according to the manufacturer's instructions. The gel was s~ained with silver stain to visualize the protein bands. The band correspondina to the cbhl protein ~as a~son~ from the sample derived from the strain P37P~CBHI, as shown in FIG. 5. This isoelec~rls ~ocu~ el shows various proteins in dif~erent supernatant cultures of T. reesei. Lane A is parti~lly ~-r ~ied CBHI; Lane B is th supernat~nt ,_a~ an untransformed T. rees2i culture; Lan~ c ls ~ne superna~ant from strain P37PACBi~I proàuced accordir.g to the m~thods o~ the pres2nt inventlon. Th~
position of various cellulase components ar2 labelled CBH}, CBHII, EGI, EGII, and EGIII. Since CBHI constitutes S0% of the total extracellular protein, it is the major secreted protein and hence is the darkest band on the gel. This isoelectric focusing gel clearly~shows depletion of the CBHI `
protein in the P37P~CBHI straln.-E~ample 8 .
:~
Preparation of pP~CBHII
The cbh2 gene of T. reesei, encoding the CBHII l , protein, has been cloned as a 4.1 kb EcoRI fraament of genomic DNA which is shown diagramatically in FIG. 6A (Chen et al., 1987, Biotèchnoloay, 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 t~e order : ~ ' . . .

.

~ 92/17572 .~. 9 ~ ~. ? ~ PCT/US92/02629 shown here: EcoRI, BamHI, SacI, SmaI, HindIII, XhoI, BalII, ClaI, ~glII, XhoI, HindIII, SmaI, SacI, BamHI, EcoRI. Using methods known in the art, a plasmid, pPaCBHII (FIG. 6B), has ~een cor.structed in which a l.7 kb central region of this gene between a HindIII site (at 74 bp 3' of the CBHII t_anslation initiation site) and a ClaI site (at 26~ bp 3' o~
the last codon of CBHII) has ~e_n ~e~o~1ed and replaced by a ~.S Xb ~ndIII- ClaI ~ . agm2nt containing the T. rees~i yrd geno.

The T. reesei Yr4 gene was excised ~.om pTpyr2 (see Example 2) on a 1.6 kb NheI-S~hI fra~m~nt and inserted between the ~E~I and XbaI sites of pUC219 (see Example 25) to create p219M (Smith et al., 1991, Curr. ~enet l2 P. 27-33). The Eyr~ gene was then removed as a ~indIII-ClaI fragment having seven bp of DNA ~t one end and six bp of DNA at the other ` end derived from the pUC219 multiple cloning site and inserted into the HindIII and ClaI sites of the cbh2 gene to form the plasmid pP~CBHII (see FIG.
6B). "

Diqestion of this plasmid with ~coRI will liberate a fragment having 0.7 kb of flanking DNA
from the cbh2 locus at one end, l.7 kb of flanking DNA from the cbh2 locus at the other end and the T.
reesei ~vr4 gene in the middle. :~

~ .
' .. ',., .' ' . . '' ' ;.' '~` '' ` ' ' - 2-1~72~8 !~

-:
Example 9 Deletion of the cbh2 aene in T. reesei strain GC69 Protoplasts of strain GC69 will be generated and transfor~ed with EcoRI diyested pP~CBHII
according to the methods outlined in Examples 3 and 4. DNA from the transformants will be digested with EcoRI and Asp718, and subjectPd to agarose gel electrophoresls. The DN~ from the gel will be , olotted to a membrane filter and hybridized with 32p .7 10 labelled ~P~C3HII according to the methods in Exampl~ ll. Transiorman.s will be identi.ied which nava a sin512 co~y of the ~coRI fragment ~rom j pP~CBHII integrated precisely at the c~h2 locus.
The transformants will also be grown in shaker flasks as in Example 7 and the protein in the culture supernatants examined by isoelectric focusing. In this manner 1 reesei GC69 ~ :~
~ transformants which do not produce the CBHII protein -, will be generated.
.
lj 20 Example 10 . . .:
Generation of a pyr4 Derivative of P37P~CBHI
Spores of the transformant (P37P~CBH~) which was deleted for the cbhl gene were spread onto medium containing FOA. A Dyr4- derivative of this transformant was subsequently obtained usiny the methods of ~xample 1. This pvr4 strain was designated P37PAC~IPyr26. ~

'~ .....

..
, :

- `392/17572 ~ 8 PCT/US92/02629 ~ .

~ .
. ..
Example 11 Deletion of the cbh2 ~ene in a strain ~reviously deleted for cbhl t' Protoplasts of strain P37P~CBHIPyr26 were generated and transformed with EcoRI digested pPACBHII according to the methods outlined in Examples 3 and 4.
.j .
, ' Purified stable transfor~ants were cultured in shaker flasks as in Example 7 and the protein in the culture supernatants was exam~ned by isoelectric ~ `' focusins. On_ transformant (designated p37pAAcBH67) ,, was identified which did not produce any CBHII
protein. Lane D of FIG. 5 shows the supernatant ~ from a transformant deleted for both the Qkhl and iS Çkh~ genes produced according to the methods of the present invention.

DNA was extracted from strain P37P~ACBH6~, digested with ~çQRI and ~718, and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with p labe}led pP~CBHII (FIG. 7). Lane A of FIG. 7 shows the hybridization pattern observed for DNA
from an untransformed T. reesei strain. The 4.1 kb EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization pattern observed for strain P3iP~CBH67. The single 4.1 kb band has been eliminated and replaced by two bands of approximately 0.9 and 3.1 kb. This is the expected pattern if a single copy of the EcoRI
fragment from pP~CB~II had integrated precisely at the cbh2 locus.
!~

., .
3 ~ ~:

2~7~
W O 92/17572 PC~r/~S92/02629 , The same DNA samples were also digested with EcoRI and Southern blot analysis was performed as above. In this Example, th~ prob~ was 32p labelled pIntC3HII. This plasmid contains a portion o~ the ` -cbh2 gene coding sequence ~rom within that se~ment of the cbh2 gene which was del~ od in plasmid ~ pP~CBHII. No hybridization was seen ~lth 3NA ~rom i~ strain P37P~CBH~7 ~howing .hat ~he cbh2 gene was deleted and that no sequenc2s d2riva~ ~rom the pUC
plasmid were pres~nt in this ~t~
.
~ 2m~
. :
, Construction of pEGI~yr4 The T. reesei e~ll gene, vhich encodes EGI, has been cloned as a 4.2 kb ~indIII fragment of genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthe ized according to the ¦~ publlshed sequence (Penttila et al., 1986, Gene 45:253-263; van Arsdell et al., 1987, Bio/Technolo~y 5:60-64). A 3.6 kb ~dIII-~3mHI fragment was taken from this clone and ligatsd with a 1.6 kb HindIII-HI fragment containing the T. reesei ~y~ gene obtained from pTpyr2 ~sae Example 2) and pUC218 (identical to pUC219, see Example 25, but with the multiple cloning site in the opposite orientation) ~ 25 cut with ~indIII to give the plasmid pEGI~vr4 (FIG.
'I 8). Digestion of pEGIEy~ with HindIII would liberate a fragment of DNA containing only T. reesei genomic DNA (the eqll and Dvr4 genes) except for 24 bp of sequenced, synthetic DNA between the two genes and 6 bp of sequenced, synthetic DNA at one end (see FIG. 8).

.:

~ .

92/17572 ? ~ n ~ ~ PCT/~S92/02629 Example 13 Purification of Cytolase 123 Cellulase into c llulas2 com~onents CYTOL~SE 123 c~llulase was ~ractionat~d in the ~ 5 following manner. The nor~al distribution of ~ cellulas~ compononts in this -ellulasa system is as ,~ . , follows: -CBH I ~-55 w~oight ~ercent CBH I~ 13-15 .~ighr ~rcent :~ 10 ~G I 11-13 weight percent EG II 8-10 wright percent ~G III 1-~ wei7h~ percent BG 0. 3-l weig~t pe_cent .
The fractionation was done using columns containing the following resins: Sephadex G-25 gel ~.
filtration resin from Sigma Chemical Company (St.
: . Louis, MO), QA Trisacryl M anion exo~ange resin and SP Trisacryl M cation exchange resin from IBF
Biotechnics (Savage, MD). CYTOLASE 123 cellulase, : 20 0.5g, was desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. ~he desalted solution, was then loaded onto a column of 20 ml of QA .
Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I.
~, These components were separated by gradient elution using an aqueous gradient containing from 0 to about 500 mM sodium chloride. The fraction not bound on this column contained CBH II and EG II. These fractions were desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM
sodium citrate, pH 3.3. This solution, 200 ml, was then loaded onto a column or 20 ml of SP Trisacryl M

:' , , .
~ ' ~-10~7~
wos2tl7s72 PCT/US92/02629 cation exchange resin. CBH II and EG II were eluted separately using an aqueous gradient containing from o to about 200 mM sodium chloride.

Following procedures similar to that of Example 13 above, other cellulase systems which can be -~ -separat~d into their components include CELLUCAST
(available from Novo Ind~stry, Copenhagen, Denmark~, RAPIDASE (available from Gist ~rocades, N.V., Delft, ~olland), and cellulase systems derived from Trichoderma koninqii, Penicillum so. and the like. --, - . ., ExamDle 14 Purification of EG III from Cvtolase ?23 Cellulase Example 13 above demonstrated the isolation of several components from Cytolase 123 Cellulase. `
However, because EG III is present in very small quantities in Cytolase 123 Cellulase, the following procedures were employed to isolate this component.

A. Large Scale ~ actio~_of EG III Cellulase Enzyme one hundred liters of cell 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 sulfate. The mixture formed a two phase liquid mixture. The phases were separated using an SA-l disk stac~ centrifuge. The phases were analyzed using silver staining isoelectric focusing gels. Separation was obtained for EG III and xylanase. The recovered composition contained a~out 20 to 50 weight percent of EG III.

' ~

, 7 ? l~ 8 .. ~92/17572 PCT/US92/02629 Regarding the above procedure, use of a polyethylene qlycol having a molecular weight of 3 less than about 8000 gave inadequate separation; -~
whereas, use of polyethylene glycol having a S molecular weight of greater than about 8000 resulted i in the exclusion of desired enzymes in the recovered ~ composition. With regard to the amount of sodium ; sulfate, sodium sulfate levels greater than about ! lO~ wt/vol caused precipitation problems; whereas, sodium sulfate levels less than about 10% wt/vol gave poor separation or the solution remained in a single phasa.

B. Purification of EG III Via Fractionation The purification of EG III is conducted by j 15 fractionation from a complete fungal cellulase ~ composition (CYTOLASE 123 cellulase, commercially Z available from Genencor International, South San Fr~ncisco, CA) which is produced by wild type Trichoderma reesei. Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel fi}tration 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, 0.5g, is desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8.
~ The desalted solution, is then loaded onto a column f~ ~ of 20 ml of QA Trisacryl M anion exchanqe resin.
The fraction bound on this column contained CBH I
and EG I. The fraction not bound on this column ! contains CBH II, EG II and EG III. These fractions , i~ are desalted using a column of Sephadex G-25 gel i~ ' ' 7 '~
wos2/17s72 PCT/~S92/02629 ~"''".'''.
filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto a column of 20 ml of SP Trisacryl ~
cation exchange r2sin. The EG III was eluted with S 100 mL of an aq~eous solution of ~00 mM sodium chloride. ~ -: In order to enhanc~ t~e e}.ici~ncy oI the isolation or ~G III, it may ~e desi~able to employ Trichoderma ro~sei ger.oticaily m~ d so as to be incapable of producing one or more of EG I, EG II, CBH I and/or CBH II. The ~bsence ef on.~ or morQ o4 : such compon2nts will n3sa~sa~ily 12ad .o mor_ ~ efficient isolation of EG III.
' ~ . ''.
Likewise, it may be desirable or the EG III
compositions described above to be further purified to provide or substantially pure EG III
compositions, i.e., co~positions containing EG III -at greater than about 80 weight percent of protein.
For example, such a substantially pure EG III
protein can be obtained by utilizing material obtained rom procedure A in procedure B or vica versa. One particular method for further purifying ;-EG I}I is~by further fractionation of an EG III
sample obtained in part b) of this Example 14. The - further raction was done on a FPLC system using a Mono-S-HR 5/5 column (available from Pharmacia LKB
Biotechnology, Piscataway, NJ). The FPLC system consists of a liquid chromatography controller, 2 pumps, a dual path monitor, a fraction collector and a chart recorder (all of which are available from Pharmacia LKB Biotechnology, Piscataway, NJ). The fractionation was conducted by desalting 5 ml of the ?7~,n8 ~`.`~92/17572 PCT/US92/02629 EG III sample prepar~d in part b) of this Example 14 I with a 20 ml Sephadex G-25 column which had been ! previously equilibrated with lo mM sodium citrate pH 4. The column was then eluted with 0-200 mM
agueous gradient of NaCl ~t a rate of 0.5 ml/minute with samples collected in 1 ~l fractions. EG III
was recover~d in fractior.i 10 ~nd 11 iand ~as determined to ~e gr~ac2r than 90~ ~ura by SDS gel electrophoresis. ~G III of this ~urity is suitable for det2rmining th2 N- ~-min21 a~;no acid sequence by known techniques.

Substantiallt~ ~u-e ~~ as ~il as ~G I and EG II components purified in Example 13 above can be - used singularly or in mixtures in the methods of this invention. These EG components have the following characteristics:
MW pI ~H optimum~
EG I -47-49 kD 4.7 -5 EG II -35 kD 5.5 -S
EG III -25-28 kD 7.4 ~5.5-6.0 :
; 1. pH optimum determined ~y RBB-CMC activity as ~ per Example 15 below.

, The use of a mixture of these components in the practice of this invention may give a synergistic response in improving softening, feel, appearance, etc., as compared to a single component. on the other hand, the use of a single component in the practice of this invention may be more stable or ~; have a broader spectrum of activity over a ran~e of ~, ~, ' .~. .

7?,~
''~W092/17572 PCT/US92/02629 ;
,......................................................................... . .
~ 52 , . .
i,~ , -pHs. For instance, Example 15 below shows that EG
III has considerable activity against RBB-CMC under alkaline conditions.
. .
Example lS
. ~.. i ., ~ 5 Activitv of Cellulase compositions - Over a ~H Ranqe The following procedure was employed to determine the pH profiles of two different cellulase -compositions. The first cellulase composition was a '~ 10 CBH I and II delet~d cellulase composition prepar2d ;; from Trichoderma reesei genetically modified in a manner similar to that described above so as to be l~ unable to produce CBH I and CBH II components.
;~', Insofar as this cellulase composition does not lS contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase composition derived from Trichoderma reeseij this cellulase composition is necessarily substantially free of CBH I type and CBH II type cellulase components and accordingly, is enriched in EG
~, components, i.e., EG I, EG II, EG III and the like.

The second cellulase composition was an approximately 20 to 40% pure fraction of EG III
isolated from a cellulase composition derived from Trichoderma 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.
,~
t J
;

`~ 7 ~
`~ 92/1~572 P~rtUS92/02629 . `

Add 5 to 20 ~1 of an appropriate enzyme ` solution at a concentration sufficient to provide the requisite amount of enzyme in the final solution. Add 250 ~1 of 2 weight percent RBB-CMC
(Remazol Brilliant Blue R-Carboxymethylcellulose --commercially available from MegaZyme, 6 Altona Place, North RocXs, N.S.W. 2151, Australia) in 0.05M
citrate/phosphate buffer at p~ 4, 5, 5.S, 6, 6.S, 7, 7.5 and 8.

Vortex and incubate at 40C for 30 minutes.
Chill in an ice bath f or S to 10 minutes. Add 1000 ~l of methyl cellosolve containing 0.3M sodium acetate and 0.02M zinc acetate. Vortex and let sit for 5-10 minutes. Centrifuge and pour supernatant ~ 15 into cuvets. Measure the optical density (OD) of ¦ the solution in each cuvet at 590 nm. Higher levels 3 ~ of optical density correspond to higher levels of ~ , ' enzyme activity.
i' ~ .
he results of this analysis are set forth in FIG. 9 which illustrates the re}ative activity of the CBH I and II deleted cellulase composition compared to the EG III cellulase composition. From this figure, $t is seen that the cellulase composition deleted in CBH I and CBH II possesses optimum cellulolytic activity against RBB-CMC at near pH 5.5 and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8. On the other hand, the cellulase composition enriched in EG III
possesses optimum cellulolytic activity at pH 5.5 - 6 and possesses significant activity at alkaline pHs.

2 1 ~
W O 92/175~2 PC~r/US92/02629 ~ `

From the above example, one sXilled in the art would merely need to adjust and maintain the pH of the aqueous textile composition so that the ~i cellulase composition is activa and pr~f2rably, possesses optimum activity. As noted:above, such .
l~ adjustments and maint2nance may involv2 the use or a j:~ suitable buffar.
;. :
~ 2~ilp~

Launderom2t=r s~renqt~ Loss Assay C ~ l l ~ l l . 5 2 ~ O~ O a i t ~ O n a i This exampla axaminas tln2 ~bill ~y or di^ferent 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.the most of the cellulase components derived from Trichoderma ~eesei is greatest at or near pH 5 and accordingly, strength ~ ~
: . losis 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 (C~TOLASE 123 cellulase, .~ commercially available from Genencor International, . South San Francisco, CA) produced by wild type Trichoderma reesei and is identified as GC010. ..

: The second cellulase composition analyzed was a . CBH II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner : similar to Examples 1 to 12 above and 22 to 30 below : ~
:~ 30 so as to be incapable of exprassing CBH II and is ~ .

,. ~ .

" 1 n r~ ~ ~ 8 ~92/17572 PCT/US92/02629 identified as CBHIId. Insofar as CBH II comprises up to about 15 percent of the celluIase composition, deletion of this component results in enriched levels of CBH I, and all of thP EG components.

; 5 The thi~d cellulas2 csmposi'isn analyzed was a CBH I and CBH II dPleted cellulas2 composition prepared from Trichod~ 2 eese~ genPtisally modi~ied in a mann2r similar '.o that desc_i~ed above so as to be incapa~l~ of e~p.~ssing c~ I and CB~ II
lo and is identiLied as CB~I/IId. InsoLar as CBH I and CBH II are not produc2d by thi~ ~odiri~d microorganism, the cellulase is necQssarily frQe of all CBH I type components as well as all CBH
components.

I 15 The last cellulase composition analyzed was a j CBH I deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner .~ similar to that described above so as to be ~ incapable of expressing CBH I and is identified as ¦ 20 CBHId. Insofar as ths modified microorganism ls i incapable of expressing CBH I, this cellulase composition is necessarily free of all CBH I type ' cellulase components.

~ The cellulase compositions described above were i 25 tested for their effect on cotton-containing fabric strength loss in a launderometer. The compositions were first normalized so that equal amounts of EG
' components were used. Each cellulase composition was then added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer, titrated to pH 5, and which contains O.S ml of a non-ionic I ?~ ~ 2 3 g WO92/17572 PCT/US92/02629 `

surfactant. ~ach of the resulting solutions was then added to a separate launderometer canister.
Into these canisters were added a quantity of marbles to facilitate strPngth 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., Niddlesex, NJ 08846). The canistsr was then closed and the canister lowered into cne 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) ~or about 1 hour. Aft~rwards, ths cloth is removed, rinsed well and dried in a standard drier.

In order to maximize strength loss results, the above procedure was repeated twice more and after the third treatment, the cotton fabrics were removed and analyzed for strength loss. Strength loss was measured by determining the tensile strength in the fill direction (nFTS") using a Instron Tester and the results compared to the FTS of the fabric treated with the same solution with the exception that no cellulase was added. The results of this analysis are repGrted as percent strength loss which is determined as follows:
_ % Strength Loss = 100 x 1 - FTS with cellulase L FTS without cellulas~
, .
The results of this analysis are set forth in FIG. 10 which shows that compositions containing CBH I, i.e., whole cellulase (GC010) and CBH II
deleted cellulase, possessed the most strength loss whereas, the compositions containing no CBH I
possessed significantly reduced strength loss as ~ )92/17572 i'~? 1 Q '~ ~ ~ 8 PC~r/US92/02629 compared to whole cellulase and CBH II deleted cellulase. From these results, it is seen that the presence of CBH I type components in a cellulase composition imparts increased strength loss to the composition as compared to a similar composition not containing CBH I type components.

LiXewise, these results show that CBH II plays some role in strength losc.

Accordingly, in view of these results, strength loss resistant cellulase compositions are those i~
i compositions free of all CBH I type cellulase components and preferably, all CBH type cellulase components. In this regard, it is contemplated that such cellulase compositions will result in even lower strength loss at pH 2 7 than those results observed at pH 5 shown in FIG 10.

During the manufacture of cotton-containinq 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 fabric/color enhancement. This is believed to -arise by removal of some of the broken and ~ 25 disordered fibers which has the effect of restoring ;~ the`appearance of the fabric prior to-becoming ~ stressed.
~ ~ .
~- The following Examples 17 and 18 illustrate ~1 this benefit of the present invention. It is noted -that these example employed worn cotton T-shirts ~:; .:

`

WO 9~ 7 7 PCT/US92/02629 s~

' (knits) as well as new cotton knits. The faded appearance of the worn cotton-containing fabric arises from the accumulation on the fabric of broken and loose sur~ac2 fi3~-s ov2r a period o~ time.
These fibers give rise to a faded and matted appearance ,'or the -a~r-c and accordingly, tha j removal of these fi~er~ is a nec~ssary prer2quisite to restoring b- o._ginal ~arp c~lor ,o the fabric.
Additionally, the accumuiation o~ oro~en sur~ace ~' 10 fibers on new cotton ~nits impa .g a dull appearance ! to such ~abrics. Arcordingly, t~es2 experiments are 3 necessarily applica~le to color enhanc2ment of - stressed cotton-containing f~b~ics bec3usQ bo'h involve removal of surface fibers from the fabric.

ExamDle 17 Color ~_ancement i~ 'rhe ability of EG components to enhance color in cotton-containing fabrics was analyzed in the following experiment-. Specifically, the first experiment measures the ability of a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, Sou ~ San Francisco, CA) produced by wild type Trichoderma reesei to remove surface fibers from a cotton-containing fabric over various p~s. This -cellulase was tested for its ability to remove surface fibers in a launderometer. An appropriate ¦~ amount of cellulase to provide for either 25 ppm or I00 ppm cellulase in the final composition was added to separate solutions of 400 ml of a 20 mM
citrate/phosphate buffer containing 0.5 ~1 of a .

.~ .

`~)92/17572 ~ ~ 7 ` ~ ~ P(~r/US92/02629 .
. , .. . .
non-ionic surfactant. Samples were prepared and titrated so as to provide for samples at pH 5, pH 6, pH 7 and pX 7.5. Each of the resulting solution was then added to a s2parat- launderomet2r canister.
~; 5 Into t~ese canisters wero added a auantity of ~ marbles to facilitate Iiber removal as well as a 7 ;3 inch x 5 inch cotton fabric (100% woven cotton, available as Style ~o. 439W L: om Tast ~abrics, Inc., 200 Blac~rord Avs., ~iddles2x, NJ 08846). The ~ 10 canister was .he~ clos2d and Lhe s2ais~er lowered t~, into the launderomet2r bath wnicn was maintained at i 43C. The canister was then rotated in the bath at ~.j a speed of ~t lezst abcut A~ o r~ olutior.s pQr minuto (rpms) for about 1 hour. Afterwards, the cloth is ~¦ 15 removed, rinsPd well and dried in a standard drier.
.
~,s : .
The so treated fabrics were then analyzed for ~ fiber removal by evaluation in a panel test. In ;~ ~ particular, the fabrics (unmarked) were rated for levels of fiber by 6 individuals. The fabrics were visually evaluated for surface fibers and rated on a 0 to 6 scale. The sral2 has six standards to allow ~li meaningful comparisons. The standards are:
Ratina Standard' 0 Fabric not treated with cellulase i . .- .
25 ~ l Fabric treatedb with 8 ppm cellulase 2 Fabric treated with 16 ppm cellulase ;
3 Fabric treated with 20 ppm cellulase 4 Fabric treated with 40 ppm cellulase ~ ~
' 5 Fabric treated with 50 ppm cellulase ~ -6 Fabric treated with 100 ppm cellulase ',J ~
~, .
s 2 ~
WO92/l7s72 PCT/US92/02629 ' .

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 BlacXford Ave., Middlesex, NJ 08846 S b) All samples were treated with the same cellulase composition. Cellulase concentrations are in total protein. The launderometer '! treatment conditions are the same as set farth in Example 16 above.
, . . .
The fabric to be rated was provided a rating which most closely matched one of the standards.
After compl~t2 analysis or the îabrics, the values assigned to eac~ rabric by all of the individuals were added and an average value generated.
. ~
The results of this analysis are set forth in ; FIG. 11. Specifically, FIG. ll illustrates that at the same pH, a dose dependent response is seen in ~ the amount of fibers removed. That is to say that ,!3 at the same p~, the fabrics treated wit~ more 3 20 cellulase provided for higher levels of fiber removal as compared to fabrics treated wit~ less `
cellulase. ~oreover, the results of this figure demonstrate that at higher pHs, fiber removal can still be effected merely by using higher concentrations of cellulase.
;l .
In a second experiment, two different cellulase compositions were compared for the ability to remove fiber. Specifically, the first cellulase composition analyzed was a complete cellulase system (CYTOLASE
3~ 30 123 cellulase, commercially available from Genencor ~ Int-rnational, South san Franci~co, CA) produced by .:

i~ ' .

7 ~! 9 iS~
92/1 7572 PCI~/IJS92/02629 .

.
wild type Trichoderma reesei and is identified as GC010.
.. ~
~he second cellulase composition analyzed was a cellulase composition substantially free of all s CBH type components (including CBH I type components~ which composition was prepared from Trichoderma reesei genetically modified in a manner ,~ similar to that described a~o~ so as to be incapable of expressing CBH I and CBH II and is -identified as C~HT/II deleted. Insofar as C8H I and - CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG
j; components.

These compositions were tested for their ability to remove surface fibers in a launderometer.
An appropriate amount of cellulase to provide for the requisite concentrations of EG components in the final compositions were added to separate solutions ;20 ~ of~400 ml of a 20 mM citrate/phosphate buffer ~ containing 0.5 ml of a non-ionic surfactant.
¦ Samples were prepared and titrated to pH 5. Each of ~;
the resulting solutions was then added to a separate launderometer canister. Into these canisters were -~
added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100$ woven cotton, available as Style No. 439W from Test~Fabrics, Inc., 200 Blackford Ave., Middlesex, ; NJ 08846). The canister was then closed and the canister lowered into the launderometer bath which was maintained at 43C. The canister was then rotated in the bath at a speed of at least about 40 - ':
. . .
' '3 o W O 92/~7572 PC~r/US92tO2629 --.
revolutions per minute (rpms) for about 1 hour.
Afterwards, th~ cloth is remov2d, rinsed well and dried in a standard drier.

The so treated fabrics were then analyzed for fiber removal ~y eYaluation in the p2nol tQst described above. The rosults of this analysis are !~ set for-h in ~G. 12 ~nich is ~lot~d ~ased on estimat2d ~G e~ncontrztior.s. S~eci.lc211y, ~IG. 12 illust~at~s ';.a 30t~ ~COlo and C~H I/I_ Deletod celll~las~ ccmp~sl~ion,i ga-~ su~stan~iall~ identical fiber removal r~i-iulti~i a-t su~stantially isqual EG
concentrations. The rosults of this fig~rP suggest that it is the-EG componPnts which provide for fiber removal. These results coupled with the results of FIG. 11 denonstrate that EG components remove !: surface f1bers.
;~ ~X~Dle 1~ ; i 1 '' i Tergo omet~r Color ~'~' , ! ~ ~ This example is further to ~xample 17 and 20substantiates that CBH type components are not necessary for color enhancement and the purpose of this example is to examine the ability of cellulase compositions deficient in CBH type components to enhance color to cotton-containing fabrics.

25Specifically, the ce}lulase composition employed in this example was substantially free of ~, all CBH type components (inc~uding CBH I type components) insofar as this composition was prepared from Trichoderma reesei genetically modifiad in a --3 92/i~572 2 ~ a~Y~ PC~r/~S92/02629 manner similar to that described above so as to be incapabl~f~ ol e~:~ ~sssing C~H I and CBH II. Insofar as CBH I and CBH II compris2s up to about 70 percent of the c211ulase iomposi~ion, d 'etion of .his -~
component results in enriched levels of all of the EG components.

The a5s2y ~ s conduc~_d ?3y adding a sufficient concentration of this cellulase composi~ion to a 50 mM citra~e~,hos~h _~ JU"ff '~ "-3 ~rovid2 ~00 ppm of cellulase. The solut~on ~3s ti,rat2d to p-I 5 and containef~ 5. ~ly~ht ~a-c;~ 0c nonio~ic surfactant (Grescot,erg GL100 -- commercially available from Gresco ~fg., Thomasville, NC 27360). A 10 inch x io ~:-inch faded cotton-containing fabric as well as a lO
inch x 10 inch new knitted fabric having loose and broken surface fibers were then placed into 1 liter of this buffer and allowed to sit at 110F for 30 minutes and then agitated for 30 minutes at 100 rotations per minute. The fabrics were then removed from the buffer, washed and dried. The resulting fabrics wers t~en compared to the fabric prior to treatment. ~he results of this analysis are as follows:

Cotton-Containin~ Material _ Result Worn Cotton T-Shirt benefit seen New Cotton ~nit benefit seen The term "benefit seen" means that the treated fabric exhibits color restoration (i.e., is less faded) as compared to the non-treated fabric which includes removal of broken surfacP fibers including broken surface fibers generated as a result of using :

''' .:

2 1 1~ 0 ~
W092/17572 PCT/US92tO2629 the tergotometer. These results substantiate the results of Example 17 that the presence of CBH ~ype components is not necessary for effecting color restoration of faded cotton-containing fabrics.

It is contemplated that the use of such cellulase compositions would be beneficial during fabric processing because such compositions would remove broken/loose fibers gen~rated du~
processing without detrimental ~trenglh loss to -ch~ -fabric.

ExamDle 19 Softness This Example demonstrates that the presence of CBH type co~ponents are not essential for imparting improved softness to cotton-containing fabrics.
Speci~ically, this Example employs a cellulase composition free of all CBH type components which composition is derived from Trichoderma raesei genetically engineered in the manner described above so as to be incapable of producing CBH I and II
components. -- .
.:
This cellulase composition was tested for its ability to soften terry wash cloth. Specifically, unsoftened 8.S ounce cotton terry cloths, 14 inches by 15 inches (available as Style No. 420NS from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846), were cut into 7 inch by 7.5 inch swatches.

;;

92/17572 2:t Q7 ~ PC~r~US92/02629 .. .
- 65 - ~ -.~ ' The cellulase composition described above was tested for its ability to soften these swatches in a launderometer. Specifically, an appropriate amount of cellulase to provide for 500 ppm, 250 ppm, lOo ppm, 50 pp~, and 10 ppm cellulase in the final cellulase solution was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.025 weight percent of a non-ionic surfactant (Triton X114). Additionally, a blan~ was lo run containing the same solution but with no added cellulase. Samples so prepared were titrated to pH 5. ~ach of the resulting solution'was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to -facilitate softness as well as cotton swatches described above. All conditions were run in tripIicate with two swatches per canister. Each canister was then closed and the canister lowered 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 revolutions per minute -(rpms) for about 1 hour. Afterwards, the swatches were removed, rinsed well and dried in a standard drier. --The swatches were then analyzed for softness by evaIuation in a preference test. Specifically, six panelists were given their own sçt of swatches and ask to rate them with respect to softness based on the softness criteria such as the pliability of the ; 30 whole fabric. Swatches obtained from treatment with the five different enzyme concentrations and the - "
blank were placed behind a screen and the panelists ~ -were asked to order them from least soft to most ,~ . , 21~ 7~ ~8 .

. , ' .
soft. scores were assigned to each swatch based on its order rslative to th~ othPr swatches; 5 being most soft and o being least so~t. The scores from each pan21ist3 ~.ier~ sumulat_d and then averaged.

The resul! â Oc thi~ avPraging are set forth in FIG. 13. Specifically, these results demonstrate that ac hi$h2- concentrations, improved softening is obtained. It is noted that this improvod sof ~ning is achiavad ~ .cut the pr~2nca of either CBH I or II in t~e cellulas2 composi~ion.

Exam~le ~o J Feel and Appearance This example demonstrates that the presence of CBH type components are not essential for imparting i 15 improved feel and appearance to cotton-containing r ~ ``
fabrics. Specifically, this example employs a cellulase composition derived from Trichoderma -~ reesei genetically engineered in the manner describ2d abova so as to be incapable of producing any CB~ 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 ,~ 25 sized 100% cotton sheeting (available as Style No.
439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the appearance aspects of this example.

~ .
.

.

.,,,,,, ., . , , . ~ , . ~ , . .. . . . . .

~ 92/i7572 21 ~7 ~ PCT/US92/02629 . .
'.~. - ' ':' The cellulase composition described above was ~ tested for i~s a~ility to improve the appearance of -~ these sampl2s in a launderometer. Specifically, an -~
-i appropriat2 a~ount or CBX I and II deletsd cellulase `~ 5 to provide for 25 ppm, 50 ppm, and lO0 ppm cellulase `,~ in the final c211ulas~ solution was added to separate solutions of 400 ml of a 20 mM
; citrat~/phospnat2 ~u,-f3ar containing 0.025 weight pæ c~nt of ~ non-ionic surL~ctant tTriton Xl14).
Additionall~, a ~lan~ ~as run contalning the same . . .
solu~ion ~ut with no added cellulase. Samples so ir prepared w2r~ titrac~d to pH ~. Each of the resulting solutions was then added to a separate , launderometer canister. Into these canisters were y~ 15 added a guantity of marbles to facilitate improvements in appearance as well as cotton samples described above. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at about 40-C. The canister wa- then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 -hour. Afterwards, the samples were removed, rinsed -~well and dried in a standard drier.

The samples were then analyzed for improved appearance by evaluation in a preference test.
Specifically, 6 panelists were given the 4 samples (not~identified) and asked to rate them with respect to appearance. The panelists were instructed that the term "appearance" refers to the physical appearance of the cotton-containing fabric to the ~ eye and is determined in part! by the presence or 3~ absence of, fuzz, surface fibers, and the like on ~ the surface of the fabric as well~as by the ability ~3, ,3 ::
,, ~ , .
.~, , "`

2~72Q8 ~ wo92/l7s72 PCT/US92/02629 ~ ~
, ~ ~
: - 68 -or inability to discern the construction (weave) of the fabric. Fabrics which have little if any fuzz and surface fibers and wherein the construction (weave) is clearly discernable possess impro~2d appearance as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weaY2.
;,, :
The panelists then assigned scores wer~
assigned to each sample based on its order relatiYe to the other samples; 4 having thP best app2a_ ~nc and 1 having the worst appearanc~. Th- sco.-s f o~
each panelists werP cumulat~d and then aYeraged.
The results of this test are as follows:
~', . . .
q- Amt Cellulase _ Averaqe Appearance J~ None 25 ppm 2 50 ppm 3 100 pp~ 4 .~
The CBH I and II deleted cellulase composition ~; was then tested for its ability to improve the feel of cotton-containing fabrics. Specifically, appropriately sized 100% cotton sheeting (available -~
as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the-feel aspects of this example.
:1:
~ 25 The cellulase composition described above was i, .
tested for its ability to improve the feel of these samples in a launderometer. Specifically, an appropriate amount of cellulase to provide for 500 ~ ~ ppm, 1000 ppm, and 2000 ppm cellulase in the final -~ 30 cellulase solution was added to separate solutions . ~ .
.,~ .
,, , .,, 92/17572 v ~ 9 8 PCT/US92/02629 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 ; 5 washer. The washer was operated at 50C, a total volume of 24 L, a liquor to cloth ratio of 50:~
(weight to weight) and the washer was run for 30 minutes. Afterwards, the samples were remo~ed and dried in an industrial dr~er.
.1 :
i 10 The samples were then analyz~d ~or improved feel by evaluation in a prsference test.
i~ Specifically, 5 panelists were given the 4 sampl_iis (not identified) and asked to rate them with respect to feel. The panelists were instructed that fabrics having improved feel are smoother and silkier to the -; touch than other fabrics and that feel is distinguished from qualities such as softness (which 1 refers to the pliability of the fabric rather than its feel), thickness, color, or other physical characteristics not involved in smoothness of the :, fabric.
3 ~ ~
3 The panelists then assigned scores to each sample based on its order relative to the other samples; 4 having the best feel and 1 having the worst~féel. The scores from each panelists were cumulated and then averaged. The results of this test are as follows:
Amt Cellulase Averaae Feel None 1.5 + 0.5 500 ppm 1.7 + 0.4 1000 ppm 3.2 + 0.4 2000 ppm 3.8 + 0.4 :

, ~

-~:
.
.,~ ~'.
i~; . . . .. ~ . . . ... . . : . . ., ., .. ,.. ,: .. ,,.. ." .. .... . ... .. . .. .. . . . .. . .

,' 9 ~
WO92/17~72 PCT/US92/02629 ~

:' The above results demonstrate that improvements in feel and appearance can ~e achieved with cellulase compositions free of all CBH type -components.
: .:
' Example 21 . .
~ ~ Ston~ Washad A~pearance "~
This 2xampl2 demonstrates that the presence of CBH .ype componen-~ aro not essential for imparting a stone washed appearance to cotton-containing lo fabrics. Sp~cirically, this example employs a lu13so ~m-osition de~iY2d from Trichoderma reesei genetically engineered in the manner déscribed above so as to ~e incapable of producing any CBH type components (i.e., incapable of producin~ CBH I and,II components) as well as a complete cellulase composition derived from , ,Trich~d~rma~~ree~ei and which is available as Cytolase 123 cellulase from Genencor International, -`
South San Francisco, California.

~These cellulase compositions were tested for , ,~
- their abllity to impait a stone washed appearance to dyed cotton-containing denims pants. Specifically, the~samples were prepared using an industrial washer and~dryer under the following conditions: ' 10 mM citrate/phosphate buffer pH 5 ~n~ 40 L total volume 110F , ' Four~pair of,denim pants l hour run time 50 ppm CBH I and-II deleted cellulase or 100 ppm ~hole cellulase (i.e., at approximately equal EG concentrations~

,~'-: ~ ' ~ 92~17S72 ~ 7 ~ n ,~ PCT/~S92/02629 .
:
Samples ~ere evaluated for their stonewashed appearance by 8 panelists. All eight panelists choose loo ppm whol~ cellulase over non-enzyme treatod ~ants as haYing the ~etter stone washed looX. ~our o~ the 8 panelists choose the CBH I and II d_letQd cellulas2 traated pants over whole cellulas* as haYing tho better stone washed look;
! ~ whereas th2 other panelists choose the whole ~ cellul~so t_ gatod pants ~s having a better stone 1 10 washed look. Th~se results indicate that the CBH I
and IT doleted col lulase t__at~d pants ~ere indisti,~gu shabi~ -LOm ~hel2 cPllulase treated pants and that CBH I and/or CBX II are not esséntial for imparting a stone washed appearance to cotton~
containing fabrics.

With regard to Examples 16 to 21, cellula~e compo~iitions free of C8H I type components and derived from microorganisms other than Trichoderma reesei could be used in place gf 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 composition containing one or more EG type components and substantially frèe of all CBH I type components can be used herein. For example, fungal cellulases for USQ in preparing the fungal cellulase compositions used in this invention can be obtained from Trichoderma oningii~ Pencillum s~., and the ~ -30~ like or commercially available cellulases can be used, i.e., CELLUCAST (available from Novo Industry, .

':

2.~.07~.98 WO92/175~2 PCT/US92/02629 -, ,. . , :
Copenhagen, Denmark), RAPIDASE (available from Gist 8rocades, N.V., Delft, Holland), and the liXe.

Example 22 Transfor~ants of Trichoderma reesei Containin~
the plasmid pEGIDyr4 A pvr4 defective derivative of T. ro~s~i str- n }~ RutC30 (Sheir-Neiss and Montonecourt, ~19~4), Micro~io~. Biotechnol. 20:46-~33 was obtalned ~ t~
i method outlined in Example 1. Protoplasts o. this strain wer2 transformed with undigested pEGIpvr~ and stable transformants were purified.
, Five of these transformants (designated EP2, EP4, EP5, EP6, EPll), as well as untransformed RutC10 were inoculated into 50 ml of YEG medi.um (yeast extract, 5 g/l; glucose, 20 g/l) in 250 ml shake f}asks and cultured with shaking for two days at 28C. The resulting mycelium was washed with sterile~water and added to 50 ml of TSF medium (0.05M citrate-phosphate buffer, pH 5.0; Avicel microcrystalline cellulose, 10 g/l; KH2P0" 2.0 g/l;
(NH4)2SO" 1.4 g~l; proteose peptone, 1.0 g/l; Urea, 0.3 g/l; MgS04.7H20, 0.3 g/l; CaCl2, 0.3 g/l;
FeSO~.~H20, 5.0 mg/l; MnS04.H20, 1.6 mg/l; ZnS04, 1.4 mg/l; CoCl2, 2.0 mg/1; 0.1% Tween 80). These cultures were incubated with shaking for a further `.
four days at 28C. Samples of the supernatant were ~: ~ taken from these cultures and assays designed to x measure the total amount of protein and of endoglucanase activity were performed as described below.
i ~ :
~ ~:

.

`'; )92~17572 ~ PC~r/US92/02629 ~3 -.
The endoglucanase assay relied on the release of soluble, dyed oligosaccharides from ~emazol Brilliant Blue-carboxymethylcellulose (~3B-CMC, ~-obtained fr~m MegaZyme, North Roc~s, NSW, --' 5 Australia). The substrate was prepared by adding 2 g of dry ~3B-CMC to 80 ml of just boiled deionized water with vigorous stirring. When cooled to room temperature, S ml of 2 M sodium acstate bufîer lpH
4.8) was addzd and the pH adjusted to 4.5. The 3 10 volume was finally adjusted ~o 100 ml wit~ deloaized water and sodium azide added to a final concentration of 0.02~. Aliquo.s of T. r2esei control culture, pEGInvr4 transformant culture supernatant or 0.1 M sodium acetate as a blan~ (10-20 ~l) were placed in tubes, 250 ~l of substrate was , added and the tubes were incubated for 30 minutes at 37C. The tubes were placed on ice for 10 minutes '~ and 1 ml of cold precipitant (3.3% sodium acetate, ~1 0.4% zinc acetate, pH 5 with HCl, 76% ethanol) was then added. The tubes were vortexed and allowed to sit for five minutes before centrifuging for three minutes at approximately 13,000 x g. The optical density was measured spectrophotometrically at a -wavelength of 590-600 nm.

The protein assay used was the BCA ~-~
(bicinchoninic acid) assay using reagents obtained I - from Pierce, Rockford, Illinois, USA. The standard was bovine serum albumin tBSA). BCA reagent was made by mixing 1 part of reagent B with 50 parts of reagent A. One ml of the BCA reagent was mixed with 50 ~l of appropriately diluted 8SA or test culture supernatant. Incubation was for 30 minutes at 3rC

".1~7~

and the optical density was finally measured spectrophoto~etrically at a wavelength of 562 nm.

The results of the ascays described above ar~
shown in Table 1. It is clear that some of the S transformants produced increas2d amounts of ` -endogll~canase activity compared to untransformed strain ~UtC3 0 . It is thought that the endoglucanases and _~o-cellobiohydrolases produced by unt-ans^or~d ~. r-es2i co~stitutP approximately lo 20 and 70 p2r _~t ~sp_ctiY~ly of tha .o~al amount of prot~ sec_~ted. Ther~.or2 a .rans.ormant such as EP5, ~hich produc~s approximately four-fold more endoglucanase than strain RutC30, would be expected to secre~e approximate}y equal amounts of endoglucanase-type and exo-cellobiohydrolase-type protein6.
.
The transformants described in this Example were obtained using intact pEGIEy~ and will contain ~ DNA sequences integrated in the genome which were ; ~ 20 ~derived from the pUC plasmid. Prior to transformation it would be possible to digest pEOIgy~ with HindIII and isolate the larger DNA
~fragment containing only T. reesei DNA.
Transformation of ~ çLsei with this isolated fragment of DNA would allow isolation of transformants which overproduced EGI and contained no heterologous DNA sequences except for the two short pieces of synthetic DNA shown in FIG. 8. It would also be possible to use pEGIpvr4 to transform a strain which was deleted for-either the cbhl gene, or the cbh2 gene, or for both genes. In this way a strain could be constructed which would over-produce :~ .
. .

~ . O 92/ 1 757 2 '1 ¦ ~ 7 ~ ~ ~ pcr/ US92/02629 EGI and produce either a limited range of, or no, exo-c~llobiohydrolases.

Th~ m~ods o~ Example 22 could be ~sed to producs T. reesei strains which would over-produce s any o~ the other c211ulase components, xylanase components or ot~er proteins normally produced by T.
reesei.
:

S~cr_c d ~ndo~lucanas2 Acti~ity of T. reesei Transformants -.
. . .
.
: ~ . A B
ENDOGLUCANASE
ACTIVITY PROTEIN
STRAIN ~O.D~ ~T 590 nm! (mg/~LL
RutC30 0.32 4.1 0 a78 ~ EP2 0.70 3.7 0 189 ,~ EP4 0.76 3.65 0.208 -EP5 1.24 4.1 0 302 : :.
: ~ EP6 0.52 2.93 0 177 EP11 0.99 . 4.11 0.241 .

~ ~ - , . : .
The above results are presented for the purpose of demonstrating the overproduction of the EGI
component relàtive to total protein and not for the :: ~
purpose of demonstrating the extent of ~-25~ :overproduction. In this regard, the extent of overproduction is expected to vary with each . experiment. ~ .

.

'd ' . . ", ', d~.. ',i,,: ' ' , , ~ ~ , ~ ,; ~ , ~

~177~ PCT/US92/02629 Exa~æle 23 ^ Construction of ~CEPC1 A plasmid, pCEPCl, was constructed in which the coding sequence for EGI was functionally fused to the promoter from the cbhl gene. This was achi2~Jed using in vitro, site-specific mutagenesis to alter the DNA sequence of the cbhl and eqll genes in or~e_ to create convenient restriction andonuclease cleavage sit2s just S' (upstream) of their respective translation initiation si~as. DNA
sequence analysis was performed to varify the expected sequence at the junction between the two DNA segments. The specific alterations made are shown in FIG. 14.

The DNA fragments which were combined to form pCEPCl were inserted between the EcoRI sites of pUC4K and were as follows (see FIG. lS):
~) A 2.1 kb fragment from the 5' flanking region of the cbhl locus. This includes the promoter region and extends to the engineered site and so contains no cbhl coding sequenc~.
B) A 1.9 kb fragment of genomic DNA from the e~ll locus starting at the 5' end with the engineered ~HI site and extending through the coding region and including approximately O.S kb beyond the translation stop codon. At the 3' end of the fragment is 18 bp derived from the pUC218 multiple cloning site and a lS bp synthetic oligonucleotide used to link this fragment with the `~
fragment below.

~`392/1~72 ~ n 7 ? ~ ~ PCT/US92/02629 C) A fragment of DNA from the 3~ flanking region of the cbhl locus, extending from a position approximately l kb downstream to approximately 2.5 kb downstream of the cbh~ translation stop codon.
D) Inserted into an NheI site in fragment (C) was a 3.1 kb NheI-S~hI fragment of DNA containing the T.
reesei EYE~ gene obtained from pTpyr2 (Example 2) and having 24 bp of D~A at one end derived from the pUCl8 multiple cloning sit~

The plasmid, pCEPCl was designed so that the EGI coding sequenc~ ~ould ~e integrated at the cbhl ~
locus, replacing the coding seguence ~or CB~I ;
without introducing any foreign DNA into the host ~ .
strain. Digestion of this plasmid with Eco~I
~15 liberates a fragment which includes the Çkhl promoter region, the egll coding sequence and transcription termination region, the T. reesei EYE~
gene and a segment of DNA from the 3' (downstream) -flanking region of the çkhL locus (see Fig. 15).

Example 24 Transforman~s containina pCEPCl DNA
A EY~ defective strain of ~ 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 EcoRI.
Stable transformants were selected and subsequently cultured in shaker flasks for cellulase production às descFibed in Example 22. In order to visualize the cellulase proteins, isoelectric focusing gel electrophoresis was performed on samples from these 2~7''~

, .. ..

cultures using the method described in Example 7. .
Of a total of 23 transformants analysed in this manner 12 were found to producP no CBHI protein, which is the exp~t~d -~sult,of integration of the pCEPCl DNA at the c~hl locus. Southern blot analysis was used to confirm that intagration had indeed occurred at th2 cbhl locus in some of these trans~ormants 3nd that no sequenc2s derivPd from the bacterial plasmid v ctor (puc4x) wPre present (see Fig. l6~. ~or ~his ~na'ysia ~e DNl rrom the transfor~ants ~as dig~st~d ~ith Ps.I before being subjectDd t3 ~lec_r3~ho.~sls and blotting to a : membrane filter. The resulting Southern blot was probed with radiolabelled plasmid pUC4X::cbhl (see Example 2). T~e probe hybridised to the cbhl gene on ~:, a 6.5 kb fragment of DNA from the untransformed control culture (FIG. 16, lane A). Integration of the pCEPCl fragment of DNA at the cb,~l locus would be expected to result in the loss of this 6.5 kb band and the appearance of three other bands corrésponding to approximately l.0 kb, 2.0 ~b and :~ , 3.~5 kb DNA frag~ents. 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 25 ~ transformant in which multiple copies of pCEPCl have integrated at sites in the genome other than the ", ,cbhl locus.

. . ; Endoglucanase activity assays were performed on samples of culture supernatant from the ~'3Q 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 '' '~
...
.~

092/~7572 ~ n 7~ PCT/USg2/02629 approximately 0.03 and 0.07 mg/ml. The results of assays performed with the untransformed control culture and four different transformants (designated CEPCl-101, C~PC1-103, CEPC1-105 and CEPCl-112) are shown in Ta~le 2. Transrormants CEPCl-103 and CEPCl-112 ar xamples in which integration of the CEPCl fragment had led to loss of CBHI production.
-~
Table 2 Secret~d endoqlucanase actiYitv of T. reesçi ~ -transformants : . :: .
~ ~ B ~-:. .
ENDOGLUCANASE
ACTIVITY PROTEIN
;(!D~_a~_~2Q_n~l (ma/ml) ~L~ ;
RutC300 0.037 2.38 0.016 CEPCl-101 0.082 2.72 0.030 CEPCl-103 0.099 1.93 0.051 CEPCl-105 0.033 2.07 0.016 20CEPC1-112 0.093 1.72 0.054 The above results are presented for the purpose of demonstrating the overproduction of the EGI
; ~compon-nt relative to total protein and not for the purpose of demonstrating the xtent of 25~ overproduction. In this regard, the extent of overproduction is expected to vary~with each - ~ -expériment. -It would be possible to constr~ct plasmids ; similar to pCEPCl but with any other T. reesei gene ~ replacing the eall gene. In this way, overexpression of other genes and simultaneous d-letion of the ~ha gene could be achieved.

~ ~ -: ,~ ' ' , .:

~.~.n7~0~
WOs2/17572 PCT/US92/02629 '-,' It would also be possible to transform E~E~
derivative strains of T. reesei which had previously been deleted for other genes, eg. for cbh2, with pCEPCl to construct transformants which would, for example, produce no exo-cellobiohydrolases and overexpress endoglucanases.

Using constructions similar to pCE~C1, ~ut in which D~A from another locus of T. r2esei ~as substituted for the DNA from the cbhl locus, it would be possibl~ to ins2rt genes under th2 contLol of another promoter at another locus in the T.
reesei genome.

Example 25 Construction of DEGII::P~
The,eal3 gene, encoding EGII (previously referred to as EGIII by others), has been cloned ;
from T. reesei and the DNA sequence published I ", (Saloheimo et al., 1988, Gene 63~ 21). We have obtained the gene from strain RL-P37 as an approximately 4 kb PstI- ~h~I fragment of genomic DNA inserted between the PstI and ~E 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 s,ites for ~glII, ClaI and XhoI.
Using methods known in the art the T. reesei EYE~ - -' ' gene, present on a 2.7 ~b SalI fragment of genomic DNA, was inserted into a ~31I site within the EGII ~, coding sequence to create plasmid pEGII::P-l (FIF.

~ ' ' ''. ~ , .' ' . ..
- ' ~ .

*~ 92/17572 PC~r/US~2/02629 ~;

17). This resulted in disruption of the EGII coding sea,uence but without deletion of any sequencs~. Th~
plasmid, pEGII::P-1 can be digested with HindIII and a3~HI to yield a linear fragment of DNA d~ri~ed exclusively from T. reesei except for 5 bp on one end and 16 bp on the other end, both of which ar~
derived from the multiple cloning site of pUC219.
,:, ,, Example 26 Transformation of T. rees2i GC63 with p~GII::P-1 .o lQ cr2at a strain unabl to produc~ EGII
T. reesei strain GC69 will be transformed with pEGII::P-1 which had been previously digested with ~indIII and ~HI and stable transformants will be selected. Total DNA will be isolated from the transformants and Southern blot analysis used to identify those transforJants in which the fragment ' ,' , of DNA containing the EY~ and eal3 genes had integrated at the eal3 locus and consequently disrupted the EGlI coding se~uence. ~he transformants will be unable to produce EGII. It would also be possible to use pEGII::P-1 to ~ transfor,m a strain which was deleted for either or ' ~ ,, '~ all of the cbhl, cbh2, or ~gL~ genes. In this way a ~' strain could be constructed which would only produce ~5 cert~in c-llul~s- c~mponents ~nd no ~II co ponent.

5 ! ~

Example 27 Transformation of T. reesei with pEGII::P-l to create a strain unable to produce C8HI.
CBHII and EGII
A pyr4 dericient deri~ative of strain P37P~aCBH67 (from Example ll) was obtained by the method outli~ed i~ ~xample l. This strain P37P~67P
l was t-ansformed with p~GII::P-l which had been - previouslv digested -~ith HindIII and 3amHI and stable transIormants were selected. Total DNA was isol~L2d f-am ~ransrcr~an~s and Southern blot analysis us2d to id~nti~y s~rains in which the fragmen~ or 3N~ containing the ~yr4 and eal3 genes had integrated at the eal3 locus and consequently disrupted the EGII coding sequence. The Southern blot illustrated in FIG. l8 was probed with an approximately 4 kb PstI fragment of T. ~eesei DNA
containing the eql3 gene which had been cloned into the ~P~I site of pUCl8 and subsequently re-isolated.
When the DNA isolated from strain P37P~67P-l was digested with PstI for Southern blot analysis the eal3 locus was subsequently visualized as a single 4 kb band on the autoradiograph ~FIG. 18, lane E).
However, for a transformant disrupted for the eal3 gene this band was lost and was replaced by two new ;
bands as expected (FIG. 18, Lane F). If the DNA was :
digested with EcoRV or BalII the size of the band -;
corresponding to the eal3 gene increased in size by approximately 2.7 kb (thè size of the inserted ~vr4 fragment) between the untransformed P37P~67P-l strain (Lanes A and C) and the transformant disrupted for eal3 (FIG. 18, Lanes B and D). The transformant containing the disrupted eal3 gene ,".
~ ...

'. ~ :

... ~92~17~72 2~ ~ 7 ~ PCT/US92/02629 illustrated in FIG. 18 ~Lanes B, D and F) was named A22. The transrormant identified in FIG. 18 is unable to produce CBHl, C8HII or EGII.
. ' ~xamDle 28 .: .
construction of ~P~EGI-l . .
Tha eqll sane of T. reesei strain RL-P37 was obtained, as àescri~ed in Example 12, as a 4.2 kb XindIII f ag~ant of genomic DNA. This fragment was inserted at the HindIII site of pUClO0 (a derivative .: lo of pUC187 Yanisch-Pe~ron et al., 1985, Gene 33:103-119, wi.h an oligonucl20~ide insarted into the multiple cloning site adding restriction sites for ~ .
, Çl~I and ~hQI). Using methodology known in the art an approximately 1 kb EQQRV fragment lS . extending from a position close to the middle:of the . .
EGI coding sequence to a position beyond the 3' end :;~ j o~ the coding sequence was removed and replaced by a , : 3.~S kb ScaI fragment of I~ reesei DNA containing the D~r4 gene. Thè resulting plasmid was called pP~EGI-1~(see Fig. 19).

~: ~ The plasmid pP~EGI-l can be digested with ; HindIII to release a DNA fragment comprising only T.
r--sei genomic DNA having a segment of the ~gL1 gene -: at either~end:and:the ~vr4 gene replacing part of .
: 25 the EGI coding sequence, in the center.
.
Transformation of a suitable T. reesei ~:
: E~L~ deficient stra$n with the pP~EGI-l digested with ~indIII will lead to integration of this DNA
fragment at tha eall locus in some proportion of the .

.

2107~Q~
wos2/l7is72 PCT/US92/02629 transformants. In this manner a strain unable to produce EGI will be obtained.
Exam~le 29 :
Construction of p~EGI~yr-3 and Transformation of a Dvr4 deficient strain of T. reesai The expectation that the EGI gene could ~e inactivated using the method out1in~d n ~ mpls ~
is str~ngthened by this experiment. ln thls cas~ 2 ` `
plasmid, paEGIpyr-3, was construc~2d w~ich wa~
similar to pP~EGI-l exc2pt that the Asp raillus niger ~vr4 gene replaced the T. reesei ~vr4 gene as selectable marker. In this case the eall gene was again pr-sent as a 4.2 kb HindIII fragment inserted at the ~indIII site of pUClO0. The same internal 1 kb EQQRv fragment was removed as during the construction of pP~EGI-l (see Example 28) but in this case it was replaced by a 2.2 kb fragment containing the cloned ~ niger EY~_ gene (Wilson et al.~, 1988, Nucl. A~id~_Res. 1~ p.2339).
20~ ~ Transformation of a EYE~ deficient strain o~ T. ~ ~ t' r~ç~ (strain GC69) with p~EGIpyr-3, after it had been;digested with Hind}II to release the fragment ;
containing the Qy~ gene with flanking regions from ; the eall locus at either end, led to transformants 2~5 ~ in which the eall gene was disrupted. These `~
transformants were recognized by Southern blot analysis of transformant DNA digested with HindIII ~`
and~probed with radiolabelled p EGIpyr-3. In the untransformed strain of T. eesei the ~gll gene was present on a 4.2 kb ~indIII fragment of DNA and this pattern of hybridization is represented by Fig. 20, lane C. However, following deletion of the eall 92/17S72 ~ PCT/US92/02629 gene by integration of the desired fragment from . .
p~EGIpyr-3 this 4.2 kb fragment disappeared and was replaced by a fraa,ment approximately l.2 ~b larger in size, FIG. 20, lane A. Also shown in FIG. 20, lane B is a~ example of a transformant in which integration of a single copy of pP~EGIpyr-3 has occurred at a site in t~e genome other t~an the e~ll .
locus.
Exam~le 30 Transformation o~ T.reesel wlth p~ GI-l to cri~at _a strain unable ~o ~,d~c CBHI, CBHII. EGI and EGTT :~
A ~vr4 deficient derivati~e of strain A22 (from Example 27) will be obtained by the method outlined in Example l. This strain will be transformed with pP~EGI-l which had been previously digested with ~ ~indIII to release a DNA fragment comprising only T.
: ' ,reesei genomic DNA having a segment of the eall gene .
at either end with part of the EGI coding sequence . . .
replaced by the EYE~ gene.

StabIe 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 fragment of DNA
containing the EY~ gene and eall sequences has integrated at the ~gll locus and consea,uently disrupted the EGI coding sequence. The ,.
transformants identified will be unable to produce CBHI, CBHII, EGI and EGII.

Claims (8)

WHAT IS CLAIMED IS:

1. An improved method for treating cotton-containing fabrics by treatment with a fungal cellulase composition wherein said improvement comprises employing a fungal cellulase composition which is substantially free of all CBH I type cellulase components.

2. The method according to Claim 1 wherein said fungal cellulase composition is free of all CBH
type components.

3. 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.

4. An improved method for the treatment of cotton-containing fabrics with an aqueous fungal cellulase solution wherein said method is conducted with agitation 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 which is substantially free of all CBH I type components.

5. A method according to Claim 4 wherein said fungal cellulase composition is free of all CBH type components.

6. A method according to Claim 4 wherein said fungal cellulase composition comprises at least ?O 92/17572 PCT/US92/02629 about 20 weight percent of EG components based on the total weight of protein in the cellulase composition.

7. A cotton-containing fabric having improved feel and/or appearance wherein said fabric is prepared by the method defined in Claim 1.

8. A cotton-containing fabric having improved feel and/or appearance wherein said fabric is prepared by the method defined in Claim 4.