CA2093425A1 - Detergent compositions containing cellulase compositions enriched in acidic endoglucanase type components - Google Patents

Abstract

Disclosed are acidic cellulase compositions enriched in endoglucanase components. Such cellulase compositions impart improved softening properties to detergent compositions when used in acidic, neutral or alkaline washing media. Additionally, such compositions impart color retention/restoration properties to the detergent composition.

Description

W O 92/06210 PC~r/US9t/~7273 -- 1 .

2 0 9 3 ~-~2 ~ - ~

DETERGENT coMposITIoNR CONTAINING
CEL~LA8E COMPO8ITION8 ENRICHED IN
ACIDIC ENDOGLUCANA8E TYP~ COMPONENT8 BACRGROUND OF THE INVENTION
.
1. Field of the Invention.
The present invention is directed to detergent compositions containing specific cellulase compositions. In particular, the present invention is directed to detergent compositions containing a cleaning effective amount of a surfactant and an acidic cellulase composition containing an enriched amount of acidic endoglucanase (EG) type components relative to exo-cellobiohydrolase (CBH) I type components and, preferably, all CBH type components.
Even more particularly, the present invention re1ates to de*ergent compositions containing a cleaning effective amount of a surfactant and a cellulase~composition wherein the cellulase composition comprises one or more acidic EG type --components and optionally CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1. Such detergent compositions provide substantial improvements in softness, feel, color retention/restoration, and the like. Additionally, when such cellulase compositions comprise one or - 25 more acidic EG type components containing at least about l~weight percent, preferably, at least about 5 weight percent, and more preferably, at least about 10 weight percent, of -.

W092~06210 PCT/US91/~7273 ".
~3~25 - 2 - .

CBH I type cellulase components (based on the total protein weight o. all of the acidic EG components), an incremental cleaning benefit is also observed.
r 2. State of the Art.
Cellulases are known in the art as enzymes that hydrolyze cellulose (B-1,4-glucan linkages) thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like.
While cellulases are produced (expressed) in fungi, -bacteria and the like, those produced by fungi have been given the most attention because certain fungi --produce a complete cellulase system capable of -degrading crystalline forms of cellulose and such ~-cellulases can be readily produced in large quantities via fermentation procedures.

In regard to the above, Wood et al., "Methods in Enzymology", 160, 25, pages 234 et seq. (1988), discloses that certain fungi produce complete cellulase systems which are comprised of several different enzyme components including those identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBHn), endoglucanases (EC 3.2.1.4) ("EG"), and glucosidases (EC 3.2.1.21) ("BG"). On the other hand, some fungi are incapable of producing complete cel}ulase systems. Generally, these systems lack CBH components. See, for instance, Coughlan et al., Biochemistry and Genetics of Cellulose Degradation, Aubert et al., Editor, pages 11 et seq., (Academic Press, 1988); and Wood et al., Methods in Enzymology, 60, 25, pages 87 et seq., (Academic Press, New York, 1988). : -: '~

~~ : -' --~ 3 ~ 2~93~

Likewise, while bacterial cellulases arerepor'2d in the literature as containing little or no CBH components, there are a few cases where CBH-like components derived from bacterial cellulases have been reported to possess exo-cellobiohydrolase activity.

The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs and EGs have been isolated from a variety of fungal sources including Trichoderma reesei which contains 2 CBHs, i.e., CBH
I and CBH II, and at least 3 EGs, i.e., EG I, EG II
and EG III.

The complete cellulase system comprising CBH, EG and BG is required to efficiently convert crystalline cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components parti-cularly if they are of different classifications. That is to say the effectiveness of a complete cellulase system is -~
significantly greater than the sum of the contributions from the isolated components of the same classification. In this regard, it is known in the art that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. See, for example, Wood, Biochem. Soc.
Trans., 13, pp. 407-410 (1985).

The substrate specificity and mode of action of the different cellulase components varies .

WO92/06210 PCT/US91/~7273 2 0 9 '~ 4 Z ~ 4 ~

significantly with classification which may account for the synergy of thc combined components. Por example, the current accepted mode of cellulase action is that endoglucanase hydrolyzes internal B-1,4-glucosidic bonds, particularly, in regions of ~ -low crystallinity of the cellulose and exo-cellobio-hydrolase hydrolyzes cellobiose from the non- -reducing end of cellulose. The action of endoglucanases greatly facilitates the action of exo-cellobio-hydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components. -:' B-Glucosidase act only on cellooligosaccharides, e.g., cellobiose, to give -glucose as the sole product. They are considered an ~; integral part of the cellulase system because they drive the overall reaction to glucose and thereby relieve the inhibitory effects of cellobiose on CBH
and EG components. -2~0~ On the other hand, cellulases are also known in~the~art to be usefuI in detergent compositions for~the purposes of enhancing the cleaning ability -~
of the composition, for use as a softening agent, and for improving the feel of cotton fabrics, and 25~ ~ the like. While the exact mechanism by which cellulase compositions soften garments is not fully understood, softening and color restoration properties of cellulase have been attributed to ~
alkaline endoglucanase components in cellulase 30~ compositions. Thus, for instance, International Application Publication No. W0 89/09259 discloses that detergent compositions containing a cellulase composition enriched in a specified alkaline :.-,~, - 5 - 2~93~2~

endoglucanase component impart color restoration and - improve~ sof.ening tG treated garments as compared to cellulase compositions not enriched in such components. Additionally, the use of such alkaline endoglucanase components in detergent compositions complements the pH requirements of the detergent composition. The endoglucanase component of this reference is defined as one exhibiting maximal activity at an alkaline pH of 7.5 to 10 and which has defined activity criteria on carboxymethylcellulose and Avicel. On the other hand, the most readily employed sources of cellulase compositions are fungally derived cellulase compositions and components which exhibit maximal activity at an acidic pHs with less activity at alkaline pHs.

Moreover, in view of the ready availability of such acidic funqal cellulase compositions, it ~-would be advantageous to use such cellulases in detergent compositions and particularly in detergent compositions employed in alkaline conditions.

It has now been found that fungal cellulase compositions enriched in acidic endoglucanase type components can be combined in detergent compositions to attain improvements in softening, color retention/restoration and feel. It has further been found that such cellulase compositions impart improvements in color retention/restoration, softening and feel in acidic, neutral or alkaline wash media. In alkaline wash media, improvements in softening, color retention/restoration, and feel are -.

.

W O 92/06210 PC~r/US91/07273 '~U934~ - 6 - ~ .~'.. .

particularly surprising because acidic endoglucanase type components possess minimal enzymatic activity in alkaline media and are considered to be unstable under alkaline conditions. However, in the present S invention, it has been unexpectedly found that even with minimal activity in alkaline media and not withstanding their instability under alkaline conditions, the acidic endoglucanase type components -still impart improvements in softening, color retention/restoration, and feel to cotton fabrics treated with an alkaline detergent medium. Such improvements are particularly noticeable either in a single wash using higher concentrations of such cellulase compositions or with repeated washings -using lower concentrations of such cellulase compositions.

In regard to the detergent compositions containing cellulase compostions which are CBHI -deficient, CBHI enriched or EGIII enriched, it has been found that it is the amount of cellulase, and not the relative rate of hydrolysis of the specific enzymatic components to produce reducing sugars from ~; cellulose, which imparts the desired detergent properties to cotton-containing fabrics, eg., one or more of improved color restoration, improved softening and improved cleaning to the detergent composition.

Accordingly, in one of its composition aspects, the present invention is directed to a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent, and preferably from a~out 0.05 to about 2 , ,.. . . . ; . . .. . . ; . . , . . : . . . . . . . . . . . . . . ` . . . . .

WQ92/062l0 PCT/USg1/~7273 . ~ 7 ~ 2~93~2:3 weight percent, of a fungal cellulase composition comprising one or more acidic EG .ype components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I
type components of greater than 5:1. In a preferred embodiment, the fungal cellulase composition employed in the detergent composition comprises one or more acidic EG type components and optionally one or more CBH type components wherein said cellulase ~
composition has a protein weight ratio of all acidic -EG type components to all CBH type components of greater than 5:1. Even more preferably, this fungal cellulase composition is free of all CBH I type -components and, still more preferably, free of all CBH type components.

In another preferred embodiment, the cellulase composition is derived from a microorganism which has been genetically modified so as to be incapable of producing any CBH I type components and more preferably any CBH type.
~:~ components, i.e., CBH I type and CBH II type ~ -components. In even a more preferred embodiment, the cellulase composition is derived from a microorganism which has been genetically modified so as to be incapable of producing any CBH I type components or any CBH type components without the introduction of any heterologous proteins.

In one of its method aspects, the present invention is directed to a method for enhancing the softness of a cotton-containing fabric which method comprises washing the fabric in a wash medium ~ :
derived from a detergent composition comprising a - : .

W092/06210 PCT/US91/~7273 '~93~Z~ - 8 -cleaning effective amount of a surfactant or a mixture o~ surfa.-tants and ~rom about 0.01 to about -5, and preferably from about 0.05 to about 2, weight percent of a fungal cellulase composition comprising - :
one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type ~, components of greater than 5:1. -In another of its method aspects, the present invention is directed to a method for :
retaining/restoring the color of a cotton-containing fabric which method comprises washing the fabric one -~
or more times in a wash medium derived from a :
detergent composition comprising a cleaning ;.
effective amount of a surfactant or a mixture of ; surfactants and from about 0.01 to about 5, and ;-preferably from about 0.05 to about 2, weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of -a}l acidic EG type components to all CBH I type components of greater than 5:1.
:::
. BRIEF DE8C~IPTION OF THE DRA~ING8 FIG. 1 is an outline of the construction of p CBHIpyr4. .
.:
FIG. 2 illustrates deletion of the T. reesei gene by integration of the larger EcoRI fragment -.
from p~CBHIpyr4 at the ~khl locus on one of the T.
: ~YY~l chromosomes.

W092/062~0 PCT/US91/072~3 g ~ i3 ~

FIG. 3 is an autoradiograph of DNA from T.
r.esei strain GC69 trans~ormed with ~çQRI digested p~CBHIEyE~ after Southern blot analysis using a 32p ::
labelled p~CBHIEyE~ as the probe. The sizes of molecular weight markers are show~ in kilobase pairs to the left of the Figure.

FIG. 4 is an autoradiograph of DNA from a T.
reesei strain GC69 transformed with EcoRI digested p~CBHIpyr4 using a 32p labelled pIntCBHI as the ;~
probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure. ~-FIG. 5 is an isoelectric focusing gel -displaying the proteins secreted by the wild type and by transformed strains of T. reesei.
Specifically, in FIG.5, Lane A of the isoelectric focusing gel employs partially purified CBHI from T.
reesei; Lane B employs a wiId type T. reesei : Lane C employs protein from a T. reesei strain with the cbhl gene deleted; and Lane D employs protein from a ZO~ T.~reçsei strain with the cbhl and cbh2 genes deleted. In FIG. 5, the right hand side of the figure is~marked to indicate the location of the single proteins found in one or more Or the secreted proteins. Specifically, BG refers to the ~-25~ glucosidase, E1 refers to endo~lucanase I, E2 refersto endoglucanase II, E3 refers to endoglucanase III, C1 refers to~exo-cellobiohydrolase I and C2 refers to exo-cellobiohydrolase II.

FIG. 6A is a representation of the T. reesei 30~ cbh2 locus, cloned as a 4.1 kb EcoRI fragment on ; genomic DNA and~FIG. 6B is a representation of the çk~;gene deletion vector pP~CBHII.

:

~ . .

W O 92/06210 PC~r/US91/07273 ' 209342~ -10-- ' '~,',' FIG. 7 is an autoradiograph of DNA from T. -reesei strain P37PACBHIPyr26 transformed with EcoRI
digested pP~CBHII after Southern blot analysis using a 32p labelled pP~C~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 pEGI~vr4.

FIG. 9 illustrates the RBB-CMC activity profile of an acidic EG enriched fungal cellulase composition (CBH I and II deleted) derived from Trichoderma reesei over a pH range at 40C; as well as the activity profile of an enriched EG III
cellulase composition derived from Trichoderma ~9Ç~i over a pH range at 40C.

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

FIG. ll 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 ree~en~ and ~or a cotton fabric treated with cellulase secreted by a strain of Trichoderma reesei :
~: :

WO92Jo6210 PCT/US91/~7273 ' 2~'93~23 genetically engineered so as to be incapable of secreting CB~ I and C~

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. : .

FIG. 14 is a diagram of the site specific alterations made in the eqll and cbhl genes to -:
create convenient restriction endonuclease cleavage :
sites. In each case, the upper line shows the original DNA sequence, the changes introduced are shown in the middle line, and the new sequence is - shown in the lower line.

FIG. 15 is a diagram of the larger EcoRI
fragment which can be obtained from pCEPCl.
. ,:
FIG. 16 is an autoradiograph of DNA, from an untransformed strain of T. reesei RutC30 and from two transformants obtained by transforming T. reesei with EcoRI digested pCEPCl. The DNA was digested with PstI, a Southern blot was obtained and .:;
hybridized with 32p labelled pUC4K::cbhl. The sizes of marker DNA fragments are shown in kilobase pairs to the left of the Figure.
, :
FIG. 17 is a diagram of the plasmid pEGII::P~
~ .
FIG 18. is an autoradiograph of DNA from T.
reesei strain P37P~67P1 transformed with HindIII
and ~HI digested pEGII::P-l. A Southern blot was ~ .

~ .

W O 92/06210 PC~rtUS91/07273 2~934~ - 12 - ~ :-prepared and the DNA was hybridized with an apprGx~mataly 4kb PstI fragment of radiolabeiled T.reesei DNA containing the eal3 gene. Lanes A, C
and E contain DNA from the untransformed strain whereas, Lanes B, D and F contain DNA from the 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.
The size of marker DNA fragments are shown in kilobase pairs to the left of the Figure.

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

FIG. 20 is an autoradiograph of a Southern blot of DNA isolated from transformants of strain GC69 obtained with HindIII digested p~EGIpyr-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 disrupted and Lane B
shows a transformant in which p~EGIpyr-3 DNA has integrated into the genome but without disrupting the eall gene. Lane D contains p~EGIpyr-3 digested with HindIII to provide appropriate size markers.
~he sizes of marker DNA fragments are shown in kilobase pairs to the right of the figure.

DETAILED DESCRIPTION OF THE INVENT~ON
,..
As noted above, the present invention generally relates to detergent compositions containing fungal cellulase compositions enriched in acidic EG type cellulase components relative to CBH
I type components as well as for methods employing - 13 - 2 ~ 2 ~

such cellulase compositio~s. When used in detergent compositions at acidic, neutral or alkdline pH's, such co~positions impart improvements in softening, color retention/restoration, and feel to the treated cotton-containing fabric.

However, prior to discussing this invention -in detail, the following terms will first be defined.

The term "fungal cellulase" refers to the enzyme composition derived from fungal sources or microorganisms genetically modified so as to ~
incorporate and express all or part of the cellulase -genes obtained from a fungal source. Fungal cellulases act on cellulose and its derivatives to hydrolyze cellulose and give primary products, glucose and cellobiose. Fungal cellulases are distinguished from cellulases produced from non-fungal sources including microorganisms such as actinomycetes, gliding bacteria (myxobacteria) and true bacteria. Fungi capable of producing cellulases useful in preparing cellulase compositions used in the detergent compositions -described herein are disclosed in British Patent No.
2 094 826A, the disclosure of which is incorporated herein by reference. -Most fungal cellulases generally have their optimum activity in the acidic or neutral pH range although some fungal cellulases are known to possess significant activity under neutral and slightly alkaline conditions, i.e., for example, cellulase derived from Humicola insolens is known to have activity in neutral to slightly alkaline conditions. -W O 92/06210 P~r/US91/~7273 ~ o 9 3 4 2 ~ - 14 -Fungal cellulases are known to be comprised of saveral enzym- ~lassifi--ations having different substrate specificity, enzymatic action patterns, and the like. Additionally, enzyme components within each classification can exhibit different - :
molecular weights, different degrees of glycosylation, different isoelectric points, different substrate specificity, different enzymatic action patterns, etc. For example, fungal cellulases can contain cellulase classifications which include endoglucanases (EGs), exo-cellobiohydrolases (CBHs), ~-glucosidases (BGs), etc. On the other hand, while bacterial cellulases are reported in the literature as containing little lS or no CBH components, there are a few cases where CBH-like components derived from bacterial cellulases have been reported to possess exo-cellobiohydrolase activity.

A fungal cellulase composition produced by a naturally occurring fungal source and which ~ i comprises one or more CBH and EG components wherein each~of these components is found at the ratio pro~uced by~the fungal source is sometimes referred to~herein as a "complete fungal cellulase system" or 2~5 ~a ncomplete fungal cellulase composition" to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase composi:tions produced by bacteria and some fungi, or from a cel}ulase 3a~ compo-ition~obtained from a microorganism genetically modified so as to overproduce, . .
- ~ underproduce~or not produce one or more of the CBH
and/or~EG COmpOneDtS of cellulase.

WO92/06210 PCT/US91/n7273 ~ 15 ~ 2~3~23 The fermentation procedures for culturing fur.g, for production of cellulase are known EE 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 collection and purification of the cellulase systems from the fermentation broth can also be effected by procedures known ~er se in the art.

"Acidic Fungal Cellulase Compositions" refer to fungally derived cellulase compositions which possess an activity profile having a pH optimum at a pH of less than 7.0, and preferably less than 6.5.
Generally but not necessarily, such acidic cellulase -compositions contain acidic EG and acidic CBH -components. -"Endoglucanase ("EG") type components" refer to all of those fungal cellulase components or combination of components which exhibit detergent activity properties similar to the endoglucanase components of Trichoderma reesei. In this regard, the endoglucanase components of Trichoderma reesei (specifically, EG I, EG II, EG III, and the like either alone or in combination) impart softening, color retention/restoration and improved feel to cotton-containing fabrics when these components are incorporated into a wash medium and the fabric is treated with this medium. Accordingly, endoglucanase type components are those fungal cellulase components which impart softening, color retention/restoration and improved feel to cotton garments when these components are incorporated into -~ a wash medium. In a preferred embodiment, the . '', '.
:.
: , .

.

2~9'~ 5 - 16 -endoglucanase type components employed in the detergent compositions of this invention also impart less strength loss to cotton-containing fabrics as compared to strength loss arising from the complete cellulase system derived from Trichoderma reesei.

Such endoglucanase type components may not include components traditionally classified as endoglucanases using activity tests such as the ability of the component (a) to hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (CMC), thereby reducing the viscosity of CMC -containing solutions, (b) to readily hydrolyze hydrated forms of cellulose such as phosphoric acid swollen cellulose (e.g., Walseth cellulose) and hydrolyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.). -on the other hand, it is believed that not all endoglucanase components, as defined by such activity tests, will provide improved softness, feel and color retention/restoration. Accordingly, it is more accurate for the purposes herein to define endoglucanase type components as those components of fungal cellulase which possess similar properties in detergent compositions as possessed by the endoglucanase components of Trichoderma reesei.

Fungal cellulases can contain more than one EG type component. The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificity, different enzymatic action patterns, etc. ~he different isoelectric points of the components allow for their separation via ion exchange chromatography WO92/06210 PCT/US9t/a7273 - 17 _ 2~ 3~2 :~

and the like. In fact, the isolation of components from different fungai sources is ~nown in the art.
See, for example, Bjork et al., U.S. Serial No.
07/422,814, Schulein et al., International Application WO 89/09259, Wood et al., Biochemistry and Genetics of Cellulose Degradation, pp. 31 to S2 (1988); Wood et al., Carbohydrate Research, Vol.
190, pp. 279 to 297 (1989); Schulein, Methods in Enzymology, Vol. 160, pp. 234 to 242 (1988); and the like. The entire disclosure of each of these references is incorporated herein by reference.

"Acidic fungal EG type components" (acidic EG
type components) refer to those fungally derived EG
type components which have a pH optimum of less than 7.0 and preferably, less than 6.5, but which also possess at least some activity on RBB-CMC at 40C
(per Example 15) in alkaline media (i.e., an aqueous solution containing the acidic EG type cellulase component buffered to a pH of from greater than 7 to about 10, and preferably at a pH of from greater than 7 to about 8). Preferably, at pH 7.5 and at 40C, the acidic EG type component possesses at least 10% of its maximal activity.

In general, it is contemplated that combinations of acidic EG type components may give a synergistic response in improving softening, color retention/restoration and feel as compared to a single acidic EG type component. On the other hand, a single acidic EG type component may be more stable or have a broader spectrum of activity over a range of pHs. Accordingly, the acidic EG type components employed in this invention can be either a single acidic EG type component or a combination of two or .
.

; ;:..

WO92/06210 PCT/US91/~7273 2ag3 ~2 ~ - 18 -more acidic EG type components. When a combination of compo..ents is employed, the acidic E& type components may be derived from the same or different fungal sources.

"Exo-cellobiohydrolase type ("CBH type") components" refer to those fungal cellulase components which exhibit detergent activity properties similar to CBH I and/or CBH II components of Trichoderma reesei. In this regard, when used in the absence of EG type components (as defined above), the CBH I and CBH II components of Trichoderma reesei alone do not impart color -retention/restoration and improved feel to the so- -treated cotton-containing fabrics. Additionally, when used in combination with EG type components, the CBH I component of Trichoderma reesei can impart enhanced strength loss and incremental cleaning effect to cotton-containing fabrics.

Accordingly, CBH I type components and CBH II
~-~ 20 type components refer to those fungal cellulase components which exhibit detergent 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 properties of enhancing strength loss of cotton-containing fabrics and/or imparting an incremental cleaning benefit when used in the presence of EG type components. In a preferred embodiment, the CBH I components also impart an incremental softening benefit when used in the ~ i presence of EG type components.

.. . . . .

WO92/06210 PCT/US91/~7273 .
- 19 - 2~93~2~

Such exo-cellobiohydrolase type components may include components not traditionally rla~s~d as exo-cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II from Trichoderma reesei. For example, such components (a) are competitively inhibited by cellobiose (K, -approximately lmM); (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., and (c) hydrolyze -phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose. On the other hand, it is believed that some fungal cellulase components which are characterized as CBH components by such activity tests, will provide improved softness, feel and color retention/restoration to cotton-containing fabrics when these components are used alone in detergent compositions. Accordingly, it is believed to be more accurate for the purposes herein to define such exo-cellobiohydrolases as EG
type components because these components possess similar functional properties in detergent -compositions as possessed by the endoglucanase components of Trichoderma reesei. -"Acidic Fungal CBH type Components" (acidic CBH type components) refer to those fungally derived CBH type components which possess an activity profile on RBB-CMC at 40C (per Example 15) having a pH optimum at a pH of less than 7.0 and preferably, less than 6.5.
`
Acidic fungal cellulases enriched in acidic EG type components can be obtained by purification techniques. Specifically, the complete cellulase system can be purified into substantially pure . . . . ; ,, ., .. =.. . .. , .. ,.. .... . . ... ~ .... ,.. , . . . , ., ., . . ,. . . . . " . , . .... ., . -. -.. .

2~3 42~ - 20 -components by recognized separation techniques well published in t~.e li-erature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchanqe chromatography (usually anion exchange chromatography), it is possible to -separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient.

It is also contemplated that mixtures of cellulase components having the requisite ratio of acidic EG type components to CBH I type cellulase components could be prepared by means other than -isolation and recombination of the components. In this regard, it may be possible to modify the fermentation conditions for a natural microorganism in order to give relatively high ratios of EG to CBH
components. Likewise, recombinant téchniques can alter the relative ratio of EG components to CBH
components so as to produce a mixture of cellulase components having a relatively high ratio of EG
components to CBH components or which can produce one or more EG components free of all CBH
components. -In regard to the above, a preferred method for the preparation of cellulase compositions enriched in acidic EG type components is by genetically modifying a microorganism so as to be incapable of producing one or more CBH type components which methods do not produce any heterologous protein. Likewise, it is also possible to genetically modify a microorganism so as to overproduce one or more acid~c EG type components.

' :

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

WO 92/06210 PCI/US9t/07273 ~ 2~3342~ -For example, U.S. Serial No. 07/593,919, filed Octcib2r 5, 1990 and which is incorporated herein by reference in its entirety, discloses methods for genetically engineering ~richoderma reesei so as to 5 be incapable of producing one or more CBH components and/or overproducing one or more EG components.
Moreover, the methods of that application create Trichoderma reesei strains which do not produce any heterologous proteins. Likewise, Miller et al., 10 "Direct and Indirect Gene Replacement in AsDeraillus nidulans", Molecular and Cellular Biology, p. 1714-1721 (1985) discloses methods for deleting genes in AsDeraillus nidulans by DNA mediated transformation using a linear fragment of homologous DNA. The 15 methods of Miller et al., would achieve gene deletion without producinq any heterologous proteins.

In view of the above, the deletion of the genes responsible for producing either CBH I type or 20 CBH II type cellulase components ~would 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 CBH I and II type components would result in a ~-25 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 celluIase composition. For example, it is known 30 that certain fungi produce cellulase compositions free of CBH components. See, for example, Coughlan et al., Biochemistry and Genetics of Cellulose Degradation, Aubert et al. Editors, pp. 11-30 .
~ - ;',.' ' W092t062l0 PCT/US91/~7273 2~93 42~ - 22 -(Academic Press, 1988), disclose that brown rot fungi do not apparently produce CB~ component~, but it may be possible that one or more of these components are CBH I type components. On the other hand, if a sufficient amount of the endoglucanases produced by such fungi are, for the purposes herein, EG type components, then it would be possible to use such cellulase in the practice of this invention without enrichment to obtain the necessary ratio of EG type components to CBH I type components.

Additionally, a requisite amount of one or more CBH type components purified by conventional procedures can be added to a cellulase composition produced from a microorganism genetically engineered so as to be incapable of producing CBH type components so as to achieve a specified ratio of acidic EG type components to one or more CBH type components, i.e., an acidic cellulase composition free of all CBH type components so as to be enriched in acidic EG type components can be formulated to contain 1 weight percent of a CBH I type component merely by adding this amount of a purified CBH I
type component to the cellulase composition.
::
"B-Glucosidase (BG) components" refer to those components of cellulase which exhibit BG
activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides ("cellobiose") and give glucose as the sole product. BG components do not adsorb onto or react with cellulose polymers.
Furthermore, such BG components are competitively inhibited by glucose (~ approximately lmM). While in a strict sense, BG components are not literally :

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

- 23 _ 2~93~Z5 cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly -cellobiose) produced by the combined action of CBH
components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur. BG components are often characterized on aryl substrates such as p-nitrophenol B-D-glucoside (PNPG) and thus are often called aryl-glucosidases. It should be noted that not all aryl glucosidases are 8G components, in that some do not hydrolyze cellobiose. ~ ~-It is contemplated that the presence or absence of BG components in the cellulase -composition can be used to regulate the activity of the CBH components. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to -glucose by BG components, the absence of BG
components in the cellulase composition will "turn-off" CBH activity when the concentration of csllobiose reaches inhibitory levels. It is also contemplated that one or more additives (e.g., cellobiose, glucose, etc.) can be added to the 0 cellulase composition to effectively "turn-off", directly or indirectly, some or all of the CBH I
type activity as well as other CBH activity. When such additives are employed, the resulting composition is considered to be a composition W O 92/06210 PC~r/US91/07273 2~ 24_ suitable for use in this invention if the amount of additiva amployed is sufficient to lower the CBH I
type activity to levels equal to or less than the CBH I type activity levels achieved by using the cellulase compositions described herein. `

On the other hand, a cellulase composition containing added amounts of BG components may increase overall hydrolysis of cellulose if the level of cellobiose generated by the C8H components becomes restrictive of such overall hydrolysis in the absence of added BG components.

Methods to either increase or decrease the amount of BG components in the cellulase composition are disclosed in U.S. Serial No. 07/625,140, filed December 10, 1990, as attorney docket no. 010055-056 and entitled "SACCE~ARIFICATION OF CELLULOSE BY
CLONING AND AMPLIFICATION OF THE ~-GLUCOSIDASE GENE
OF TRICHODERMA REESEI", which application is incorporated herein by reference.

Fungal cellulases can contain more than one BG component. The different components generally have different isoelectric points which allow for ~; their separation via ion exchange chromatography and the like. Either a single BG component or a combination of BG components can be employed.
':
When employed in the detergent composition, the BG component is generally added in an amount sufficient to prevent inhibition of the CBH and EG
components and particularly, CBH I type cellulase components, by cellobiose. The amount of BG
component added depends upon the amount of - 25 - ~ 2~ :-cellobiose produced in the detergent wash which can be readily determined by the sk~lled artisan.
However, when employed, the weight percent of BG
component relative to CBH type components in the cellulase composition is preferably from about 0.2 to about 10 weight percent, and more preferably, 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., and the like. Certain fungal cellulases 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, California) and the like. Other fungal cellulases can be readily isolated by art recognized fermentation and isolation procedures.

The term "cotton-containing fabric" refers to sewn or unsewn fabrics made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, and the like. When cotton blends are employed, the amount of cotton in the fabric should be at least about 40 percent by weight percent cotton; preferably, more than about 60 percent by weight cotton; and most preferably, more than about 75 percent by weight cotton. When employed as blends, the companion material employed in the fabric can include one or more non-cotton fibers including synthetic fibers such as polyamide fibers (for example, ny}on 6 and nylon 66), acrylic fibers (for example, polyacrylonitrile fibers), polyester -~

20g3 ~25 - 26 - ~ ;~

fibers (for example, polyethylene terephthalate), polyvinyl alcohol ,,bers (,or 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 cotton containing fabrics.

The term "surface active agent or surfactant"
refers to anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions. -The term "wash medium~ refers to an aqueous wash solution prepared by adding a requisite amount of a detergent (surfactant) composition to water.
The wash medium generally contains a cleaning effective amount of the detergent.

The wash medium is defined as an "acidic wash medium~ if the pH of the medium is from about 4 to less~than about 7. The wash medium is defined as a "neutral wash medium" if the pH of the medium is about 7. The wash medium is defined as an "alkaline ;
wash medium~ if the pH of the medium is from above 7 to about lO. Preferably, the alkaline wash medium will have a pH of from above 7 to about 9 and, even more preferably, from above 7 to about 8.

.
The present invention is directed to the discovery that acidic EG type components can be used in detergent compositions to effect softening of cotton-containing fabrics regardless of whether such ~ 30 compositions are employed in an acidic, neutral or ; ~ alkaline wash medium. In particular, it has been .

' .

W O 92/06210 P~r/US91/~7273 - 27 - ~93~2~ -found that when so employed, the detergent - co.. p~s.ti~n will also ef,ect color retention/restoration and will improve the feel of the treated fabric.

Although the presence of EG type components are necessary to effect color retention/restoration, softening and improved feel, specified mixtures of EG type components and CBH type components (including CBH I type components) also show the same benefits or even some incremental benefits. In particular, when mixtures of such components are employed, the mixture contains one or more acidic EG
type components and one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1 and preferably a ratio of all acidic EG
type components to all CBH components of greater than 5:1.
, .~
Additionally, while the use of EG components will result in some cleaning benefit, an incremental cleaning benefit is observed for cotton-containing fabrics washed with a detergent composition containing a cellulase composition enriched in .
acidic EG type components and which contains at least about 1 weight percent, and preferably, at least about 5 weight percent, and even more preferably, at least about 10 weight percent, of CBH
I type cellulase components (based on the total protein weight of all of the acidic EG components). - .
on the other hand, in addition to imparting color retention/restoration, softness, and improved feel, detergent compositions containing a cellulase -.
- .

;. ' .' . " . ' ' .. . " ' ' . ' .' , ',' . .' . '' .. . . .. .

WO92~062l0 pcT/us9l/n7273 ~at~3 ~2~ - 28 -composition enriched in acidic EG type components and which is free of CBH I type component~ (and preferably all CBH type components) also result in significantly reduced strength loss of the cotton-containing fabric as compared to cellulasecompositions enriched in acidic EG type components but which contain CBH I type components. That is to say that repeated washings with detergent compositions containing cellulase compositions lo enriched in acidic EG type components and free of CBH I type components will result in significantly -~
less strength loss of cotton-containing fabrics as compared to detergent compositions containing a cellulase composition comprising both acidic EG type components and CBH type components.

Accordingly, in selecting the specific cellulase composition for use in the detergent composition, the skilled artisan would need to balance the desire for an incremental cleaning benefit against that for strength loss resistance.
That is to say that if the primary emphasis for incorporating cellulase into the detergent composition is for improved softness, color -- retention/restoration, and improved feel of cotton-containing fabrics without significant strength loss in the cotton-containing fabric, then the cellulase employed in the detergent composition should preferably comprise one or more acidic EG
type components free of all CBH I type components.
on the other hand, if the primary emphasis for incorporating cellulase into the detergent composition is for color retention/restoration, ~ improved softness and improved feel of cotton-: containing fabrics, in conjunction with an :

W092/062tO PCT/US91/07273 ~ 2~3~2~ -incremental cleaning benefit, then the cellulase employed in .he detergent composition should contain a cellulase comprising one or more acidic EG type components as well as at least about 1 weight percent of a CBH I type component (based on the total protein weight of all of the acidic EG
components).

In regard to the above, the amount of cellulase generally employed in the detergent compositions of this invention is an amount sufficient to impart color retention/restoration and softness to the cotton garments. Preferably, the cellulase compositions are employed from about 0.01 weight percent to about 5 weight percent relative to the weight of the total detergent composition. More -preferably, the cellulase compositions are employed from about 0.05 weight percent to abGut 2 weight percent relative to the weight of the total detergent composition. The specific concentration of cellulase employed in the detergent composition -is selected so that upon dilution into a wash medium, the concentration of acidic EG type components will range from about 0.5 to about 500 ppm, and preferably from about 2 to about 100 ppm.
The specific amount of cellulase employed in the detergent composition will depend on the amount of EG type components in the cellulase compositions as well as the extent the detergent composition will be diluted upon addition to water to form a wash medium. These factors are readily ascertained by the skilled artisan.
:.

Preferably, the cellulase compositions employed in the detergent compositions of this -Zog 3 42~ _ 30 _ invention contain at least about 20 weight percent of endoglucanase type components based on the total weight of protein.

At lower cellulase concentrations (i.e., concentrations of acidic EG type components of less ~ -than about 5 ppm in the wash medium), softness, color retention/restoration and improved feel achieved by use of the detergent compositions of this invention is more evident over repeated washings. At higher concentrations (i.e., -concentrations of acidic EG type components of about 5 ppm and above in the wash medium), the improvements can become noticeable in a single wash. ;

One of the important aspects of the present invention is that by tailoring the cellulase composition to contain enriched amounts of acidic EG
components, it is possible to achieve the desired effects of softening, color retention/restoration and~improved~feel with the use of lower 20~ concentrations of cellulase in the detergent co~position. In turn, with consu ers who are allergic to proteins, the use of lower -¢oncentrations of cellulase in the detergent compositions should lead to lower allergenic levels `25 ~than compositions containing higher concentrations of cellulase - The cellulase composition as described above can be added to the detergent composition either in a~liquid diluent, in granules, in emulsions, in 30~ gels,~ in pastes, and the like. Such forms are well known to the skilled artisan. When a solid ~: ::: ::: :

W O 92/06210 PC~r/US91/07273 ~*~
- 31 - 2 ag3 ~w ~ ` -detergent composition is employed, the cellulase - composition is preferably formulated as granules.
Preferably, the granules can be formulated so as to contain a cellulase protecting agent. See, for instance, U.S. Serial No. 07/642,669 filed January 17, 1991 as Attorney Docket No. 010055-073 and entitled "GRANULES CONTAINING ~OTH AN ENZYME AND AN
ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS
CONTAINING SUCH GRANULES" which application is incorporated herein by reference in its entirety.
Likewise, the granule can be formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium. Such materials and granules are disclosed in U.S. Serial No.
07/642,596 filed on January 17, 1991 as Attorney Docket No. GCS-171-US1 and entitlèd "GRANULAR
COMPOSITIONS" which application is incorporated herein by reference in its entirety. ~ -The detergent compositions of this invention employ a surface active agent, i.e., surfactant, including anionic, non-ionic and ampholytic surfactants well known for their use in detergent ~ -compositions.

Suitable anionic surfactants for use in the detergent composition of this invention include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates and the like. Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and -potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines ... .
'~ .

,:
:, ' .

.. . .

WO92/06210 PCT/US91/072~3 @~
2~9~ ~2~ _ 32--having 1 to 3 alkanol groups of carbon number 2 or ~.
' ' 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.

Suitable surfactants for use in this invention are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.
:
Mixtures of such surfactants can also be used.

~ he surfactant or a mixture of surfactants is generalIy employed in the detergent compositions of this invention in an amount from about 1 weight ` percent to about 95 weight percent of the total ~detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition. Upon dilution in the wash medium, the surfactant concentration is generally about 500 ppm or more; and preferably, from about 1000 ppm to 15,000 ppm.

In addition to the cellulase composition and the surfactant(s), the detergent compositions of - --:: . :

. .. ,: ., .: i . . .. -:, . . .. ., - , . . . : .. . ~ .,,,, :; .. .. , ,. ,. .. - . , :, , - , .. .

_ 33 _ ~ ~ 9:3 ~

this invention can optionally contain one or more of the follow.ng components:

Hydrolases except cellulase - . .
Such hydrolases include carboxylate ester 5 hydrolase, thioester hydrolase, phosphate monoester -hydrolase, and phosphate diester hydrolase which act cn the ester bond; glycoside hydrolase which acts on glycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds; thioether hydrolase which acts on the ether bond; and ~-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptide hydrolase which act on the peptide bond.
Preferable among them are carboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptide hydrolase. Suitable hydrolases include (1) proteases belonging to petidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, e}astase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, c}ostridiopeptidase B, kallikrein, gastrisin, cathepsin D., bromelin, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidase A and B, and aminopeptidase; ~2) glycoside hydrolases (cellulase which is an ' essential ingredient is excluded from this group) ~-amylase, B-amylase, gluco amylase, invertase, lysozyme, pectinase, chitinase, and dextranase. ~
Preferab}y among them are ~-amylase and B-amylase. -They function in acid to neutral systems, but one vhich is obtained from bacteria exhibits high .
:~ -- , :

2~9342~ 34 _ ~

activity in an alkaline system; (3) carboxylate ester hydrolase including carbo~yl esterase, lipase, pectin esterase, and chlorophyllase. Especially effective among them is lipase.

Trade names of commercial products and producers are as follows: "Alkalase", "Esperase", "Savinase", "AMG", "BAN", "Fungamill", "Sweetzyme", "Thermamyl" (Novo Industry, Copenhagen, Denmark);
"Maksatase", "High-alkaline protease", "Amylase THC", "Lipase" ~Gist Brocades, N.V., Delft, - -Holland); "Protease B-400", "Protease B-4000", "Protease AP", "Protease AP 2100" (Schweizerische Ferment A.G., Basel, Switzerland); "CRD Protease"
(Monsanto Company, St. Louis, Missouri); "Piocase"
(Piopin Corporation, Monticello, Illinois); "Pronase pn , "Pronase AS", "Pronase AF" (Kaken Chemical Co., Ltd., Japan); "Lapidase P-2000" (Lapidas, Secran, France); protease products (Tyler standard sieve, 100% pass 16 mesh and 100% on l50 mesh) (Clington ; 20 ~Corn Products, Division of Standard Brands Corp., New York); "Takamine", "Bromelain 1: ld" , "HT
Protease 200", "Enzyme L-W" (obtained from fungi, ; not from bacteria) (Miles Chemical Company, Elkhart, Ind~.); "Rhozyme P-ll Conc.", "Pectinol", "Lipase B", ~25 nRhozyme PFn, NRhozyme J-25" (Rohm & Haas, Genencor, South San Francisco, CA); "Ambrozyme 200" (Jack Wolf & Co., Ltd., Subsidiary of Nopco Chemical Company, Newark, N.J.); "ATP 40", "ATP l20", "ATP 160"
(Lapidas, Secran, France); "Oripase" (Nagase h Co., Ltd., Japan).

The hydrolase other than cellulase is incorporated into the detergent composition as much as reguired according to the purpose. It should -- .
, W O 92~06210 P~r/US91/~7273 '.~ `:i - 35 - 2393~2~ ~

preferably be incorporated in an amount of 0.001 to 5 weight percent, and more preferably 0.02 to 3 - ;
weight percent, in terms of purified one. This enzyme should be used in the form of granules made of crude enzyme alone or in combination with other ~
components in the detergent composition. Granules ~--of crude enzyme are used in such an amount that the purified enzyme is 0.001 to 50 weight percent in the granules. The granules are used in an amount of 0.002 to 20 and preferably 0.1 to 10 weight percent. -As with cellulases, these granules can be formulated so as to contain an enzyme protecting agent and a dissolution retardant material.

Cationic surfactants and lona-chain fattv acid salts Such cationic surfactants and long-chain fatty acid salts include saturated or unsaturated fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, ~-sulfofatty acid salts or esters,amino ~-acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substituents and up to 1 phenyl substituted alkyl substituents. Suitable cationic surfactants and long-chain fatty acid salts are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. The composition may contain from about 1 to about 20 weight percent of such cationic surfactants and long-chain fatty acid salts.
.
Builders A. Divalent sequestering agents.

. '..' '' .
- , W O 92~06210 PC~r/US91/~7273 2~9342~ - 36 -The composition may contain from about 0 to about 50 waigh~ per-ent of one or more builder components selected from the group consisting of alkali metal salts and alkanolamine salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, aminopolyacetates high molecular electrolytes, non-dissociating polymers, salts of dicarboxylic acids, and aluminosilicate salts. Suitable divalent sequestering gents are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

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

Antiredeposition agents The composition may contain from about 0.1 to about 5 weight percent of one or more of the following compounds as antiredeposition agents:
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose.

Among them, a combination of carboxymethyl-cellulose or/and polyethylene glycol with thecellulase composition of the present invention ~ .
~ .

WO92/06210 PCT/US91/0~273 ~' :
~ ~ ~ 3 ~ v provides for an especially useful dirt removing -cor..pcsition.

Bleachinq aaents The use of the cellulase of the present ~ --invention in combination with a bleaching agent such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct or/and a photo-sensitive bleaching dye such as zinc or aluminum salt of sulfonated phthalocyanine further improves the deterging effects. --Bluinq aqents and fluorescent dYes Various bluing agents and fluorescent dyes ~
may be incorporated in the composition, if -necessary. Suitable bluing agents and fluorescent ;
dyes are disclosed in British Patent Application No.
2 094 826 A, the disclosure of which is incorporated herein by reference.

Cakina inhibitors The following caking inhibitors may be incorporated in the powdery detergent:
p-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium silicate (such as Micro-Cell of Johns Manville Co.), calcium carbonate and magnesium oxide.
, Maskinq aaents for factors inhibitinq the cellulase -activitv ' . .

:.

, .. ,, _.. ~ . . . . ... .. . .. .. ..
r WO92/06210 PCT/US~ 7273 2~3425 - 38 - ~

The cellulase composition of this invention are deactivated in some cases in the presence of copper, zinc, chromium, mercury, lead, manganese or silver ions or their compounds. Various metal chelating agents and metal-precipitating agents are effective against these inhibitors. They include, for example, divalent metal ion sequestering agents as listed in the above item with reference to optional additives as well as magnesium silicate and magnesium sulfate.

Cellobiose, glucose and gluconolactone act sometimes as the inhibitors. It is preferred to avoid the co-presence of these saccharides with the cellulase as far as possible. In case the co-presence in unavoidable, it is necessary to avoidthe direct contact of the saccharides with the cellulase by, for example, coating them. -Long-chain-fatty acid salts and cationic surfactants act as the inhibitors in some cases.
However, the co-presence of these substances with the cellulase is allowable if the direct contact of them is prevented by some means such as tableting or coating.

The above-mentioned masking agents and methods may be employed, if necessary, in the present invention.

Cellulase-activators ~

The activators vary depending on variety of -the cellulases. In the presence of proteins, cobalt -and its salts, magnesium and its salts, and calcium :

. . , . ,~ . ~ . - : . " .. .. : . . , . .... . , , . . .. -. . - .. , .. ,,, . , .. , , , : ,, . .: . . .

wos2/o62lo PCT/US9t/07273 2~3~23 and its salts, potassium and its salts, sodium and its salts or monosaccharid~s such as mannose and xylose, the cellulases are activated and their deterging powers are improved remarkably.
':
Antioxidants -~ -The antioxidants include, for example, tert- ~
butyl-hydroxytoluene, 4,4'-butylidenebis(6-tert-butyl-3-methylphenol), 2,2'-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol, distyrenated cresol, monostyrenated phenol, distyrenated phenol and 1,1-bis(4-hydroxy-phenyl)cyclohexane.

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

The detergent composition of the present invention can be used in a broad pH range of from , acidic to alkaline pH. In a preferred embodiment, the detergent composition of the present invention can be used in alkaline detergent wash media and more preferably, alkaline detergent wash media ;~ having a pH of from above 7 to no more than about 8.

Aside from the above ingredients, perfumes, buffers, preservatlves, dyes and the like can be .
- ;~
~~~~~~ ~~~~ '~~ ~ - ~~r . ; __, __,_ ._~_ , "~ ,,_, W O 92J06210 PC~r/US91/07273 ~3~ 40 -used, if desired, with the detergent compositions of.his inven.ion. Such components are conventionally employed in amounts heretofore used in the art.

When a detergent base used in the present invention is in the form of a powder, it may be one which is prepared by any known preparation methods including a spray-drying method and a granulation method. The detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred. The detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions.
The detergent base obtained by the spray-drying method is hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space. The granules have a size of from 50 to 2000 micrometers. After the spray-drying, perfumes, enzymes, bleaching agents, inorganic alkaline builders may be added. With a highly dense, granular detergent base obtained such as by the spray-drying-granulation method, various ingredients may also be added after the preparation of the base.
.~ .
When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion. For removing the decomposition of carboxymethylcellulose by the cellulase in the detergent, it is desirable that carboxymethyl-cellulose is granulated or coated before the incorporation in the composition.

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

' -, ~ 41 - 2~3~2~

The detergent compositions of this invention are used in industrial and household uses at temperatures and liquor ratios conventionally -~
employed in these environments.

In addition to their use in laundry detergents, the cellulase compositions described herein can additionally be used in a pre-washing step in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements in color retention/restoration, --softening and feel. The cellulase could also be employed as a pre-soak either as a liquid, spray, gel or paste for uses, such as, as a spot remover.
When so used, the cellulase composition described herein is generally employed from about 0.002 to :
about 0.5 weight percent based on the total weight of the pre-soak composition. In such compositions, a surfactant may optionally be employed and when employed, is generally present at a concentration of from about O.Ol to about 20 weight percent based on the total weight of the pre-soak. The remainder of the composition comprises conventional components used in the pre-soak, i.e., diluent, buffers, other enzymes (proteases), and the like at their conventional concentrations.

Also, it is contemplated that the cellulase compositions described herein can also be used in home use as a stand alone composition suitable for restoring color to faded fabrics. See, for example, U.S. Patent No. 4,738,682, which is incorporated herein by reference in its entirety.
.

- ' ' .

. .

- ::: ~ ; :: . . - . - : ~. : . . . : . - ,:
.. -........ . : . . . . . . .. .

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

S B~A~P~E8 Examples 1-12 and 20-28 demonstrate the preparation of Trichoderma reesei genetically engineered so as to be incapable of producing one or more cellulase components or so as to overproduce -specific cellulase components.

Exam~le 1 Selection for pyr4 derivatives of Trichoderma reesei The pvr4 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 EyE_ gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for ~ ~ EYL~ derivative strains using FOA. In practice, - spores of T. reesei strain RL-P37 (Sheir-Neiss, G.
and Montenecourt, B.S., ADD1. Microbiol. Biotechnol.
20, p. 46-53 (1984)) were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resistant colonies ~; appeared within three to four days and it was possible to subsequently identify those FOA-resistant derivatives which required uridine for growth. In order to identify those derivatives whicb specifically had a defective EYE~ gene, protoplasts were generated and transformed~ith a plasmid containing a wild-type EY~ gene (see Examples 3 and 4). Following transformation, protoplasts were plated on medium lacking uridine.
Subsequent growth of transformed colonies demonstrated complementation of a defective ~vr4 gene by the plasmid-borne EYE~ gene. In this way, strain GC69 was identified as a ~vr4- derivative of strain RL-P37.
. ..
Exam~le 2 Preparation of C~HI Deletion Vector A cbhl gene encoding the CBHI protein was cloned from the genomic DNA of T. reesei strain RL-P37 by hybridization with an oligonucleotide probe :
lS designed on the basis of the published sequence forthis gene using known probe synthesis methods ~- (Shoemaker et al., 1983b). ~he 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 Kanl gene 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, pUC4K::cbhl was then cut with HindIII and ;~ 25 the larger fragment of about 6 kb was isolated and religated to give pUC4K::cbhl~H/H (see FIG. 1).
This procedure removes the entire cbhl coding sequence and approximately 1.2 kb upstream and 1.5 kb downstream of flanking sequences. Approximately, - -1 kb of flanking DNA from either end of the original PstI fragment remains.

~ . - - ' ~ ~ :

~,~93~
The T. reesei vr4 gene was cloned as a 6.5 kb HindIII fragment of genomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991) following the methods of Maniatis et al., supra. The plasmid pUC4K::cbhI~H/H
was cut with HindIII and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was ligated with the 6.5 kb HindIII fragment containing the T. Eeesei ~vr4 gene to give p~CBHI~yE~. FIG. 1 illustrates the construction of this plasmid.
~' Example 3 Isolation of ProtoDlasts - Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2~ glucose) in a 500 ml flask with about 5 x 107 T. reesei GC69 spores (the EY3~L derivative strain). The flask was then incubated at 37C with shaking for about 16 hours.
The mycelium was harvested by centrifugation at -2,750 x g. The harvested mycelium was further washed in a 1.2 M sorbitol solution and resuspended ~-in 40 ml of a solution containing 5 mg/ml 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 Biolabs, Danbury, CT); 5 mg/ml MgSO4.7H2O; 0.5 mg/ml bovine 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. The protoplasts were washed three times in 1.2 M
sorbitol and once in 1.2 M sorbitol, 50 mM CaCl2, centrifuged and resuspended at a density of .

':

W O 92/06210 PC~r/US91/07273 - 45 - ~ ~9~

approximately 2 x lo8 protoplasts per ml of 1 2 M
sorbitol, 50 mM CdC12- -Exam~le 4 Transformation of Funaal Protoplasts with p~CBHI~Yr4 ;
200 ~1 of the protoplast suspension prepared in Example 3 was added to 20 ~1 of EcoRI digested p~CBHI~yE~ (prepared in Example 2) in TE buffer (10 -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 PEG
solution was added thereto, the solution was further mixed and incubated at room temperature for 5 minutes. After this second incubation, 4.0 ml of a solution containing 1.2 M sorbitol and 50 mM CaCl was added thereto and this solution was further -mixed. The protoplast solution was then immediately ` added to molten aliquots of Vogel's Medium N (3 grams sodium citrate, 5 grams ~H2P04, 2 grams NH4N03, 0.2 grams MgS0~.7H20, 0.1 gram CaCl2.2H20, 5 ~g ~--~ biotin, 5 mg citric acid, 5 mg ZnS04.7H20, 1 mg Fe(NH~)2.6H20, 0.25 mg CuS04.5H20, 50 ~g MnS04.4H20 per ~ liter) containing an additional 1% glucose, 1.2 M
- 25 sorbitol and 1% agarose. The protoplast/medium ; mixture was then poured onto a solid medium containing the same Vogel's medium as stated above.
~` No uridine was present in the medium and therefore onIy~transformed colonies were able to grow as a result of complementation of the EYE~ mutation of strain GC69 by the wild type pyr4 gene insert in p~CBEIEyE~. These colonies were subsequently transferred and purified on a solid Vogel's medium N

:: ~ :.

W092/06210 PCT/US91/~7273 ~ 3~2~ - 46 -containing as an additive, 1% glucose and stable transformants were chosen for further analysis.

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

Exam~le s ', ' ' Anal~sis of the Transformants DNA was isolated from the transformants obtained in Example 4 after they were grown in liquid Vogel's medium N containing 1% glucose. , , These transformant DNA samples were further cut with a PstI restriction enzyme and subjected to agarose gel electrophoresis. The gel was then blotted onto a Nytran membrane filter and hybridized with a 32p labelled p~CBHIEyE~ probe. The probe was selected to ,identify the native cb~1 gene as a 6.5 kb PstI
fragment, the native Eyr~ gene and any DNA seguences derived from the transforming DNA fragment.

The radioactive bands from the hybridization were visua}ized by autoradiography. The ' ~' ' autoradiograph is seen in FIG. 3. Five samples were ~-run~as described above, hence samples A, B, C, D, ~, and E. Lane E is the untransformed strain GC69 and ' - ~ .
~ ,, ~ . - . .

WO92/06210 PCT/US9t/07273 :
- 47 - 2~93~2~

; .
was used as a control in the present analysis.
Tanes A-D represent transformants obtained by the methods described above. The numbers on the side of the autoradiograph represent the sizes of molecular weight markers. As can be seen from this autoradiograph, lane D does not contain the 6.5 kb CBHI band, indicating that this gene has been totally deleted in the transformant by integration of the DNA fragment at the cbhl gene. The cbhl deleted strain is called P37P~CBHI. Figure 2 outlines the deletion of the T. reesei cbhl gene by integration through a double cross-over event of the -larger EcoRI fragment from p~CBHIEy~ at the cbhl locus on one of the T. reesei chromosomes. The lS other transformants analyzed appear identical to the untransformed control stra~n.
, ExamDle 6 AnalYsis of the Transformants with ~IntCBHI
:
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 pUC-type plasmid containing a 2 kb BalII
fragment from the cbhl locus within the region that was de}eted in pUC4K::cbhl~H/H. Two samples were run in this example including a control, sample A, which is the untransformed strain GC69 and the transformant P37P~CBHI, sample B. As can be seen in F}G. 4, sample A contained the cbhl gene, as indicated by the band at 6.5 kb; however the -transformant, sample B, does not contain this 6.5 kb band and therefore does not contain the cbhl gene and does not contain any sequences derived from the pUC plasmid.

W O 92/062tO PC~r/US91/07273 4~ 48- ~ ~

Example 7 Protein Secretion by Strain P37PACBHI
- Spores from the produced P37P~CBHI strain were inoculated into 50 ml of a Trichoderma basal medium containing 1% glucose, 0.1496 (NH4)2SO4, 0.2%
KH2PO4, 0.03% MgS0~, 0.03% urea, 0.75% bactotryptone, 0.05~ Tween 80, 0.000016% CuSO4.5H20, 0.001% --FeS04.7H2O, 0.000128% ZnSO4.7H2O, 0.0000054% -Na2MoO4.2H2O, 0.0000007% MnCl.4H20). The medium was incubated with shaking in a 250 ml flaslc at 37C for about 48 hours. The resulting mycelium was collected by filtering through Miracloth (Calbiochem Corp.) and washed two or three times with 17 mM
potassium phosphate. The mycelium was finally suspended in 17 mM potassium phosphate with 1 mM
sophorose and further incubated for 24 hours at 30C --with shaking. The supernatant was then collected from these cultures and the mycelium was discarded.
Samples of the culture supernatant were analyzed by isoelectric focusing using a Pharmacia Phastgel system and pH 3-9 precast gels according to the manufacturer's instructions. The gel was stained with silver stain to visualize the protein bands.
The band corresponding to the cbhl protein was absent from the sample derived from the strain P37PACBHI, as shown in FIG. 5. This isoelectric focusing gel shows various proteins in different supernatant cultures of T. reesei. Lane A is partially purified CBHI; Lane B is the supernatant from an untransformed T. reesei culture; Lane C is the supernatant from strain P37P~CBHI produced according to the methods of the present invention; ~ --. ., .. - . . ., . ~ - -. ... ., ~ . . - - .. -, . . - .. , . . : . .. ,- . . -W O 92/06210 P(~r/US9t/07273 ~ 2 ~ v The position of various cellulase components are labelled CBHI, CBHII, EGI, EGII, and EGIII. Since CBHI constitutes 50% of the total extracellular protein, it is the major secreted protein and hence is the darkest band on the gel. This isoelectric focusing gel clearly shows depletion of the CBHI
protein in the P37P~CBHI strain.

Exam~le 8 Pre~aration of DP~CBHII
The cbh2 gene of T. reesei, encoding the ;
CBHII protein, has been cloned as a 4.1 kb EcoRI
fragment of genomic DNA which is shown diagramatically in FIG. 6A (Chen et al., 1987, Biotechnoloay, 5:274-278). This 4.1 kb fragment was inserted between the EcoRI sites of pUC4XL. The latter plasmid is a pUC derivative (constructed by R.M. Berka, Genencor International Inc.) which contains a multiple cloning site with a symetrical pattern of restriction endonuclease sites arranged -in the order shown here: EcoRI, BamHI, SacI, SmaI, - -HindIII, XhoI, BalII, ClaI, BalII, XhoI, ~_dIII, SmaI, SacI, BamHI, EcoRI. Using methods known in the art, a plasmid, pP~CBHII (FIG. 6B), has been constructed in which a 1.7 kb central region of this gene between a HindIII site (at 74 bp 3' of the CBHII translation initiation site) and a ClaI site (at 265 bp 3' of the last codon of CBHII) has been removed and replaced by a 1.6 kb HindIII~ I DNA
fragment containing the T. reesei ~vr4 gene.
, -.
The T. reesei pYr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb NheI-S~hI
fragment and inserted between the ~E_I and XbaI

. . . .. . ... . ... . .. .

W092/062l0 PCT/US91/07273 2~9 3 42~ _ 50 _ ~

sites of pUC219 (see Example 23) to create p219M -(Smith et al., 1991, Curr. Genet 19 p. 27-33). The pyr4 gene was then removed as a HindIII-ClaI
fragment having seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the HindIII
and ClaI sites of the cbh2 gene to form the plasmid ~-pP~CBHII (see FIG. 6B).
~:
Digestion of this plasmid with EcoRI will liberate a fragment having 0.7 kb of flanking DNA ;
from the cbh2 locus at one end, 1.7 kb of flanking DNA from the cbh2 locus at the other end and the T.
reesei EY~ gene in the middle. ~ ~ -Example 9 Deletion of the cbh2 gene in T. reesei strain GC69 Protoplasts of strain GC69 will be generated and transformed with EcoRI digested pPACBHII
according to the methods outlined in Examples 3 and 4. DNA from the transformants will be digested with EcoRI and Asp718, and subjected to agarose gel electrophoresis. The DNA from the gel will be blotted to a membrane filter and hybridized with 32p labelled pPACBHII according to the methods in Example 11. Transformants will be identified which have a single copy of the EcoRI fragment from pP CBHII integrated precisely at the cbh2 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 T. reesei GC69 transformants which do not produce the CBHII protein -will be generated. --- ...

:

- 51 - 2~93~23 Example lo Generation of a Dyr4- Derivative of P37P~CBHI
Spores of the transformant (P37P~CBHI) which was deleted for the cbhl gene were spread onto medium containing FOA. A Dvr4- derivative of this transformant was subsequently obtained using the ~ -methods of Example 1. This Dvr4- strain was designated P37PACBHIPyr26.

ExamDle 11 Deletion of the cbh2 qene in a stra~n DreviouslY deleted for cbhl Protoplasts of strain P37P~CBHIPyr-26 were generated and transformed with EcoRI digested pP~CBHII according to the methods outlined in Examples 3 and 4.

Purified stable transformants were cultured in shaker flasks as in Example 7 and the protein in the culture supernatants was examined by isoelectric focusing. One transformant (designated P37P~CBH67) 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 cbh2 genes produced according to the methods of the - present invention.
.
DNA Was extracted from strain P37P~CBH67, digested with EcoRI and ASD718, and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with labelled pP~CBHII (FIG. 7). Lane A of FIG. 7 ', ~ .

W O 92/06210 PC~r/US91/~7273 `~933~2~ - 52 -shows the hybridization pattern observed for DNA
from an un.ransformed T. reesei strain. The 4.1 kb EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization ;
5 pattern observed for strain P37P~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 pPt~CBHII had integrated precisely at lO the cbh2 locus.

The same DNA samples were also digested with EcoRI and Southern blot analysis was performed as ~--àbove. In this Example, the probe was 32p labelled pIntCBHII. This plasmid contains a portion of the cbh2 gene coding sequence from within that segment ~-of the cbh2 gene which was deleted in plasmid pP~CBHII. No hybridization was seen with DNA from strain P37P/~/~CBH67 showing that the cbh2 gene was -;
deleted and that no sequences derived from the pUC
20 plasmid were present in this strain.

Example 12 .

Construction of pEGIpyr4 -~;
` The . reesei eall gene, which encodes EGI, has been cloned as a 4.2 kb E~adIII fragment of 25 genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthesized according to the ; ; published sequence (Penttila et al., 1986, Gene 45:253-263; van Arsdell et al., 1987, Bio/Technoloay 5:60-64). A 3.6 kb ~dIII-BamHI fraament was taken 30 from this clone and ligated with a 1.6 kb HindIII-~II fragment containing the T. reesei }2Y~ gene ~ -obtained from pTpyr2 (see Example 2) and pUC218 ' ': .

W092/06210 PCT/US9t/07273 _ 53 _ 2~93~

(identical to pUC219, see Example 23, but with the multiple cloning site in the opposite orientatlon) cut with HindIII to give the plasmid pEGI~y~ (FIG.
8). Digestion of pEGIEyE~ with HindIII would liberate a fragment of DNA containing only T. reesei genomic DNA (the eall and EYE~ 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).

Exam~le 13 Purification of CYtolase 123 Cellulase into Cellulase ComDonents CYTOLASE 123 cellulase was fractionated in the following manner. The normal distribution of 15 cellulase components in this cellulase system is as follows:
CBH I45-55 weight percent -CBH II13-15 weight percent EG I11-13 weight percent EG II8-10 weight percent EG III1-4 weight percent BG0.5-1 weight percent.

The fractionation was done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St.
~ Louis, MO), QA Trisacryl M anion exchange resin and ; SP Trisacryl M cation exchange resin from IBF
Biotechnics (Savage, MD). CYTOLASE 123 cellulase, 0.5g, was desalted using a column of 3 liters of ~;30 Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, was then loaded onto a column of 20 ml of QA
Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I.
These components were separated by gradient elution 2~93~ 54 _ ~:
using an aqueous gradient containing from 0 to about 500 mM sodium chlcr.de. ~he fraction not bound on this column contained CBH II and EG II. These fractions were desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM
sodium citrate, pH 3.3. This solution, 200 ml, was then loaded onto a column of 20 ml of SP Trisacryl M
cation exchange resin. CBH II and EG II were eluted separately using an aqueous gradient containing from 0 to about 200 mM sodium chloride.

Following procedures similar to that of -~
Example 13 above, other cellulase systems which can be separated into their components include CELLUCAST
(available from Novo Industry, Copenhagen, Denmark), -RAPIDASE (available from Gist Brocades, N.V., Delft, Holland), and cellulase systems derived from Trichode_ma koningii, Penicillum s~. and the like. ;

Exam~le 14 Purification of ~G 1~1 f~
2U Cvtolase 123 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 25 procedures were employed to isolate this component.
A. Large Scale Extraction of EG III
Cellulasç En~yme one hundred liters of cell free cellulase filtrate were heated to about 30C. The heated 30 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 ~ .
~ ~:
~ ' ' W O 92/06210 P ~ /US9t/07273 ~ 2 ~ ~ 3 ~ 2 ~

separated using an SA-1 disk stack centrifuge. The phases were ar.alyzed using silver staining isoelectric focusing gels. Separation was obtained for EG III and xylanase. The recovered composition contained about 20 to 50 weight percent of EG III.

Regarding the above procedure, use of a polyethylene glycol having a molecular weight of less than about 8000 gave inadequate separation;
whereas, use of polyethylene glycol having a molecular weight of greater than about 8000 resulted in the exclusion of desired enzymes in the recovered composition. With regard to the amount of sodium sulfate, sodium sulfate levels greater than about 10% wt/vol caused precipitation problems; whereas, sodium sulfate levels less than about 10%wt/vol gave poor separation or the solution remained in a single phase.
:
B. Purification of EG III Via Fractionation The purification of EG III is conducted by fractionation from a complete fungal cellulase composition (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) which is produced by wild type Trichoderma reesei. Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Mo), QA Trisacryl M anion exchange resin and SP Trisacryl M cation -exchange resin from IBF Biotechnics (Savage, Md).
~ 30 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 -,~

WOg2/06210 PCT/US91/~7273 2^~
of 20 ml of QA Trisacryl M anion exchange resin.
The fraction bound on this column contained CBX I
and EG I. The fraction not bound on this column contains CBH II, EG II and EG III. These fractions are desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto a column of 20 ml of SP Trisacryl M -cation exchange resin. The EG III was eluted with 100 mL of an aqueous solution of 200 mM sodium chloride. `~

In order to enhance the efficiency of the isolation of EG III, it may be desirable to employ ~ric~oderma reesei genetically modified so as to be -incapable of producing one or more of EG I, EG II, CBH I and/or CBH II. The absence of one or more of such components will necessarily lead to more efficient isolation of EG III.

Likewise, it may be desirable for the EG III
compositions described above to be further purified to provide for substantially pure EG III
compositions, i.e., compositions 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 from procedure A in procedure B or vica versa. One particular method for further purifying EG III is by further fractionation of an EG III
sample obtained in part b) of this Example 14. The further fraction was done on a FPLC 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 ~ . .' ~' ' -W O 92t06210 PC~r/US91/07273 pumps, a dual path monitor, a fraction collector and a chart recorder ~all cf which ar2 ~vailable from Pharmacia LXB Biotechnology, Piscataway, NJ). The fractionation was conducted by desalting 5 ml of the EG III sample prepared in part b) of this Example 14 with a 20 ml Sephadex G-25 column which had been previously equilibrated with 10 mM sodium citrate pH
4. The column was then eluted with 0-200 mM aqueous gradient of NaCl at a rate of 0.5 ml/minute with samples collected in 1 ml fractions. EG III was recovered in fractions 10 and 11 and was determined to be greater than 90% pure by SDS gel electrophoresis. EG III of this purity is suitable for determining the N-terminal amino acid sequence by known techniques.

Substantially pure EG III as well as EG 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 DH o~timum~
EG I -47-49 kD 4.7 -5 ~ EG II -35 kD 5.5 -5 EG III-25-28 kD 7.4 -5.5-6.0 1. pH optimum determined by RBB-CMC activity as per Example 15 below.
Accordingly, each of EG I, EG II and EG III
are acidic EG components.

The use of a mixture of these components in the practice of this invention may give a .
' - .

WO92/06210 PCTtUS91/07273 c.
~9'33~5 - 58 -synergistic response in improving softening, color ratantion/res~oration and/or feel as compared to ~
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 range of pHs. For instance, Example 15 below shows that EG III has considerable activity against RBB-CMC under alkaline conditions. , .
Example 15 ActivitY of Cellulase Compositions --Over a pH Range The following procedure was employed to determine the pH profiles of two different cellulase compositions. The first cellulase composition was a CBH I and II deleted cellulase composition prepared j ~ from Trichoderma,reesei genetically modified in a ' manner similar to that described above so as to be unable to produce CBH I and CBH II components.
Insofar as this cellulase composition does not contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase '~
composition derived from Tric~,o,derma Feesç~, this cellulase composition is necessarily enriched in ',', acidic EG components, i.e., EG I, EG II, EG III and the like.

The second cellulase composition was an approximately 20-40% pure fraction of EG III
isolated from a cellulase composition derived from Trichoderma reesei via purification methods similar to part b) of Example 14.

W O 92/06210 PC~r/US91/07273 .
- 59 - ~93~2 The activity of these cellulase compositions was determined at 40C and the determinations were made using the following procedures.

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

Vortex and incubate at 40C for 30 minutes.
Chill in an ice bath for 5 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 into cuvets. Measure the optical density (OD) of ~ -the solution in each cuvet at 590 nm. Higher levels of optical density correspond to higher levels of enzyme activity.
.
The results of this analysis are set forth in FIG. 9 which illustrates the relative activity of the CBH I and II deleted cellulase composition compared to the EG III cellulase composition. From this figure, it is seen that the cellulase composition deleted in CBH I and CBH II possesses optimum cellulolytic activity against RBB-CMC at near pH 5.5 and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8. On the other hand, the cellulase composition enriched in EG III
posgesses optimum cellulolytlc activity at about pH

: WO~2/06210 PCT/US91/07273 ~ ......

~o33 42S _ 60 -5.5 - 6 and possesses significant activity at -alkaline pHs.

Exam~le 16 .

Launderometer Strenath Loss Assay .. 5 Cellulase Compositions -The purpose of this example is to examine the .~ ability of different cellulase compositions to :
s reduce the strength of cotton-containing fabrics.
Because the activity of the most of the cellulase ..
. 10 components derived from Trichoderma reesei is greatest at or near pH 5, strength loss results will be most evident when the assay is conducted at about ....
this pH. However, the results achieved at pH 5 are .. -believed to correlate with strength loss results ~:: 15 which would occur at other pHs and accordingly, are . indicative of the relative strength loss capacity of ~: .
¦ the cellulase components analyzed. .
,.
.
Specifically, in this example, the first ~ -cel~lulase composition analyzed was a complete fungal .
cellulase composition (CYTOLASE 123 cellulase, -.
commercially available from Genencor International, .:
South San Francisco, CA) produced by wild type . .
Tricboderma rçesei 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 it:~ similar to Examples 1-12 above and 20-28 below so as -~ to be~incapable of expressing CBH II and is ::` identified as CBHIld. Insofar as CBH II comprises ~ 30 up to about 15 percent of the cellulase composition, WO92/06210 PCT/US9t/07273 ~Q~3~

deletion of this component results in enriched levels of CBH I, and all of the EG components.

The third cellulase composition analyzed was a CBH I and CBH II deleted cellulase composition prepared from Trichoderma reesei genetically `~ modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II
and is identified as CBHI/IId. Insofar as CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG
components.
' ~.
The last cellulase composition analyzed was a 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 CBHId. Insofar as CBH I comprises up to about 55 - percent of the cellulase composition, deletion of this component results in enriched levels of CBH II -and all of the EG components.

The cellulase compositions described above were tested for their effect on cotton-containing fabric strength loss in a launderometer. The compositions were first normalized so that equal amounts of EG components were used. Each cellulase composition was then added to separate solutions of -400 ml of a 20 mM citrate/phosphate buffer, titrated to pH 5, and which contains 0.5 ml of a non-ionic surfactant. Each of the resulting solutions was then added to a separate launderometer canister.
Into these canisters were added a quantity of ; WO92/06210 PCT/US91/07273 ~ , .

~93~2~ ' ~
marbles to facilitate strength loss as well as a 16 inch x 20 inch cotton fabric (100~ woven co'ton, available as Style No. 467 from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846). The canister was then closed and the canister lowered into the launderometer bath which was maintained at 43C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
. . . .
In order to maximize strength loss results, the above procedure was repeated twice more and after the third treatment, the cotton fabrics were removed and analyzed for strength loss. Strength loss was measured by determining the tensile strength in the fill direction ("FTS") using a Instron Tester and the results compared to the FTS
of the fabric treated in the same solution with the exception that no cellulase was added. The results of this analysis are reported as % strength loss which is determined as follows: -Strength Loss = 100 x ~ - 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 C8H II deleted cellulase, possessed the most strength loss whereas, the compositions containing no CBH I possessed significantly reduced strength loss as compared to whole cellulase and CBH II deleted cellulase. From these results, it is seen that the presence of CBH I
in a cellulase composition imparts increased .~ .
.
. ~ .
; ~ - ~

. ~ .

W O 92/06210 PC~r/US9t/~7273 - 63 - 2~93~2 ':
strength loss to the composition as compared to a similar composition not containing CBH I.
: . , Likewise, these results show that CBH II
plays some role in strength loss.
-Accordingly, in view of these results, when strength loss resistant cellulase compositions are desired, such compositions should be free of all C8H I type cellulase components and preferably, all `- CBH type cellulase components. In this regard, it is contemplated that acidic EG enriched cellulase compositions will result in even lower strength loss at pH 2 7 than those results observed at pH 5 shown in FIG. 10.

Example 17 Color Restoration The ability of acidic EG enriched cellulase to restore color in cotton-containing fabrics was analyzed in the following experiments.
~i Specifically, color restoration of a worn cotton fabric arises from the accumulation on the fabric of surface fibers over a period of time. These fibers give rise to a faded and matted appearance for the fabric and accordingly, the removal of these fibers is a necessary prerequisite to restoring the original sharp color to the fabric. In view of the above, these experiments determine the ability of a cellulase composition to restore color by measuring the ability of the cellulase composition to remove surface fibers.
, ~ 30 The first experiment measures the ability of :

~, !~" ` ', ` ` .. ' .. ,.. . ~: . ,. ` . ',` i . ' ` . ' ' ' W O 92/06210 P(~r/US91/07273 2~93~2~
a complete cellulase system (CYTOLASE 123 cellulase, co~mercialiy available from Genencor International, South San ~rancisco, CA) produced by wild type Trichoderma reesei to remove surface fibers from a cotton-containing fabric over various pHs. This cellulase was tested for its ability to remove -surface fibers in a launderometer. An appropriate amount of cellulase to provide for either 25 ppm or 100 ppm cellulase in the final composition was added to separate solutions of 400 ml of a 20 mM
citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated so as to provide for samples at pH 5, pH 6, pH 7 and pH 7.5. Each of the resulting solution was -then added to a separate launderometer canister.
Into these canisters were added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100% woven cotton, available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846). The canister was then closed and the canister lowered into the launderometer bath which was maintained at 43C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

The 80 treated fabrics were then analyzed for fiber removal by evaluation in a panel test. In particular, the fabrics (unmarked) were rated for levels of fiber by 6 individuals. The fabrics were , visually evaluated for surface fibers and rated on a 0 to 6 scale. The scale has six standards to allow meaningful comparisons. The standards are as follows:

: -.

W O 92/06210 P ~ /US91/~7273 - 65 _ 2Q 93~ ~ ~

Ratina Standard2 0 Fabric not treated with cellulase 1 Fabric treated3 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 Fabric treated with 50 ppm cellulase 6 Fabric treated with 100 ppm cellulase ' 2 In all of the standards, the fabric was a 100% cotton sheeting standardized test fabric (Style No. 439W) available from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ -3 For all samples treated with the same cellulase composition. Cellulase concentrations are in total protein. The launderometer treatment conditions are the same as set forth in Example 16 above.
~j .
¦ The fabric to be rated was provided a rating ~which most closely matched one of the standards.
After complete analysis of the fabrics, the values assigned to each fabric by all of the individuals were added and an average value generated.
,, 1~ The results of this analysis are set forth in -FIG. lI. Specifically, FIG. 11 illustrates that at :
the same pH, a dose dependent response is seen in the amount of fibers removed. That is to say that at the same pH, the fabrics treated with more -~
cellulase provided for higher levels of fiber removal as compared to fabrics treated with less ; cellulase. Moreover, the results of this figure demonstrate that at higher pHs, fiber removal can : ~ -W O 92/06210 P(~r~US91/0~273 93~1rj ~

still be effected merely by using higher concentrations of cellulase.

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 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma reesei and is identified as GC010. -. ~
m e second cellulase composition analyzed was a CBN I and CBH II deleted cellulase composition ~ prepared from T~ichoderma reesei genetically ;~ ~ modlfied in a manner similar to that described above sd~as~to be incapable of producing CBH I and CBH II
and is identified as CBHI/IId. Insofar as CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component r-sults in enriched leve}s of all of the EG
20~ ~components.

The cellulase compositions were first normalized so~that similar amounts of EG components were~used. ~`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 of 400~ml of a 2~0 mM citrate/phosphate buffer containing 0.5 ml of a~non-ionic surfactant.
30~ ~ Samples~were prepared~and titrated t;o pH 5. Each of ~-the~resulting~solutions was then added to a separate launderometer canister. Into these canisters were '`' """" ' ;'.,;'.,~''."~''' ,'.""''',''' ''"',,"'' ',"''.,`",''''."'`''' '''''` ,```,',.''' ,'''''' W O 92/062~0 P(~r/US91/07273 2a93~23 added a quantity of marbles to facilitate fiber .e;;irval as w211 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 revolutions per minute (rpms) for about 1 hour.
Afterwards, the cloth is removed, rinsed well and dried in a standard drier.

The so treated fabrics were then analyzed for fiber removal by evaluation in the panel test described above. The results of this analysis are set forth in FIG. 12. Specifically, FIG. 12 illustrates that both GC010 and CBH I/IId cellulase compositions gave substantially identical fiber removal results. The results of this figure suggést that it is the EG components which provide for fiber removal. These results coupled with the results of FIG. 11 demonstrate that acidic EG components can remove fibers even under alkaline conditions.

Exam~le 18 ~-Ter~otometer Color Restoration This example is further to Example 17 and substantiates that CBH type components are not necessary for color restoration. The purpose of this example is to examine the ability of cellulase compositions deficient in CBH type components to restore color to cotton-containing fabrics. Because the activity of most of the EG components derived from Trichoderma reesei is greatest at or near pH 5, :.
. : .-2~93~2~ - 68 - ~
.~
color restoration results will be most evident when the assay is conducted at about this p~. However, the results achieved at pH 5 are believed to correlate with color restoration results which would occur at other pHs and accordingly, are indicative of the relative color restoration capacity of the cellulase components analyzed.

Specifically, the cellulase composition employed in this example was a CBH I and CBH II
deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner ~! similar to that described above so as to be incapable of producing CBH I and CBH II. Insofar as CBH I and CBH II comprises up to about 70 percent of lS the cellulase composition, deletion of this component results in enriched levels of all of the EG components.
'``:
The assày was conducted by adding a ~`~ sufficient concentration of this cellulase composition to a 50 mM citrate/phosphate buffer to provide 500 ppm of cellulase. The solution was titrated to pH 5 and contained 0.1 weight percent of nonionic surfactant (Grescoterg G1100 - commercially ~ ~ available from Gresco Mfg., Thomasville, NC 27360).
I ~ 25 A 10 inch x lO inch faded cotton containing fabric was then placed into 1 liter of this buffer and allowed to sit at 110F for 30 minutes and then ¦ agitated for 30 minutes at lO0 rotations per minute.
The fabrics were then removed from the buffer, washed and dried. The resulting fabrics were then ~ compared to the fabric prior to treatment. The l~ results of this analysis are as follows:

W O 92/06210 PC~r/US91/07273 ~ ~ ~ 3 ~ 2 v Cotton Containina Material Result Worn Cotton T-Shirtbenefit seen Cotton Knit benefit seen The term ~benefit seen" means that the treated fabric exhibits color restoration (i.e., is less faded) as compared to the non-treated fabric.
These results substantiate the results of Example 17 that the presence of CBH type components is not necessary for effecting color restoration of faded cotton containing fabrics.

ExamDle 19 Softness This example demonstrates that the presence of CBH type components are not essential for imparting improved softness to cotton-containing fabrics. Specifically, this example employs a cellulase composition derived from Trichoderma ~-reesei genetically engineered in the manner described above so as to be incapable of producing CBH I and II components.
;,~ :. .;
This cellulase composition was tested for its ability to soften terry wash cloth. Specifically, unsoftened 8.5 ounce cotton terry cloths, 14 inches by 15 inches (available as Style No. 420NS from~ 25 Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846), were cut into 7 inch by 7.5 inch swatches.
:: :
The cellulase composition described above was tested for its ability to soften these swatches in a launderometer. Specifically, an appropriate amount --.
: ~ - ,.' W O 92/06210 PC~r/US91/07273 ~93~ 70 -; of cellulase to provide for 500 ppm, 250 ppm, 100 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 blank was run containing the same solution but with no added cellulase. Samples so prepared were titrated to pH

5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate softness as well as cotton swatches -described above. All conditions were run in triplicate with two swatches per canister. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at 37~. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the swatches were removed, rinsed well and dried in a standard drier.

' ~ The swatches were then analyzed for softness by evaluation in a preference test. -~
Specifically, six panelists were given their own set of swatches and ask to rate them with respect to softness based on softness criteria such as the pliability of the whole fabric. Swatches obtained from treatment with the five different enzyme ~-concentrations and the blank were placed behind a scrèen and the panelists were asked to order them from least soft to most soft. Scores were assigned to each swatch based on its order relative to the other swatches; 5 being most soft and 0 being least ~ ' ' .. . .

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

:

W O 92t06210 PC~r/US91/07273 2~3~2~

soft. The scores from each panelists were cumulated and then averaged.
.
The results of this averaging are set forth in FIG. 13. Specifically, these results demonstrate that at higher cellulase concentrations, improved softening is obtained. It is noted that this improved softening is achieved without the presence of either CBH I or II in the cellulase composition.
: ' With regard to these results, it is contemplated that with lower concentrations of the cellulase composition, improved softening will manifest itself over repeated washings. -,:
With regard to Examples 16 to 19, cellulase compositions enriched in acidic EG type components -derived from microorganisms other than Trichoderma reesei could be used in place of the cellulase compositions described in these examples. In particular, the source of the cellulase composition enriched in acidic EG type components is not important to this invention and any fungal cellulase composition enriched in acidic EG type components can be used. For example, fungal cellulases for use in preparing the fungal cellulase compositions used in this invention can be obtained from Trichoderma koninaii, Pencillum s~., and the like or commercially available cellulases can be used, i.e., CELLUCLAST (available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist Brocades, N.V., Delft, Holland), and the like. -:. ':
: .., :..

. - , .
, .

W O 92/06210 PC~r/US91/07273 2~93~'3 - 72 -Example 20 Transformants of Trichoderma reesei Containinq tlle plasmid pEGIpyr4 A ~vr4 defective derivative of T. reesei strain RutC30 (Sheir-Neiss and Montenecourt, (1984), Appl. Microbiol. Biotechnol. 20:46-53) was obtained by the method outlined in Example 1. Protoplasts of this strain were transformed with undigested pEGIE~ and stable transformants were purified.

Five of these transformants (designated EP2, EP4, EP5, EP6, EPll), as well as untransformed RutC30 were inoculated into 50 ml of YEG medium (yeast extract, 5 g/l; glucose, 20 g/l) in 250 ml shake flasks and cultured with shaking for two days at 28C. The resulting mycelium was washed with sterile water and added to 50 ml of TSF medium (0.05M citrate-phosphate buffer, pH 5.0; Avicel microcrystalline cellulose, 10 g/l; KH2PO4, 2.0 g/l; ~-` ~ (NH4)~2S04, 1.4 gll; proteose peptone, 1.0 g/l; Urea, 0.3 g/l; MgS04.7H20, 0.3 g/l; CaCl2, 0.3 g/l; -FeS0~.7H20, 5.0 mg/l; MnS04.H20, 1.6 mg/l; ZnS04, 1.4 mg/l; CoC12, 2.0 mg/l; 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 desigr.ed to measure the total amount of protein and of endoglucanase activity were performed as described below.
~ . ..
The endoglucanase assay relied on the release of soluble, dyed oligosaccharides from Remazol Brilliant Blue-carboxymethylcellulose (RBB-CNC, obtained from MegaZyme, North Rocks, NSW, :

-~ ~ .

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

~ . 2~93~

Australia). The substrate was prepared by adding 2 g of dry RBB-C~C to 80 ml of just boiled deionized - water with vigorous stirring. When cooled to room temperature, 5 ml of 2 M sodium acetate buffer (pH
4.8) was added and the pH adjusted to 4.5. The volume was finally adjusted to 100 ml with deionized - water and sodium azide added to a final concentration of 0.02%. Aliquots of T. reesei control culture, pEGI~y~ transformant culture supernatant or 0.1 M sodium acetate as a blank (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, 0.4% zinc acetate, pH 5 with HCl, 76% ethanol) was ~5 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 from Pierce, Rockford, Illinois, USA. The standard was bovine serum albumin (BSA?. 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 BSA or test culture supernatant. Incubation was for 30 minutes at 37C
and the optical density was finally measured spectrophotometrically at a wavelength of 562 nm.

. ' ~
The results of the assays described above are shown in ~able 1. It is clear that some of the transformants produced increased amounts of ~ '~ ";' ' '-, W O 92/06210 PC~r/US91/07273 ~ 34~ 74 ~

endoglucanase activity compared to untransformed strain RutC30. It is thought that the endoglucanases and exo-cellobiobydrolases produced by untransformed T. reesei constitute approximately 20 and 70 percent respectively of the total amount of protein secreted. Therefore a transformant such as EP5, which produces approximately four-fold more endoglucanase than strain RutC30, would be expected to secrete approximately equal amounts of endoglucanase-type and exo-cellobiohydrolase-type proteins.

The transformants described in this Example were obtained using intact pEGIDvr4 and will contain DNA sequences integrated in the genome which were derived from the pUC plasmid. Prior to transformation it would be possible to digest pEGIE ~ with ~ dIII and isolate the larger DNA
fragment containing only ~. reesei DNA.
Transformation of T. reesei 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 pEGIpyr4 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 EGI and produce either a limited range of, or no, exo-cellobiohydrolases.
.
The methods of Example 20 could be used to produce ~. reesei strains which would over-produce any of the other cellulase components, xyl~nase .

components or other proteins normally produced by T.
reesei.
., , - . ;

Secreted Endoalucanase Activity of T. reesei Transformants -A B
ENDOGLUCANASE
ACTIVITY PROTEIN
STRAIN(O.D. AT 590 nm) ~ma/ml) A/B
10 RutC300.32 4.1 0.078 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 15 EP11 0.99 4.11 0.241 The above results are presented for the purpose of demonstrating the overproduction of the EGI component relative to total protein and not for the purpose of demonstrating the extent of - -overproduction. In this regard, the extent of - -overproduction is expected to vary with each ,' experiment.
:: .: . .
Examnle 2I

Construction of pCEPC1 . .
A plasmid, pCEPCl, was constructed in which the coding sequence for EGI was functionally fused to the promoter from the Çkhl gene. This was achieved using in vitro, site-specific mutagenesis to-alter the DNA seguence of the cbhl and eall genes in order to create convenient restriction -~: .:, , 2~ 9~ ~27 - 76 -endonuclease cleavage sites just 5' (upstream) of thair respect.~e translation initiation sites. DNA
sequence analysis was performed to verify 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. 15):
A) A 2.1 kb fragment from the 5~ flanking region of the cbhl locus. This includes the promoter region and extends to the engineered ~clI site and so contains no cbhl coding sequence.
~) A 1.9 kb fragment of genomic DNA from the eqll locus starting at the 5' end with the engineered BamHI site and extending through the coding region and including approximately 0.5 kb beyond the translation stop codon. At the 3~ end of the fragment is 18 bp derived from the pUC218 multiple c}oning site and a 15 bp synthetic oligonucleotide used to link this fragment with the fragment below.
C) A fragment of DNA from the 3~ flanking region of the cbhl locus, extending from a position -approximately 1 kb downstream to approximately 2.5 kb downstream of the cbhl translation stop codon.
D) Inserted into an NheI site in fragment (C) was a 3.1 kb NheI-SphI fragment of DNA containing the T.
reesei Dvr4 gene obtained from pTpyr2 (Example 2) and having 24 bp of DNA at one end derived from the pUC18 multiple cloning site.
,: . .
The plasmid, pCEPCl was designed so that the EGI coding sequence would be integrated at the cbhl locus~, replacing the coding sequence for CBHI

.

WO92/06210 PCT/US91/~7273 _ 77 - ~3~12~ . -without introducing any foreign DNA into the host strain. Digestion of this plasmid with EcoRI
liberates a fragment which includes the cbhl promoter region, the egll coding sequence and transcription termination region, the T. reesei Dvr4 gene and a segment of DNA from the 3' (downstream) flanking region of the cbhl locus (see Fig. 15).

Example 22 Transformants containina ~CEPCl DNA
A pyr4 defective strain of T. reesei RutC30 (Sheir-Neiss, su~ra) was obtained by the method outlined in Example 1. 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 as described in Example 20. In order to visualizè -~
the cellulase proteins, isoelectric focusing gel electrophoresis was performed on samples from these -~
` ~ cultures using the method described in Example 7.
Of a total of 23 transformants analysed in this manner 12 were found to produce no CBHI protein, which is the expected result of integration of the --CEPCl DN.~ at the cbhl locus. Southern blot analysis was used to confirm that integration had indeed occurred at the cbhl locus in some of these transformants and that no seguences derived from the bacterial plasmid vector (pUC4K) were present (see Fig. 16). For this analysis the DNA from the transformants was digested with E~I before being subjected to electrophoresis and blotting to a ;
membrane filter. The resulting Southern blot was ;
probed with radiolabelled plasmid pUC4K::cbhl (see Example 2). The probe hybridised to the cbhl gene on - - ' ':.

?~

W O 92/06210 PC~r/US91/07273 2 ~ g 3 4 2 ~ - 78 -a 6.5 kb fragment of DNA from the untransformedcontrol culture (FI~. 16, lane A). Integration of the CEPC1 fragment of DNA at the cbhl locus would be expected to result in the loss of this 6.5 kb band and the appearance of three other bands corresponding to approximately 1.0 kb, 2.0 kb and : 3.5 kb DNA fragments. This is exactly the pattern observed for the transformant shown in FIG. 16, lane C. Also shown in FIG. 16, lane B is an example of a transformant in which multiple copies of pCEPCl have integrated at sites in the genome other than the cbhl locus.

Endoglucanase activity assays were performed on samples of culture supernatant from the untransformed culture and the transformants exactly .: as described in Example 20 except that the samples were diluted 50 fold prior to the assay so that the protein concentration in the samples was between approximately 0.03 and 0. 07 mg/ml. The results of . 20 :assays:performed with the untransformed control ~ culture and four different transformants (designated ! CEPC1-101, CEPCl-103, CEPC1-105 and CEPCl-112) are shown in Table 2. Transformants CEPC1-103 and ~:~ CEPCl-112 are examples in which integration of the CEPC1 fragment had led to 105s o~ ca~I production.

~' ,.'.-:
,, ~VO 92/06210 PC~r/US91/07273 ~ ', ~'S~3~s~ . ;' ' Table 2 Secreted endoqlucanase activitv of T. reesei transformants A B
ENDOGLUCANASE
ACTIVITY PROTEIN
STRAIN(O.D. at 590 nm)tma/ml~ A/B
RutC300 0.037 2.38 0.016 CEPC1-101 0.082 2.72 0.030 ;~-CEPCl-103 o.oss 1.93 0.051 CEPC1-105 0.033 2.07 0.016 CEPCl-112 0.093 1.72 0.054 The above results are presented for the -purpose of demonstrating the overproduction of the EGI component relative to total protein and not for the purpose of demonstrating the extent of l~ overproduction. In this regard, the extent of `~ overproduction is expected to vary with each experiment. ;
- . .
It would be possible to construct plasmids s~imilar~to pCEPCl but with any other T. reesei gene replacing the ~gl~ gene. In this way, overexpression of other genes and simultaneous :
deletion of the cbhl gene could be achieved.
~ ~ , '", It would also be possible to transform EY~
derivative strains of T. reesei which had previously -~
.
been deleted for other genes, eg. for cbh2, with pCEPC1 to construct transformants which would, for example, produce no exo-cellobiohydrolases and overexpress endoglucanases.
' ,- ~ ~ , . .

-, :
~' ' ' ., WO92/06210 PCT/US91/n7273 2~34~ - 80 -Using constructions similar to pCEPCl, but in which DNA from another locus of T. reesei was substituted for the DNA from the cbhl locus, it would be possible to insert genes under the control of another promoter at another locus in the T.
reesei genome.

Example 23 Construction of ~EGII::P-l ;

The eql3 gene, encoding EGII (previously 10 referred to as EGIII by others), has been cloned from T. reesei and the DNA sequence published (Saloheimo et al., 1988, Gene 63~ 21). We have obtained the gene from strain RL-P37 as an approximately 4 kb PstI- XhoI fragment of genomic 15 DNA inserted between the PstI and XhoI sites of pUC219. The latter vector, pUC219, is derived from pUCll9 (described in Wilson et al., 1989, Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for BglII, ClaI and XhoI.
20 Using methods known in the art the T. reesei pvr4 ~-gene, present on a 2.7 kb SalI fragment of genomic -DNA, was inserted into a SalI site within the EGII
coding sequence to create plasmid pEGII::P-l (FIG.
17). This resulted in disruption of the EGII coding 25 sequence but without deletion of any sequences. The plasmid, pEGII::P-l can be digested with ~i~dIII and HI to yield a linear fragment of DNA derived exclusively from T. reesei except for 5 bp on one , end and 16 bp on the other end, both of which are 30 derived from the multiple cloning site of pUC219.

~ , .

.
, -~

W O 92/06210 PC~r/US9t/07273 ~ '.
- 81 - ~
~ ~ ~ 3 4 ~7 ~ : :
Example 24 ----Transformation of T. reesei GC69 with PEGII::P-1 to create a strain unable to Droduce EGII6.75 .~
T. reesei strain GC69 will be transformed with pEGII::P-l which had been previously digested with HindIII and BamHI and stable transformants will be selected. Total DNA will be isolated from the transformants and Southern blot analysis used to identify those transformants in which the fragment of DNA containing the EYE~ and eal3 genes had integrated at the eql3 locus and consequently disrupted the EGII coding sequence. The transformants will be unable to produce EGII. It would also be possible to use pEGII::P-l to , 15 transform a strain which was deleted for either or I all of the cbhl, cbh2, or eall genes. In this way a strain could be constructed which would only produce certain cellulase components and no EGII component.
, ! ~ ; 20 Exam~le 25 Transformation of T. reesei with ~EGII::P-l 1, to create a strain unable to Droduce CBHI. , CBHII and EGII
A ~YE~ deficient derivative of strain P37P~CBH67 tfrom Example 11) was obtained by the method outlined in Example 1. This strain P37P~67P
~ 1 was transformed with pEGII::P-l which had been '~ previously digested with HindIII and BamHI and If~ stable transformants were selected. Total DNA was 1 30 isolated from transformants and Southern blot 1~ analysis used to identify strains in which the ;~ fragment of DNA containing the Dvr4 and eal3 genes had integrated at the eal3 locus and consequently - -~ .
': ' ' .

- - . , . . . ... , ;: ,,. - :. : : . , .

WO92/06210 PCT/US91/~273 ~9'~ 4~ - 82 -disrupted the EGII coding sequence. The Southern blot illustrated in FIG. 18 was probed with an approximately 4 kb PstI fragment of T. reesei DNA
containing the eql3 gene which had been cloned into the PstI site of pUC18 and subsequently re-isolated.
When the DNA isolated from strain P37P~67Pl 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 BglII the size of the band corresponding to the egl3 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37P~A67P1 strain (Lanes A and C) and the transformant disrupted for eal3 (FIG. 18, Lanes B and D). The transformant containing the disrupted eal3 gene illustrated in FIG. 18 (Lanes B, D and F) was named A22. The transformant identified in FIG. 18 is -unable to produce CBHI, CBHII or EGII.

Example 26 . .
Construction of pP~EGI-~

The eall gene of T. ~eesei strain RL-P37 was obtained, as described in Example 12, as a 4.2 kb ~indIII fragment of genomic DNA. This fragment was inserted at the ~i_dIII site of pUC100 (a derivative of pUC18; Yanisch-Perron et al., 1985, Gene 33:103-119, with an oligonucleotide inserted into the multiple cloning site adding restriction sites for ~glII, ClaI and ~hQI). Using methodology known in ~-~

' ~
- 83 - ~3ll2~ - -the art an approximately 1 kb EcoRV fragment extending from a position close to the middle of the EGI coding sequence to a position beyond the 3' end of the coding sequence was removed and replaced by a 3.5 kb ScaI fragment of T. reesei DNA containing the EYE~ gene- The resulting plasmid was called pPAEGI-1 (see Fig. 19).

The plasmid pP~EGI-l can be digested with HindIII to release a DNA fragment comprising only T.
reesei genomic DNA having a segment of the eall gene at either end and the ~y~ gene replacing part of the EGI coding sequence, in the center.

Transformation of a suitable T. reesei Dvr4 deficient strain with the pPAEGI-l digested with HindIII will lead to integration of this DNA
fragment at the eall locus in some proportion of the transformants. In this manner a strain unable to produce EGI will be obtained. - -,:, Example 27 Construction of_pAEGIpyr-3 and Transformation of a pvr4 deficient strain of T. reesei The expectation that the EGI gene could be inactivated using the method outlined in Example 26 is strengthened by this experiment. In this case a plasmid, pAEGIpyr-3, was constructed which was similar to pP~EGI-l except that the As~eraillus niaer EYE~ gene replaced the T. reesei EYE~ gene as -selectable marker. In this case the eqll gene was again present as a 4.2 kb HindIII fragment inserted at the HindIII site of pUC100. The same internal 1 kb EcoRV fragment was removed as during the :

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

~ ~ ~ 3 ~ 84 -construction of pP~EGI-l (see Example 26) but in this case it was replaced by a 2.2 kb fragment containing the cloned A. niger ~y~ gene (Wilson et - al., 1988, Nucl. Acids Res. 16 p.2339).
Transformation of a ~Yr4 deficient strain of T.
reesei (strain GC69) with p~EGIpyr-3, after it had -been digested with HindIII to release the fragment containing the ~YE~ gene with flanking regions from the eqll locus at either end, led to transformants 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. reesei the egll gene was present on a 4.2 kb HindIII fragment of DNA and this pattern of hybridization is represented by Fig. 20, lane C. However, following deletion of the eall gene by inteqration of the desired fragment from ~ p~EGIpyr-3 this 4.2 kb fragment disappeared and was replaced by a fragment approximately 1.2 kb larger in size, FIG. 20, lane A. Also shown in FIG. 20, lane B is an example of a transformant in which integration of a single copy of pP~EGIpyr-3 has occurred at a site in the genome other than the eqll locus.

i Example 28 .
Transformation of T.reesei with pP~EGI-1 to create a strain unable to roduce CBHI. CBHII, EGI and EGII
A Dyr4 deficient derivative of strain A22 (from Example 25) will be obtained by the method outlined in Example 1. This strain will be transformed with pP~EGI-1 which had been previously .

,- .

.. . . . : . ~ : . . , - : :: ~ .. . ; -: , . . .. . .. .: . ..

WO92/06210 PCT/US91/~7273 ~ .
- 85 ~ 3 diqested with ~i_dIII to release a DNA fragment compris.ng 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. :
. .
s Stable PYr4+ 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 ~y~ gene and eall sequences has :' -integrated at the eqll locus and consequently disrupted the EGI coding sequence. The transformants identified will be unable to produce CBHI, CBHII, EGI and EGII.
'~'~'''"
' : ' ' ' - ' , . ' ,: '. , , ,, ' , ' . . '' ' ' :

Claims (39)

WHAT IS CLAIMED IS:

1. A detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I
type components of greater than 5:1.

2. A detergent composition according to Claim 1 wherein said cellulase composition comprises from about 0.05 to about 2 weight percent of said detergent composition.

3. A detergent composition according to Claim 1 wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH type components of greater than 5:1.

4. A detergent composition according to Claim 1 wherein said cellulase composition is free of all CBH I type components.

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

6. A detergent composition according to Claim 1 wherein said cellulase composition comprises at least about 1 weight percent of CBH I type cellulase components based on the total protein weight of all of the acidic EG components.

7. A detergent composition according to Claim 1 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH I type components and/or overexpressing one or more EG type components.

8. A detergent composition according to Claim 1 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH type components.

9. A detergent composition according to Claim 7 wherein said cellulase composition does not contain any heterologous proteins.

10. A detergent composition according to Claim 8 wherein said cellulase composition does not contain any heterologous proteins.

11. A method for enhancing the softness of a cotton-containing fabric which method comprises washing the fabric in a wash medium derived from a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.

12. A method according to Claim 11 wherein said cellulase composition comprises from about 0.05 to about 2 weight percent of said detergent composition.

13. A method according to Claim 11 wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH
type components of greater than 5:1.

14. A method according to Claim 11 wherein said cellulase composition is free of all CBH I type components.

15. A method according to Claim 14 wherein said cellulase composition is free of all CBH type components.

16. A method according to Claim 11 wherein said cellulase composition comprises at least about 1 weight percent of CBH I type cellulase components based on the total protein weight of all of the acidic EG components.

17. A method according to Claim 11 wherein said cellulase composition is derived from a genetically modified microorganism incapable of producing any CBH I type components and/or overexpressing one or more EG type components.

18. A method according to Claim 11 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH type components.

19. A method according to Claim 17 wherein said cellulase composition does not contain any heterologous proteins.

20. A method according to Claim 18 wherein said cellulase composition does not contain any heterologous proteins.

21. A method for retaining/restoring the color of a cotton-containing fabric which method comprises washing the fabric one or more times in a wash medium derived from a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I
type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.

22. A method according to Claim 21 wherein said cellulase composition comprises from about 0.05 to about 2 weight percent of said detergent composition.

23. A method according to Claim 21 wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH
type components of greater than 5:1.

24. A method according to Claim 21 wherein said cellulase composition is free of all CBH I type components.

25. A method according to Claim 24 wherein said cellulase composition is free of all CBH type-components.

26. A method according to Claim 21 wherein said cellulase composition comprises at least about 1 weight percent of CBH I type cellulase components based on the total protein weight of all of the acidic EG components.

27. A method according to Claim 21 wherein said cellulase composition is derived from a genetically modified microorganism incapable of producing any CBH I type components and/or overexpressing one or more EG type components.

28. A method according to Claim 21 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH type components.

29. A method according to Claim 27 wherein said cellulase composition does not contain any heterologous proteins.

30. A method according to Claim 28 wherein said cellulase composition does not contain any heterologous proteins.

31. A pre-soak composition comprising from about 0 to about 20 weight percent of a surfactant and from about 0.002 to about 0.5 weight percent of a of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.

32. A pre-soak composition according to Claim 31 wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH type components of greater than 5:1.

33. A pre-soak composition according to Claim 31 wherein said cellulase composition is free of all CBH I type components.

34. A pre-soak composition according to Claim 33 wherein said cellulase composition is free of all CBH type components.

35. A pre-soak composition according to Claim 31 wherein said cellulase composition comprises at least about 1 weight percent of CBH I
type cellulase components based on the total protein weight of all of the acidic EG components.

36. A pre-soak composition according to Claim 31 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH I type components and/or overexpressing one or more EG type components.

37. A pre-soak composition according to Claim 31 wherein said cellulase composition is derived from a genetically modified microorganism incapable of expressing any CBH type components.

38. A pre-soak composition according to Claim 36 wherein said cellulase composition does not contain any heterologous proteins.

39. A pre-soak composition according to Claim 37 wherein said cellulase composition does not contain any heterologous proteins.