WO1993022414A1 - Degradation resistant detergent compositions based on cellulase enzymes - Google Patents

Abstract

Disclosed are detergent compositions containing a combination of exo-cellobiohydrolase 1 type cellulase components and endoglucanase type components wherein the exo-cellobiohydrolase 1 type cellulase components are enriched relative to the endoglucanase type components. The detergent compositions provide cleaning and softening of cotton garments while also providing substantially reduced degradation of the cotton fabric in the garment.

Description

BACKGROUND OFTHE INVENTION

DEGRADATI_ON RESISTANT DETERGENT COMPOSITIONS BA_SED ON CELLULASE ENZYMES

Field of invention

The present invention relates to detergent compositions which have improved degradation resistance to cotton fabrics. More particularly, the present invention relates to detergent compositions containing a cellulase composition comprising a combination of exo- cellobiohydrolase I type cellulase components and endoglucanase cellulase components wherein the exo-cellobiohydrolase I type cellulase components are enriched relative to the endoglucanse type cellulase components. Such detergent compositions provide excellent cleaning, especially of cotton garments, while also providing substantially reduced degradation of the cotton fabric in the garment.

State of the Art

Cellulases are known in the art as enzymes that hydroiyze cellulose (β-1 ,4-glucan linkages) thereby resulting in the formation of glucose, cellobiose, cellooligosaccharide, and the like. While cellulases are produced in fungi, bacteria and the like, those produced by fungi have been given the most attention because fungi typically 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, "Methods in Enzyrπology", 160. 25, pages 234-242 (1988) discloses that certain fungi produce cellulase systems which are comprised of several different enzyme components including those identified as exo-cellobiohydroiases (EC 3.2.1.91 ) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG"), β-glucosidase (EC 3.2.1.21 ) ("BG"). Moreover, these classes can be further separated into individual components. For example, multiple CBHs and EGs have been isolated from a variety of bacterial and fungal sources including T. 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 ratio of CBH I type components to EG type components (including ail of the EG type components) in naturally occurring cellulases does not appear to exceed about 5:1 . For example, see Brown et al., Genetic Control of Environmental Pollutants, Gilbert S. Omenn Editor, Chapter -- "Microbial Enzymes and Ligno- Celluiase Utilization", Hollaeπder Publishing Corp. Variations in this ratio can result from the use of different microorganisms, depending upon the characteristics of the strain, but in any event such ratios still do not exceed about 5:1.

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. That is to say the effectiveness of the complete system is significantly greater than the sum of the contributions from the isolated components. It has also been suggested by Wood, "Properties of Ceiiulolytic Systems", Biochem. Soc. Trans., 13, 407- 410 (1985), that CBH I and CBH II derived from either I. reesei or P.. funiculosum synergϊstically interact in solubilizing cotton fibers. On the other hand. Shoemaker et al., Bio/Technology, October 1983, disclose that CBH I (derived from T. reesei). by itself, has the highest binding affinity but the lowest specific activity of all forms of ceilulase.

The substrate specificity and mode of action of the different cellulase components varies from component to component which may account for the synergy of the combined components. For example, the current accepted mechanism of cellulase action is that endoglucanase components hydrolyze internal β-1 , 4-glucosidic bonds particularly in regions of low crystallinity of the cellulose and cellobiohydrolase components hydrolyse cellobiose from the non- reducing end of the cellulose. β-Glucosidase components act on ceilooligosaccharides, e.g., cellobiose, to give glucose as the sole product.

Cellulases are also known in the art to be useful in detergent compositions either for the purpose of enhancing the cleaning ability of the composition or as a softening agent. When so used, the cellulase will degrade a portion of the cellulosic material, e.g., cotton fabric, in the wash, which in one manner or another facilitates the cleaning and/or softening of the cotton fabric. While the exact cleaning mechanism of cotton fabrics by cellulase is not fully understood, the cleaning of cotton fabrics by cellulase has been attributed to its cellulolytic activity. Thus, for instance, U.S. Patent No. 4,822,516 discloses that detergent compositions containing a cellulase having iow activity on highly crystalline cellulose and high activity on low crystalline cellulose possesses good detergency and a low degree of damage on cotton garments. As noted by Wood, supra, the presence of CBH components is the distinguishing feature of cellulases that are able to degrade crystalline cellulose. Accordingly, these references would suggest that CBH components are in some form involved in the degradation of cotton fabric.

However, regardless of its cleaning and/or softening mechanϊsm(s), the use of cellulases in detergent compositions is complicated by the fact that exposure of cotton garments to cellulase results in partial degradation of the cotton fabric in these garments. After repeated washing and drying, the integrity of the cotton garment is compromised resulting in the tearing, weakening and/or thinning of the cotton garment. When its integrity has been so compromised by repeated exposure to cellulase containing detergents, the cotton garment is no longer of any practical utility. Needless to say, such degradation greatly impairs the commercial utility of cellulases in detergent compositions. Accordingly, cellulase compositions have been sought which possess reduced cotton degradation while retaining enhanced cleaning capabilities.

Accordingly, it is an object of this invention to develop a detergent composition containing cellulase which is resistant to degrading cotton fabrics. It is a further object of this invention that such detergent compositions provide excellent cleaning of such cotton fabrics. These and other objects are achieved by the present invention as evidenced by the attached Summary of the Invention, Detailed Description of the Invention and Claims. SUMMARY OF THE INVENTION

The present invention is directed to the discovery that detergent compositions containing cellulase compositions enriched in CBH I type cellulase components relative to the EG components provide cleaning and softening of cotton garments while at the same time having a reduced capacity for degrading cotton fabrics. Accordingly, in its composition aspects, the present invention is directed to detergent compositions comprising at least one surface active agent and a cleaning effective amount of a cellulase composition wherein said cellulase composition contains a weight ratio of CBH I type cellulase components to EG type components of greater than about 10: 1 . Such compositions are particularly useful as laundry detergents.

In one of its method aspects, the present invention is directed to a method for enhancing the softening properties of a detergent composition which comprises adding a softening effective amount of a cellulase composition containing a weight ratio of CBH I type cellulase components to EG type components of greater than about 10: 1 .

In another of its method aspects, the present invention is directed to a method for enhancing the degradation resistance to cotton fabric of a detergent composition containing cellulase which comprises employing a cellulase composition containing a weight ratio of CBH I type cellulase components to EG type components of greater than about 10: 1. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outline of the construction of pΔCBHIrj r4.

FIG. 2 is an outline of the construction of pΔEGIpyrG-3.

FIG. 3 illustrates deletion of the eα l gene by integration of the Hindlll fragment from pΔEGlpyrG-3 at the eαll locus on one of the T. reesei chromosomes.

FIG. 4 is an outline of the construction of pAΔEGH-1.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention generally relates to detergent compositions containing a cellulase composition enriched in

CBH l type cellulase components relative to the EG type components. Such detergent compositions possess excellent cleaning abilities and softening potential while exhibiting reduced degradation potential against cotton fabrics relative to detergent compositions containing a whole cellulase composition. The softening potential and reduced degradation potential against cotton fabrics possessed by the detergent compositions of this invention is surprising in view of the fact that the detergent compositions contain cellulase compositions enriched in CBH I type cellulase components. Specifically, while the art discloses that EG type components impart softening to cotton-containing fabrics there appears to be no teaching that CBH components can also impart softening in the presence of EG components. Additionally, with regard to the reduced degradation potential of the compositions described herein, the presence of CBH I is the distinguishing feature of cellulases that are able to degrade crystalline cellulose which in turn has been implicated in the degradation of cotton fabric. Moreover, the excellent cleaning properties of the compositions of this invention are also surprising because CBH I (derived from T. reesei) has been shown to have the lowest specific activity of all cellulase components derived from T. reesei on ail forms of cellulose.

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

Definitions

The term "cellulase" or "cellulase composition" refers to an 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 (sometimes referred to hereafter as "fungal cellulases"). Cellulases act on cellulose or one or more of its degradation products to hydroiyze 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 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, for example, cellulase derived from Humicola insolens is known to have activity in neutral to slightly alkaline conditions.

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

A cellulase composition produced by a naturally occurring fungal source, and which comprises one or more CBH and EG components, wherein each of these components is found at the ratio produced by the fungal source is sometimes referred to herein as a "complete cellulase system" or a "complete cellulase composition" to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism genetically modified so as to overproduce, underproduce or not produce one or more of the CBH and/or EG components of cellulase. The fermentation procedures for culturing fungi for production of cellulase are known rjer 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 r__r sj_ in the art.

"Endoglucanase ("EG") type components" refer to all of those ceilulase 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 cellulase components which impart softening, color retention/restoration and improved feel to cotton garments when these components are incorporated into a wash medium. The 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 classified as endoglucanases using traditional biochemical activity tests. For example, such traditional activity tests are based on the ability of the component (a) to hydroiyze soluble cellulose derivatives such as carboxymethylcellulose (CMC), thereby reducing the viscosity of CMC containing solutions, and (b) to readily hydroiyze hydrated forms of cellulose such as phosphoric acid swollen cellulose (e.g., Walseth cellulose) and hydroiyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.). In contrast, it is believed that not all endoglucanase components, as defined by such activity tests, will provide improved softness, feel and coior 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.

Cellulases can contain more than one EG type component. The different components generally have different isoeiectric points, different molecular weights, different degrees of glycosylation, different substrate specificity, different enzymatic action patterns, etc. The different isoeiectric points of the components allow for their separation via ion exchange chromatography and the like. In fact, the isolation of components from different fungal sources is known in the art. See, for example, Schulein et al., International Patent Application WO 89/09259, Wood et al., Biochemistry and Genetics of Cellulose

Degradation, pp. 31 to 52 (1988); Wood et al.. Carbohydrate Research, Vol. 190, pp. 279 to 297 (1989); Schulein, Methods in Enzymology, Vol. 160, pp. 234 to 242 (1988); and the like. The entire disclosure of each of these references is incorporated herein by reference.

In general, it is contemplated that combinations of EG type components may give a synergistic response in improving softening, coior retention/restoration and feel as compared to a single EG type component. On the other hand, a single EG type component may be more stable or have a broader spectrum of activity over a range of pHs. Accordingly, the EG type components employed in this invention can be either a single EG type component or a combination of two or more EG type components. When a combination of components is employed, the EG type 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 significant color retention/restoration and improved feel to the so-treated cotton- containing fabrics. Additionally, when used in combination with EG type components at concentrations of about 5:1 and greater, the CBH I component of Trichoderma reesei imparts an incremental cleaning effect to cotton-containing fabrics. However, as the concentration of EG type components is increased so that the CBH I type components to EG type components approach that of whole cellulase which has a ratio of about 2:5:1 enhanced strength loss occurs as compared to cellulase compositions containing a ratio of CBH I type to EG type components of greater than 5:1.

Accordingly, CBH I type components and CBH II 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. Further, "CBH I type components" refer to those components which exhibit similar cleaning performance as that exhibited by CBH I derived from T. reesei when combined with EG components. Preferably, this includes the properties of reducing strength loss of cotton-containing fabrics and/or imparting an incremental cleaning benefit when used in the presence of EG type components at the ratios recited above. In a preferred embodiment, the CBH I components also impart an incremental softening benefit when used in the presence of EG type components.

"CBH II type cellulase components" refer to those components which exhibit exocellobiohydrolase activity similar to that of CBH II derived from T. reesei. Accordingly, the cellulase composition employed in the detergent compositions of the present invention can contain CBH II type cellulase components in addition to CBH I type cellulase components and EG components. When so employed, the amount of CBH II type cellulase components is generally from about 0.001 to about 30 weight percent relative to the CBH I type cellulase component in the detergent compositions. However, in the preferred embodiment, the cellulase composition contains no CBH II type cellulase components. In fact, our results indicate that CBH II, when employed at the same concentrations as CBH I, will not demonstrate the same cleaning benefits when combined with EG components that CBH I type cellulase components do. It is, however, contemplated that CBH II may provide softening when combined with EG components.

Such exocellobiohydrolase type components may include components not traditionally classified as exo-cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II from Trichoderma reesei. For example, using such traditional classification tests, such components are: (a) competitively inhibited by ceilobiose (Kj approximately 1 mM); (b) unable to hydroiyze to any significant degree substituted celluloses, such as carboxy-methylcellulose, etc.; and (c) able to hydroiyze phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose. In contrast, 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.

"^β-GIucosidase (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 cellooiigosaccharides ("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 (IC, approximately 1 mM). While in a strict sense, BG components are not literally cellulases because they cannot degrade cellulose; such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the combined action of CBH components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur. BG components are often characterized on aryl substrates, such as p-nitrophenol B-D-glucoside (PNPG) and, thus, are often called aryl-glucosidases. It should be noted that hot all aryi-giucosidases are BG components, in that some do not hydroiyze 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 hydroiyzed to glucose by BG components, the absence of BG components in the cellulase composition will "turn-off" CBH activity when the concentration of cellobiose reaches inhibitory leveis. It is also contemplated that one or more additives (e.g., cellobiose, glucose, etc.) can be added to the cellulase composition to effectively "turn-off" directly or indirectly, some or ail of the CBH I type activity, as well as other CBH activity. On the other hand, a cellulase composition containing added amounts of BG components may increase overall hydrolysis of cellulose, if the level of cellobiose generated by the CBH components becomes restrictive of such overall hydrolysis in the absence of added BG components.

Methods to either increase or decrease the amount of BG components in the cellulase composition are disclosed in U.S. Serial No. 07/807,028 filed December 10, 1991 , as Attorney Docket No.

010055-077, and entitled "IMPROVED SACCHARIFICATION OF CELLULOSE BY CLONING AND AMPLIFICATION OF THE β- GLUCOSIDASE GENE FROM TRICHODERMA REESEI", which application is incorporated herein by reference.

Fungal cellulases can contain more than one BG component. The different components generally have different isoeiectric points which allows 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 cellobiose produced in the detergent wash which can be readily determined by the skilled artisan.

"Degradation Resistant" refers to the diminished capacity of a detergent composition containing a cellulase composition of this invention to degrade cotton fabric. In general, degradation of cotton fabric by a cellulase containing detergent is measured by the degree of thinning, weakening and/or tearing produced in the cotton fabric over a repeated number of washings with the cellulase containing detergent followed after each washing with drying in a mechanical dryer. In this regard, it appears that the use of a mechanical dryer after washing facilitates this analysis insofar as the movement of the dryer during its operation stretches and puils the garment, which, if substantially degraded, can result in tearing of the fabric. The degradation resistanc of detergent compositions containing the cellulase components, as per this invention, can be readily determined by measuring the degradation of identical sets of cotton clothing or cotton swatches after a repeated number of washing/drying cycles under identical conditions. One set being washed with the detergent composition of this invention, and the other being washed with a detergent composition containing a cellulase system (preferably produced from the same organism) having a ratio of

CBH I type cellulase components to EG components of about 2.5:1. At the completion of at least 20 washing/drying cycles, the sets of cotton clothing are evaluated for degradation. Degradation is measured by testing the tensile strength of each garment/swatch for each set, and a summation of ail of the ratings for each set is then divided by the number of garments/swatches in the set so as to provide an average tensile strength. In this regard, in a preferred embodiment, the term "degradation resistant" means that the average tensile strength after at least 20 washing/drying cycles for the set of garments/swatches treated with the detergent composition of this invention is significantly higher than the average tensile strength of the set of garments/swatches treated with a detergent composition containing the cellulase system described above. Preferably, the detergent compositions of this invention will result in at least a 10% increase, and more preferably, a 20% increase in the average tensile strength for the set of garments/swatches treated with a detergent composition of this invention, as compared to the average tensile strength of the set of garments/swatches treated with a detergent composition containing the cellulase system described above.

Additionally, the degradation resistance of detergent compositions containing the cellulase components, as per this invention, can be determined by measuring the tensile strength in the warp and fill direction by the test method described in ASTM D 1682- 64 which is incorporated herein by refrence in its entirety.

Methodology

In accordance with the present invention, detergent compositions which employ a cellulase composition will be rendered degradation resistant if the cellulase composition employed in the detergent contains a weight ratio of CBH I type cellulase components to EG type components from greater than about 10:1 to 400:1 . More preferably, the weight ratio of CBH I type cellulase components to EG type components is from about 20:1 to 100:1 , and even more preferably, about 40:1 or more.

The detergent compositions of this invention will also result in reduced harshness (i.e., softening) of the washed garments. At higher weight ratios of CBH I components to EG components, the softening and cleaning effects of the cellulase compositions becomes more evident with repeated treatments.

Surprisingly, it has been found that it is the amount of cellulase and the ratio of CBH I type cellulase components to EG type components employed in detergent compositions and not the relative rate of hydrolysis of the individual enzymatic components in producing reducing sugars from cellulose which imparts the improved cleaning of cotton garments. Even more surprisingly, is the fact that CBH II type cellulase components do not substitute for CBH I type cellulase components (at the levels tested) in providing cleaning benefits when combined with EG type cellulase components in detergent compositions. Accordingly, when used to enhance the cleaning of fabrics, the amount of the ceilulase composition generally employed in the detergent compositions of this invention is an amount sufficient to impart improved cleaning of cotton garments. Preferably, the cellulase compositions are employed from about 0.002 weight percent to about 10 weight percent relative to the total detergent composition. More preferably, the cellulase compositions are employed from about 0.01 weight percent to about 5 weight percent relative to the total detergent composition. The cellulase composition can be added to such detergent compositions either in a liquid diluent, or as granules, or as an emulsion. Such forms are well known to the skilled artisan. Preferably, the amount of cellulase composition employed in the detergent composition will be at least about 50 ppm, more preferably at least about 100 ppm.

The term "softening effective amount" means a sufficient amount of the cellulase composition employed in the detergent compositions of this invention to impart softening over one or more treatments with the cellulase containing detergent composition. Preferably, the amount of cellulase composition employed in the detergent compositions will be at least about 50 ppm, more preferably, at least about 100 ppm, and most preferably, at least about 250 ppm. At lower concentrations, it is contemplated that the softening effect of cellulase will be evident over repeated treatments of the fabric with the cellulase containing detergent composition. As is apparent, these concentrations also provide enhanced cleaning of the fabrics. It is contemplated that the CBH II component, when employed at the same concentrations as CBH I, may provide softening. As a further embodiment of this invention, the CBH II component is substituted for the CBH I component, when softening is desired. In the event that CBH II is used in the invention, in place of or in addition to the CBH I component, to provide a softening effective amount, the ratio of CBH I and CBH II components to EG components is preferably 10:1 and more preferably 20:1 .

Without being limited to any theory, it is beiieved that the EG type components and/or CBH II type cellulase components in combination with the CBH I type components are primarily responsible for degrading cotton fabric. On the other hand, EG type components are required to provide the synergistic mixture of enzymes which result in improved cleaning and softening. However, the present invention is directed to the discovery that the desired increase in cleaning and softening can be achieved by using a detergent composition containing only small amounts of EG type component(s), i.e., less than that found in cellulases naturally produced by fungal microorganisms. Thus, by carefully controlling the amount of EG type components used in the cellulase employed in the detergent composition, one achieves a high level of cleaning and softening while at the same time reducing the degradation potential of the composition.

Cellulase compositions having the requisite ratio of CBH I type cellulase components to EG type cellulase components can be prepared by purifying the cellulase system into its components and then recombining the requisite amount of the components to achieve the desired ratio of components. In this manner, it is also possible to create cellulase compositions having little or no amounts of certain components, i.e., one can prepare a cellulase composition to be free of CBH II type cellulase components, or free of all EG type components except either EG-I type cellulase components (i.e., an cellulase component having properties similar to EG-I derived from T. reesei), EG-

II type cellulase components (i.e., an cellulase component having endoglucanase properties similar to EG-II derived from T. reesei). or EG

III type cellulase components (i.e., an ceilulase component having endoglucanase properties similar to EG 111 derived from T. reesei) or free of BG components, merely by not recombining that (those) component(s). The cellulase compositions employed in the detergent compositions of this invention may be free of CBH II type cellulase components. In particular, CBH II type cellulase components, when employed at the same levels as CBH I, do not significantly enhance the cleaning properties of the detergent composition when enriched relative to the EG components.

The particular ceilulase system employed to isolate the respective components is not critical, although certain cellulase systems may be preferred over others, i.e., an alkaline cellulase may be preferred over an acidic cellulase for use in laundry detergent compositions wherein the detergent wash solution is generally alkaline. On the other hand, an acid cellulase can be used in a pre-washing step in the appropriate solution or at an intermediate pH where sufficient activity to provide cleaning benefits still exists. Alternatively, the cellulase could be employed as a pre-soak either as a liquid or a spray, for example, as a spot remover. Preferred cellulases for use in this invention are those obtained from Trichoderma reesei. T. koninαii. Pencitlum so., and the like. Certain cellulases are commercially available, i.e., CELLUCAST (available from Novo Industry, Copenhagen, Denmark), RAPIDASE (available from Gist Brocades, N.V., Delft, Holland), and the like. Other cellulases can be readily isolated by art recognized fermentation and isolation procedures.

Fungal cellulases enriched in CBH type components can be obtained by purification techniques. Specifically, the complete cellulase system can be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion, and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient.

Mixtures of cellulase components having the requisite ratio of CBH I type components to EG type cellulase components can 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 CBH to EG components.

However, in this regard, many attempts to modify the fermentation conditions for a natural microorganism in order to give relatively high ratios of CBH to EG components have failed, likely because CBH and EG components are coordinately regulated by the microorganism.

Likewise, recombinant techniques, as set forth in the examples, can alter the relative ratio of CBH I components to EG components so as to produce a mixture of cellulase components having a relatively high ratio of CBH I component to EG components.

In regard to the above, a preferred method for the preparation of cellulase compositions enriched in CBH type components is by genetically modifying a microorganism so as to be incapable of producing one or more EG type components and/or overproducing CBH

I type components preferably without producing any heterologous protein. For example, U.S. Serial No. 07/770,049, filed October 4, 1991 which is a continuation-in-part of U.S. Serial No. 07/593,919, filed October 5, 1990, both of which are incorporated herein by reference in their entirety, disclose methods for genetically engineering

Trichoderma reesei so as to be incapable of producing one or more EG components and/or overproducing one or more CBH components. Moreover, the methods of that application create Trichoderma reesei strains which do not produce any heterologous proteins. U.S. Serial No. 07/ , , filed April 3, 1992, Attorney Docket No. 010055-106, entitled "METHODS FOR PRODUCING SUBSTANTIALLY PURE EG III USING POLYETHYLENE GLYCOL," incorporated herein by reference, disclose methods of producing a T. reesei EG I and EG II deleted strain. Likewise, Miller et at., "Direct and Indirect Gene Replacement in Asperoillus nidulans". Molecular and Cellular Biology, p. 1714-1721

(1985) disclose methods for deleting genes in Asperoillus nidulans by DNA mediated transformation using a linear fragment of homologous DNA.

Additionally, a requisite amount of one or more EG 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 EG type components, so as to achieve a specified ratio of CBH I type components to one or more EG type components, i.e., a cellulase composition free of ail EG type components, so as to be enriched in CBH I type components, can be formulated to contain 1 weight percent of an EG type component merely by adding this amount of a purified EG type component to the ceilulase composition.

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 aikenyl ether sulfates having linear or branched alkyl groups or aikenyl groups, alkyl or aikenyl sulfates, olefinsulfonates, aikanesuifonates, 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 alkanoiamines having 1 to 3 alkanol groups of carbon number 2 or 3. Ampholytic surfactants include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.

Nonionic surfactants generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or aikyiene 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.

The surfactant is generally 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.

In addition to the cellulase components and the surface active agent, the detergent compositions of this invention can additionally contain the following components:

Hydrolase Except Cellulase

Such hydrolases include carboxylate ester hydrolase, thioester hydrolase, phosphate monoester hydrolase, and phosphate diester hydrolase which act on the ester bond; glycoside hydrolase which acts on giycosyi compounds; an enzyme that hydrolyzes N-glycosyl compounds; thioether hydrolase which acts on the ether bond; and σ- amino-acyi-peptide hydrolase, peptidyl-amino acid hydroiase, acyl-amino acid hydrolase, dipeptide hydroiase, 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, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinoiysin, renin, subtiiisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kaliikrein, 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) σ-amylase, β-amylase, giuco amyiase, inverτase, lysozyme, pectinase, chitinase, and dextranase. Preferably among them are σ-amylase and β-amyiase. They function in acid to neutral systems, but one which is obtained from bacteria exhibits high activity in an alkaline system; and (3) carboxylate ester hydrolase including carboxyl esterase, lipase, pectin esterase, and chlorophyllase. Especially effective among them is lipase.

Trade names of commercial products and producers are as follows: "Alkaiase", "Esperase", "Savinase", "AMG", "BAN", "Fungamill", "Sweetzyme", "Thermamyl" (Novo Industry, Copenhagen, Denmark); "Maksatase", "High-alkaline protease", "Amyiase THC", "Lipase" (Gist Brocades, N.V., Delft, Holland); "Protease B-400",

"Protease B-4000", "Protease AP", "Protease AP 2100" (Scheweizerische Ferment A.G., Basel, Switzerland); "CRD Protease" (Monsanto Company, St. Louis, Missouri); "Piocase" (Piopin Corporation, Monticello, Illinois); "Pronase P", "Pronase AS", "Pronase AF" ( aken Chemical Co., Ltd., Japan); "Lapidase P-2000" (Lapidas, Secran, France); protease products (Tyler standard sieve, 100% pass 16 mesh and 100% on 150 mesh) (Clington Corn Products, Division of Standard Brands Corp., New York); "Takamine", "Bromelain 1 :10", "HT

Protease 200", "Enzyme L-W" (obtained from fungi, not from bacteria) (Miles Chemical Company, Elkhart, Indiana); "Rhozyme P-1 1 Cone", "Pectinol", "Lipase B", "Rhozyme PF", "Rhozyme J-25" (Rohm & Haas, Genencor, South San Francisco, California); "Ambrozyme 200" (Jack Wolf &. Co., Ltd., Subsidiary of Nopco Chemical Company, Newark,

New Jersey); "ATP 40", "ATP 120", "ATP 160" (Lapidas, Secran, France); "Oripase" (Nagase & Co., Ltd., Japan).

The hydroiase other than cellulase is incorporated into the detergent composition as much as required according to the purpose. It should 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 enzyme. 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.

Cationic Surfactants and Long-Chain Fattv Acid Salts

Such cationic surfactants and long-chain fatty acid salts include saturated or unsaturated fatty acid salts, alkyl or aikenyi ether carboxylic acid salts, σ-sulfofatty acid salts or esters, amino acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substitueπts 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 saits.

Builders

A. Divalent Sequestering Agents

The composition may contain from about 0 to about 50 weight percent 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 dicarboxyiic acids, and aluminosiiicate salts. Suitable divalent sequestering agents 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 saits of the following compounds as the alkalis or inorganic electrolytes: silicates, carbonates and sulfates as well as organic alkalis such as triethanoiamine, diethanofamine, monoethanoiamine and trilsopropanolamiπe.

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 carboxymethylcellulose or/and polyethylene glycol with the ceilulase composition of the present invention provides for an especially useful dirt removing composition.

For removing the decomposition of carboxymethylcellulose by the ceilulase in the detergent, it is desirable that carboxymethylcellulose is granulated or coated before the incorporation in the composition.

Bleaching Agents

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 suifonated phthalocyanine further improves the deterging effects.

Bluing Agents and Fluorescent Dves

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.

Caking Inhibitors

The following caking inhibitors may be incorporated in the powdery detergent: p-toluenesuifonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium silicate (such as Micro-Cell of Johns Manvilie Co.), calcium carbonate and magnesium oxide.

Masking Agents for Factors Inhibiting the Cellulase Activity

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 inhibi¬ tors. 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 giuconoiactone 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 is unavoidable, it is necessary to avoid the direct contact of the saccharides with the cellulase by, for example, coating them.

Long-chain-fatty acid saits and cationic surfactants act as the inhibitors in some cases. However, the co-presence of these substances with the ceilulase is allowable if the direct contact of them is prevented by some means such as tabieting 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 saits, and calcium and its salts, potassium and its salts, sodium and its salts or monosaccharides 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'-butγlidenebis(6-tert-butyi-3-methylphenol), 2,2'-butγlidenebis(6- tert-butyl-4-methylphenoi), monostyrenated cresol, distyrenated cresoi, monostyrenated phenol, distyrenated phenol and 1 ,1 -bis(4- hydroxyphenyOcyciohexane.

Solubilizers

The solubilizers include, for example, lower alcohols such as ethanol, benzenesulfonate salts, lower alkylbenzenesulfonate saits such as p-toluenesulfonate salts, glycols such as propylene glycol, acetylbenzenesulfonate salts, acetamides, pyridinedicarboxyiic 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.

Aside from the above ingredients, perfumes, preservatives, dyes and the like can be used, if desired, with the detergent compositions of this invention.

When a detergent base used in the present invention is in the form of a powder the cellulase composition is preferably formulated as granules. Preferably, the granules can be formulated so as to contain cellulase protecting agent. See, for instance, U.S. Serial No. 07/642,669 filed January 17, 1991 , as Attorney Docket No. 010055- 073, and entitled "GRANULES CONTAINING BOTH AN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES." which application is incorporated herein by reference in its entirety. Likewise, the granules can be formulated so as to contain materials to reduce the rate of dissolution of the granules into the wash medium. Such materials and granules are disclosed in U.S. Serial No. 07/642,596 filed on January 17, 1991 as Attorney Docket No. GCS-171-US1 and entitled "GRANULAR COMPOSITIONS" which application is incorporated herein by reference in its entirety. With a highly dense, granular detergent base obtained such as by the spray-drying-granuiatioπ method, various ingredients may also be added after the preparation of the base. The granules or other detergent formulations containing the ceilulase composition can then be used for cleaning fabrics, imparting softening properties to the fabric and the like.

When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion.

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.

E X A M P L E S

Example 1 -- Fractionation of Cellulase Components

CYTOLASE 123 cellulase, a commercially available cellulase system (from Genencor international, Inc., South San Francisco, California) derived from wild type Trichodermia reesei, was fractionated. The normal distribution of cellulase components in this ceilulase system is as follows:

The fractionation was done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma

Chemical Company (St. Louis, Mo), QA Trisacryi M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Maryland ). CYTOLASE 123 ceilulase, 0.5g, was desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, was then loaded onto a column of 20 ml of QA Trisacryl M anion exchange resin equilibrated with 10 mM sodium phosphate buffer pH = 6.8. The fraction bound on this coiumn contained CBH I and EG I. These components were separated by gradient elution using an aqueous gradient containing from 0 to about 500 mM sodium chloride. The fraction not bound on this column contained CBH II, EG II and EG HI. These fractions were desaited using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 mi, was then ioaded onto a column of 20 ml of SP Trisacryl M cation exchange resin. CBH II, EG II, and EG III were eluted separately using an aqueous gradient containing from 0 to about 200 mM sodium chloride.

Following procedures similar to that of Example 1 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 T. koninqii, Penicillum so. and the like.

Example 2 -- Cleanliness Assay of Cellulase Compositions

Certain of the cellulase components isolated above were combined so as to provide for cellulase compositions having known ratios of CBH I components to EG components. These combinations were then employed in the swatch washing procedure set forth below. This procedure tests the ability of different cellulase detergent compositions to clean cotton swatches. In this procedure, the degree of cleaning is measured by the change (increase) in reflectance of the cotton swatches after washing as compared to its reflectance prior to washing. The iarger increase in reflectance is indicative of cleaner swatches. Also in this procedure, other than the use of different cellulase compositions, the conditions are identical. Materials

50 ml cap tubes

3 inch by 4 inch ciay soiled Swatches cut in quarters (depending upon stain, use 1 /4 size for ciay) Cellulase sample

Detergent (commercially available powder or liquid detergents)

Shakers

37°C room

50 mM sodium citrate or 50 mM sodium

Acetate, pH 4.8-5.0

Procedure

Gloves are worn when handling swatches in order to avoid introducing any foreign components onto the swatches.

Calculate ppm cellulase to add to each swatch tube.

Label swatches, include duplicates and controls.

Measure reflectance of each swatch.

Load 1 swatch per tube.

Pipet 25 mis of sodium citrate buffer per tube.

Pipet the calculated ppm cellulase into each tube.

Cap tubes. Shake each tube hard once.

Place tubes on shakers in 37°C room for 30 minutes.

Prepare a 1 :20 dilution of detergent in distilled water.

After 30 minute incubation with ceilulase, add 1 mi of the 1 :20 dilution of detergent to each tube.

Shake each tube hard once.

Place tubes back on shakers in 37°C room for 20 minutes.

Prepare a 1 :500 dilution of detergent in distilled water.

After incubation, rinse swatches in the tubes one time each with distilled water.

To each tube add 25 mis of the 1 :500 dilution of detergent in distilled water.

Shake each tube hard once.

Place tubes back on shakers in 37°C room for 20 minutes.

After incubation, rinse swatches in the tubes 2-3 times with distilled water. With tube partially filled with distilled water and capped, shake the tube vigorously a few times. Remove swatches from tube and rinse lightly one final time. Place swatch on paper towel and dry.

Measure reflectance of each swatch.

The results of this procedure are set forth in Table I below. This table indicates the increase in reflectance for detergent compositions employing the cellulase compositions having the amounts of EG II component indicated by the x-axis and the amounts of CBH I component indicated by the y-axis. TABLE I

(Values Reported are Reflectance Values)

The above data demonstrate that ratios of CBH I component to EG II component greater than 5:1 provide excellent cleaning of the cotton swatches at a level almost as good as ratios of CBH I component to E II component of 5:1 or less. In fact, a 50:1 ratio of CBH I component to EG II component provides about 91 percent of the cleaning ability o _ 5:1 ratio of these two cellulase components. Moreover, because the amount of EG components (in the presence of CBH components) are reduced relative to the cellulase system, the degradation potential of the detergent composition containing this ceilulase composition is reduced relative to detergent compositions containing cellulase compositions having greater amounts of EG components.

In comparison to the results set forth in Table I above, Table II below sets forth the increase in reflectance resulting from the use of a ceilulase system derived from Trichodermia reesei in the procedure set forth above. As noted in Example 1 above, such cellulase has an approximate ratio of 2.5: 1 of CBH I component to EG components (i.e EG I, EG II and EG III). TABLE II

ppm cellulase 0 50 100 ^" 200 500 1000 refit.¹ 17.75 52.05 61.55 63.9 66.15 70.55

^a = refit, means reflectance values.

The above data shows that the detergent compositions of this invention provide excellent cleaning of cotton swatches at a level almost on par with detergent compositions containing a cellulase system. For example, the reflectance resulting from using 500 ppm

CBH I component and 10 ppm EG II component in the above procedure was 56.85 (Table I) or about 86 percent of the reflectance resulting from using 500 ppm of the ceilulase system. This data further shows that excellent cleaning can be obtained in spite of the fact that a sizeable portion of the EG components have been removed from the composition.

Example 3 -- Cleanliness Assay of Cellulase Compositions

Certain of the cellulase components isolated above were combined so as to provide for cellulase compositions having known ratios of CBH I component to EG components. These combinations were then employed in the swatch washing procedure set forth in Example 2 above. As in Example 2 above, other than the use of different cellulase compositions, the conditions are identical. The results of this procedure are set forth in Table 111 below. Thi table indicates the increase in reflectance for cellulase compositions used in this procedure and which have the amounts of EG I and EG II components (comprised of equal amounts of EG I and EG II components) indicated by the x-axis and the amounts of CBH I component indicated by the y-axis. The CBH I composition may contain approximately 1 % of EG component impurities, but the amount of EG component has been indicated as O. The indicated amounts of EG I and EG ll should be added to this background contamination of EG components.

TABLE III

(Values Reported are Reflectance Values)⁰

ppm ppm EG I plus EG II^e

CBH I 0 5 10 20 40 100 200 400

10 17.5 14.7 20.2 17.3

20 28.4 25.7 31.1 30.1 30 32.75

50 55.4 56.7 55.7 50.5 62

100 63.3 68.3 60.1 51.2 200 58.l^d 60.8 61.7 61.1 57.4

42

500 36.4^e 62.1 66.1 66 63.5

1000 44.8^e

b = all reflectance values are the average of two duplicate runs; certain of the reflectance values reported herein have been rounded to the nearest tenth. c = 500 ppm EG I and EG II without CBH I gave a reflectance value of 17. d = the duplicate runs for this combination of CBH I component and EG components varied so substantially that both results are reported herein.

e = these cleaning results are possibly due to EG component impurities in the CBH I component of approximately 1 weight percent or less.

The above data together with the data taken from Example 2 demonstrates that ratios of CBH I component to EG components greater than 5: 1 provide excellent cleaning of the cotton swatches at a level on par with ratios of CBH I components to EG components of 5:1 or less. For example, in Table III, a 10:1 ratio of CBH I component to EG components, i.e., 100 ppm CBH I to 10 ppm EG I plus EG II, provides about 92 percent of the cleaning ability of a 5:1 ratio of these two cellulase components, i.e., 100 ppm CBH I to 20 ppm EG I plus EG II. Likewise, a 25:1 ratio of CBH I component to EG component, i.e., 500 ppm CBH I to 20 ppm EG I plus EG II, provides substantially the same level of cleaning as a 5:1 ratio of these two cellulase components i.e., 500 ppm CBH I to 100 ppm EG 1 plus EG II. Moreover, because the amount of EG components are reduced (in the presence of CBH components) relative to the ceilulase system, the degradation potential of the detergent composition containing this cellulase composition is reduced relative to detergent compositions containing cellulase compositions having greater amounts of EG components.

In comparison to the results set forth in Table III above, Table IV below sets forth the increase in reflectance resulting from the use of a cellulase system derived from Trichodermia reesei in the procedure set forth above. As noted in Example 1 above, such cellulase has an approximate ratio of 2.5: 1 of CBH I component to EG components, i.e., EG l, EG II and EG III.

TABLE IV

ppm cellulase

20 50 100 reflectance 32.5 42.2 57.7 values

The above data shows that the detergent compositions of this invention (e.g., containing an enriched fraction of CBH I type cellulase component relative to the EG components) are capable of providing a level of cleaning on par with a cellulase system in spite of the fact that a sizeable portion of the EG components have been removed from the composition.

Similarly, a CBH I type ceilulase component and EG components could be substituted in place of CBH I component and EG I and II components employed in Examples II and III to provide a degradation resistant detergent composition having excellent cleaning. Such CBH I type cellulase components can be obtained from T. koningii, Penicillium s_>. and the like. Example 4 ~ Softness Assay of Cellulase Compositions

Certain cellulase compositions were produced by genetically modifying the T. reesei microorganism so as to be incapable of producing one or more EG-type components which methods do not produce any heterologous protein. The method used to produce the cellulase compositions is disclosed in Examples 6-1 1 and U.S. Serial No. 07/770,049 filed October 4, 1991 , which is a continuation-in-part of U.S. Serial No. 07/593,919 filed October 5, 1990, both of which are incorporated herein by reference in their entirety. These combinations were then employed in the swatch washing procedure set forth below.

This procedure tests the ability of different cellulase detergent compositions to soften cotton swatches. In this procedure, the degree of softness is measured by whole fabric feel by a group of panelists.

The washing machine (Unimac 50 lb. capacity, rotary drum) was filled with 9.5 gallons of cold water. The buffer (42 grams of citric acid anhydrous and 101 grams of sodium phosphate dibasic) and 50 mis Triton™ X-114 were added to the washing machine. The temperature of the wash liquor was adjusted to 40°C and the test ceilulase composition added. Specifically, an appropriate amount to provide for 2000 ppm, 1000 ppm, 500 ppm, 250 ppm and 100 ppm of certain cellulase compositions was added in the final detergent wash. If required, the pH was then adjusted to pH 5.0 by adding citric acid or sodium phosphate. Three 100% cotton terry towels, 25" x 46", were washed for 45 minutes at 37 rpm at 60-80° C and then dried for 2 minutes at a maximum rpm of 460. The towels were rinsed in 24 gallons of water at 34°C for 5 minutes. The towels were again dried for 2 minutes at a maximum rpm of 460. The toweis were dried in a conventional drier for 50 minutes on the high temperature setting of approximately 60-80°C.

After washing, the towels were then labeled (to prevent panelists from ascertaining how the fabric had been treated) and tested for softness by a group of panelists by whole fabric feel and by mechanized test methods. The panelists evaluated the fabrics by a preference for "softer" and "rougher" fabric.

The first set of fabrics analyzed was treated with an EG I and EG II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to that described below, so as to be incapable of expressing EG I and EG II. Insofar as EG I and EG II comprises up to about 23 percent of the cellulase composition, deletion of this component results in enriched levels of all of the CBH components. Moreover, because EG components, other than EG I and

II, comprise no more than about 5 weight percent of the complete cellulase composition, the CBH I to EG ratio in the EG I/ll deleted cellulase composition was at least about 12:1 .

The second set of fabrics analyzed were tested with a control solution which did not contain a cellulase composition.

The test fabrics were judged for softness by six panelists, in two different tests, on the basis of whole fabric feel. The results of this test demonstrated that at least 250 ppm of an EG l/EG II deleted cellulase composition provides softening of the cotton fabric as compared to the control solution which did not contain a cellulase composition. This data shows that enhanced softening as compared to the control can be obtained even though a sizeable portion of the EG components have been removed from the ceilulase composition. Therefore, a cellulase composition comprising CBH I and EG components in a ratio of at least 5:1 will provide some softening.

The test fabrics were further judged for softness by another 8 panelists, also on the basis of whole fabric feel. It was found that while 100 ppm of EG l/EG II deleted ceilulase composition did not significantly soften the cotton fabric in a single treatment over that of the control, increasing the amount of EG l/EG II deleted cellulase in the treatment of the cotton fabric resulted in increased softening over the control. It is also contemplated that repeated treatments at these lower concentrations will also result in enhanced softening.

The fabrics treated with the cellulase compositions were also tested for stiffness of fabric by the cantilever test method as described in American Standard Test Manual Methods, Designation D 1388-64

(Reapproved 1975), which is incorporated herein by reference in its entirety. In summary, a strip of fabric is slid in a direction parallel to its long dimension so that its end projects from the edge of a horizontal surface. The length of overhang is measured when the tip of the test specimen is depressed under its own weight to the point where the line joining the tip to the edge of the platform makes an angle of 41.5° with the horizontal. One half of this length is the bending length of the specimen. The cube of this quantity multiplied by the weight per unit area of the fabric is the fiexural rigidity.

The results of this procedure demonstrated that cotton fabric treated with the EG I/ll deleted cellulase composition overall had reduced fiexural rigidity when compared to cotton fabric treated with the control solution which did not contain a cellulase composition. These results indicate that the fiexural rigidity of the fabric is decreased and thus softness is increased in fabric treated with a cellulase composition comprising at least a 10:1 ratio of CBH I to EG components over the control.

Example 5 -- Tensile Testing of Cotton Fabric treated with Cellulase

Cotton terry towels were treated with ceilulase compositions by the method described in Example 4 except a set of fabrics treated with whole cellulase was included in the analysis. Specifically, the composition analyzed was a complete fungal ceilulase composition (CYTOLASE 123 ceilulase, commercially available from Genencor International, Inc., South San Francisco, California) produced by wild- type Trichoderma reesei.

The test fabrics were tested for breaking load and elongation of the fabric by the test method as described in American Standard Test Manual Methods, Designation D 1682-64 (Reapproved 1975) which is incorporated herein by reference in its entirety. In summary, a specimen of fabric is cut to the width to be tested, a continually increasing load is applied longitudinally to the specimen, and the test is carried to rupture in a specific time. Values for the breaking load and elongation of the test specimen are obtained from machine scales or dials or autographic recording charts. The results of this procedure demonstrated that cotton fabric treated with the same total weight of the EG I/II deleted ceilulase composition had reduced strength loss compared to fabrics treated with the same total weight of the complete cellulase composition. Contrarily, as expected both cellulase compositions showed increased strength loss as compared to the control. Therefore, treatment of fabric with a ceilulase composition comprising at least a 10:1 ratio of CBH I to EG components will result in cleaning and some softening of the fabric with reduced strength loss.

Example 6 -- Selection for pyr4^' Derivatives of Trichoderma reesei

The PVΓ4 gene encodes orotidine-5'-monophosphate decarboxyiase, an enzyme required for the biosynthesis of uridiπe. 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 oyr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for oyr4 derivative strains using FOA. In practice, spores of T. reesei strain RL-P37 (Sheir-Neiss, G. and Montenecourt, B.S., Appl. Microbioi. Biotechnol., 20, p. 46-53 (1984)) were spread on the surface of a solidified medium containing 2 mg/mi 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 which specifically had a defective oyr4 gene, protoplasts were generated and transformed with a piasmid containing a wiid-type PVΓ4 gene (see

Examples 8 and 9). Following transformation, protoplasts were plated on medium lacking uridine. Subseαuent growth of transformed colonies demonstrated complementation of a defective oyr4 gene by the piasmid-borne oyr4 gene. In this way, strain GC69 was identified as a ^pyr4^" derivative of strain RL-P37.

Example 7 -- Preparation of CBHI 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 designed on the basis of the published sequence for this gene using known probe synthesis methods (Shoemaker et al., 1983b). The cbhl gene resides on a 6.5 kb Pstl fragment and was inserted into Psjl cut pUC4K (purchased from Pharmacia inc., Piscataway, New Jersey) replacing the Kan^r gene of this vector using techniques known in the art, which techniques are set forth in Maniatis et al. (1989) and incorporated herein by reference. The resulting plasmid, pUC4K::cbh1 was then cut with Hindlll and the larger fragment of about 6 kb was isolated and relegated to give pUC4K::cbh1 ΔH/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 Pstl fragment remains.

The T. reesei oyr4 gene was cloned as a 6.5 kb Hindlll fragment of genomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991 ) following the methods of Maniatis et ai., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour (1989). The plasmid pUC4K::cbhlΔH/H was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was iigated with the 6.5 kb Hindlll fragment containing the T. reesei oyr4 gene to give pΔCBHIβγr4. FIG. 1 illustrates the construction of this plasmid.

Example 8 ~ Isolation of Protoplasts

Mycelium was obtained by inoculating 100 mi of YEG (0.5% yeast extract, 2% glucose) in a 500 ml flask with about 5 x 10⁷ T. reesei GC69 spores (the oyr4^" derivative strain). The flask was then incubated at 37°C with shaking for about 16 hours. The myceiium was harvested by centrifugation at 2,750 x g. The harvested myceiium was further washed in a 1.2 M sorbitol solution and resuspended in 40 ml of a solution containing 5 mg/mi Novozym^R 234 solution (which is the trade name for a multicomponent enzyme system containing 1 ,3- aipha-giucanase, 1 ,3-beta-giucanase, lamiπarinase, xyiaπase, chitinase and protease from Novo Biolabs, Danbury, Connecticut); 5 mg/mf

MgS0₄.7H₂0; 0.5 mg/ml bovine serum albumin; 1 .2 M sorbitol. The protoplasts were removed from the cellular debris by filtration through Miracioth (Calbiochem Corp, La Jolla, California) and collected by centrifugation at 2,000 x g. The protoplasts were washed three times in 1.2 M sorbitol and once in 1.2 M sorbitol, 50 mM CaCI₂, centrifuged and resuspended at a density of approximately 2 x 10^β protoplasts per ml of 1 .2 M sorbitol, 50 mM CaCL. Example 9 -- Transformation of Fungal Protoplasts with PΔCBHIPVΓ4

200 μ\ of the protoplast suspension prepared in Example 8 was added to 20 μl of EcoRI digested DΔCBH1PVΓ4 (prepared in Example 7) in TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) and 50 μ\ of a polyethylene glycol (PEG) solution containing 25% PEG 4000, 0.6 M

KCI and 50 mM CaCl₂. This mixture was incubated on ice for 20 minutes. After this incubation period 2.0 mi 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

CaCI₂ was added thereto and this solution was further mixed. The protoplast solution was then immediately added to molten aiiquots of Vogel's Medium N (3 grams sodium citrate, 5 grams KH₂P0₄, 2 grams NH₄N0₃, 0.2 grams MgS0₄.7H₂0, 0.1 gram CaCl₂.2H₂0, 5 μg σ-biotin, 5 mg citric acid, 5 mg ZnS0₄.7H₂0, 1 mg Fe(NH ₂.6H₂0, 0.25 mg

CuSO,.5H₂0, 50 μg MnS04.4H20 per liter) containing an additional 1 % glucose, 1 .2 M sorbitol and 1 % agarose. The protoplast/medium mixture was then poured onto a solid medium containing the same Vogel's medium as stated above. No uridine was present in the medium and therefore only transformed colonies were able to grow as a result of complementation of the pyr4 mutation of strain GC69 by the wild type oyr4 gene insert in oΔCBHIpyr4. These colonies were subsequently transferred and purified on a solid Vogel's medium N containing as an additive, 1 % glucose and stable transformants were chosen for further analysis.

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

Example 10 -- Construction of PΔEGIPVΓ-3 and Transformation of a pyr4 Deficient Strain of T. reesei

The T. reesei eol gene, which encodes EGI has been cloned as a 4.2 kb Hindlll fragment of genomic DNA from strain RL-P37 by hybridization with oiigonucleotides synthesized according to the published sequence (Peπtilla et al., 1986, Gene, 45.: 253-263; van Arsdell et al., 1987, Bio/Technology, 5_: 60-64).

This DNA fragment was inserted at the Hindlll site of pUCI OO. An internal 1 kb EcoRV fragment which extended 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 by enzyme digestion and was replaced by ligation with a 2.2 kb BamHI - Hindlll fragment containing the cloned A. niger PVΓG gene (Wilson et al., 1988, Nucl. Acids Res., 16, p. 2339) to give pΔEGlpyrG-3 (FIG. 2). Transformation of a oyr4 deficient strain of T. reesei (strain GC69) by the method set forth in Examples 8 and 9, with pΔEGlpyr-3, after it had been digested with Hindlll to release the fragment containing the oyrG gene with flanking regions from the eoll locus at either end, led to transformants in which the genomic eoll gene was disrupted by a mechanism outlined in FIG. 3. DNA was extracted from transformants, digested with Hindlll. subjected to agarose gel electrophoresis and blotted onto a membrane filter. The fiiter was hybridized with radiolabeiled pΔEGlpyr-3. In the untransformed strain of T. reesei the eαll gene was present on a 4.2 k Hindlll fragment of DNA. However, following deletion of the eαll gene by integration of the desired fragment from pΔEGIpyr-3 this 4.2 kb Hindlll fragment disappeared and was replaced by a Hindlll fragment approximately 1.2 kb larger in size. This pattern was observed for one transformant which was designated ΔEGl-3.

Example 11 -- Construction of PAΔEGH-1 and Deletion of the EG II Gen

The eql3 gene, encoding EG II (also referred to in the literature a EG III), was cloned from T. reesei strain RL-P37 as a 4 kb Pstl genomic DNA fragment by hybridization with oligonucieotides synthesized according to the published sequence (Saioheimo et al., 1988, Gene, 63:1 1 -21 ). This DNA fragment was inserted into the Pstl site of pUC18. This plasmid, pEGlI, was subsequently digested with EcoRV t remove the entire EG II coding region on an approximately 2 kb segment extending from a position approximately 180 bp 5' of the EG coding region to a position a few hundred base pairs beyond the end o the coding region. This segment was replaced with an Ssol fragment of Asperoillus nidulans genomic DNA containing the amdS gene (Corric et al., 1987, Gene, 53:63-71 ) to create plasmid PAΔEGII-1 (See FIG. 4). Wild-type strains of T. reesei are unable to grow on acetamide as a sole nitrogen source. Transformation with the amdS gene confers this ability and this is the basis for the selection system for transformants containing this gene.

Protoplasts of strain ΔEGI-3 were transformed, by the methods described in Examples 8 and 9, with pAΔEGH-1 which had been digested with Hindlll and EcoRI and transformants able to grow on acetamide were selected. Subsequently, DNA was extracted from stable transformants, digested with Pstl. subjected to agarose gel electrophoresis and blotted onto a membrane filter. The filter was hybridized with radiolabelled pAΔEGll-1. Homologous integration of the Hjndlll-EcoRI fragment from pAΔEGII-1 , which contained eo!3 flanking regions and amdS. at the genomic eq!3 locus in a transformant lead to the 4 kb genomic Pstl fragment containing the eα!3 gene being replaced by smaller Pstl fragments including two which would be approximately

1.0 and 2.8 kb in length. This pattern of hybridization was observed for one transformant which was designated strain ΔΔEG-1. This strain has deletions in both the EGI and EGII encoding genes and consequently is unable to produce either of these proteins.

The methods described in Examples 6 to 11 and in U.S. Serial

No. 07/770,049, filed October 4, 1991 (incorporated herein by reference in its entirety) may be used to obtain T. reesei transformants which are unable to produce any or all of the following cellulase components; EG I, EG II, EG HI and CBH II components.

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit and scope thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof.

Claims

WHAT IS CLAIMED IS:

1. A detergent composition comprising at least one surface active agent and a cleaning effective amount of a cellulase composition wherein said cellulase composition contains a weight ratio of CBH I type cellulase components to EG type components of greater than about 10:1.

2. The detergent composition according to Claim 1 wherein said detergent composition is substantially free of CBH II type cellulase components.

3. The detergent composition according to Claim 2 wherein said weight ratio of CBH I type cellulase components to EG type components is about 20:1 or more.

4. The detergent composition according to Claim 3 wherein the weight ratio of said CBH I type ceilulase components to said EG type components is about 40:1 or more.

5. The detergent composition according to Claim 1 wherein said composition is a liquid.

6. The detergent composition according to Claim 1 wherein said composition is a powder.

7. The detergent composition according to Claim 1 wherein said CBH I type ceilulase components and said EG type components are derived from a microorganism selected from the group consisting of Trichoderma reesei. Penicillium so. and T. koningii.

8. The detergent composition according to Claim 7 wherein said CBH I type cellulase components and said EG type components are derived from Trichoderma reesei.

9. The detergent composition according to Claim 8 wherein said CBH I type cellulase components and said EG type components are derived from CYTOLASE 123 cellulase.

10. The detergent composition according to Claim 1 wherein said composition is used as a laundry detergent composition.

1 1 . The detergent composition according to Claim 1 wherein said composition is used as a spot remover.

12. The detergent composition according to Claim 1 wherein said composition is used as a presoak.

13. A method for enhancing the degradation resistance to cotton fabric of a detergent composition containing a cellulase which comprises employing a cellulase composition containing a weight ratio of CBH I type cellulase component to EG type components of greater than about 10:1.

14. The method according to Claim 13 wherein said CBH I type cellulase components are substantially free of CBH II type cellulase components.

15. The method according to Claim 14 wherein the weight ratio of said CBH I type cellulase components to said EG type components is about 20:1 or more.

16. The method according to Claim 15 wherein the weight ratio of said CBH I type cellulase components to said EG type components is about 40:1 or greater.

17. The method according to Claim 13 wherein said detergent composition is a liquid.

18. The method according to Claim 13 wherein said detergent composition is a powder.

19. The method according to Claim 13 wherein said CBH I type cellulase components and said EG type components are derived from a microorganism selected from the group consisting of Trichoderma reesei. Penicillium so. and T. koningii.

20. The method according to Claim 19 wherein said CBH I type cellulase components and said EG type components are derived from Trichoderma reesei.

21. The method according to Claim 20 wherein said CBH I type and said EG type components are derived from CYTOLASE 123 ceilulase.

22. The method according to Claim 13 wherein said detergent composition is a laundry detergent composition.

23. The method according to Claim 13 wherein said detergent composition is a presoak detergent composition.

24. The method according to Claim 13 wherein said detergent composition is a spot removing detergent composition.

25. A method for softening cotton fabric which comprises contacting said fabric with an aqueous solution containing a detergent composition comprising at least one surface active agent and a softening effective amount of a cellulase composition having a weight ratio of CBH I type cellulase component to EG type components of greater than about 10:1.

26. The method of Claim 25 wherein said CBH I type cellulase components are substantially free of CBH II type cellulase components.

27. The method of Claim 25 wherein the weight ratio of said CBH I type ceilulase components to said EG type components is about 20:1 or more.