Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Definitions

• the present invention is directed to improved methods for treating cotton-containing fabrics with cellulase as well as the fabrics produced from these methods.
• the improved methods of the present invention are directed to contacting cotton-containing fabrics with an aqueous solution containing a fungal cellulase composition substantially free of all CBH I type cellulase components.
• the resulting fabric possesses the expected enhancements in, for example, feel, appearance, and/or softening, etc., as compared to the fabric prior to treatment and the fabric also possesses decreased strength loss as compared to the fabric treated with a cellulase composition containing CBH I type cellulase components.
• cotton-containing fabrics can be treated with cellulase in order to impart desirable properties to the fabric.
• cellulase has been used to improve the feel and/or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, for imparting a stone washed appearance to cotton- containing denims and the like.
• a common problem associated with the treatment of such cotton-containing fabrics with a cellulase solution is that the treated fabrics exhibit significant strength loss as compared to the untreated fabric. Strength loss arises because the cellulase hydrolyzes cellulose ( ⁇ -l,4-glucan linkages) which, in turn, can result in a breakdown of a portion of the cotton polymer. As more and more cotton polymers are disrupted (broken down) , the tensile strength of the fabric is reduced.
• fungal sources of cellulase are known to secrete very large quantities of cellulase and further because fermentation procedures for such fungal sources as well as isolation and purification procedures for isolating the cellulase are well known in the art, it would be particularly advantageous to use such fungal cellulases in the methods for improving feel and/or appearance.
• the present invention is directed to the discovery that heretofore known methods for treating cotton-containing fabrics with fungal cellulases can be improved by employing a fungal cellulase composition which is substantially free of all CBH I type components.
• EG type components are capable of imparting enhancements to the treated fabric with regard to feel, appearance, softness, color enhancement, and/or stone washed appearance as compared to the fabric before treatment with such a cellulase composition.
• the present invention is directed to an improved method for the treatment of cotton- containing fabrics with a fungal cellulase composition wherein said improvement comprises employing a fungal cellulase composition which is substantially free of all CBH I type components.
• the fungal cellulase composition is free of all CBH I type and all CBH II type components.
• the fungal cellulase composition comprises at least about 10 weight percent and preferably at least about 20 weight percent of EG type components based on the total weight of protein in the cellulase composition.
• the present invention is directed to an improved method for the treatment of cotton-containing fabrics with an aqueous fungal cellulase solution wherein said method is conducted with agitation under conditions so as to produce a cascading effect of the cellulase solution over the fabric wherein said improvement comprises employing a fungal cellulase composition which is substantially free of all CBH I type components.
• the fungal cellulase composition is free of all CBH I type components and all CBH II type components.
• the fungal cellulase composition comprises at least about 10 weight percent and preferably at least about 20 weight percent of EG type components based on the total weight of protein in the cellulase composition.
• Cotton-containing fabrics treated by the methods of this invention have the expected enhancement(s) as compared to the fabric prior to treatment while exhibiting reduced strength loss as compared to the fabric treated with a fungal cellulase composition which contains CBH I type cellulase components.
• the reduced strength loss evidences that methods of this invention are strength loss resistant.
• the present invention is directed to a cotton-containing fabric treated in the methods of this invention as defined above.
• FIG. 1 is an outline of the construction of p ⁇ CBHIpyr.4.
• FIG. 2 illustrates deletion of the Tj. reesei gene by integration of the larger EcoRI fragment from p ⁇ CBHIpyr4 at the cbhl locus on one of the T. reesei chromosomes.
• FIG. 3 is an autoradiograph of DNA from T. reesei strain GC69 transformed with EcoRI digested p ⁇ CBHIpyr4 after Southern blot analysis using a 32 P labelled p ⁇ CBHIp_y_r4 as the probe.
• the sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.
• FIG. 4 is an autoradiograph of DNA from a l ⁇ . reesei strain GC69 transformed with EcoRI digested p ⁇ CBHIpyr4 using a 32 P labelled plntCBHI as the probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.
• FIG. 5 is an isoelectric focusing gel displaying the proteins secreted by the wild type and by transformed strains of Tj. reesei. Specifically, in FIG.5, Lane A of the isoelectric focusing gel employs partially purified CBHI from T.
• Lane B employs a wild type T ⁇ _ reesei
• Lane C employs protein from a T ⁇ _ reesei strain with the cbhl gene deleted
• Lane D employs protein from a T. reesei strain with the cbhl and cbh2 genes deleted.
• FIG. 5 the right hand side of the figure is marked to indicate the location of the single proteins found in one or more of the secreted proteins.
• BG refers to the ⁇ - glucosidase
• El refers to endoglucanase I
• E2 refers to endoglucanase II
• E3 refers to endoglucanase III
• Cl refers to exo-cellobiohydrolase I
• C2 refers to exo-cellobiohydrolase II.
• FIG. 6A is a representation of the T. reesei cbh2 locus, cloned as a 4.1 kb EcoRI fragment on genomic DNA and FIG. 6B is a representation of the cbh2 gene deletion vector pP ⁇ CBHII.
• FIG. 8 is a diagram of the plasmid pEGIpyr4.
• 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 40 Q C; as well as the activity profile of an enriched EG III cellulase composition derived from Trichoderma reesei over a pH range at 40°C.
• CBH I and II deleted acidic EG enriched fungal cellulase composition
• FIG. 10 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. 12 illustrates fiber removal results (based on panel test scores) for cotton-containing fabrics treated with varying concentrations (in ppm) of cellulase secreted by a wild type Trichoderma reesei and for a cotton fabric treated with cellulase secreted by a strain of Trichoderma reesei genetically engineered so as to be incapable of secreting CBH I and CBH II.
• FIG 18. is an autoradiograph of DNA from T ⁇ _ reesei strain P37P ⁇ 67P"1 transformed with Hindlll and BamHI digested pEGII::P-l.
• a Southern blot was prepared and the DNA was hybridized with an approximately 4kb PstI fragment of radiolabelled T.reesei DNA containing the ec.13 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 BgJ.II 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. 19 is a diagram of the plasmid pP ⁇ EGI-1.
• FIG. 20 is an autoradiograph of a Southern blot of DNA isolated from transformants of strain GC69 obtained with Hindlll 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 e ll gene has been disrupted
• Lane B shows a transformant in which p ⁇ EGIpyr-3 DNA has integrated into the genome but without disrupting the eqll gene.
• Lane D contains p ⁇ EGIpyr-3 digested with Hindlll to provide appropriate size markers. The sizes of marker DNA fragments are shown in kilobase pairs to the right of the figure.
• the methods of this invention are improvements in prior art methods for treating cotton-containing fabrics with cellulase.
• the improvement comprises using a specific cellulase composition which imparts the desired enhancemen (s) to the fabric while minimizing strength loss in the fabric.
• the companion material employed in the fabric can include one or more non-cotton fibers including synthetic fibers such as polyamide fibers (for example, nylon 6 and nylon 66) , acrylic fibers (for example, polyacrylonitrile fibers) , and polyester fibers (for example, polyethylene terephthalate) , polyvinyl alcohol fibers (for example, Vinylon) , polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers and aramid fibers. It is contemplated that regenerated cellulose, such as rayon, could be used as a substitute for cotton in the methods of this invention.
• synthetic fibers such as polyamide fibers (for example, nylon 6 and nylon 66) , acrylic fibers (for example, polyacrylonitrile fibers) , and polyester fibers (for example, polyethylene terephthalate) , polyvinyl alcohol fibers (for example, Vinylon) , polyvinyl chloride fibers, polyvinylidene chlor
• finishing means the application of a sufficient amount of finish to a cotton-containing fabric so as to substantially prevent cellulolytic activity of the cellulase on the fabric. Finishes are generally applied at or near the end of the manufacturing process of the fabric for the purpose of enhancing the properties of the fabric, for example, softness, drapability, etc. , which additionally protects the fabric from reaction with cellulases. Finishes useful for finishing a cotton-containing fabric are well known in the art and include resinous materials, such as melamine, glyoxal, or ureaformaldehyde, as well as waxes, silicons, fluorochemi ⁇ als and quaternaries. When so finished, the cotton-containing fabric is substantially less reactive to cellulase.
• 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 described herein are disclosed in British Patent No. 2 094 826A, the disclosure of which is incorporated herein by reference.
• Fungal cellulases are known to be comprised of several enzyme classifications having different substrate specificity, enzymatic action patterns, and the like. Additionally, enzyme components within each classification can exhibit different molecular weights, different degrees of glyco- sylation, different isoelectric points, different substrate specificity, etc.
• fungal cellulases can contain cellulase classifications which include endoglucanases (EGs) , exo-cellobiohydrolases (CBHs) , /3-glucosidases (BGs) , etc.
• a fungal cellulase composition produced by a naturally occurring fungal source and which comprises one or more CBH and EG components wherein each of these components is found at the ratio produced by the fungal source is sometimes referred to herein as a "complete fungal cellulase system" or a “complete fungal cellulase composition” to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism genetically modified so as to overproduce, underproduce, or not produce one or more of the CBH and/or EG components of cellulase.
• endoglucanase type components are those fungal cellulase components which impart improved feel, improved appearance, softening, color enhancement, and/or a stone washed appearance to cotton-containing fabrics (as compared to the fabric before treatment) when these components are incorporated into a medium used to treat the fabrics and which impart reduced strength loss to cotton- containing fabrics as compared to the strength loss arising from treatment with a similar cellulase composition but which additionally contains CBH I type components.
• Such endoglucanase type components may not include components traditionally classified as endoglucanases using activity tests such as the ability of the component (a) to hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (CMC) , thereby reducing the viscosity of CMC containing solutions, (b) to readily hydrolyze hydrated forms of cellulose such as phosphoric acid swollen cellulose (e.g., Walseth cellulose) and hydrolyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.).
• 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.
• endoglucanase type components as those components of fungal cellulase which possess similar textile activity properties as possessed by the endoglucanase components of Trichoderma reesei.
• combinations of EG type components may give a synergistic response in imparting enhancements to the cotton-containing fabrics as well as imparting reduced strength loss as compared to a single EG component.
• a single EG type component may be more stable or have a broader spectrum of activity over a range of pHs.
• 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.
• the EG type component may be derived from the same or different fungal sources.
• EG type components can be derived from bacterially derived cellulases.
• Exo-cellobiohydrolase type (“CBH type”) components refer to those fungal cellulase components which exhibit textile activity properties similar to CBH I and/or CBH II cellulase components of Trichoderma reesei.
• CBH type Exo-cellobiohydrolase type
• the CBH I and CBH II components of Trichoderma reesei alone do not impart any significant enhancements in feel, appearance, color enhancement and/or stone washed appearance to the so treated cotton-containing fabrics.
• the CBH I component of Trichoderma reesei imparts enhanced strength loss to the cotton-containing fabrics.
• CBH I type components and CBH II type components refer to those fungal cellulase components which exhibit textile activity properties similar to CBH I and CBH II components of
• Trichoderma reesei Trichoderma reesei. respectively.
• this includes the property of enhancing strength loss of cotton- containing fabrics when used in the presence of EG type components.
• the CBH I type components of Trichoderma reesei can impart an incremental cleaning benefit.
• the CBH I components of Trichoderma reesei. when used alone or in combination with EG type components, can impart an incremental softening benefit.
• exo-cellobiohydrolase type components could possibly not include components traditionally classed as exo-cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II from Trichoderma reesei.
• such components (a) are competitively inhibited by cellobiose (K; approximately ImM) ; (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.
• Fungal cellulases substantially free of all CBH I type components can be obtained by purification techniques.
• 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.
• ion exchange chromatography usually anion exchange chromatography
• cellulase composition substantially free of all CBH I type cellulase components means that the cellulase composition, based on the weight of protein, will contain less than 1 weight percent CBH I type cellulase components.
• cellulase compositions substantially free of all CBH I type components can be prepared by means other than isolation and recombination of the components.
• recombinant techniques can be used to prepare microorganisms which are incapable of producing any CBH I type components or which are incapable of producing any CBH type components.
• a preferred method for the preparation of cellulase compositions substantially free of CBH I type components is by genetically modifying a microorganism so as to be incapable of expressing CBH I type components which methods do not express any heterologous protein.
• the deletion of the genes responsible for producing CBH I type and/or CBH II type cellulase components would also have the effect of enriching the amount of EG type components present in the cellulase composition.
• the deletion of those genes responsible for producing CBH I and II type components would result in a cellulase composition free of CBH type components.
• fungal cellulase compositions can be used herein from fungal sources which produce an incomplete fungal cellulase composition.
• 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 (Academic Press, 1988) , disclose that brown rot fungi do not apparently produce CBH components, but it may be possible that one or more of these components are CBH I type components.
• ⁇ -Glucosidase (BG) components refer to those components of cellulase which exhibit BG activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides ("cellobiose”) and give glucose as the sole product.
• BG components do not adsorb onto or react with cellulose polymers. Furthermore, such BG components are competitively inhibited by glucose (K ; approximately ImM) .
• 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 not all aryl glucosidases are BG components, in that some do not hydrolyze cellobiose.
• PNPG p-nitrophenol B-D-glucoside
• the presence or absence of BG components in the cellulase composition can be used to regulate the activity of any CBH components in the composition (i.e., non-CBH I type components) .
• CBH components i.e., non-CBH I type components
• the absence of BG components in the cellulase composition will "turn-off" CBH activity when the concentration of cellobiose reaches inhibitory levels.
• one or more additives can be added to the cellulase composition to effectively "turn-off", directly or indirectly, CBH I type activity as well as other CBH activity.
• the resulting composition is considered to be a composition free of all CBH I type components if the amount of additive is sufficient to result in effectively no CBH I type activity.
• 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.
• 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.
• the BG component When employed in textile treatment solutions, the BG component is generally added in an amount sufficient to prevent inhibition by cellobiose of any CBH and EG components found in the cellulase composition.
• the amount of BG component added depends upon the amount of cellobiose produced in the textile composition which can be readily determined by the skilled artisan.
• the weight percent of BG component relative to any CBH type components present in the cellulase composition is preferably from about 0.2 to about 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 koningii. Pencillum sp. ,
• fungicola insolens 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.
• buffer refers to art recognized acid/base reagents which stabilize the cellulase solution against undesired pH shifts during the cellulase treatment of the cotton-containing fabric.
• cellulase activity is pH dependent. That is to say that a specific cellulase composition will exhibit cellulolytic activity within a defined pH range with optimal cellulolytic activity generally being found within a small portion of this defined range.
• the specific pH range for cellulolytic activity will vary with each cellulase composition. As noted above, while most cellulases will exhibit cellulolytic activity within an acidic to neutral pH profile, there are some cellulase compositions which exhibit cellulolytic activity in an alkaline pH profile.
• the pH of the initial cellulase solution could be outside the range required for cellulase activity. It is further possible for the pH to change during treatment of the cotton-containing fabric, for example, by the generation of a reaction product which alters the pH of the solution. In either event, the pH of an unbuffered cellulase solution could be outside the range required for cellulolytic activity. When this occurs, undesired reduction or cessation of cellulolytic activity in the cellulase solution occurs.
• a cellulase having an acidic activity profile is employed in a neutral unbuffered aqueous solution, then the pH of the solution will result in lower cellulolytic activity and possibly in the cessation of cellulolytic activity.
• the use of a cellulase having a neutral or alkaline pH profile in a neutral unbuffered aqueous solution should initially provide significant cellulolytic activity.
• the pH of the cellulase solution should be maintained within the range required for cellulolytic activity.
• One means of accomplishing this is by simply monitoring the pH of the system and adjusting the pH as required by the addition of either an acid or a base.
• the pH of the system is preferably maintained within the desired pH range by the use of a buffer in the cellulase solution.
• a sufficient amount of buffer is employed so as to maintain the pH of the solution within the range wherein the employed cellulase exhibits activity.
• the specific buffer employed is selected in relationship to the specific cellulase composition employed.
• the buffer(s) selected for use with the cellulase composition employed can be readily determined by the skilled artisan taking into account the pH range and optimum for the cellulase composition employed as well as the pH of the cellulase solution.
• the buffer employed is one which is compatible with the cellulase composition and which will maintain the pH of the cellulase solution within the pH range required for optimal activity.
• Suitable buffers include sodium citrate, ammonium acetate, sodium acetate, disodium phosphate, and any other art recognized buffers.
• the tensile strength of cotton-containing fabrics can be measured in a warp and fill direction which are at right angles to each other. Accordingly, the term "warp tensile strength” as used herein refers to the tensile strength of the cotton-containing fabric as measured along the length of the cotton-containing fabric whereas the term “fill tensile strength” refers to the tensile strength of the cotton-containing fabric as measured across the width of the cotton-containing fabric.
• the tensile strength of the resulting cotton- containing fabric treated with a cellulase solution is compared to its tensile strength prior to treatment with the cellulase solution so as to determine the strength reducing effect of the treatment.
• the tensile strength of cotton-containing fabrics is readily conducted following ASTM D1682 test methodology.
• Equipment suitable for testing the tensile strength of such fabrics include a Scott tester or an Instron tester, both of which are commercially available.
• Enhancements to the cotton-containing fabric is achieved by those methods heretofore used.
• cotton-containing fabrics having improved feel can be achieved as per Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al. , "Reformation of Cotton Fabric by Cellulase” and JTN December 1988 journal article “What's New — Weight Loss Treatment to Soften the Touch of Cotton Fabric”.
• the teachings of each of these references is incorporated herein by reference.
• methods for improving both the feel and appearance of cotton-containing fabrics include contacting the fabric with an aqueous solution containing cellulase under conditions so that the solution is agitated and so that a cascading effect of the cellulase solution over the cotton-containing fabric is achieved.
• Such methods result in improved feel and appearance of the so treated cotton- containing fabric and are described in U.S. Serial No. 07/598,506, filed October 16, 1990 and which is incorporated herein by reference in its entirety.
• the present invention is an improvement over prior art methods for treating cotton-containing fabrics insofar as the present invention employs a specific cellulase composition which minimizes strength loss in the treated fabric.
• the cellulase composition employed herein is a fungal cellulase composition substantially free of CBH I type components and preferably, substantially free of all CBH type components.
• the use of the cellulase compositions described herein also result in fabric/color enhancement of stressed cotton- containing fabrics.
• the fabric can become stressed and when so stressed, it will contain broken and disordered fibers. Such fibers detrimentally impart a worn and dull appearance to the fabric.
• the so stressed fabric is subject to fabric/color enhancement. This is believed to arise by removal of some of the broken and disordered fibers which has the effect of restoring the appearance of the fabric prior to becoming stressed.
• these cellulase compositions will cause less redeposition of dye. It is also contemplated that these anti-redeposition properties can be enhanced for one or more specific EG type component(s) as compared to other components.
• the fungal cellulase compositions described above are employed in an aqueous solution which contains cellulase and other optional ingredients including, for example, a buffer, a surfactant, a scouring agent, and the like.
• concentration of the cellulase composition employed in this solution is generally a concentration sufficient for its intended purpose. That is to say that an amount of the cellulase composition is employed to provide the desired enhancement(s) to the cotton-containing fabric.
• the amount of the cellulase composition employed is also dependent on the equipment employed, the process parameters employed (the temperature of the cellulase solution, the exposure time to the cellulase solution, and the like) , the cellulase activity (e.g., a cellulase solution will require a lower concentration of a more active cellulase composition as compared to a less active cellulase composition) , and the like.
• concentration of the cellulase composition can be readily determined by the skilled artisan based on the above factors as well as the desired effect.
• the concentration of the cellulase composition in the cellulase solution employed herein is from about 0.01 gram/liter of cellulase solution to about 10.0 grams/liter of cellulase solution; and more preferably, from about 0.05 grams/liter of cellulase solution to about 2 gram/liter of cellulase solution.
• the cellulase concentration recited above refers to the weight of total protein
• the concentration of buffer in the aqueous cellulase solution is that which is sufficient to maintain the pH of the solution within the range wherein the employed cellulase exhibits activity which, in turn, depends on the nature of the cellulase employed.
• concentration of buffer employed will depend on several factors which the skilled artisan can readily take into account.
• the buffer as well as the buffer concentration are selected so as to maintain the pH of the cellulase solution within the pH range required for optimal cellulase activity. In general, buffer concentration in the cellulase solution is about 0.005 N and greater.
• the concentration of the buffer in the cellulase solution is from about 0.01 to about 0.5 N, and more preferably, from about 0.05 to about 0.15 N. It is possible that increased buffer concentrations in the cellulase solution may enhance the rate of tensile strength loss of the treated fabric.
• the cellulase solution can optionally contain a small amount of a surfactant, i.e., less than about 2 weight percent, and preferably from about 0.01 to about 2 weight percent.
• a surfactant include any surfactant compatible with the cellulase and the fabric including, for example, anionic, non-ionic and ampholytic surfactants.
• Suitable anionic surfactants for use herein include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates and the like.
• Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having l to 3 alkanol groups of carbon number 2 or 3.
• Ampholytic surfactants include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.
• Nonionic surfactants generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like. Mixtures of such surfactants can also be used.
• the liquor ratios i.e., the ratio of weight of cellulase solution to the weight of fabric, employed herein is generally an amount sufficient to achieve the desired enhancement in the cotton-containing fabric and is dependent upon the process used and the enhancement to be achieve.
• the liquor ratios are generally from about 0.1:1 and greater, and more preferably greater than about 1:1 and even more preferably greater than about 10:1.
• reaction temperatures for cellulase treatment are governed by two competing factors. Firstly, higher temperatures generally correspond to enhanced reaction kinetics, i.e., faster reactions, which permit reduced reaction times as compared to reaction times required at lower temperatures. Accordingly, reaction temperatures are generally at least about 30°C and greater.
• cellulase is a protein which loses activity beyond a given reaction temperature which temperature is dependent on the nature of the cellulase used. Thus, if the reaction temperature is permitted to go too high, then the cellulolytic activity is lost as a result of the denaturing of the cellulase. As a result, the maximum reaction temperatures employed herein are generally about 65°C. In view of the above, reaction temperatures are generally from about 30°C to about 65°C; preferably, from about 35°C to about
• reaction times are generally from about 0.1 hours to about 24 hours and, preferably, from about 0.25 hours to about 5 hours.
• the cotton-containing fabrics treated in the methods described above using such cellulase compositions possess reduced strength loss as compared to the same cotton-containing fabric treated in the same manner with a complete fungal cellulase composition.
• a concentrate can be prepared for use in the methods described herein.
• Such concentrates would contain concentrated amounts of the cellulase composition described above, buffer and surfactant, preferably in an aqueous solution.
• the concentrate can readily be diluted with water so as to quickly and accurately prepare cellulase solutions having the requisite concentration of these additives.
• such concentrates will comprise from about 0.1 to about 20 weight percent of a cellulase composition described above (protein) ; from about 10 to about 50 weight percent buffer; from about 10 to about 50 weight percent surfactant; and from about 0 to 80 weight percent water.
• aqueous concentrates When aqueous concentrates are formulated, these concentrates can be diluted by factors of from about 2 to about 200 so as to arrive at the requisite concentration of the components in the cellulase solution. As is readily apparent, such concentrates will permit facile formulation of the cellulase solutions as well as permit feasible transportation of the concentration to the location where it will be used.
• the cellulase composition as described above can be added to the concentrate either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the like. Such forms are well known to the skilled artisan.
• the cellulase composition is generally a granule, a powder, an agglomeration, and the like.
• the granules can preferably be formulated so as to contain a cellulase protecting agent. See, for instance, U.S. Serial No.
• cellulase compositions described herein can additionally be used in a pre-wash and as a pre-soak either as a liquid or a spray. It is still further contemplated that the cellulase compositions described herein can also be used in home use as a stand alone composition suitable for enhancing color and appearance of fabrics. See, for example, U.S. Patent No. 4,738,682, which is incorporated herein by reference in its entirety.
• the pyr4 gene encodes orotidine-5'- monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine.
• the toxic inhibitor 5- fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells.
• FAA 5- fluoroorotic acid
• cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for p ⁇ r4 derivative strains using FOA.
• spores of T. reesei strain RL-P37 (Sheir-Neiss, G. and Montenecourt, B.S. , Appl. Microbiol. Biotechnol. 20, p.
• 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 PstI fragment and was inserted into PstI cut pUC4K (purchased from Pharmacia Inc.
• the T. reesei pyr4 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 al. , supra.
• the plasmid pUC4K: :cbhI ⁇ H/H was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was ligated with the 6.5 kb Hindlll fragment containing the T. reesei pyr4 gene to give p ⁇ CBHlp ⁇ r4.
• FIG. 1 illustrates the construction of this plasmid.
• Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2% glucose) in a 500 ml flask with about 5 x 10 7 T. reesei GC69 spores (the pyr4" derivative strain) . The flask was then incubated at 37 ⁇ C with shaking for about 16 hours. The mycelium was harvested by centrifugation at 2,750 x g.
• the harvested mycelium was further washed in a 1.2 M sorbitol solution and resuspended in 40 ml of a solution containing 5 mg/ml Novozym R 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 MgS0 4 .7H 2 0; 0.5 mg/ml bovine serum albumin; 1.2 M sorbitol.
• Novozym R 234 solution which is the tradename for a multicomponent enzyme system containing 1,3-alpha- glucanase, 1,3-beta-glucanase, laminarinase, xylanase, chitinase and prote
• 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 CaCl 2 , centrifuged and resuspended at a density of approximately 2 x 10 s protoplasts per ml of 1.2 M sorbitol, 50 mM CaCl 2 .
• the radioactive bands from the hybridization were visualized by autoradiography.
• the autoradiograph is seen in FIG. 3.
• Five samples were run as described above, hence samples A, B, C, D, and E.
• Lane E is the untransformed strain GC69 and was used as a control in the present analysis.
• Lanes A-D represent transformants obtained by the methods described above.
• the numbers on the side of the autoradiograph represent the sizes of molecular weight markers.
• 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.
• FIG. 2 outlines the deletion of the T.
• 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.
• the medium was incubated with shaking in a 250 ml flask at 37°C for about 48 hours.
• the resulting mycelium was collected by filtering through Miracloth (Calbiochem Corp.) and washed two or three times with 17 mM potassium phosphate.
• the mycelium was finally suspended in 17 mM potassium phosphate with 1 mM sophorose and further incubated for 24 hours at 30°C with shaking.
• the supernatant was then collected from these cultures and the mycelium was discarded.
• Samples of the culture supernatant were analyzed by isoelectric focusing using a Pharmacia Phastgel system and pH 3- 9 precast gels according to the manufacturer's instructions. The gel was stained with silver stain to visualize the protein bands.
• the band corresponding to the cbhl protein was absent from the sample derived from the strain P37P ⁇ CBHI, 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. 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.
• 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, Biotechnology. 5:274-
• T. reesei pyr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb Nhel-SphI fragment and inserted between the SphI and Xbal sites of pUC219 (see Example 25) to create p219M (Smith et al., 1991, Curr. Genet 19 p. 27-33) .
• the pyr4 gene was then removed as a Hindlll-Clal fragment having seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pP ⁇ CBHII (see FIG. 6B) .
• Protoplasts of strain GC69 will be generated and transformed with EcoRI digested pP ⁇ CBHII according to the methods outlined in Examples 3 and 4.
• DNA from the transformants will be digested with EcoRI and Asp718. and subjected to agarose gel electrophoresis.
• the DNA from the gel will be blotted to a membrane filter and hybridized with 32 P labelled pP ⁇ CBHII 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.
• 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.
• DNA was extracted from strain P37P ⁇ CBH67, digested with EcoRI and Asp718. and subjected to agarose gel electrophoresis. The DNA from this gel was blotted to a membrane filter and hybridized with 32 p labelled pP ⁇ CBHII (FIG. 7). Lane A of FIG. 7 shows the hybridization pattern observed for DNA from an untransformed T. reesei strain. The 4.1 kb EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization pattern observed for strain P37P ⁇ CBH67. The single 4.1 kb band has been eliminated and replaced by two bands of approximately 0.9 and 3.1 kb.
• the same DNA samples were also digested with EcoRI and Southern blot analysis was performed as above.
• the probe was 32 P labelled plntCBHII.
• This plasmid contains a portion of the cbh2 gene coding sequence from within that segment of the cbh2 gene which was deleted in plasmid 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 plasmid were present in this strain.
• the T. reesei egll gene which encodes EGI, has been cloned as a 4.2 kb Hindlll fragment of genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthesized according to the published sequence (Penttila et al., 1986, Gene 45:253-263; van Arsdell et al., 1987, Bio/Technology 5:60-64).
• a 3.6 kb Hindlll-BamHI fragment was taken from this clone and ligated with a 1.6 kb Hindlll- BamHI fragment containing the T.
• reesei pyr4 gene obtained from pTpyr2 (see Example 2) and pUC218 (identical to pUC219, see Example 25, but with the multiple cloning site in the opposite orientation) cut with Hindlll to give the plasmid pEGIpyr4 (FIG. 8) .
• Digestion of pEGIpyr4 with Hindlll would liberate a fragment of DNA containing only T. reesei genomic DNA (the egll and pyr4 genes) except for 24 bp of sequenced, synthetic DNA between the two genes and 6 bp of sequenced, synthetic DNA at one end (see FIG. 8) .
• CYTOLASE 123 cellulase was fractionated in the following manner.
• the normal distribution of cellulase components in this cellulase system is as follows:
• cellulase systems which can be separated into their components include CELLUCAST (available from Novo Industry, Copenhagen, Denmark) , RAPIDASE (available from Gist Brocades, N.V. , Delft, Holland) , and cellulase systems derived from Trichoderma koningii. Penicillu sp. and the like.
• Example 13 above demonstrated the isolation of several components from Cytolase 123 Cellulase. However, because EG III is present in very small quantities in Cytolase 123 Cellulase, the following procedures were employed to isolate this component.
• A. Large Scale Extraction of EG III Cellulase Enzyme One hundred liters of cell free cellulase filtrate were heated to about 30°C. The heated material was made about 4% wt/vol PEG 8000 (polyethylene glycol, MW of about 8000) and about 10% wt/vol anhydrous sodium sulfate. The mixture formed a two phase liquid mixture. The phases were separated using an SA-1 disk stack centrifuge. The phases were analyzed using silver staining isoelectric focusing gels. Separation was obtained for EG III and xylanase. The recovered composition contained about 20 to 50 weight percent of EG III.
• 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) .
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• 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) .
• CYTOLASE 123 cellulase 0.5g is desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, is then loaded onto a column of 20 ml of QA Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I. The fraction not bound on this column contains CBH II, EG II and EG III. These fractions are desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto a column of 20 ml of SP Trisacryl M cation exchange resin. The EG III was eluted with 100 mL of an aqueous solution of 200 mM sodium chloride.
• Trichoderma 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.
• the EG III compositions described above may 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.
• 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 pumps, a dual path monitor, a fraction collector and a chart recorder (all of which are available from Pharmacia LKB Biotechnology, Piscataway, NJ) .
• the fractionation was conducted by desalting 5 ml of the EG III sample prepared in part b) of this Example 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.
• 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.
• EG components have the following characteristics:
• the first cellulase composition was a CBH I and II deleted cellulase composition prepared 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.
• this cellulase composition does not contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase composition derived from Trichoderma reesei.
• this cellulase composition is necessarily substantially free of CBH I type and CBH II type cellulase components and accordingly, is enriched in EG components, i.e., EG I, EG II, EG III and the like.
• the second cellulase composition was an approximately 20 to 40% pure fraction of EG III isolated from a cellulase composition derived from Trichoderma reesei via purification methods similar to part b) of Example 14.
• FIG. 9 illustrates the relative activity of the CBH I and II deleted cellulase composition compared to the EG III cellulase composition. From this figure, it is seen that the cellulase composition deleted in CBH I and CBH II possesses optimum cellulolytic activity against RBB-CMC at near pH 5.5 and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8. On the other hand, the cellulase composition enriched in EG III possesses optimum cellulolytic activity at pH 5.5 - 6 and possesses significant activity at alkaline pHs.
• This example examines the ability of different cellulase compositions to reduce the strength of cotton-containing fabrics.
• This example employs an aqueous cellulase solution maintained at pH 5 because the activity of the most of the cellulase components derived from Trichoderma reesei is greatest at or near pH 5 and accordingly, strength loss results will be most evident when the assay is conducted at about this pH.
• the first cellulase composition analyzed was a complete fungal cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma reesei and is identified as GC010.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the second cellulase composition analyzed was a CBH II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to Examples 1 to 12 above and 22 to 30 below so as to be incapable of expressing CBH II and is identified as CBHIId.
• CBH II comprises up to about 15 percent of the cellulase composition, deletion of this component results in enriched levels of CBH I, and all of the EG components.
• the third cellulase composition analyzed was a
• the cellulase compositions described above were tested for their effect on cotton-containing fabric strength loss in a launderometer.
• the compositions were first normalized so that equal amounts of EG components were used.
• Each cellulase composition was then added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer, titrated to pH 5, and which contains 0.5 ml of a non-ionic surfactant.
• Each of the resulting solutions was then added to a separate launderometer canister.
• Into these canisters were added a quantity of marbles to facilitate strength loss as well as a 16 inch x 20 inch cotton fabric (100% woven cotton, available as Style No. 467 from Test Fabrics, Inc. , 200 Blackford Ave.
• the canister was then closed and the canister lowered into the launderometer bath which was maintained at 43°C.
• the canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
• strength loss resistant cellulase compositions are those compositions free of all CBH I type cellulase components and preferably, all CBH type cellulase components.
• cellulase compositions will result in even lower strength loss at pH > 7 than those results observed at pH 5 shown in FIG 10.
• the fabric can become stressed and when so stressed, it will contain broken and disordered fibers. Such fibers detrimentally impart a worn and dull appearance to the fabric.
• the methods of this invention will result in fabric/color enhancement. This is believed to arise by removal of some of the broken and disordered fibers which has the effect of restoring the appearance of the fabric prior to becoming stressed.
• Examples 17 and 18 illustrate this benefit of the present invention. It is noted that these example employed worn cotton T-shirts (knits) as well as new cotton knits.
• the faded appearance of the worn cotton-containing fabric arises from the accumulation on the fabric of broken and loose surface fibers over a period of time. These fibers give rise to a faded and matted appearance for the fabric and accordingly, the removal of these fibers is a necessary prerequisite to restoring the original sharp color to the fabric. Additionally, the accumulation of broken surface fibers on new cotton knits imparts a dull appearance to such fabrics. Accordingly, these experiments are necessarily applicable to color enhancement of stressed cotton-containing fabrics because both involve removal of surface fibers from the fabric.
• the ability of EG components to enhance color in cotton-containing fabrics was analyzed in the following experiments. Specifically, the first experiment measures the ability of a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma 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.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the canister was then closed and the canister lowered into the launderometer bath which was maintained at 43°C.
• the canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
• the 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.
• 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 still be effected merely by using higher concentrations of cellulase.
• 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.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the second cellulase composition analyzed was a cellulase composition substantially free of all CBH type components (including CBH I type components) which composition was prepared from Trichoderma 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/II deleted.
• 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.
• 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 20 mM citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100% woven cotton, available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) .
• the canister was then closed and the canister lowered into the launderometer bath which was maintained at 43°C.
• the canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
• FIG. 12 is plotted based on estimated EG concentrations. Specifically, FIG. 12 illustrates that both GC010 and CBH I/II Deleted cellulase compositions gave substantially identical fiber removal results at substantially equal EG concentrations. The results of this figure suggest that it is the EG components which provide for fiber removal. These results coupled with the results of FIG. 11 demonstrate that EG components remove surface fibers.
• Example 17 This example is further to Example 17 and substantiates that CBH type components are not necessary for color enhancement and the purpose of this example is to examine the ability of cellulase compositions deficient iri CBH type components to enhance color to cotton-containing fabrics.
• the cellulase composition employed in this example was substantially free of all CBH type components (including CBH I type components) insofar as this composition was prepared from Trichoderma reesei genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II.
• CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG components.
• the assay was conducted by adding a sufficient concentration of this cellulase composition to a 50 mM citrate/phosphate buffer to provide 500 ppm of cellulase.
• the solution was titrated to pH 5 and contained 0.1 weight percent of nonionic surfactant (Grescoterg GL100 — commercially available from Gresco Mfg., Thomasville, NC 27360).
• a 10 inch x 10 inch faded cotton-containing fabric as well as a 10 inch x 10 inch new knitted fabric having loose and broken surface fibers were then placed into 1 liter of this buffer and allowed to sit at 110°F for 30 minutes and then agitated for 30 minutes at 100 rotations per minute.
• the fabrics were then removed from the buffer, washed and dried. The resulting fabrics were then compared to the fabric prior to treatment.
• the results of this analysis are as follows:
• cellulase compositions would be beneficial during fabric processing because such compositions would remove broken/loose fibers generated during processing without detrimental strength loss to the fabric.
• 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
• the cellulase composition described above was tested for its ability to soften these swatches in a launderometer. Specifically, an appropriate amount of cellulase to provide for 500 ppm, 250 ppm, 100 ppm, 50 ppm, and 10 ppm cellulase in the final cellulase solution was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.025 weight percent of a non-ionic surfactant (Triton X114) .
• Triton X114 Triton X114
• 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 solution was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate softness as well as cotton swatches described above. All conditions were run in triplicate with two swatches per canister. Each canister was then closed and the canister lowered into the launderometer bath which was maintained at 37°C. The canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the swatches were removed, rinsed well and dried in a standard drier.
• rpms revolutions per minute
• FIG. 13 Specifically, these results demonstrate that at higher concentrations, improved softening is obtained. It is noted that this improved softening is achieved without the presence of either CBH I or II in the cellulase composition.
• This example demonstrates that the presence of CBH type components are not essential for imparting improved feel and appearance to cotton-containing fabrics.
• this example employs a cellulase composition derived from Trichoderma reesei genetically engineered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and II components) .
• This cellulase composition was tested for its ability to improve the appearance of cotton- containing fabrics. Specifically, appropriately sized 100% cotton sheeting (available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the appearance aspects of this example. The cellulase composition described above was tested for its ability to improve the appearance of these samples in a launderometer.
• CBH I and II deleted cellulase an appropriate amount of CBH I and II deleted cellulase to provide for 25 ppm, 50 ppm, and 100 ppm cellulase in the final cellulase solution was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.025 weight percent of a non-ionic surfactant (Triton X114) . Additionally, a blank was run containing the same solution but with no added cellulase. Samples so prepared were titrated to pH 5. Each of the resulting solutions was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate improvements in appearance as well as cotton samples described above.
• Each canister was then closed and the canister lowered into the launderometer bath which was maintained at about 40°C.
• the canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the samples were removed, rinsed well and dried in a standard drier.
• the samples were then analyzed for improved appearance by evaluation in a preference test.
• the CBH I and II deleted cellulase composition was then tested for its ability to improve the feel of cotton-containing fabrics. Specifically, appropriately sized 100% cotton sheeting (available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846) were employed in the feel aspects of this example.
• the cellulase composition described above was tested for its ability to improve the feel of these samples in a launderometer. Specifically, an appropriate amount of cellulase to provide for 500 ppm, 1000 ppm, and 2000 ppm cellulase in the final cellulase solution was added to separate solutions of 24 L of a 20 nH citrate/phosphate buffer. Additionally, a blank was run containing the same solution but with no added cellulase. All tests were conducted at pH 5.8 and run in an industial washer. The washer was operated at 50°C, a total volume of 24 L, a liquor to cloth ratio of 50:1 (weight to weight) and the washer was run for 30 minutes. Afterwards, the samples were removed and dried in an industrial dryer.
• the samples were then analyzed for improved feel by evaluation in a preference test. Specifically, 5 panelists were given the 4 samples (not identified) and asked to rate them with respect to feel. The panelists were instructed that fabrics having improved feel are smoother and silkier to the touch than other fabrics and that feel is distinguished from qualities such as softness (which refers to the pliability of the fabric rather than its feel) , thickness, color, or other physical characteristics not involved in smoothness of the fabric.
• the panelists then assigned scores to each sample based on its order relative to the other samples; 4 having the best feel and 1 having the worst feel.
• the scores from each panelists were cumulated and then averaged. The results of this test are as follows:
• this example demonstrates that the presence of CBH type components are not essential for imparting a stone washed appearance to cotton-containing fabrics.
• this example employs a cellulase composition derived from Trichoderma reesei genetically engineered in the manner described above so as to be incapable of producing any CBH type components (i.e., incapable of producing CBH I and II components) as well as a complete cellulase composition derived from Trichoderma reesei and which is available as Cytolase 123 cellulase from Genencor International, South San Francisco, California.
• cellulase compositions free of CBH I type components and derived from microorganisms other than Trichoderma reesei could be used in place of the cellulase compositions described in these examples.
• the source of the cellulase composition containing the EG type components is not important to this invention and any fungal cellulase composition containing one or more EG type components and substantially free of all CBH I type components can be used herein.
• fungal cellulases for use in preparing the fungal cellulase compositions used in this invention can be obtained from Trichoderma koningii. Pencilium sp.
• BCA bovine serum albumin
• the transformants described in this Example were obtained using intact pEGlpyr4 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 pEG!pyr4 with Hindlll and isolate the larger DNA fragment containing only T. reesei DNA. Transformation of T. reesei with this isolated fragment of DNA would allow isolation of transformants which 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 pEGlpyr4 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,
• Example 22 could be used to produce T. reesei strains which would over-produce any of the other cellulase components, xylanase components or other proteins normally produced by T. reesei.
• a plasmid, pCEPCl was constructed in which the coding sequence for EGI was functionally fused to the promoter from the cbhl gene. This was achieved using in vitro, site-specific mutagenesis to alter the DNA sequence of the cbhl and egll genes in order to create convenient restriction endonuclease cleavage sites just 5' (upstream) of their respective 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 plasmid, pCEPCl was designed so that the
• EGI coding sequence would be integrated at the cbhl locus, replacing the coding sequence for CBHI without introducing any foreign DNA into the host strain. Digestion of this plasmid with EcoRI liberates a fragment which includes the cbhl promoter region, the egll coding sequence and transcription termination region, the j. reesei pyr4 gene and a segment of DNA from the 3' (downstream) flanking region of the cbhl locus (see Fig. 15) .
• a pyr4 defective strain of T_ s _ reesei RutC30 (Sheir-Neiss, supra) 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 22.
• isoelectric focusing gel electrophoresis was performed on samples from these cultures using the method described in Example 7.
• 12 were found to produce no CBHI protein, which is the expected result of integration of the pCEPCl DNA 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 sequences derived from the bacterial plasmid vector (pUC4K) were present (see Fig. 16) .
• the DNA from the transformants was digested with PstI 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 a 6.5 kb fragment of DNA from the untransformed control culture (FIG. 16, lane A). Integration of the pCEPCl fragment of DNA at the 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.
• lane C 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 22 except that the samples were diluted 50 fold prior to the assay so that the protein concentration in the samples was between approximately 0.03 and 0.07 mg/ml.
• the results of assays performed with the untransformed control culture and four different transformants are shown in Table 2.
• CEPC1-112 are examples in which integration of the CEPC1 fragment had led to loss of CBHI production.
• the eg!3 gene encoding EGII (previously referred to as EGIII by others) , has been cloned from T. reesei and the DNA sequence published (Saloheimo et al., 1988, Gene 63:11-21).
• the latter vector, pUC219 is derived from pUC119 (described in Wilson et al., 1989, Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for BglII r Clal and Xhol.
• the T ⁇ . reesei pyr4 gene present on a 2.7 kb Sail fragment of genomic DNA, was inserted into a Sail site within the EGII coding sequence to create plasmid pEGII::P-l (FIG. 17) .
• the plasmid, pEGII::P-l can be digested with Hindlll and BamHI to yield a linear fragment of DNA derived exclusively from ⁇ _ reesei except for 5 bp on one end and 16 bp on the other end, both of which are derived from the multiple cloning site of pUC219.
• T. reesei strain GC69 will be transformed with pEGII::P-l which had been previously digested with Hindlll 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 p ⁇ r4 and eg!3 genes had integrated at the eg!3 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 transform a strain which was deleted for either or all of the cbhl. cbh2. or egll genes. In this way a strain could be constructed which would only produce certain cellulase components and no EGII component.
• Example 27
• P37P ⁇ CBH67 (from 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 Hindlll and BamHI and stable transformants were selected. Total DNA was isolated from transformants and Southern blot analysis used to identify strains in which the fragment of DNA containing the pyr4 and eg!3 genes had integrated at the eg!3 locus and consequently disrupted the EGII coding sequence. The Southern blot illustrated in FIG. 18 was probed with an approximately 4 kb PstI fragment of T ⁇ .
• the size of the band corresponding to the eg!3 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37P ⁇ 67P"1 strain (Lanes A and C) and the transformant disrupted for egl3 (FIG. 18, Lanes B and D) .
• the transformant containing the disrupted eg!3 gene illustrated in FIG. 18 was named A22.
• the transformant identified in FIG. 18 is unable to produce CBHI, CBHII or EGII.
• the egll gene of T. reesei strain RL-P37 was obtained, as described in Example 12, as a 4.2 kb Hindlll fragment of genomic DNA. This fragment was inserted at the Hindlll site of pUClOO (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 Bglll. Clal and Xhol) .
• the plasmid pP ⁇ EGI-1 can be digested with Hindlll to release a DNA fragment comprising only T. reesei genomic DNA having a segment of the egll gene at either end and the pyr4 gene replacing part of the EGI coding sequence, in the center.
• Example 28 The expectation that the EGI gene could be inactivated using the method outlined in Example 28 is strengthened by this experiment.
• a plasmid, p ⁇ EGIpyr-3 was constructed which was similar to pP ⁇ EGI-1 except that the Aspergillus niger p ⁇ r4 gene replaced the T reesei p ⁇ r4 gene as selectable marker.
• the egll gene was again present as a 4.2 kb Hindlll fragment inserted at the Hindlll site of pUClOO.
• Stable pyr4-t- transformants will be selected and total DNA isolated from the transformants.
• the DNA will be probed with 32 P labelled pP ⁇ EGI-1 after Southern blot analysis in order to identify transformants in which the fragment of DNA containing the pyr4 gene and egll sequences has integrated at the egll locus and consequently disrupted the EGI coding sequence.
• the transformants identified will be unable to produce CBHI, CBHII, EGI and EGII.

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