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WO1994023113A1 - Procedes de reduction de la formation de coton egraine lors du traitement de tissus cellulosiques contenant du coton et ne contenant pas de coton - Google Patents

Procedes de reduction de la formation de coton egraine lors du traitement de tissus cellulosiques contenant du coton et ne contenant pas de coton Download PDF

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Publication number
WO1994023113A1
WO1994023113A1 PCT/US1994/003407 US9403407W WO9423113A1 WO 1994023113 A1 WO1994023113 A1 WO 1994023113A1 US 9403407 W US9403407 W US 9403407W WO 9423113 A1 WO9423113 A1 WO 9423113A1
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WIPO (PCT)
Prior art keywords
cellulase
fabric
cotton
cbh
components
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PCT/US1994/003407
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English (en)
Inventor
Kathleen A. Clarkson
Edmund A. Larenas
Geoffrey L. Weiss
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Genencor International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genencor International, Inc. filed Critical Genencor International, Inc.
Priority to EP94912897A priority Critical patent/EP0692041A1/fr
Publication of WO1994023113A1 publication Critical patent/WO1994023113A1/fr

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    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms

Definitions

  • the present invention is directed to improved methods for treating cotton-containing and non-cotton-containing cellulosic fabric during manufacture of the fabric without excessive lint generation and excessive fabric weight loss during treatment.
  • the improved methods of the present invention comprise contacting the fabric with an effective amount of a fungal cellulase composition comprising CBH type components and EG type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein lint production is reduced.
  • cotton-containing fabrics can be treated with a complete cellulase composition in order to impart desirable properties to the fabric.
  • a complete cellulase composition has been used to improve the feel and/or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, and to impart a stone washed appearance to cotton-containing denims and the like.
  • Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al., "Reformation of Cotton Fabric by Cellulase” and JTN December 1988 journal article "What's New — Weight Loss Treatment to Soften the Touch of Cotton Fabric” each disclose that treatment of cotton- containing fabrics with complete cellulase compositions results in an improved feel for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing and/or surface fibers. The combination of these effects imparts improved feel to the fabric, i.e., the fabric feels more like silk.
  • a common problem associated with the treatment of such cotton-containing fabrics with an aqueous solution containing a complete cellulase composition is that such treatment can result in the production of an excessive amount of lint which can cause problems with further handling of the fabric, such as cutting and sewing of the fabric. Also, excessive lint can result in equipment damage and lint released into the atmosphere of the plant can pose a serious health problem to workers.
  • 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 treatment of cotton-containing fabric.
  • the present invention is directed to the novel and unexpected discovery that known methods for treating cotton- containing and non-cotton-containing cellulosic fabrics with complete fungal cellulases can be improved to reduce lint production by contacting said fabric with an e fective amount of a fungal cellulase composition comprising a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions which further inhibit lint production.
  • EG type components are capable of imparting enhancements to the treated fabric with regard to feel, appearance, softness, color enhancement, and/or a stone- washed appearance as compared to fabric before treatment with such a cellulase composition. Additionally, it has been found that it is the CBH type components in combination with the EG type components which account for a sizable portion of the production of lint and the loss of fabric weight during treatment of the fabric. Further, it has been found that while use of EG type components reduces lint production, further reductions in lint production can be achieved by using conditions which result in the reduced production of lint. Accordingly, in the present invention, the cellulase composition employed to treat cotton-containing fabrics and non-cotton-containing cellulosic fabrics is tailored so as to be enriched for EG type components and the treatment conditions are tailored to reduce lint production.
  • the present invention is directed to a method for treating cotton- containing and non-cotton-containing cellulosic fabric during manufacture of the fabric, with a fungal cellulase composition naturally comprising CBH type and EG type components, wherein the improvement comprises contacting the fabric with an effective amount of a fungal cellulase composition having a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 under conditions wherein excess lint production is reduced.
  • the fungal cellulase composition comprises at least 30 weight percent of EG type components based on the total weight of protein in the cellulase composition.
  • the present invention is directed to a method for treating cotton-containing and non-cotton-containing cellulosic fabric with a fungal cellulase composition naturally comprising CBH type and EG type components in order to impart enhancement with regard to feel, appearance, softness, color enhancement, and/or a stone- washed appearance to the fabric
  • a fungal cellulase composition naturally comprising CBH type and EG type components in order to impart enhancement with regard to feel, appearance, softness, color enhancement, and/or a stone- washed appearance to the fabric
  • said cellulase composition comprises a protein weight ratio of all EG type components to all CBH type components of greater than 5:1 wherein said contacting is conducted at a predetermined place in a continuous manufacturing process for said cotton-containing fabric and under conditions wherein excess lint production is reduced.
  • the fungal cellulase composition comprises at least 30 weight percent of EG type components based on the total weight of protein in the cellulase composition.
  • Cotton-containing and non-cotton-containing cellulosic fabrics treated by the methods of this invention have the desired enhancement(s) of improved softness, feel and appearance, and/or a stone-washed appearance and/or surface polishing, with a reduction in the amount of lint and the amount of fabric weight loss produced during treatment of the fabric as compared to the amount of lint and the amount of fabric weight loss produced when fabrics are treated with fungal cellulase compositions containing greater amounts of CBH type components.
  • FIG. 1 is an outline of the construction of p ⁇ CBHIpyr..
  • FIG. 2 illustrates deletion of the T. longibrachiatum gene by integration of the larger .EcoRI fragment from p ⁇ CBHIpyr4 at the cJb l locus on one of the T. longibrachiatum chromosomes.
  • FIG. 3 is an autoradiograph of DNA from T. longibrachiatum strain GC69 transformed with EcoRI digested p ⁇ CBHIpyr- after Southern blot analysis using a 32 P labelled p ⁇ CBHIpyr- 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 T. longibrachiatum strain GC69 transformed with .EcoRI digested p ⁇ CBHIpy 4 using a P labeled 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 r. longibrachiatum.
  • Lane A of the isoelectric focusing gel employs partially purified CBH I from T. longibrachiatum; Lane B employs a wild type T. longibrachiatum: Lane C employs protein from a T. longibrachiatum strain with the cbhl gene deleted; and Lane D employs protein from a T. longibrachiatum strain with the cbhl and cbh2 genes deleted.
  • 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 3-glucosidase
  • El refers to endoglucanase I
  • E2 refers to endoglucanase II
  • E3 refers to endoglucanase III
  • CI refers to exo-cellobiohydrolase I
  • C2 refers to exo-cellobiohydrolase II.
  • FIG. 6A is a representation of the T. longibrachiatum 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. 7 is an autoradiograph of DNA from T. longibrachiatum strain P37P ⁇ CBHIPyr " 26 transformed with EcoRI digested pP ⁇ CBHII after Southern blot analysis using a 32 P labelled pP ⁇ CBHII as the probe.
  • the sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.
  • FIG. 8 is a diagram of the plasmid pEGIpyr..
  • FIG. 9 is an outline of the construction of plasmid pTEX.
  • FIG. 10 illustrates the RBB-CMC activity profile of an acidic EG enriched fungal cellulase composition (CBH I and II deleted) derived from _rric_- ⁇ _.er__.a longibrachiatum over a pH range at 40°C; as well as the activity profile of an enriched EG III cellulase composition derived from Trichoderma longibrachiatum over a pH range at 0°C.
  • FIG. 11 illustrates the effect of different cellulase compositions on the redeposition of colorant during the stonewashing process.
  • FIG. 12 illustrates strength loss results after three wash cycles in a laundero eter for cotton-containing fabrics treated with cellulase compositions having varying amounts of CBH components.
  • FIG. 13 illustrates fiber removal results (based on panel test scores) for cotton-containing fabrics treated with cellulase secreted by a wild type Trichoderma longibrachiatum (whole cellulase) at various pHs.
  • FIG. 14 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 longibrachiatum and for a cotton fabric treated with cellulase secreted by a strain of Trichoderma longibrachiatum genetically engineered so as to be incapable of secreting CBH I and CBH II.
  • FIG. 15 illustrates the softness panel test results for varying concentrations (in ppm) of an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be incapable of producing CBH I and CBH II.
  • FIG. 16 illustrates the softness panel test results for a non-cotton-containing cellulosic fabric treated with an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be incapable of producing CBHI&II.
  • FIG. 17 illustrates an appearance panel test results for a non-cotton-containing cellulosic fabric treated with an EG enriched cellulase composition derived from a strain of Trichoderma longibrachiatum genetically modified so as to be incapable of producing CBHI&II.
  • the methods of this invention are directed to treating cotton-containing and non-cotton- containing cellulosic fabrics during manufacture with an effective amount of a cellulase composition comprising CBH type components and EG type components wherein said cellulase composition has a protein weight ratio of all EG type components to all CBH type components of greater than 5:1.
  • the improvement comprises using a specific cellulase composition which imparts the desired enhancement(s) to the fabric and reduces lint production while treating the fabric under conditions which still further reduce the production of lint and the amount of fabric weight loss.
  • the fungal cellulase compositions of this invention can be used in a continuous treatment process as well as a batch treatment process. However, prior to discussing this invention in detail, the following terms will first be defined.
  • cotton-containing fabric refers to sewn or unsewn fabrics made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, cotton toweling and the like.
  • the amount of cotton in the fabric should be at least about 40 percent by weight cotton; preferably, more than about 60 percent by weight cotton; and most preferably, more than about 75 percent by weight cotton.
  • 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 ara id fibers. It is contemplated that regenerated cellulose or a cellulosic fabric, 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,
  • cellulosic-containing or cellulosic fabric refers to any non-cotton-containing cellulosic fabric or non- cotton-containing cellulosic blend including natural cellulosics (such as jute, flax, ramie and the like) and manmade cellulosics. Included under the heading of manmade cellulosics are the regenerated cellulosics that are well known in the art such as rayon. Other manmade cellulosics include chemical modification of cellulose fibers (e.g, cellulose derivatized by acetate and the like) and solvent- spun cellulose fibers (e.g.lyocell) .
  • non-cotton-containing cellulosics can also be employed as blends that include lyocell-rayon, lyocell-linen, viscose rayon acetate, rayon-wool, silk-acetate, and the like.
  • finishing means the application of a sufficient amount of finish to a cotton- containing or non-cotton-containing cellulosic 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 ureafor aldehyde, as well as waxes, silicons, fluorochemicals and quaternaries. When so finished, the cotton-containing fabric is substantially less reactive to cellulase.
  • loose fiber refers to small loose threads and fibers, typically less than 1 cm in length, which form as a result of treatment of cotton containing fabrics with cellulase. Without being limited to a theory, it is believed that treatment with a complete cellulase composition causes excessive degradation of the fabric and excessive formation of loose fibers. The resulting loose fibers are no longer associated with the fabric and, accordingly, form lint which remains suspended in the treatment liquor or collects on the surface of the fabric or in machine filters. On the other hand, without being limited to any theory, it is believed that the specific cellulase compositions recited herein when used under the conditions of the present invention do not excessively degrade the fabric and thus do not create excessive amounts of lint.
  • the amount of lint produced by this method will be at least 20% less than the lint produced during treatment with a complete cellulase, more preferably the amount of lint produced will be at least 30% less than the lint produced during treatment with a complete cellulase.
  • fabric weight loss refers to the decrease in the weight of a piece of fabric after treatment with a cellulase composition.
  • the amount of fabric weight loss during treatment with a complete cellulase composition can be from 5 to 25% of the weight of the total fabric.
  • a reduction in fabric weight loss means that the amount of fabric weight lost during treatment with the cellulase composition of this invention when used under the conditions of the present invention is at least 20% less than that loss observed during treatment of the same fabric with a complete cellulase composition when used under the same conditions.
  • the amount of fabric weight loss will be at least 20% less than the fabric weight loss observed during treatment with a complete cellulase composition, more preferably, the amount of fabric weight loss will be at least 30% less than the fabric weight loss observed during treatment with a complete cellulase composition.
  • feel refers to the physical smoothness of a cotton- containing and non-cotton-containing cellulosic fabric to touch. Fabrics having improved feel are smoother and silkier to the touch than other fabrics and accordingly are viewed as higher quality products. As defined, the term “feel” is distinguished from qualities such as thickness, color, or other physical characteristics not involved in smoothness of the fabric.
  • the term "appearance” as used herein refers to the physical appearance of the cotton-containing and non-cotton- containing cellulosic fabric to the eye and is determined in part, by the presence or absence of, fuzz, surface fibers, and the like on the surface of the fabric as well as by the ability or inability to discern the construction (weave) of the fabric. Fabrics, which have little if any fuzz and surface fibers and wherein the construction (weave) is clearly discernable, possess improved appearance as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weave. As defined, the term “improved appearance" may include surface polishing of the fabric.
  • the improvements in feel and appearance of cotton-containing and non-cotton-containing cellulosic fabrics after treatment by the methods of the present invention are readily ascertained by simple analytical tests which provide a numerical rating to the fabric both before and after treatment by the methods of this invention.
  • the test procedure is conducted as a side-by-side comparison of a fabric sample before treatment by the process of this invention with a sample of that fabric after treatment by the process of this invention.
  • Specific test procedures useful herein for ranking the improvements in feel and appearance are disclosed in U.S. Application Serial No. 07/598,506 filed October 16, 1990 and entitled "Methods for Improving the Appearance and Feel Characteristics of Cotton Woven Fabrics" the disclosure of which is incorporated herein by reference in its entirety.
  • 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 as 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. 2094826A, the disclosure of which is incorporated herein by reference.
  • fungal cellulases generally have their optimum activity in the acidic or neutral pH range although some fungal cellulases are known to possess significant activity under neutral and slightly alkaline conditions, i.e., for example, cellulase derived from Humicola insolens is known to have activity in neutral to slightly alkaline conditions.
  • Fungal cellulases are known to be comprised of several enzyme classifications having different substrate specificity, enzymatic action patterns, and the like. Additionally, enzyme components within each classification can exhibit different molecular weights, different degrees of glycosylation, different isoelectric points, different substrate specificities, 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 one or more 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.
  • cellulase or “cellulase composition” refers to cellulase expressed by fungal sources or microorganisms genetically modified to incorporate and express all or part of the cellulase genes obtained from fungal sources which composition may also contain non-cellulase proteins.
  • 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 per se in the art.
  • Endoglucanase (“EG”) type components refer to all of those fungal cellulase components or combination of components which exhibit textile activity properties similar to the endoglucanase components of Trichoderma longibrachiatum (previously classified as T. reesei) .
  • the endoglucanase components of Trichoderma longibrachiatum specifically, EG I, EG II, EG III, and the like either alone or in combination
  • treatment of cotton-containing fabrics with endoglucanase components of Trichoderma longibrachiatum result in less strength loss as compared to the strength loss arising from treatment with a similar composition but which additionally contains CBH type components in a ratio of EG components to CBH components approaching that found in naturally complete cellulase compositions which contain both CBH and EG components.
  • treatment of cotton- containing fabrics with endoglucanase components of _T_ric-- ⁇ ⁇ -7er_-ia longibrachiatum result in less production of excessive lint and less fabric weight loss compared to treatment with a similar composition but which additionally contains CBH type components.
  • 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.
  • a cellulase composition containing endogluconose type components imparts 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 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 longibrachiatum.
  • Fungal cellulases can contain more than one EG type component.
  • the different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities, different enzymatic action patterns, etc.
  • the different isoelectric points of the components allow for their separation via ion exchange chromatography and the like.
  • the isolation of components from different fungal sources is known in the art. See, for example, Bjork et al., U.S. Patent No. 5,120,463, Jrin et al... International 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); Hydrin, 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
  • enriched EG type components refers to a cellulase composition containing at least 30 weight percent, preferably at least 70 weight per-cent and most preferably at least 90 weight percent of EG type cellulase components or a particular EG type component specified based on the total weight of the proteins in the cellulase composition.
  • EG I cellulase refers to the component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pi) of from about 4.5 to 5.3, and a molecular weight of about 47 to 50 Kdaltons.
  • EG I cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride .
  • EG I cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.0, an isoelectric point (pi) of about 4.7 and a molecular weight of about 47 to 49 Kdaltons.
  • EG I cellulase derived from Trichoderma viride has a pH optimum of about 5.0, an isoelectric point (pi) of about 5.3 and a molecular weight of about 50 Kdaltons.
  • EG I type cellulase refers to fungal cellulase components exhibiting textile activity properties similar to EG I cellulase.
  • EG II has been previously referred to by the nomenclature "EG III” by some authors but current nomenclature uses the term EG II.
  • the EG II protein is substantially different from the EG III protein defined below in its molecular weight, pi and pH optimum.
  • EG II cellulase refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pl) of from about 5.4 to 6.0 and a molecular weight of about 35 to 50 Kdaltons.
  • EG II cellulase refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pl) of from about 5.4 to 6.0 and a molecular weight of about 35 to 50 Kdaltons.
  • pl isoelectric point
  • cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride.
  • EG II type cellulase refers to fungal cellulase components exhibiting textile activity properties similar to EG II cellulase.
  • EG III cellulase refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 5.5 to 6.0, an isoelectric point (pi) of from about 7.2 to 8.0, and a molecular weight of about 23 to 28 Kdaltons.
  • EG III cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride .
  • Trichoderma longibrachiatum has a pH optimum of about 5.5 to 6.0, an isoelectric point (pl) of about 7.4, and a molecular weight of about 25 to 28 Kdaltons.
  • EG III cellulase derived from Trichoderma viride has a pH optimum of about 5.5, an isoelectric point (pi) of about 7.7, and a molecular weight of about 23.5 Kdaltons.
  • EG III type cellulase refers to fungal cellulase components exhibiting textile activity properties similar to EG III cellulase.
  • EG type components can be derived from bacterially derived cellulases.
  • combinations of EG type components may give a ⁇ ynergistic response in reducing lint production and fabric weight loss.
  • a single EG type component may be more stable or have a broader spectrum of activity over a range of pHs.
  • enriched EG I type cellulase retards lint formation and reduces fabric weight loss.
  • 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.
  • proteins other than CBH type cellulase components present in the whole cellulase composition may produce lint or cause fabric weight loss alone or in combination with EG , ⁇ or other proteins during the treatment of cotton fabric with cellulases. Therefore, it is contemplated that the use of enriched EG I type, EG II type or EG III type components may result in a reduction of some or all of these proteins present in the whole cellulase composition and may result in a further reduction in lint.
  • 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 longibrachiatum.
  • CBH type Exo-cellobiohydrolase type
  • the CBH I and CBH II components of Trichoderma longibrachiatum 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 components of Trichoderma longibrachiatum impart enhanced strength loss and enhanced weight loss to the cotton-containing fabrics and production of excess lint.
  • 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 longibrachiatum, respectively.
  • this includes the property of enhancing strength loss of cotton-containing fabrics when used in the presence of EG type components.
  • the CBH components of Trichoderma longibrachiatum 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 longibrachiatum.
  • such components (a) are competitively inhibited by cellobiose (K f approximately lmM) ; (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., and (c) hydrolyze phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose.
  • Fungal cellulase compositions having enriched EG type components can be obtained by purification techniques.
  • the complete cellulase system can be purified into enriched 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
  • the requisite amount of the desired components could be recombined.
  • mixtures of cellulase components enriched for EG type components could be prepared by means other than isolation and recombination of the components.
  • recombinant techniques can alter the relative ratio of EG type components to CBH type components so as to produce a mixture of cellulase components having a relatively high ratio of EG type components to CBH type components.
  • a preferred method for the preparation of cellulase compositions described herein is to genetically modify a microorganism so as to overproduce one or more EG type components.
  • the deletion of the genes responsible for producing CBH I type and/or CBH II type cellulase components would have the effect of enriching the amount of EG components present in the cellulase composition.
  • An increase in the number of genes responsible for producing EG type components would also have the effect of enriching the amount of EG components in the cellulase composition.
  • fungal cellulase compositions used herein can be derived from fungal sources which produce low concentrations of CBH type components.
  • a requisite amount of one or more CBH type components purified by conventional procedures can be added to a cellulase composition produced from a microorganism genetically engineered so as to be incapable of producing CBH type components so as to achieve a specified ratio of EG type components to CBH type components, i.e., a cellulase composition free of all CBH type components so as to be enriched in EG type components can be formulated to contain 2 weight percent of a CBH type component merely by adding this amount of a purified CBH type component to the cellulase composition.
  • ⁇ -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-nitfophenol 0-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-nitfophenol 0-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. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG components in the cellulase composition will be used to regulate the activity of any CBH components in the composition. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG components in the cellulase composition will be used to regulate the activity of any CBH components in the composition. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because
  • “turn-off” CBH activity when the concentration of cellobiose reaches inhibitory levels.
  • one or more additives e.g., cellobiose, glucose, etc.
  • the resulting composition is considered to be a composition suitable for use in this invention if the amount of additive employed is sufficient to lower the CBH type activity to levels equal to or less than the CBH type activity levels achieved by using the cellulase compositions described herein.
  • 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.
  • Preferred fungal cellulases for use in preparing the fungal cellulase compositions used in this invention are those obtained from Trichoderma longibrachiatum, Trichoderma koningii, Pencillum sp., Humicola insolens, and the like. Certain fungal cellulases are commercially available, i.e.,
  • RAPIDASE available from Gist Brocades, N.V. .
  • CYTOLASE 123 available from Genencor International, South San Francisco, California
  • 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.
  • Enhancements to the cotton-containing and non-cotton- containing cellulosic fabric are achieved by those fabric treatment methods heretofore used with the improvements of the invention being incorporated into such methods.
  • 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 and non-cotton-containing cellulosic 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.
  • agitation means any mechanical and/or physical force which agitates the cellulase solution so as to result in agitation of the cotton-containing or non- cotton-containing cellulosic fabric. Without being limited to any theory, it is believed that such agitation facilitates the removal (clipping) of loose fibers, surface fibers (fuzz) and the like from the cotton-containing or non-cotton-containing cellulosic fabric.
  • the agitation required in the methods described herein defines a vigorous action of the cellulase solution against the fabric surface which is substantially greater than mere mechanical stirring of the cellulase solution in order to achieve uniform cellulase concentration throughout the cellulase solution. Agitation may also include fabric to fabric contact.
  • Agitation suitable for use in the methods described herein can be achieved, for example, by employing a laundrometer, a jig, a jet, a mercerizer, a beck, a paddle machine, continuous bleach range, continuous wash range and the like.
  • the agitation employed herein is either repetitive (e.g., intermittent) or continuous agitation.
  • the cellulase solution can be continuously agitated by employing a laundrometer, a jet and the like.
  • a laundrometer the cotton-containing or non-cotton-containing cellulosic fabric is loaded into stainless steel water-tight canisters.
  • Continuous agitation is achieved by rotation of the fixed canisters on a frame within a temperature adjustable water bath.
  • the degree of agitation is defined by the speed at which the canisters rotate.
  • canisters rotated at a speed of at least about 40 revolutions per minute (rpms) achieve the agitation effect required in the herein described methods.
  • Laundrometers are well known in the textile art and are generally employed as laboratory equipment. Suitable laundrometers are commercially available from, for example. Custom Scientific Instruments, Inc., Cedar Knolls, N.J.
  • jets In a jet, the cotton-containing or non-cotton-containing cellulosic fabric, in a rope form, continuously rotates through and with the cellulase solution.
  • jets are based on a venturi tube in which the circular movement of liquor carries the fabric with it in a totally enclosed tubular chamber, annular in shape.
  • the tubular chamber is filled in part with solution and the fabric is rotated through the chamber via a lifter roller so that at any given time a portion of the fabric is being lifted upward.
  • the venturi tube is a constriction in the annular passage through which the speed of the flow of the liquor must be increased, thus causing suction which imparts movement to the fabric.
  • the primary flow is given by a centrifugal pump, but it is usual to incorporate also a few inclined steam jets to boost the movement of both the fabric and the liquor.
  • the movement of the fabric through the jet preferably at a rate of at least about 6 ft/sec, provides the agitation required in the herein described methods.
  • a jet is a well known apparatus found in mills and are generally used for the purpose of dyeing and after treating fabrics.
  • Repetitive agitation can be achieved by employing a jig, a mercerizer, a beck, and the like.
  • a jig is a well known apparatus found in mills manufacturing cotton-containing and non-cotton-containing cellulosic fabrics and is generally used for the purpose of scouring fabrics prior to dyeing.
  • a defined length of cotton-containing or non-cotton- containing cellulosic fabric, in its open width position, is maintained on and between two rollers wherein the fabric is passing from one roller which is in the unwinding stage to a second roller which is in the winding stage.
  • the process is reversed so that the previous unwinding roll becomes the winding roll and the previous winding roll becomes the unwinding roll.
  • This process is continuously conducted during the entire cellulase treatment time.
  • a trough containing the cellulase solution is placed between the two rollers and the rollers are adjusted so that the cotton-containing or non- cotton-containing cellulosic fabric becomes immersed in the cellulase solution as it passes from one roller to the other.
  • Repetitive agitation is achieved in the jig by continuously rolling and unrolling the cotton-containing and non-cotton-containing cellulosic fabric from the rolls, preferably at a rate of speed of at least about 1 yd/sec and more preferably at least about 1.5 yd/sec so that at any given time, part of the length of the fabric is moving through the cellulase solution at this defined rate of speed.
  • a mercerizer unit is similar to a jig in that the cotton- containing or non-cotton-containing cellulosic fabric, in its open width position, is passed through a trough of solution, e.g., cellulase solution, at a set speed. Passing the cotton- containing or non-cotton-containing cellulosic fabric through the trough, preferably at a speed of at least 1 yd/sec, and more preferably at a rate of at least 1.5 yd/sec, provides the agitation required in the herein described methods.
  • a trough of solution e.g., cellulase solution
  • the mercerizer unit operates in only one direction and the length of time the fabric is exposed to the cellulase solution can be varied by modifying the mercerizer so as to contain more than one trough.
  • the length of time the fabric is exposed in such a modified mercerizer depends on the number of troughs and the speed the fabric is moving through the troughs.
  • each portion of the cotton-containing or non-cotton-containing cellulosic fabric is preferably exposed to the cellulase solution under agitating conditions at least once every minute on average. and more preferably at least 1.5 times every minute on average.
  • this required degree of repetitive agitation can be achieved by limiting the length of the fabric so that when conducted at the requisite speed, each portion of the cotton-containing or non-cotton- containing cellulosic fabric is exposed to the cellulase solution under agitating conditions at least once every minute on average.
  • the desired degree of repetitive agitation can be achieved by adding a sufficient number of troughs appropriately spaced so that the fabric repetitively passes through different troughs.
  • cascading means the rapid flow of cellulase solution across and eventually away from the surface of the cotton-containing or non-cotton-containing cellulosic fabric. That is to say that cascading occurs when a stream of cellulase solution (liquid) is moving on and relative to at least part of the surface of the cotton- containing or non-cotton-containing cellulosic fabric and this stream eventually moves away from this part of the surface of the fabric.
  • a cascading effect can be achieved, for example, by use of a laundrometer, a jig, a jet, a mercerizer and the like.
  • the canister when a laundrometer is employed, rotation of a partially filled canister will result in movement of the cellulase solution relative to the surface of the cotton- containing or non-cotton-containing cellulosic fabric thereby creating a flow of cellulase solution across and eventually off part of the surface of the cotton-containing or non- cotton-containing cellulosic fabric thereby resulting in a cascading effect.
  • the canister is rotated at the requisite rpms needed to achieve agitation, the flow of cellulase solution will be sufficiently rapid so as to cause agitation and additionally create a cascading effect of the cellulase solution.
  • the canister should be filled to no greater than about 75 percent of capacity, and preferably no greater than about 50 percent capacity.
  • Cascading can also be accomplished with the use of a jig.
  • a jig is employed to achieve the requisite agitation described above, the cotton-containing fabric rapidly departs from the trough containing the cellulase solution and is lifted somewhat upward in order to be wound onto the winding roller.
  • any cellulase solution remaining on the surface of the cotton-containing or non-cotton-containing cellulosic fabric as it exits from the cellulase solution rapidly flows across and eventually off this part of the fabric surface.
  • cascading in a jig is achieved by the passage of the cotton-containing and non-cotton-containing cellulosic fabric through the cellulase solution, preferably at a speed of at least 1 yd/sec and more preferably at a speed of at least 1.5 yd/sec, coupled with the gravitational effect of the upward lift of the fabric as it is being rolled which results in the rapid flow of the cellulase solution across and eventually away from the surface of the cotton-containing and non-cotton-containing cellulosic fabric theretofore covered with the cellulase solution.
  • Cascading can also be accomplished by use of a jet.
  • movement of the fabric relative to the cellulase solution provides agitation whereas rotation of the fabric upward and downward during rotation in the circular jet results in solution cascading over and from the fabric.
  • the fabric is so moved, preferably at a rate of at least about 6 ft/sec, through the jet, cascading of the cellulase solution on the fabric is achieved.
  • a predetermined place in said manufacturing process means the place in the process where the fabric is treated with cellulase.
  • the fabric is treated with cellulase at a point in manufacturing which will not interfere with the other manufacturing processes but will allow the cellulase to act on the fabric.
  • the particular place in the manufacturing process is not critical, but in general the cellulase treatment will occur after scouring of the fabric and before finishing of the fabric. It may occur either before or after the bleaching or dying of the fabric. In the case of stonewashing, the treatment may occur after the denim clothes have been finished and sewn so as to achieve the stonewashed "stressed" look.
  • the present invention is an improvement over previously disclosed methods for treating cotton- containing non-cotton-containing cellulosic fabrics insofar as the present invention employs an effective amount of a specific cellulase composition used under conditions which minimize the production of lint and fabric weight loss during treatment.
  • the cellulase composition described herein reduces lint formation. Further lint formation can be achieved by using reaction conditions which reduce lint production. Suitable conditions for further reducing lint production are set forth below and include using one or more of a specific EG type component (e.g.
  • the cellulase composition employed herein is a fungal cellulase composition which comprises a protein weight ratio of all EG type components to all CBH type components of greater than 5:1.
  • the use of the cellulase compositions described herein may 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 because some of the broken and disordered fibers are removed which has the effect of restoring the appearance of the fabric to its appearance prior to becoming stressed and yet, not excessively degrade the fabrics which typically results in the production of excessive lint.
  • cellulase composition described herein with pigment type dyed fabrics may cause a stone-washed appearance with less redeposition of dye.
  • pigment type dyed fabrics e.g., denims
  • These anti-redeposition properties can be enhanced for one or more specific EG type component(s) as compared to other components. See U.S. Serial No. 07/954,113 filed September 30, 1992 and entitled “Stonewashing of Denim Garments using Endoglucanase I and III" which is incorporated herein by reference.
  • an "effective amount" of the cellulase composition is that amount which will provide one or more of the desired enhancements of improved feel, softness, hand, color enhancement and/or a stone-washed appearance with reduced lint formation and reduced fabric weight loss.
  • it has been found that it is the amount of cellulase, and not the relative rate of hydrolysis of the specific enzymatic components to produce reducing sugars from cellulose, which imparts the desired enhancement(s) to cotton-containing and non-cotton-containing cellulosic fabrics, e.g., one or more of improved color restoration, improved softening, improved stonewashing, etc.
  • 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 in imparting the desired enhancement (e.g., a lower concentration of a more active cellulase composition may be required as compared to the concentration required for 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 0.005 grams of cellulase composition per liter; to about 5.0 grams/liter; and more preferably, from about 0.01 grams/liter to about 2 grams/liter.
  • 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.
  • 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.
  • 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 a pholytic 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 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 alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.
  • the liquor ratios i.e., the ratio of weight of liquid 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 achieved.
  • the liquor ratios are generally from about 3:1 to 40:1, and more preferably from about 5:1 to 20:1. Use of liquor ratios of greater than about 50:1 are usually not preferred.
  • 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.
  • the maximum reaction temperatures employed herein are generally about 70°C. In view of the above, reaction temperatures are generally from about 30 ⁇ C to about 70 ⁇ C; preferably, from about 35°C to about 60 ⁇ C; and more preferably, from about 35°C to about 55°C.
  • Reaction times are generally from about 0.1 hours to about 24 hours and, preferably, from about 0.25 hours to about 5 hours.
  • 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 greater than about 0 to about 50 weight percent buffer; from greater than 0 to about 50 weight percent surfactant; and from greater than 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 40,000 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 agglomerate and the like.
  • the granules are preferably formulated so as to contain a cellulase protecting agent such as ammonium sulfate.
  • a cellulase protecting agent such as ammonium sulfate.
  • the ammonium sulfate employed as part of the granule is from about 0.1 to 40 percent based on the weight of the granule
  • the cellulase is from about 0.001 to 50 percent based on the weight of the granule and the balance comprises inert materials and the like. See, for instance, U.S. Serial No.
  • the granule can be formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium.
  • 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.
  • Examples 1 - 14 demonstrate the preparation of Trichoderma longibrachiatum genetically engineered so as to be incapable of producing one or more cellulase components ⁇ r so as to overproduce specific cellulase components.
  • 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 pyr4 derivative strains using FOA.
  • spores of T. longibrachiatum previously classified as T. reesei
  • strain RL-P37 Sheir- Neiss, G. and Montenecourt, B.S., Apol. Microbiol. Biotechnol. 20, p.
  • a cbhl gene encoding the CBH I protein was cloned from the genomic DNA of T. longibrachiatum 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., Piscataway, New Jersey) replacing the Kan r gene of this vector using techniques known in the art, which techniques are set forth in Sambrook et al.
  • the T. longibrachiatum pyr4 gene was cloned as a 6.5 kb Hindi11 fragment of genomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991) following the methods of Sambrook et al., supra .
  • the plasmid pUC4K: :cbhl ⁇ H/H was cut with Hindi! I and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was ligated with the 6.5 kb ifindlll fragment containing the T. longibrachiatum pyr4 gene to give p ⁇ CBHIpyr..
  • 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 IO 7 T. longibrachiatum 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" 234 solution (which is the trade name for a multicomponent enzyme system containing 1,3-alpha-glucanase, 1,3-beta-glucanase, la inari- nase, 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 Novozym
  • the protoplasts were removed from the cellular debris by filtration through Miracloth (Calbioche 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 CaCl 2 , centrifuged and resuspended at a density of aapppprrooxxiimmately 2 x 10 protoplasts per ml of 1.2 M sorbitol, 50 mM CaCl 2 .
  • 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 pyr4 gene inserted in p ⁇ CBHIpyr.. 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.
  • DNA was isolated from the transformants obtained in Example 4 after they were grown in liquid Vogel's medium N containing 1% glucose. These transformant DNA samples were further cut with a PstI restriction enzyme and subjected to agarose gel electrophoresis. The gel was then blotted onto a Nytran membrane filter and hybridized with a 32 P labelled p ⁇ CBHIpyr. probe. The probe was selected to identify the native cbhl gene as a 6.5 kb PstI fragment, the native pyr4 gene and any DNA sequences derived from the transforming DNA fragment.
  • 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 CBH I band, indicating that this gene has been totally deleted in the transformant by integration of the DNA fragment at the cjbj-l gene.
  • the cbhl deleted strain is called P37P ⁇ CBHI.
  • Figure 2 outlines the deletion of the T.
  • longibrachiatum cbhl gene by integration through a double cross-over event of the larger EcoRI fragment from p ⁇ CBHIpyr4 at the cJbhl locus on one of the T. longibrachiatum chromosomes.
  • the other transformants analyzed appear identical to the untransformed control strain.
  • sample A contained the cJbhl 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.
  • Spores from the produced P37P ⁇ CBHI strain were inoculated into 50 ml of a Trichoderma basal medium containing 1% glucose, 0.14% (NH 4 ) 2 S0 4 , 0.2% KH 2 P0 4 , 0.03% MgS0 4 , 0.03% urea, 0.75% bactotryptone, 0.05% Tween 80, 0.000016% CuS0 4 * 5H 2 0, 0.001% FeS0 4 '7H 2 0, 0.000128% ZnS0_ 7H 2 0, 0.0000054% Na ⁇ oO ⁇ O, 0.0000007% MnCl'4H 2 0.
  • 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., La Jolla, CA) 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. longibrachiatum. Lane A is partially purified CBH I; Lane B is the supernatant from an untransformed T. longibrachiatum 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 CBH I, CBH II, EG I, EG II, and EG III. Since CBH I constitutes about 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 CBH I protein in the P37P ⁇ CBHI strain.
  • the cbh2 gene of T. longibrachiatum, encoding the CBH II protein has been cloned as a 4.1 kb EcoRI fragment of genomic DNA which is shown diagrammatically in FIG. 6A (Chen et al., 1987, Biotechnology. 5:274-278). This 4.1 kb fragment was inserted between the .EcoRI sites of pUC4XL. The latter plasmid is a pUC derivative (constructed by R.M.
  • the T. longibrachiatum 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 to create p219M (Smith et al., 1991, Curr. Genet 19 p. 27-33).
  • the vector pUC219 is derived from pUC119 (described by Wilson et al. (1984) Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for Bglll, Clall and Xhol.
  • the pyr4 gene was removed from p219M as a Hindlll-CIal 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 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. longibrachiatum 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. This is the expected pattern if a single copy of the EcoRI fragment from pP ⁇ CBHII had integrated precisely at the cbh2 locus.
  • EXAMPLE 11 Selection of a pyr4 null mutant of strain P37P ⁇ CBH67 Spores of the transformant (P37P ⁇ CBH67) which was deleted for both the cJbhl and cbh2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in Example 1. This pyr4 deficient strain was designated P37P ⁇ CBH67Pyr * l. Southern analysis has shown that a spontaneous deletion had occurred when strain P37P ⁇ CBH67Pyr * l was selected. This deletion completely removed the pyr4 gene which had integrated at the cbh2 locus beyond the extent of the 4.1 kb EcoRI fragment of genomic DNA which was originally cloned. The short (6 bp and 7 bp) fragments of DNA derived from the pUC219 multiple cloning site which were present at either end of the pyr4 gene would also have been removed from the genome by this deletion.
  • EXAMPLE 12 Construction of pEGIpyr4
  • the T. longibrachiatum egll gene which encodes EG I, 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 Hi_.dIII-Ba.__HI fragment containing the T.
  • longibrachiatum pyr4 gene obtained from pTpyr2 (see Example 2) and pUC218 (identical to pUC219, see Example 8, but with the multiple cloning site in the opposite orientation) cut with Hindlll to give the plasmid pEGIpyr. by standard molecular techniques as outlined in Sambrook et al. (1989), supra (FIG. 8). Digestion of pEGIpyr. with Hindlll would liberate a fragment of DNA containing only T. longibrachiatum 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) . Both these pieces of synthetic DNA were obtained from the multiple cloning site of pUC-type vectors.
  • the plasmid, pTEX was constructed following the methods of Sambrook et al. (1989), supra, and is illustrated in FIG. 9.
  • This plasmid has been designed as a multi-purpose expression vector for use in the filamentous fungus Trichoderma longibrachiatum.
  • the expression cassette has several unique features that make it useful for this function. Transcription is regulated using the strong CBH I gene promoter and terminator sequences for T. longibrachiatum. Between the CBH I promoter and terminator there are unique Pmel and Sst I restriction sites that are used to insert the gene to be expressed. The T.
  • longibrachiatum pyr4 selectable marker gene has been inserted into the CBH I terminator and the whole expression cassette (CBH I promoter-insertion sites- CBH I terminator-pyr4 gene-CBH I terminator) can be excised utilizing the unique NotI restriction site or the unique NotI and Nhel restriction sites.
  • This vector is based on the bacterial vector, pSLll ⁇ O (Pharmacia Inc., Piscataway, New Jersey) , which is a pUC-type vector with an extended multiple cloning site.
  • pTEX was digested with Sstll and Pmel and then ligated with a synthetic DNA linker designed to join the cJbhl promoter with the egll coding sequence and with an approximately 2 kb Sfil - Seal fragment of T. longibrachiatum DNA containing most of the egll coding sequence and the terminator region. This ligation produced the vector pTEX-EGI.
  • This vector was digested with WotI and Whel to release the expression cassette which comprised the following components: a) 11 bp of linker DNA derived from the multiple cloning site of pSLll ⁇ O. b) An approximately 2.2 kb Pstl-Sstll fragment of T. longibrachiatum DNA from the promoter region of the cJb l gene. The Sstll site is at a position 15 bp 5' of the translation initiation codon (ATG) . c) A synthetic DNA linker used to join the cbhl promoter with the egll coding sequence, having single-stranded overhanging ends compatible with Ss til and Sfil digested DNA, and having the following sequence:
  • longibrachiatum DNA containing the egll coding sequence, starting at an Sfil site 30 bp after the first ATG codon, and approximately 300 bp of 3' flanking DNA containing the transcription termination and polyadenylation signals.
  • the T. longibrachiatum pyr4 gene on a 1.6 kb Bglll fragment which has 13 bp on one end and 17 bp on the other end of synthetic linker DNA.
  • Both linkers were derived from the multiple cloning site of the pUC219 vector.
  • the latter synthetic linker was additionally modified by insertion of a Bglll site at the Hindlll site using the linker shown in FIG. 9.
  • Oligonucleotide directed mutagenesis (Sambrook et al. (1989) Molecular Cloning a Laboratory Manual, Cold Spring Harbor Press) was used to change a single nucleotide within the pyr4 coding region (FIG. 9) .
  • the third nucleotide of the codon coding for the arginine residue at amino acid position 251 of the protein was changed from a C nucleotide to an A nucleotide by this method.
  • the liquid medium used for these experiments had the following composition: alpha-lactose, 30 g/1; (NH 4 ) 2 S0 4 , 6.5 g/1; KH 2 P0 4 , 2.0 g/1; MgS0 4 '7H 2 0, 0.3 g/1; CaCl 2 , 0.2 g/1; lOOOx trace salt solution, 1.0 ml/1; 10 % Tween 80, 2.0 ml/1; Proflo, 22.5 g/1; CaC0 3 , 0.72g/l. Source for Tween 80 and Proflo.
  • the lOOOx trace salt solution had the following composition: FeS0 4 "7H 2 0, 5.0 g/1; MnS0_ H 2 0, 1.6 g/1; ZnS0 4 , 1.4 g/1. These shake flask cultures were incubated with shaking for seven days at 30°C. Samples of the supernatant were taken from these cultures and assays designed to measure the endoglucanase activity were performed as described below.
  • the endoglucanase assay relied on the release of soluble, dyed oligosaccharides from Remazol Brilliant Blue- carboxymethylcellulose (RBB-CMC, obtained from MegaZyme, North Rocks, NSW, Australia) .
  • the substrate was prepared by adding 2 g of dry RBB-CMC to 80 ml of just boiled deionized water with vigorous stirring. When cooled to room temperature, 5 ml of 2 M sodium acetate buffer (pH 4.8) was added and the pH adjusted to 4.5. The volume was finally adjusted to 100 ml with deionized water and sodium azide added to a final concentration of 0.02%. Aliquots of T. longibrachiatum control culture, pEGIpyr.
  • pTEX-EGI transformant culture supernatants or 0.1 M sodium acetate as a blank (10-20 ⁇ l) were placed in tubes, 250 ⁇ l of substrate was added and the tubes were incubated for 30 minutes at 37°C. The tubes were placed on ice for 10 minutes and 1 ml of cold precipitant (3.3% sodium acetate, 0.4% zinc acetate, pH 5 with HC1, 76% ethanol) was then added. The tubes were vortexed and allowed to sit for five minutes before centrifuging for three minutes at approximately 13,000 x g. The optical density was measured spectrophotometrically at a wavelength of 590-600 nm.
  • CYTOLASE 123 cellulase was fractionated in the following manner.
  • the normal distribution of cellulase components in this cellulase 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 Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Maryland) .
  • CYTOLASE 123 cellulase, 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. The fraction bound on this column contained CBH I and EG I.
  • 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, Penicillium sp. and the like.
  • EXAMPLE 16 Purification of EG III from CYTOLASE 123 Cellulase Example 15 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. See also U.S. Serial No. 07/862,846 filed April 3, 1992 and entitled "METHODS FOR PRODUCING SUBSTANTIALLY PURE EG III CELLULASE USING POLYETHYLENE GLYCOL", which is incorporated herein in its entirety by reference.
  • 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.
  • EG III cellulase may be extracted by the method described in U.S. Serial No. 07/862,641 filed April 3, 1992 which is entitled “METHODS FOR PRODUCING SUBSTANTIALLY PURE EG III CELLULASE USING ALCOHOL,” which reference is incorporated herein in its entirety.
  • 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, California) which is produced by wild type Trichoderma longibrachiatum. Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Missouri) , QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Maryland) .
  • CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, California
  • 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 longibrachiatum 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.
  • EG III compositions described above may be further purified to provide for enriched EG III compositions, i.e., compositions containing EG III at greater than about 80 weight percent of protein.
  • enriched EG III protein can be obtained by utilizing material obtained from procedure A in procedure B or vice 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 16. 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 16 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.
  • Enriched EG III as well as EG I and EG II components purified in Example 15 above can be used singularly or in mixtures in the methods of this invention. These EG components have the following characteristics: MW Dl PH optimum
  • EG III has both a higher pH optimum and a higher pi as compared to the other endoglucanase components of Trichoderma longibrachiatum.
  • Example 17 it is seen that EG III also retains significant RBB-CMC activity under alkaline pHs.
  • EG III cellulase from other strains of Trichoderma spp. can be purified.
  • EG III cellulase derived from Trichoderma viridae has been described by Voragen et al.. Methods in Enzymology, 160:243-251, 1988. This reference describes the EG III cellulase as having a molecular weight of about 23.5 kdaltons, a pH optimum of 5.5 and a pi of 7.7.
  • the first cellulase composition was a CBH I and II deleted cellulase composition prepared from Trichoderma longibrachiatum 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 longibrachiatum
  • this cellulase composition is necessarily 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 longibrachiatum via purification methods similar to part b) of Example 16.
  • FIG. 10 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 demonstrates that the presence of CBH type components is not essential for imparting a stonewashed appearance to denim fabrics.
  • this example employs a cellulase composition derived from Trichoderma longibrachiatum 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 longibrachiatum and which is available as Cytolase 123 cellulase from Genencor International, South San Francisco, California.
  • Samples were evaluated for their stonewashed appearance, but not the level of redeposition of dye, by 8 panelists. All eight panelists choose 100 ppm whole cellulase over non-enzyme treated pants as having the better stonewashed look. Four of the 8 panelists choose the CBH I and II deleted cellulase treated pants over whole cellulase as having the better stonewashed look; whereas the other four panelists choose the whole cellulase treated pants as having the better stonewashed look. These results indicate that the CBH I and II deleted cellulase treated pants were indistinguishable from whole cellulase treated pants and that CBH I and/or CBH II are not essential for imparting a stonewashed appearance to denim fabrics.
  • EXAMPLE 19 Reduced Redeposition of dye
  • This example demonstrates that the use of EG type components substantially free of CBH type components results in a reduced redeposition of dye onto the fabric during stonewashing.
  • this example employs the following cellulase compositions: a) a complete cellulase composition derived from Trichoderma longibrachiatum and which is available as CYTOLASE 123 cellulase from Genencor International, South San Francisco, California.
  • These cellulase compositions were tested for their ability to impart a stonewashed appearance to dyed denim pants and their ability to prevent the redeposition of dye onto the fabric.
  • the samples were prepared using an industrial washer and dryer under the following conditions:
  • the garments were rinsed according to a standardized protocol in three consecutive cycles of clean liquor.
  • Rinse ⁇ l 24 gallons hot water, approximately 50°C, plus -100 grams standard detergent WOR (from American Association of Textile Chemists and Colorists [AATCC], WOB - without brighteners) . Agitation was for 12 minutes at 36 rpms.
  • the bath was dropped.
  • Rinse 02 24 gallons warm water, -40°C, with no additional detergents, agitated for 5 minutes.
  • the bath was dropped.
  • Rinse #3 24 gallons cold water, -30°C, with no additional detergents, agitated for 5 minutes.
  • Garments were extracted and dried in a standard electric clothes dryer.
  • FIG. 11 shows the results obtained with the different cellulase compositions.
  • Example 20 Laundero eter Strength Loss Assay Cellulase Compositions 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 most of the cellulase components derived from Trichoderma longibrachiatum 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 longibrachiatum 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 longibrachiatum genetically modified in a manner similar to Examples 1 to 14 above so as to be incapable of expressing CBH II and is identified as CBH lid.
  • 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 CBH I and CBH II deleted cellulase composition prepared from Trichoderma longibrachiatum 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 CBH I/IId.
  • CBH I and CBH II are not produced by this modified microorganism, the cellulase is necessarily free of all CBH I type components as well as all CBH components.
  • the last cellulase composition analyzed was a CBH I deleted cellulase composition prepared from Trichoderma longibrachiatum genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and is identified as CBH Id. Insofar as the modified microorganism is incapable of expressing CBH I, this cellulase composition is necessarily free of all CBH I type cellulase components.
  • the cellulase compositions described above were tested for their effect on cotton-containing fabric strength loss in a launderometer.
  • the compositions were first normalized so that equal amounts of EG components were used.
  • Each cellulase composition was then added to separate solutions of 400 ml of a.20 mM 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 12.
  • Example 21 Color Enhancement The ability of EG components to enhance color in cotton- containing fabrics was analyzed in the following experiments. Specifically, the first experiment measures the ability of a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type
  • Trichoderma longibrachiatum to remove surface fibers from a cotton-containing fabric at various pHs was tested for its ability to remove surface fibers in a launderometer.
  • An appropriate amount of cellulase to provide for either 25 ppm or 100 ppm cellulase in the final composition was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated so as to provide for samples at pH 5, pH 6, pH 7 and pH 7.5. Each of the resulting solution was then added to a separate launderometer canister.
  • the so treated fabrics were then analyzed for fiber removal by evaluation in a panel test.
  • the fabrics (unmarked) were rated for levels of fiber by 6 individuals.
  • the fabrics were visually evaluated for surface fibers and rated on a 0 to 6 scale.
  • the scale has six standards to allow meaningful comparisons. The standards are:
  • the fabric was a 100% cotton sheeting standardized test fabric (Style No. 439W) available from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846 b All samples were treated with the same cellulase composition. Cellulase concentrations are in total protein. The launderometer treatment conditions are the same as set forth in Example 16 above.
  • 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. 13 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 stowed 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 longibrachiatum 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
  • CBH I type components which composition was prepared from Trichoderma longibrachiatum 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 CBH I/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 was added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer containing 0.5 ml of a non-ionic surfactant. Samples were prepared and titrated 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. 14 is plotted on estimated EG concentrations. Specifically, FIG. 14 illustrates that both GC010 and CBH I/II deleted cellulase compositions gave substantially identical fiber removal results at substantially equal endoglucanase 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. 13 demonstrate that EG components remove surface fibers.
  • Example 21 is further to Example 21 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 in 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 longibrachiatum 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., Thomasvilie, N.C. 27360).
  • a 10 inch x 10 inch faded cotton-containing fabric as well as a 10 inch x 10 inch new knitted fabric having loose and broken surface fibers were then placed into 1 liter of this 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.
  • Example 23 Softness This example demonstrates that the presence of CBH type components are not essential for imparting improved softness to cotton-containing fabrics. Specifically, this example employs a cellulase composition free of all CBH type components which composition is derived from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing CBH I and II components.
  • This cellulase composition was tested for its ability to soften terry wash cloth. Specifically, unsoftened 8.5 ounce cotton terry cloths, 14 inches by 15 inches (available as Style No. 420NS from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846), were cut into 7 inch by 7.5 inch swatches.
  • the cellulase composition described above was tested for its ability to soften these swatches in a launderometer. Specifically, an appropriate amount of CBH I and II deleted 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). 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.
  • Triton X114 non-ionic surfactant
  • the swatches were then analyzed for softness by evaluation in a preference test. Specifically, six panelists were given their own set of swatches and asked to rate them with respect to softness based on the softness criteria such as the pliability of the whole fabric. Swatches obtained from treatment with the five different enzyme concentrations and the blank were placed behind a screen and the panelists were asked to order them from least soft to most soft. Scores were assigned to each swatch based on its order relative to the other swatches; 5 being most soft and 0 being least soft. The scores from each panelists were cumulated and then averaged.
  • 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 longibrachiatum 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. Specifically, 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.
  • Triton X114 non-ionic surfactant
  • the samples were then analyzed for improved appearance by evaluation in a preference test. Specifically, 6 panelists were given the 4 samples (not identified) and asked to rate them with respect to appearance. The panelists were instructed that the term "appearance" refers to the physical appearance of the cotton-containing fabric to the eye and is determined in part, by the presence or absence of, fuzz, surface fibers, and the like on the surface of the fabric as well as by the ability or inability to discern the construction (weave) of the fabric. Fabrics which have little if any fuzz and surface fibers and wherein the construction (weave) is clearly discernable possess improved appearance as compared to fabrics having fuzz and/or loose fibers and/or an indiscernible weave.
  • 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. 39W 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 mM citrate/phosphate buffer. Additionally, a blank was run containing the same solution but with no added cellulase. All tests were conducted at pH 5.8 and run in an industrial 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.
  • each of the cellulase compositions employed were dosed to give equal stone-washing performance and, in the case of the EGI expressed by pEGIpyr4 transformants, this cellulase actually contained more EG components than whole cellulase.
  • the garments were rinsed according to a standardized protocol in three consecutive cycles of clean liquor.
  • Rinse ⁇ l 24 gallons hot water, approximately 50 ⁇ C, plus -100 grams standard detergent WOR (from American Association of Textile Chemists and Colorists [AATCC] , WOB - without brighteners) . Agitation was for 12 minutes at 36 rpms.
  • the bath was dropped.
  • the bath was dropped.
  • Rinse #3 24 gallons cold water, -30 ⁇ C, with no additional detergents, agitated for 5 minutes.
  • Garments were extracted and dried in a standard electric clothes dryer for 70 minutes.
  • the lint screen of the dryer was thoroughly cleaned prior to drying the denim pants. After the pants were dried, the lint was removed from the lint screen and weighed on a tared weigh boat.
  • Enriched EG I is the supernatant from a pEGIpyr. transformant prepared by the method disclosed in Example 14 and contains substantially more EG components than whole cellulase. The following results were obtained:
  • the feel and appearance of the whole cellulase treated and EG I treated fabric were compared. It was determined that EG I enriched cellulase provided as good an appearance as could be achieved with an equivalent amount of whole cellulase.
  • the EGI treated fabric had a softer feel as compared to treatment with whole cellulase. However, the amount of fabric weight loss was less with EGI enriched cellulase.
  • Weight % Loss Weight % Loss (g/sq. yd.) (g/sq. yd.)
  • the fabric upon completing treatment of the cotton- containing fabric with cellulase, the fabric is then treated in a manner to remove and/or inactivate the cellulase.
  • One method of removing the cellulase is by thoroughly rinsing the so treated fabric with a cellulase free aqueous solution (i.e., an aqueous solution containing no cellulase).
  • the cotton-containing fabric is then dried at elevated temperatures to inactivate any cellulase remaining.
  • the cotton-containing fabric is first treated to inactivate the cellulase by heating to sufficiently high temperatures (i.e., at temperatures from about 75°C or more, e.g., 75° to 125 ⁇ C) for a sufficiently long period of time to inactivate the enzyme.
  • sufficiently high temperatures i.e., at temperatures from about 75°C or more, e.g., 75° to 125 ⁇ C
  • the cotton-containing fabric can subsequently be thoroughly rinsed and dried.
  • subsequent treatment steps for the cotton-containing fabric treated by the methods of this invention can further inactivate any active cellulase remaining either by the use of treatment conditions and/or reagents which deactivate cellulase.
  • Example 27 Enhanced properties of non-cotton-containing cellulosic fabrics This example demonstrates the ability of EG cellulase composition to enhance appearance, softness and surface polishing of non-cotton-containing cellulosic fabrics.
  • a 200 kg Jet Dyer machine was used to evaluate the enhanced properties of the non-cotton-containing cellulosic fabric TENCELTM. Approximately 10 kg of 100% TENCELTM mid-weight woven fabric was loaded into the machine in rope form and sewn end- to-end. This process may be performed on greige or dyed fabric. The jet machine was filled with 150 - 200 liters of water (which represents approximately 15-20:1 liquor to fabric ratio) and heated to 120 - 140OF (50O - 60OC) .
  • the pH was adjusted to 4.5 - 5.0 by the addition of 3.6 g/1 (56%) acetic acid and 1.9 g/1 (50%) sodium hydroxide.
  • the sodium hydroxide was added slowly to a dilute acetic acid solution before putting into the machine.
  • 0.25 - 0.5 ml/1 of a nonionic wetting agent (Triton X-100) was added to the liquor.
  • the pH and temperature was checked to ensure that the pH was between 4.5 and 5.0, and the temperature was between 500 - ⁇ ooc.
  • 3 - 4 g/1 of an enriched EG cellulase composition was added.
  • the enriched EG cellulase composition comprised a cellulase composition free of all CBH type components, which composition is derived from Trichoderma longibrachiatum genetically engineered in the manner described above so as to be incapable of producing CBH I and II components and over ⁇ produces EG I.
  • the jet was run for 30 - 60 minutes.
  • 0.25 g/1 soda ash was added to the liquor and run for 10 minutes.
  • the liquor was dropped from the jet, then the jet was filled again with water and the fabric rinsed one more time.
  • the fabric was removed from the jet and dried. Finally, a silicone-based finish was exhausted onto the fabric.
  • Swatches were analyzed for softness and surface appearance by evaluation in a preference test. Specifically, four panelists were given their own set of swatches and asked to rate them with respect to softness and surface appearance. Softness was based on the softness criteria such as pliability of the whole fabric. Surface appearance was based on the amount of loose fibers or fuzz present on the fabric. Swatches were compared to a non-enzyme treated fabric control and in the measurement of softness, an additional control was included i.e. a fabric treated with a complete fungal cellulase composition. Scores were assigned to each swatch and the average score was tabulated from the four panalists. The highest score for softness and surface appearance was assigned the value 5.0.
  • FIG. 16 An additional comparison of the EG enriched cellulase composition treated TENCELTM fabric was compared to whole cellulase treated TENCELTM fabric.
  • swatches were analyzed for softness by evaluation in a preference test. Four panelists were given their own set of swatches and asked to rate them with respect to softness. Softness was based on the above-mentioned criteria and panel score scale. Swatches were compared to a whole cellulase treated fabric control. Scores were assigned to each swatch and an average score was tabulated from the four panelists. The results of this averaging are set forth in FIG. 16. Specifically, these results demonstrate that EG enriched cellulase treated TENCELTM fabric was on average softer than the whole cellulase treated fabric control.
  • Example 28 on-cotton-containing cellulosic fabric showing less lint
  • the example demonstrates the ability of an enriched EG cellulase composition to enhance appearance and softness while generating less lint.
  • a 45 kilogram rotary drum washing machine was used to generate the non-cotton-containing cellulosic fabrics which were evaluated for enhanced properties with accompanying lower lint generation following EG enriched cellulase treatment.
  • approximately 20 kilograms of a light weight (4.5 ounce per sq. yd) , 100% TENCELTM woven fabric was loaded into the machine.
  • the rotary drum machine was filled with 300 liters of water representing a 15:1 liquor to fabric ratio and maintained at a temperature of 50 C. The fabric was run in the machine for 45 minutes.
  • the EG enriched enzyme is comprised of a cellulase composition free of all CBH type components, and is derived from Trichoderma longibrachiatum genetically engineered in a manner described above so as to be incapable of producing CBH I and II components and overproduces EG I (See Example 14). After adding the enriched EG cellulase composition, the rotary drum machine was run for 45 minutes.
  • Swatches were analyzed for softness and appearance by evaluation in a preference test.
  • the enriched EG I cellulase composition provided equal or improved softness and satisfactory appearance compared to the whole cellulase by this evaluation.
  • An additional panel rating the amount of lint present on the dried fabric was completed. Three swatches were analyzed in this panel. In the panel score for lint a rating of 5 indicated the least amount of visible lint and a rating of 0 indicated the highest level of visible lint.
  • the results are set forth in the following table. Specifically, these results demonstrate that EG enriched cellulase treatment imparted enhanced fabric properties while generating less lint versus a whole cellulase treated fabric control.
  • Example 29 Non cotton-containing cellulosic fabric showing less strength loss
  • a 100 kg jet dyer machine may be used to generate the non- cotton-containing cellulosic fabrics to evaluate for enhanced appearance, softness and surface-polishing properties of non- cotton-containing cellulosic fabrics along with corresponding fabric strength following EG enriched cellulase treatment.
  • a non-cotton-containing cellulosic fabric such as
  • TM as mid-weight, 100% TENCEL woven fabric maybe loaded into the machine in rope form and sewn end-to-end.
  • the jet machine maybe filled with approximately 600 liters of water representing a liquor to fabric ratio of approximately 15:1.
  • the liquor maybe heated to 50 - 55 C and the pH was adjusted to 5.0 by the addition of 3.6 grams per liter of (56%) acetic acid and 1.9 grams per liter of (50%) sodium hydroxide.
  • 0.5 % weight to volume of a nonionic wetting agent may be was added to the liquor.
  • 3 - 4 grams per liter of an enriched EG cellulase composition is added to the machine.
  • the enriched EG cellulase comprise a cellulase composition free of all CBH type components and derived from Trichoderma longibrachiatum genetically engineered in a manner described above so as to incapable of producing CBH I and II components and overproduces EG I (See Example 14) .
  • the jet is run for 60 minutes.
  • 0.25 grams per liter of soda is added to the liquor and the machine run an additional 10 minutes.
  • the liquor is drained from the jet and refilled to rinse the fabric.
  • the fabric is rinsed a second time before being removed from the jet and dried.
  • a silicone-based finish is applied to the fabric.
  • a whole cellulase treated fabric control is run under identical conditions with an identical dose of enzyme.
  • Swatches are analyzed for softness and appearance by evaluation in a preference test (See Example 27) .
  • Corresponding fabric tensile strengths are measured in accordance with the AATCC procedure to measure the breaking force and elongation of textile fabrics (Grab test- ASTM D 5034) . It is expected that treatment of the non-cotton cellulosic fabric with an EG I enriched cellulase composition will provide the enhancing properties (e.g., appearance, softness and/or surface polishing) as previously found in Example 27 with the additional property of lowered fabric strength loss versus whole cellulase treatment of non-cotton- containing cellulosic fabrics.

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Abstract

On décrit des procédés permettant de traiter des tissus cellulosiques contenant du coton et ne contenant pas de coton avec de la cellulase, lors de leur fabrication. Les procédés décrits consistent notamment à mettre en contact le tissu avec une composition de cellulase fongique comprenant un rapport pondéral protéinique entre tous les composants du type EG et tous les composants du type CBH supérieurs à 5:1 dans des conditions où la formation de coton égrainé est réduite. On décrit également des procédés de traitement en continu de tissus contenant du coton et ne contenant pas de coton, selon lesdits procédes consistant à mettre en contact les tissus avec une composition de cellulase fongique présentant un rapport pondéral protéinique entre tous les composants du type EG et tous les composants du type CBH supérieurs à 5:1 à un moment prédéterminé dans le processus de fabrication pendant une durée de contact efficace. Lorsque les tissus contenant du coton sont traités avec la composition de cellulase selon l'invention dans les conditions selon l'invention, les tissus ont l'avantage de présenter un toucher plus agréable et d'avoir une plus belle apparence et/ou l'apparence d'un tissu lavé à la pierre, sans formation excessive de coton égrainé ou de perte de poids excessive, cela comparativement au traitement utilisant une composition de cellulase entière.
PCT/US1994/003407 1993-03-30 1994-03-29 Procedes de reduction de la formation de coton egraine lors du traitement de tissus cellulosiques contenant du coton et ne contenant pas de coton WO1994023113A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021284A1 (fr) * 1994-02-03 1995-08-10 Genencor International, Inc. Procede d'application d'enzymes sur des tissus cellulosiques non finis afin d'ameliorer leur apparence et leur toucher
WO1995025841A1 (fr) * 1994-03-18 1995-09-28 Genencor International, Inc. Procedes pour traiter avec de la cellulase des tissus ne contenant pas de coton
EP0708819A1 (fr) * 1993-07-12 1996-05-01 Novo Nordisk A/S Composition detersive comprenant deux cellulases
US5770104A (en) * 1990-10-05 1998-06-23 Genencor International, Inc. Detergent compositions containing substantially pure EG III cellulase
EP0866165A1 (fr) * 1997-03-18 1998-09-23 Iogen Corporation Procédé et composition enzymatique pour améliorer le déboulochage des articles en coton
US5858767A (en) * 1996-11-25 1999-01-12 Rohm Enzyme Finland Oy Cellulase composition for biofinishing cellulose-containing textile materials
US5874293A (en) * 1996-11-25 1999-02-23 Rohm Enzyme Finland Oy Cellulase composition for treating cellulose-containing textile material
WO1999057257A1 (fr) * 1998-05-01 1999-11-11 The Procter & Gamble Company Detergent de lavage et/ou compositions respectant les tissus comprenant une cellulase modifiee
US6162782A (en) * 1990-10-05 2000-12-19 Genencor International, Inc. Detergent compositions containing cellulase compositions deficient in CBH I type components
US6184019B1 (en) 1995-10-17 2001-02-06 Röhm Enzyme Finland OY Cellulases, the genes encoding them and uses thereof
US6723549B2 (en) 1995-10-17 2004-04-20 Ab Enzymes Oy Cellulases, the genes encoding them and uses thereof
EP1047827B2 (fr) † 1997-12-19 2010-10-13 Novozymes North America, Inc. Biopolissage en continu de textiles contenant de la cellulose
US9963802B2 (en) 2014-03-04 2018-05-08 Basf Se Method of delinting cotton seeds

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WO1992007134A1 (fr) * 1990-10-16 1992-04-30 Genencor International, Inc. Procedes d'amelioration des caracteristiques de l'aspect et du toucher de tissu tisse en coton
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770104A (en) * 1990-10-05 1998-06-23 Genencor International, Inc. Detergent compositions containing substantially pure EG III cellulase
US6162782A (en) * 1990-10-05 2000-12-19 Genencor International, Inc. Detergent compositions containing cellulase compositions deficient in CBH I type components
EP0708819A1 (fr) * 1993-07-12 1996-05-01 Novo Nordisk A/S Composition detersive comprenant deux cellulases
WO1995021284A1 (fr) * 1994-02-03 1995-08-10 Genencor International, Inc. Procede d'application d'enzymes sur des tissus cellulosiques non finis afin d'ameliorer leur apparence et leur toucher
WO1995025841A1 (fr) * 1994-03-18 1995-09-28 Genencor International, Inc. Procedes pour traiter avec de la cellulase des tissus ne contenant pas de coton
US6723549B2 (en) 1995-10-17 2004-04-20 Ab Enzymes Oy Cellulases, the genes encoding them and uses thereof
US7323326B2 (en) 1995-10-17 2008-01-29 Ab Enzymes Oy Cellulases, the genes encoding them and uses thereof
US7273748B2 (en) 1995-10-17 2007-09-25 Ab Enzymes Oy Cellulases, the genes encoding them and uses thereof
US6184019B1 (en) 1995-10-17 2001-02-06 Röhm Enzyme Finland OY Cellulases, the genes encoding them and uses thereof
US5858767A (en) * 1996-11-25 1999-01-12 Rohm Enzyme Finland Oy Cellulase composition for biofinishing cellulose-containing textile materials
US5874293A (en) * 1996-11-25 1999-02-23 Rohm Enzyme Finland Oy Cellulase composition for treating cellulose-containing textile material
EP0866165A1 (fr) * 1997-03-18 1998-09-23 Iogen Corporation Procédé et composition enzymatique pour améliorer le déboulochage des articles en coton
EP1047827B2 (fr) † 1997-12-19 2010-10-13 Novozymes North America, Inc. Biopolissage en continu de textiles contenant de la cellulose
WO1999057259A1 (fr) * 1998-05-01 1999-11-11 The Procter & Gamble Company Compositions de detergent a lessive et/ou de produits d'entretien des tissus contenant une cellulase modifiee
WO1999057257A1 (fr) * 1998-05-01 1999-11-11 The Procter & Gamble Company Detergent de lavage et/ou compositions respectant les tissus comprenant une cellulase modifiee
US9963802B2 (en) 2014-03-04 2018-05-08 Basf Se Method of delinting cotton seeds

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