Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • 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
• • 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
  • D06M2200/00—Functionality of the treatment composition and/or properties imparted to the textile material
  • D06M2200/50—Modified hand or grip properties; Softening compositions

Definitions

• the present invention is directed to detergent compositions containing specific cellulase compositions.
• the present invention is directed to detergent compositions containing a cleaning effective amount of a surfactant and an acidic cellulase composition containing an enriched amount of acidic endoglucanase (EG) type components relative to exo-cellobiohydrolase (CBH) I type components and, preferably, all CBH type components.
• EG acidic endoglucanase
• CBH exo-cellobiohydrolase
• the present invention relates to detergent compositions containing a cleaning effective amount of a surfactant and a cellulase composition wherein the cellulase composition comprises one or more acidic EG type components and optionally CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.
• Such detergent compositions provide substantial improvements in softness, feel, color retention/restoration, and the like.
• cellulase compositions comprise one or more acidic EG type components containing at least about 1 weight percent, preferably, at least about 5 weight percent, and more preferably, at least about 10 weight percent, of CBH I type cellulase components (based on the total protein weight of all of the acidic EG components) .
• Cellulases are known in the art as enzymes that hydrolyze cellulose ( ⁇ -l,4-glucan linkages) thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. While cellulases are produced (expressed) in fungi, bacteria and the like, those produced by fungi have been given the most attention because certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose and such cellulases can be readily produced in large quantities via fermentation procedures.
• Wood et al. "Methods in Enzymology", 160. 25, pages 234 et seq. (1988), discloses that certain fungi produce complete cellulase systems which are comprised of several different enzyme components including those identified as exo-cellobiohydrolases (EC 3.2.1.91) (“CBH”), endoglucanases (EC 3.2.1.4) (“EG”), and ⁇ -glucosidases (EC 3.2.1.21) (“BG”) .
• CBH exo-cellobiohydrolases
• EG endoglucanases
• BG ⁇ -glucosidases
• some fungi are incapable of producing complete cellulase systems. Generally, these systems lack
• CBH components See, for instance, Coughlan et al., Biochemistry and Genetics of Cellulose Degradation, Aubert et al., Editor, pages 11 et seq., (Academic Press, 1988); and Wood et al., Methods in Enzymology, 160. 25, pages 87 et seq., (Academic Press, New York, 1988) .
• bacterial cellulases are reported in the literature as containing little or no CBH components, there are a few cases where CBH- like components derived from bacterial cellulases have been reported to possess exo-cellobiohydrolase activity.
• the fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification.
• multiple CBHs and EGs have been isolated from a variety of fungal sources including Trichoderma reesei which contains 2 CBHs, i.e., CBH I and CBH II, and at least 3 EGs, i.e., EG I, EG II and EG III.
• the complete cellulase system comprising CBH,
• EG and BG is required to efficiently convert crystalline cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components parti-cularly if they are of different classifications. That is to say the effectiveness of a complete cellulase system is significantly greater than the sum of the contributions from the isolated components of the same classification. In this regard, it is known in the art that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. See, for example, Wood, Bioche . Soc. Trans., 13, pp. 407-410 (1985) .
• the substrate specificity and mode of action of the different cellulase components varies significantly with classification which may account for the synergy of the combined components.
• the current accepted mode of cellulase action is that endoglucanase hydrolyzes internal ⁇ -l,4-glucosidic bonds, particularly, in regions of low cr stallinity of the cellulose and exo-cellobio- hydrolase hydrolyzes cellobiose from the non- reducing end of cellulose.
• the action of endoglucanases greatly facilitates the action of exo-cellobio-hydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components.
• ⁇ -Glucosidase act only on cellooligosaccharides, e.g., cellobiose, to give glucose as the sole product. They are considered an integral part of the cellulase system because they drive the overall reaction to glucose and thereby relieve the inhibitory effects of cellobiose on CBH and EG components.
• cellulases are also known in the art to be useful in detergent compositions for the purposes of enhancing the cleaning ability of the composition, for use as a softening agent, and for improving the feel of cotton fabrics, and the like. While the exact mechanism by which cellulase compositions soften garments is not fully understood, softening and color restoration properties of cellulase have been attributed to alkaline endoglucanase components in cellulase compositions. Thus, for instance, International Application Publication No.
• WO 89/09259 discloses that detergent compositions containing a cellulase composition enriched in a specified alkaline endoglucanase component impart color restoration and improved softening to treated garments as compared to cellulase compositions not enriched in such components. Additionally, the use of such alkaline endoglucanase components in detergent compositions complements the pH requirements of the detergent composition.
• the endoglucanase component of this reference is defined as one exhibiting maximal activity at an alkaline pH of 7.5 to 10 and which has defined activity criteria on carboxymethylcellulose and Avicel.
• the most readily employed sources of cellulase compositions are fungally derived cellulase compositions and components which exhibit maximal activity at an acidic pHs with less activity at alkaline pHs.
• fungal cellulase compositions enriched in acidic endoglucanase type components can be combined in detergent compositions to attain improvements in softening, color retention/restoration and feel. It has further been found that such cellulase compositions impart improvements in color retention/restoration, softening and feel in acidic, neutral or alkaline wash media. In alkaline wash media, improvements in softening, color retention/restoration, and feel are particularly surprising because acidic endoglucanase type components possess minimal enzymatic activity in alkaline media and are considered to be unstable under alkaline conditions.
• the acidic endoglucanase type components still impart improvements in softening, color retention/restoration, and feel to cotton fabrics treated with an alkaline detergent medium. Such improvements are particularly noticeable either in a single wash using higher concentrations of such cellulase compositions or with repeated washings using lower concentrations of such cellulase compositions.
• the present invention is directed to a detergent composition
• a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent, and preferably from about 0.05 to about 2 weight percent, of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.
• the fungal cellulase composition employed in the detergent composition comprises one or more acidic EG type components and optionally one or more CBH type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH type components of greater than 5:1. Even more preferably, this fungal cellulase composition is free of all CBH I type components and, still more preferably, free of all CBH type components.
• the cellulase composition is derived from a microorganism which has been genetically modified so as to be incapable of producing any CBH I type components and more preferably any CBH type components, i.e., CBH I type and CBH II type components.
• the cellulase composition is derived from a microorganism which has been genetically modified so as to be incapable of producing any CBH I type components or any CBH type components without the introduction of any heterologous proteins.
• the present invention is directed to a method for enhancing the softness of a cotton-containing fabric which method comprises washing the fabric in a wash medium derived from a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5, and preferably from about 0.05 to about 2, weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.
• the present invention is directed to a method for retaining/restoring the color of a cotton-containing fabric which method comprises washing the fabric one or more times in a wash medium derived from a detergent composition comprising a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5, and preferably from about 0.05 to about 2, weight percent of a fungal cellulase composition comprising one or more acidic EG type components and optionally one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1.
• FIG. 1 is an outline of the construction of p ⁇ CBHlEy 4.
• FIG. 2 illustrates deletion of the T_ reesei gene by integration of the larger EcoRI fragment from p ⁇ CBHIpyr . at the cbhl locus on one of the T. reesei chromosomes.
• FIG. 3 is an autoradiograph of DNA from _,_ reesei strain GC69 transformed with EcoRI digested p ⁇ CBHIp ⁇ r4 after Southern blot analysis using a 32 P labelled p ⁇ CBHIp ⁇ r4 as the probe. The sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.
• FIG. 4 is an autoradiograph of DNA from a T. reesei strain GC69 transformed with EcoRI digested p ⁇ CBHIp ⁇ r4 using a 32 P labelled plntCBHI as the probe.
• the sizes of molecular weight markers are shown in kilobase pairs to the left of the Figure.
• FIG. 5 is an isoelectric focusing gel displaying the proteins secreted by the wild type and by transformed strains of _ _ reesei.
• Lane A of the isoelectric focusing gel employs partially purified CBHI from T. reesei; Lane B employs a wild type T_ reesei : Lane C employs protein from a __ reesei strain with the cbhl gene deleted; and Lane D employs protein from a Tj_ reesei 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 j ⁇ - glucosidase
• El refers to endoglucanase I
• E2 refers to endoglucanase II
• E3 refers to endoglucanase III
• Cl refers to exo-cellobiohydrolase I
• C2 refers to exo-cellobiohydrolase II.
• FIG. 6A is a representation of the T. reesei cbh2 locus, cloned as a 4.1 kb EcoRI fragment on genomic DNA and FIG. 6B is a representation of the cbh2 gene deletion vector pP ⁇ CBHII.
• FIG. 7 is an autoradiograph of DNA from T. reesei strain P37P ⁇ CBHIPyr"26 transformed with EcoRI digested pP ⁇ CBHII after Southern blot analysis using a 3 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 pEGlpyr4.
• FIG. 9 illustrates the RBB-CMC activity profile of an acidic EG enriched fungal cellulase composition (CBH I and II deleted) derived from
• Trichoderma reesei over a pH range at 40°C Trichoderma reesei over a pH range at 40°C; as well as the activity profile of an enriched EG III cellulase composition derived from Trichoderma reesei over a pH range at 40°C.
• FIG. 10 illustrates strength loss results after three wash cycles in a launderometer for cotton-containing fabrics treated with cellulase compositions having varying amounts of CBH components.
• FIG. 11 illustrates fiber removal results
• FIG. 12 illustrates fiber removal results
• FIG. 13 illustrates the softness panel test results for varying concentrations (in ppm) of an EG enriched cellulase composition derived from a strain of Trichoderma reesei genetically modified so as to be incapable of producing CBHI & II.
• FIG. 14 is a diagram of the site specific alterations made in the ectll and cbhl genes to create convenient restriction endonuclease cleavage sites.
• the upper line shows the original DNA sequence
• the changes introduced are shown in the middle line
• the new sequence is shown in the lower line.
• FIG. 15 is a diagram of the larger EcoRI fragment which can be obtained from pCEPCl.
• FIG. 16 is an autoradiograph of DNA, from an untransformed strain of 3 ⁇ _ reesei RutC30 and from two transformants obtained by transforming T_ s . reesei with EcoRI digested pCEPCl. The DNA was digested with Pstl. a Southern blot was obtained and hybridized with 32 P labelled pUC4K: :cbhl. The sizes of marker DNA fragments are shown in kilobase pairs to the left of the Figure.
• FIG. 17 is a diagram of the plasmid pEGII::P-l.
• FIG 18. is an autoradiograph of DNA from T. reesei strain P37P ⁇ 67P"1 transformed with Hindlll and BamHI digested pEGII::P-l.
• a Southern blot was prepared and the DNA was hybridized with an approximately 4kb Pstl fragment of radiolabelled T.reesei DNA containing the ec ⁇ l3 gene.
• Lanes A, C and E contain DNA from the untransformed strain whereas, Lanes B, D and F contain DNA from the untransformed T ⁇ reesei strain.
• the T.reesei DNA was digested with Bc ⁇ lll in Lanes A and B, with EcoRV in Lanes C and D and with Pstl in Lanes E and F.
• the size of marker DNA fragments are shown in kilobase pairs to the left of the Figure.
• FIG. 19 is a diagram of the plasmid pP ⁇ EGI-1.
• FIG. 20 is an autoradiograph of a Southern blot of DNA isolated from transformants of strain GC69 obtained with Hindlll digested p ⁇ EGIpyr-3.
• the pattern of hybridisation with the probe, radiolabelled p ⁇ EGIpyr-3, expected for an untransformed strain is shown in Lane C.
• Lane A shows the pattern expected for a transformant in which the eqll gene has been disrupted
• Lane B shows a transformant in which p ⁇ EGIpyr-3 DNA has integrated into the genome but without disrupting the ecrll gene.
• Lane D contains p ⁇ EGIpyr-3 digested with Hindlll to provide appropriate size markers. The sizes of marker DNA fragments are shown in kilobase pairs to the right of the figure.
• the present invention generally relates to detergent compositions containing fungal cellulase compositions enriched in acidic EG type cellulase components relative to CBH I type components as well as for methods employing such cellulase compositions.
• detergent compositions When used in detergent compositions at acidic, neutral or alkaline ⁇ H*'s, such compositions impart improvements in softening, color retention/restoration, and feel to the treated cotton-containing fabric.
• fungal cellulase refers to the enzyme composition derived from fungal sources or microorganisms genetically modified so as to incorporate and express all or part of the cellulase genes obtained from a fungal source.
• Fungal cellulases act on cellulose and its derivatives to hydrolyze cellulose and give primary products, glucose and cellobiose.
• Fungal cellulases are distinguished from cellulases produced from non-fungal sources including microorganisms such as actinomycetes, gliding bacteria (myxobacteria) and true bacteria.
• Fungi capable of producing cellulases useful in preparing cellulase compositions used in the detergent compositions described herein are disclosed in British Patent No. 2 094 826A, the disclosure of which is incorporated herein by reference.
• 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 specificity, different enzymatic action patterns, etc.
• fungal cellulases can contain cellulase classifications which include endoglucanases (EGs) , exo- cellobiohydrolases (CBHs) , ⁇ -glucosidases (BGs) , etc.
• EGs endoglucanases
• CBHs exo- cellobiohydrolases
• BGs ⁇ -glucosidases
• bacterial cellulases are reported in the literature as containing little or no CBH components, there are a few cases where CBH-like components derived from bacterial cellulases have been reported to possess exo- cellobiohydrolase activity.
• a fungal cellulase composition produced by a naturally occurring fungal source and which comprises one or more CBH- and EG components wherein each of these components is found at the ratio produced by the fungal source is sometimes referred to herein as a "complete fungal cellulase system" or a “complete fungal cellulase composition” to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism genetically modified so as to overproduce, underproduce or not produce one or more of the CBH and/or EG components of cellulase.
• 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.
• Acidic Fungal Cellulase Compositions refer to fungally derived cellulase compositions which possess an activity profile having a pH optimum at a pH of less than 7.0, and preferably less than 6.5. Generally but not necessarily, such acidic cellulase compositions contain acidic EG and acidic CBH components.
• Endoglucanase (“EG”) type components refer to all of those fungal cellulase components or combination of components which exhibit detergent activity properties similar to the endoglucanase components of Trichoderma reesei.
• the endoglucanase components of Trichoderma reesei specifically, EG I, EG II, EG III, and the like either alone or in combination
• endoglucanase type components are those fungal cellulase components which impart softening, color retention/restoration and improved feel to cotton garments when these components are incorporated into a wash medium.
• the endoglucanase type components employed in the detergent compositions of this invention also impart less strength loss to cotton-containing fabrics as compared to strength loss arising from the complete cellulase system derived from Trichoderma reesei.
• Such endoglucanase type components may not include components 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 properties in detergent compositions as possessed by the endoglucanase components of Trichoderma reesei.
• Fungal cellulases can contain more than one EG type component.
• the different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificity, different enzymatic action patterns, etc.
• 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. Serial No. 07/422,814, 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
• Acidic fungal EG type components refer to those fungally derived EG type components which have a pH optimum of less than 7.0 and preferably, less than 6.5, but which also possess at least some activity on RBB-CMC at 40°C (per Example 15) in alkaline media (i.e., an aqueous solution containing the acidic EG type cellulase component buffered to a pH of from greater than 7 to about 10, and preferably at a pH of from greater than 7 to about 8) .
• alkaline media i.e., an aqueous solution containing the acidic EG type cellulase component buffered to a pH of from greater than 7 to about 10, and preferably at a pH of from greater than 7 to about 8 .
• the acidic EG type component possesses at least 10% of its maximal activity.
• combinations of acidic EG type components may give a synergistic response in improving softening, color retention/restoration and feel as compared to a single acidic EG type component.
• a single acidic EG type component may be more stable or have a broader spectrum of activity over a range of pHs.
• the acidic EG type components employed in this invention can be either a single acidic EG type component or a combination of two or more acidic EG type components.
• the acidic EG type components may be derived from the same or different fungal sources.
• Exo-cellobiohydrolase type (“CBH type”) components refer to those fungal cellulase components which exhibit detergent activity properties similar to CBH I and/or CBH II components of Trichoderma reesei.
• CBH type components when used in the absence of EG type components (as defined above) , the CBH I and CBH II components of Trichoderma reesei alone do not impart color retention/restoration and improved feel to the so- treated cotton-containing fabrics.
• the CBH I component of Trichoderma reesei can impart enhanced strength loss and incremental cleaning effect to cotton-containing fabrics.
• CBH I type components and CBH II type components refer to those fungal cellulase components which exhibit detergent activity properties similar to CBH I and CBH II components of Trichoderma reesei. respectively.
• this includes the properties of enhancing strength loss of cotton-containing fabrics and/or imparting an incremental cleaning benefit when used in the presence of EG type components.
• the CBH I components also impart an incremental softening benefit when used in the presence of EG type components.
• exo-cellobiohydrolase type components may include components not traditionally classed as exo-cellobiohydrolases using activity tests such as those used to characterize CBH I and CBH II from Trichoderma reesei.
• such components are competitively inhibited by cellobiose (K ; approximately lmM) ; (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., and (c) hydrolyze phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose.
• K cellobiose
• CBH cellobiose
• some fungal cellulase components which are characterized as CBH components by such activity tests, will provide improved softness, feel and color retention/restoration to cotton-containing fabrics when these components are used alone in detergent compositions.
• exo-cellobiohydrolases as EG type components because these components possess similar functional properties in detergent compositions as possessed by the endoglucanase components of Trichoderma reesei.
• Acidic Fungal CBH type Components refer to those fungally derived CBH type components which possess an activity profile on RBB-CMC at 40°C (per Example 15) having a pH optimum at a pH of less than 7.0 and preferably, less than 6.5.
• EG type components can be obtained by purification techniques.
• the complete cellulase system can be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like.
• ion exchange chromatography usually anion exchange chromatography
• mixtures of cellulase components having the requisite ratio of acidic EG type components to CBH I type cellulase components could be prepared by means other than isolation and recombination of the components.
• recombinant techniques can alter the relative ratio of EG components to CBH components so as to produce a mixture of cellulase components having a relatively high ratio of EG components to CBH components or which can produce one or more EG components free of all CBH components.
• a preferred method for the preparation of cellulase compositions enriched in acidic EG type components is by genetically modifying a microorganism so as to be incapable of producing one or more CBH type components which methods do not produce any heterologous protein.
• U.S. Serial No. 07/593,919, filed October 5, 1990 and which is incorporated herein by reference in its entirety discloses methods for genetically engineering Trichoderma reesei so as to be incapable of producing one or more CBH components and/or overproducing one or more EG components.
• the deletion of the genes responsible for producing either CBH I type or CBH II type cellulase components would have the effect of enriching the amount of EG type components present in the cellulase composition.
• the deletion of those genes responsible for producing CBH I and II type components would result in a cellulase composition free of CBH type components.
• fungal cellulase compositions can be used herein from fungal sources which produce an incomplete fungal cellulase composition.
• certain fungi produce cellulase compositions free of CBH components. See, for example, Coughlan et al. , Biochemistry and Genetics of Cellulose Degradation, Aubert et al. Editors, pp. 11-30 (Academic Press, 1988) , disclose that brown rot fungi do not apparently produce CBH components, but it may be possible that one or more of these components are CBH I type components.
• a requisite amount of one or more CBH type components purified by conventional procedures can be added to a cellulase composition produced from a microorganism genetically engineered so as to be incapable of producing CBH type components so as to achieve a specified ratio of acidic EG type components to one or more CBH type components, i.e., an acidic cellulase composition free of all CBH type components so as to be enriched in acidic EG type components can be formulated to contain 1 weight percent of a CBH I type component merely by adding this amount of a purified CBH I type component to the cellulase composition.
• ⁇ -Glucosidase (BG) components refer to those components of cellulase which exhibit BG activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides (“cellobiose”) and give glucose as the sole product.
• BG components do not adsorb onto or react with cellulose polymers. Furthermore, such BG components are competitively inhibited by glucose (K; approximately ImM) .
• BG components are not literally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the combined action of CBH components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur.
• BG components are often characterized on aryl substrates such as p- nitrophenol B-D-glucoside (PNPG) and thus are often called aryl-glucosidases. It should be noted that not all aryl glucosidases are BG components, in that some do not hydrolyze cellobiose.
• PNPG p- nitrophenol B-D-glucoside
• the presence or absence of BG components in the cellulase composition can be used to regulate the activity of the CBH components. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG components in the cellulase composition will "turn- off" CBH activity when the concentration of cellobiose reaches inhibitory levels. It is also contemplated that one or more additives (e.g.
• cellobiose, glucose, etc. can be added to the cellulase composition to effectively "turn-off", directly or indirectly, some or all of the CBH I type activity as well as other CBH activity.
• 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 I type activity to levels equal to or less than the CBH I 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
• 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 the detergent composition, the BG component is generally added in an amount sufficient to prevent inhibition of the CBH and EG components and particularly, CBH I type cellulase components, by cellobiose.
• the amount of BG component added depends upon the amount of cellobiose produced in the detergent wash which can be readily determined by the skilled artisan.
• the weight percent of BG component relative to CBH type components in the cellulase composition is preferably from about 0.2 to about 10 weight percent, and more preferably, from about 0.5 to about 5 weight percent.
• Preferred fungal cellulases for use in preparing the fungal cellulase compositions used in this invention are those obtained from Trichoderma reesei. Trichoderma koningii, Pencillum sp. , and the like.
• Certain fungal cellulases are commercially available, i.e., CELLUCAST (available from Novo Industry, Copenhagen, Denmark) , RAPIDASE (available from Gist Brocades, N.V. , Delft, Holland), CYTOLASE 123 (available from Genencor International, South San Francisco, California) and the like.
• Other fungal cellulases can be readily isolated by art recognized fermentation and isolation procedures.
• cotton-containing fabric refers to sewn or unsewn fabrics made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, and the like.
• the amount of cotton in the fabric should be at least about 40 percent by weight percent cotton; preferably, more than about 60 percent by weight cotton; and most preferably, more than about 75 percent by weight cotton.
• 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) , polyester fibers (for example, polyethylene terephthalate) , polyvinyl alcohol fibers (for example, Vinylon) , polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers and aramid fibers. It is contemplated that regenerated cellulose, such as rayon, could be used as a substitute for cotton in cotton containing fabrics.
• synthetic fibers such as polyamide fibers (for example, nylon 6 and nylon 66) , acrylic fibers (for example, polyacrylonitrile fibers) , polyester fibers (for example, polyethylene terephthalate) , polyvinyl alcohol fibers (for example, Vinylon) , polyvinyl chloride fibers, polyvinylidene chloride fibers
• surface active agent or surfactant refers to anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions.
• wash medium refers to an aqueous wash solution prepared by adding a requisite amount of a detergent (surfactant) composition to water.
• the wash medium generally contains a cleaning effective amount of the detergent.
• the wash medium is defined as an "acidic wash medium” if the pH of the medium is from about 4 to less than about 7.
• the wash medium is defined as a “neutral wash medium” if the pH of the medium is about 7.
• the wash medium is defined as an "alkaline wash medium” if the pH of the medium is from above 7 to about 10.
• the alkaline wash medium will have a pH of from above 7 to about 9 and, even more preferably, from above 7 to about 8.
• the present invention is directed to the discovery that acidic EG type components can be used in detergent compositions to effect softening of cotton-containing fabrics regardless of whether such compositions are employed in an acidic, neutral or alkaline wash medium.
• the detergent composition will also effect color retention/restoration and will improve the feel of the treated fabric.
• the mixture contains one or more acidic EG type components and one or more CBH I type components wherein said cellulase composition has a protein weight ratio of all acidic EG type components to all CBH I type components of greater than 5:1 and preferably a ratio of all acidic EG type components to all CBH components of greater than 5:1.
• a detergent composition containing a cellulase composition enriched in acidic EG type components and which contains at least about l weight percent, and preferably, at least about 5 weight percent, and even more preferably, at least about 10 weight percent, of CBH I type cellulase components (based on the total protein weight of all of the acidic EG components) .
• detergent compositions containing a cellulase composition enriched in acidic EG type components and which is free of CBH I type components also result in significantly reduced strength loss of the cotton- containing fabric as compared to cellulase compositions enriched in acidic EG type components but which contain CBH I type components. That is to say that repeated washings with detergent compositions containing cellulase compositions enriched in acidic EG type components and free of CBH I type components will result in significantly less strength loss of cotton-containing fabrics as compared to detergent compositions containing a cellulase composition comprising both acidic EG type components and CBH type components.
• the skilled artisan would need to balance the desire for an incremental cleaning benefit against that for strength loss resistance. That is to say that if the primary emphasis for incorporating cellulase into the detergent composition is for improved softness, color retention/restoration, and improved feel of cotton-containing fabrics without significant strength loss in the cotton-containing fabric, then the cellulase employed in the detergent composition should preferably comprise one or more acidic EG type components free of all CBH I type components.
• the cellulase employed in the detergent composition should contain a cellulase comprising one or more acidic EG type components as well as at least about 1 weight percent of a CBH I type component (based on the total protein weight of all of the acidic EG components) .
• the amount of cellulase generally employed in the detergent compositions of this invention is an amount sufficient to impart color retention/restoration and softness to the cotton garments.
• the cellulase compositions are employed from about 0.01 weight percent to about 5 weight percent relative to the weight of the total detergent composition. More preferably, the cellulase compositions are employed from about 0.05 weight percent to about 2 weight percent relative to the weight of the total detergent composition.
• the specific concentration of cellulase employed in the detergent composition is selected so that upon dilution into a wash medium, the concentration of acidic EG type components will range from about 0.5 to about 500 ppm, and preferably from about 2 to about 100 ppm.
• the specific amount of cellulase employed in the detergent composition will depend on the amount of EG type components in the cellulase compositions as well as the extent the detergent composition will be diluted upon addition to water to form a wash medium. These factors are readily ascertained by the skilled artisan.
• the cellulase compositions employed in the detergent compositions of this invention contain at least about 20 weight percent of endoglucanase type components based on the total weight of protein.
• One of the important aspects of the present invention is that by tailoring the cellulase composition to contain enriched amounts of acidic EG components, it is possible to achieve the desired effects of softening, color retention/restoration and improved feel with the use of lower concentrations of cellulase in the detergent composition.
• the use of lower concentrations of cellulase in the detergent compositions should lead to lower allergenic levels than compositions containing higher concentrations of cellulase.
• the cellulase composition as described above can be added to the detergent composition either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the like. Such forms are well known to the skilled artisan.
• the cellulase composition is preferably formulated as granules.
• the granules can be formulated so as to contain a cellulase protecting agent. See, for instance, U.S. Serial No. 07/642,669 filed January 17, 1991 as Attorney Docket No.
• the granule can be formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium. Such materials and granules are disclosed in U.S. Serial No. 07/642,596 filed on January 17, 1991 as Attorney Docket No. GCS-171-US1 and entitled “GRANULAR COMPOSITIONS” which application is incorporated herein by reference in its entirety.
• the detergent compositions of this invention employ a surface active agent, i.e., surfactant, including anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions.
• a surface active agent i.e., surfactant, including anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions.
• Suitable anionic surfactants for use in the detergent composition of this invention include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates and the like.
• Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines 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.
• Suitable surfactants for use in this invention are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.
• the surfactant or a mixture of surfactants is generally employed in the detergent compositions of this invention in an amount from about 1 weight percent to about 95 weight percent of the total detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition.
• the surfactant concentration is generally about 500 ppm or more; and preferably, from about 1000 ppm to 15,000 ppm.
• the detergent compositions of this invention can optionally contain one or more of the following components:
• Such hydrolases include carboxylate ester hydrolase, thioester hydrolase, phosphate monoester hydrolase, and phosphate diester hydrolase which act on the ester bond; glycoside hydrolase which acts on glycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds; thioether hydrolase which acts on the ether bond; and ⁇ -amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl- peptide hydrolase which act on the peptide bond.
• carboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptide hydrolase Preferable among them are carboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptide hydrolase.
• Suitable hydrolases include (1) proteases belonging to petidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein, gastrisin, cathepsin D.
• proteases belonging to petidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain
• glycoside hydrolases (cellulase which is an essential ingredient is excluded from this group) ⁇ - amylase, ⁇ -amylase, gluco amylase, invertase, lysozyme, pectinase, chitinase, and dextranase.
• ⁇ -amylase and ⁇ -amylase are ⁇ -amylase and ⁇ -amylase. They function in acid to neutral systems, but one which is obtained from bacteria exhibits high activity in an alkaline system; (3) carboxylate ester hydrolase including carboxyl esterase, lipase, pectin esterase, and chlorophyllase. Especially effective among them is lipase.
• the hydrolase other than cellulase is incorporated into the detergent composition as much as required according to the purpose. It should preferably be incorporated in an amount of 0.001 to 5 weight percent, and more preferably 0.02 to 3 weight percent, in terms of purified one.
• This enzyme should be used in the form of granules made of crude enzyme alone or in combination with other components in the detergent composition. Granules of crude enzyme are used in such an amount that the purified enzyme is 0.001 to 50 weight percent in the granules.
• the granules are used in an amount of 0.002 to 20 and preferably 0.1 to 10 weight percent. As with cellulases, these granules can be formulated so as to contain an enzyme protecting agent and a dissolution retardant material.
• Such cationic surfactants and long-chain fatty acid salts include saturated or unsaturated fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, ⁇ -sulfofatty acid salts or esters, amino acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substituents and up to 1 phenyl substituted alkyl substituents.
• Suitable cationic surfactants and long-chain fatty acid salts are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.
• the composition may contain from about
• the composition may contain from about 0 to about 50 weight percent of one or more builder components selected from the group consisting of alkali metal salts and alkanolamine salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, aminopolyacetates high molecular electrolytes, non- dissociating polymers, salts of dicarboxylic acids, and aluminosilicate salts.
• Suitable divalent sequestering gents are disclosed in British Patent
• the composition may contain from about 1 to about 50 weight percent, preferably from about 5 to about 30 weight percent, based on the composition of one or more alkali metal salts of the following compounds as the alkalis or inorganic electrolytes: silicates, carbonates and sulfates as well as organic alkalis such as triethanolamine, diethanolamine, monoethanolamine and triisopropanolamine.
• composition may contain from about 0.1 to about 5 weight percent of one or more of the following compounds as antiredeposition agents: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose.
• a combination of carboxymethyl- cellulose or/and polyethylene glycol with the cellulase composition of the present invention provides for an especially useful dirt removing composition.
• cellulase of the present invention in combination with a bleaching agent such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct or/and a photo ⁇ sensitive bleaching dye such as zinc or aluminum salt of sulfonated phthalocyanine further improves the deterging effects.
• a bleaching agent such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct or/and a photo ⁇ sensitive bleaching dye such as zinc or aluminum salt of sulfonated phthalocyanine
• bluing agents and fluorescent dyes may be incorporated in the composition, if necessary. Suitable bluing agents and fluorescent dyes are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.
• caking inhibitors may be incorporated in the powdery detergent: p-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium silicate (such as Micro-Cell of Johns Manville Co.), calcium carbonate and magnesium oxide.
• the cellulase composition of this invention are deactivated in some cases in the presence of copper, zinc, chromium, mercury, lead, manganese or silver ions or their compounds.
• Various metal chelating agents and metal-precipitating agents are effective against these inhibitors. They include, for example, divalent metal ion sequestering agents as listed in the above item with reference to optional additives as well as magnesium silicate and magnesium sulfate.
• Cellobiose, glucose and gluconolactone act sometimes as the inhibitors. It is preferred to avoid the co-presence of these saccharides with the cellulase as far as possible. In case the co- presence in unavoidable, it is necessary to avoid the direct contact of the saccharides with the cellulase by, for example, coating them.
• Long-chain-fatty acid salts and cationic surfactants act as the inhibitors in some cases. However, the co-presence of these substances with the cellulase is allowable if the direct contact of them is prevented by some means such as tableting or coating.
• the activators vary depending on variety of the cellulases. In the presence of proteins, cobalt and its salts, magnesium and its salts, and calcium and its salts, potassium and its salts, sodium and its salts cr monosaccharides such as annose and xylose, the cellulases are activated and their deterging powers are improved remarkably.
• the antioxidants include, for example, tert- butyl-hydroxytoluene, 4,4'-butylidenebis(6-tert- butyl-3-methylphenol) , 2,2'-butylidenebis(6-tert- butyl-4-methylphenol) , monostyrenated cresol, distyrenated cresol, monostyrenated phenol, distyrenated phenol and 1,l-bis(4-hydroxy- phenyl)cyclohexane.
• the solubilizers include, for example, lower alcohols such as ethanol, benzenesulfonate salts, lower alkylbenzenesulfonate salts such as p- toluenesulfonate salts, glycols such as propylene glycol, acetylbenzenesulfonate salts, acetamides, pyridinedicarboxylic acid amides, benzoate salts and urea.
• lower alcohols such as ethanol
• benzenesulfonate salts lower alkylbenzenesulfonate salts such as p- toluenesulfonate salts
• glycols such as propylene glycol
• acetylbenzenesulfonate salts acetamides
• pyridinedicarboxylic acid amides pyridinedicarboxylic acid amides
• the detergent composition of the present invention can be used in a broad pH range of from acidic to alkaline pH.
• the detergent composition of the present invention can be used in alkaline detergent wash media and more preferably, alkaline detergent wash media having a pH of from above 7 to no more than about 8.
• perfumes can be used, if desired, with the detergent compositions of this invention.
• Such components are conventionally employed in amounts heretofore used in the art.
• a detergent base used in the present invention is in the form of a powder, it may be one which is prepared by any known preparation methods including a spray-drying method and a granulation method.
• the detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred.
• the detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions.
• the detergent base obtained by the spray-drying method is hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space.
• the granules have a size of from 50 to 2000 micrometers.
• perfumes, enzymes, bleaching agents, inorganic alkaline builders may be added.
• various ingredients may also be added after the preparation of the base.
• the detergent base When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion.
• carboxymethyl- cellulose For removing the decomposition of carboxymethylcellulose by the cellulase in the detergent, it is desirable that carboxymethyl- cellulose is granulated or coated before the incorporation in the composition.
• the detergent compositions of this invention are used in industrial and household uses at temperatures and liquor ratios conventionally employed in these environments.
• the cellulase compositions described herein can additionally be used in a pre-washing step in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements in color retention/restoration, softening and feel.
• the cellulase could also be employed as a pre-soak either as a liquid, spray, gel or paste for uses, such as, as a spot remover.
• the cellulase composition described herein is generally employed from about 0.002 to about 0.5 weight percent based on the total weight of the pre-soak composition.
• a surfactant may optionally be employed and when employed, is generally present at a concentration of from about 0.01 to about 20 weight percent based on the total weight of the pre-soak.
• the remainder of the composition comprises conventional components used in the pre-soak, i.e., diluent, buffers, other enzymes (proteases) , and the like at their conventional concentrations.
• cellulase compositions described herein can also be used in home use as a stand alone composition suitable for restoring color to faded fabrics. See, for example, U.S. Patent No. 4,738,682, which is incorporated herein by reference in its entirety.
• the following examples are offered to illustrate the present invention and should not be construed in any way as limiting the scope of this invention.
• Examples 1-12 and 20-28 demonstrate the preparation of Trichoderma reesei genetically engineered so as to be incapable of producing one or more cellulase components or so as to overproduce specific cellulase components.
• the pyr4 gene encodes orotidine-5'- monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine.
• the toxic inhibitor 5- fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells.
• FAA 5- fluoroorotic acid
• cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth. It is, therefore, possible to select for p ⁇ r4 derivative strains using FOA.
• spores of T. reesei strain RL-P37 (Sheir-Neiss, G. and Montenecourt, B.S. , Appl. Microbiol. Biotechnol. 20, p.
• a cbhl gene encoding the CBHI protein was cloned from the genomic DNA of _. reesei strain RL- P37 by hybridization with an oligonucleotide probe designed on the basis of the published sequence for this gene using known probe synthesis methods (Shoemaker et al., 1983b).
• the cbhl gene resides on a 6.5 kb Pstl fragment and was inserted into Pstl cut pUC4K (purchased from Pharmacia Inc. , Piscataway, NJ) replacing the Kan r gene of this vector using techniques known in the art, which techniques are set forth in Maniatis et al., (1989) and incorporated herein by reference.
• pUC4K::cbhl was then cut with Hindlll and the larger fragment of about 6 kb was isolated and religated to give pUC4K: :cbhl ⁇ H/H (see FIG. 1).
• This procedure removes the entire cbhl coding sequence and approximately 1.2 kb upstream and 1.5 kb downstream of flanking sequences. Approximately, 1 kb of flanking DNA from either end of the original Pstl fragment remains. The T.
• reesei pyr4 gene was cloned as a 6.5 kb Hindlll fragment of genomic DNA in pUCIS to form pTpyr2 (Smith et al., 1991) following the methods of Maniatis et al., supra.
• the plasmid pUC4K: :cbhl ⁇ H/H was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase. This end dephosphorylated DNA was ligated with the 6.5 kb Hindlll fragment containing the T. reesei pyr4 gene to give p ⁇ CBHIpyr4.
• FIG. 1 illustrates the construction of this plasmid.
• Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract, 2% glucose) in a 500 ml flask with about 5 x 10 7 T. reesei GC69 spores (the pyr4 ' derivative strain) . The flask was then incubated at 37°C with shaking for about 16 hours. The mycelium was harvested by centrifugation at 2,750 x g.
• the harvested mycelium was further washed in a 1.2 M sorbitol solution and resuspended in 40 ml of a solution containing 5 mg/ml Novozym R 234 solution (which is the tradename for a multicomponent enzyme system containing 1,3-alpha- glucanase, 1,3-beta-glucanase, laminarinase, xylanase, chitinase and protease from Novo Biolabs, Danbury, CT) ; 5 mg/ml MgS0 4 .7H 2 0; 0.5 mg/ml bovine serum albumin; 1.2 M sorbitol.
• Novozym R 234 solution which is the tradename for a multicomponent enzyme system containing 1,3-alpha- glucanase, 1,3-beta-glucanase, laminarinase, xylanase, chitinase and proteas
• the protoplasts were removed from the cellular debris by filtration through Miracloth (Calbiochem Corp, La Jolla, CA) and collected by centrifugation at 2,000 x g.
• the protoplasts were washed three times in 1.2 M sorbitol and once in 1.2 M sorbitol, 50 mM CaCl 2 , centrifuged and resuspended at a density of approximately 2 x 10 8 protoplasts per ml of 1.2 M sorbitol, 50 M CaCl 2 .
• Transformation of Fungal Protoplasts with p ⁇ CBHIpyr4 200 ⁇ l of the protoplast suspension prepared in Example 3 was added to 20 ⁇ l of EcoRI digested p ⁇ CBHIpyr4 (prepared in Example 2) in TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) and 50 ⁇ l of a polyethylene glycol (PEG) solution containing 25% PEG 4000, 0.6 M KC1 and 50 mM CaCl 2 . This mixture was incubated on ice for 20 minutes. After this incubation period 2.0 ml of the above-identified PEG solution was added thereto, the solution was further mixed and incubated at room temperature for 5 minutes.
• PEG polyethylene glycol
• 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 Pstl 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 ⁇ CBHIpyr4 probe. The probe was selected to identify the native cbhl gene as a 6.5 kb Pstl 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 CBHI band, indicating that this gene has been totally deleted in the transformant by integration of the DNA fragment at the cbhl gene.
• the cbhl deleted strain is called P37P ⁇ CBHI.
• Figure 2 outlines the deletion of the T.
• sample A contained the cbhl gene, as indicated by the band at 6.5 kb; however the transformant, sample B, does not contain this 6.5 kb band and therefore does not contain the cbhl gene and does not contain any sequences derived from the pUC plasmid.
• Example 7
• 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
• This isoelectric focusing gel shows various proteins in different supernatant cultures of T. reesei. Lane A is partially purified CBHI; Lane B is the supernatant from an untransformed T. reesei culture; Lane C is the supernatant from strain P37P ⁇ CBHI produced according to the methods of the present invention. The position of various cellulase components are labelled CBHI, CBHII, EGI, EGII, and EGIII. Since CBHI constitutes 50% of the total extracellular protein, it is the major secreted protein and hence is the darkest band on the gel. This isoelectric focusing gel clearly shows depletion of the CBHI protein in the P37P ⁇ CBHI strain.
• CBHII protein has been cloned as a 4.1 kb EcoRI fragment of genomic DNA which is shown diagramatically in FIG. 6A (Chen et al., 1987, Biotechnology , 5:274-278). This 4.1 kb fragment was inserted between the EcoRI sites of pUC4XL.
• the latter plasmid is a pUC derivative (constructed by R.M. Berka, Genencor International Inc.) which contains a multiple cloning site with a symetrical pattern of restriction endonuclease sites arranged in the order shown here: EcoRI. BamHI. Sad. Smal. Hindlll, Xhol. Bglll, Clal. Bglll, Xhol.
• T. reesei pyr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb Nhel-Sphl fragment and inserted between the Sphl and Xbal sites of pUC219 (see Example 23) to create p219M (Smith et al. , 1991, Curr. Genet 19 p. 27-33).
• the pyr4 gene was then removed as a Hindlll-Clal fragment having seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pP ⁇ CBHII (see FIG. 6B) .
• Protoplasts of strain GC69 will be generated and transformed with EcoRI digested pP ⁇ CBHII according to the methods outlined in Examples 3 and 4.
• DNA from the transformants will be digested with EcoRI and Asp718. and subjected to agarose gel electrophoresis.
• the DNA from the gel will be blotted to a membrane filter and hybridized with 32 P labelled pP ⁇ CBHII according to the methods in Example 11.
• Transformants will be identified which have a single copy of the EcoRI fragment from pP ⁇ CBHII integrated precisely at the cbh2 locus.
• the transformants will also be grown in shaker flasks as in Example 7 and the protein in the culture supematants examined by isoelectric focusing. In this manner Ti reesei GC69 transformants which do not produce the CBHII protein will be generated.
• Example 10
• 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.
• FIG. 7 shows the hybridization pattern observed for DNA from an untransformed T. reesei strain. The 4.1 kb EcoRI fragment containing the wild-type cbh2 gene was observed. Lane B shows the hybridization pattern observed for strain P37P ⁇ CBH67. The single 4.1 kb band has been eliminated and replaced by two bands of approximately 0.9 and 3.1 kb. This is the expected pattern if a single copy of the EcoRI fragment from pP ⁇ CBHII had integrated precisely at the cbh2 locus.
• the T. reesei egll gene which encodes EGI, has been cloned as a 4.2 kb Hindlll fragment of genomic DNA from strain RL-P37 by hybridization with oligonucleotides synthesized according to the published sequence (Penttila et al., 1986, Gene 45:253-263; van Arsdell et al. , 1987, Bio/Technology 5:60-64).
• a 3.6 kb Hindlll-BamHI fragment was taken from this clone and ligated with a 1.6 kb Hindlll- BamHI fragment containing the T.
• reesei pyr4 gene obtained from pTpyr2 (see Example 2) and pUC218 (identical to pUC219, see Example 23, but with the multiple cloning site in the opposite orientation) cut with Hindlll to give the plasmid pEGIpyr4 (FIG. 8) .
• Digestion of pEGIpyr4 with Hindlll would liberate a fragment of DNA containing only T. reesei genomic DNA (the egll and pyr4 genes) except for 24 bp of sequenced, synthetic DNA between the two genes and 6 bp of sequenced, synthetic DNA at one end (see FIG. 8) .
• CYTOLASE 123 cellulase was fractionated in the following manner.
• the normal distribution of cellulase components in this cellulase system is as follows:
• 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. These components were separated by gradient elution using an aqueous gradient containing from 0 to about 500 mM sodium chloride. The fraction not bound on this column contained CBH II and EG II.
• 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. Penicillum sp. and the like.
• Example 13 above demonstrated the isolation of several components from Cytolase 123 Cellulase.
• the mixture formed a two phase liquid mixture.
• the phases were separated using an SA-1 disk stack centrifuge.
• the phases were analyzed using silver staining isoelectric focusing gels. Separation was obtained for EG III and xylanase.
• the recovered composition contained about 20 to 50 weight percent of EG III.
• the purification of EG III is conducted by fractionation from a complete fungal cellulase composition (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) which is produced by wild type Trichoderma reesei. Specifically, the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Mo) , QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Md) .
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the fractionation is done using columns containing the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (St. Louis, Mo) , QA Trisacryl M anion exchange resin and SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage, Md) .
• CYTOLASE 123 cellulase 0.5g is desalted using a column of 3 liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8. The desalted solution, is then loaded onto a column of 20 ml of QA Trisacryl M anion exchange resin. The fraction bound on this column contained CBH I and EG I. The fraction not bound on this column contains CBH II, EG II and EG III. These fractions are desalted using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto a column of 20 ml of SP Trisacryl M cation exchange resin. The EG III was eluted with 100 mL of an aqueous solution of 200 mM sodium chloride.
• Trichoderma reesei genetically modified so as to be incapable of producing one or more of EG I, EG II, CBH I and/or CBH II.
• the absence of one or more of such components will necessarily lead to more efficient isolation of EG III.
• the EG III compositions described above may be further purified to provide for substantially pure EG III compositions, i.e., compositions containing EG III at greater than about 80 weight percent of protein.
• substantially pure EG III protein can be obtained by utilizing material obtained from procedure A in procedure B or vica versa.
• One particular method for further purifying EG III is by further fractionation of an EG III sample obtained in part b) of this Example 14. The further fraction was done on a FPLC system using a Mono-S-HR 5/5 column (available from Pharmacia LKB Biotechnology, Piscataway, NJ) .
• the FPLC system consists of a liquid chromatography controller, 2 pumps, a dual path monitor, a fraction collector and a chart recorder (all of which are available from Pharmacia LKB Biotechnology, Piscataway, NJ) .
• the fractionation was conducted by desalting 5 ml of the EG III sample prepared in part b) of this Example 14 with a 20 ml Sephadex G-25 column which had been previously equilibrated with 10 mM sodium citrate pH 4. The column was then eluted with 0-200 mM aqueous gradient of NaCl at a rate of 0.5 ml/minute with samples collected in 1 ml fractions.
• EG III was recovered in fractions 10 and 11 and was determined to be greater than 90% pure by SDS gel electrophoresis. EG III of this purity is suitable for determining the N-terminal amino acid sequence by known techniques.
• EG III as well as EG I and EG II components purified in Example 13 above can be used singularly or in mixtures in the methods of this invention.
• EG components have the following characteristics:
• each of EG I, EG II and EG III are acidic EG components.
• the use of a mixture of these components in the practice of this invention may give a synergistic response in improving softening, color retention/restoration and/or feel as compared to a single component.
• the use of a single component in the practice of this invention may be more stable or have a broader spectrum of activity over a range of pHs. For instance, Example 15 below shows that EG III has considerable activity against RBB-CMC under alkaline conditions.
• the first cellulase composition was a CBH I and II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to that described above so as to be unable to produce CBH I and CBH II components.
• this cellulase composition does not contain CBH I and CBH II which generally comprise from about 58 to 70 percent of a cellulase composition derived from Trichoderma reesei.
• this cellulase composition is necessarily enriched in acidic EG components, i.e., EG I, EG II, EG III and the like.
• the second cellulase composition was an approximately 20-40% pure fraction of EG III isolated from a cellulase composition derived from Trichoderma reesei via purification methods similar to part b) of Example 14.
• the activity of these cellulase compositions was determined at 40°C and the determinations were made using the following procedures.
• FIG. 9 illustrates the relative activity of the CBH I and II deleted cellulase composition compared to the EG III cellulase composition. From this figure, it is seen that the cellulase composition deleted in CBH I and CBH II possesses optimum cellulolytic activity against RBB-CMC at near pH 5.5 and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8. On the other hand, the cellulase composition enriched in EG III possesses optimum cellulolytic activity at about pH 5.5 - 6 and possesses significant activity at alkaline pHs.
• the purpose of this example is to examine the ability of different cellulase compositions to reduce the strength of cotton-containing fabrics. Because the activity of the most of the cellulase components derived from Trichoderma reesei is greatest at or near pH 5, strength loss results will be most evident when the assay is conducted at about this pH. However, the results achieved at pH 5 are believed to correlate with strength loss results which would occur at other pHs and accordingly, are indicative of the relative strength loss capacity of the cellulase components analyzed.
• the first cellulase composition analyzed was a complete fungal cellulase composition (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma reesei and is identified as GC010.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the second cellulase composition analyzed was a CBH II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to Examples 1-12 above and 20-28 below so as to be incapable of expressing CBH II and is identified as CBHIId.
• CBH II comprises up to about 15 percent of the cellulase composition, deletion of this component results in enriched levels of CBH I, and all of the EG components.
• the third cellulase composition analyzed was a CBH I and CBH II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and CBH II and is identified as CBHI/IId.
• CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG components.
• the last cellulase composition analyzed was a CBH I deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to that described above so as to be incapable of expressing CBH I and is identified as CBHId.
• CBH I comprises up to about 55 percent of the cellulase composition, deletion of this component results in enriched levels of CBH II and all of the EG components.
• the cellulase compositions described above were tested for their effect on cotton-containing fabric strength loss in a launderometer.
• the compositions were first normalized so that equal amounts of EG components were used.
• Each cellulase composition was then added to separate solutions of 400 ml of a 20 mM citrate/phosphate buffer, titrated to pH 5, and which contains 0.5 ml of a non-ionic surfactant.
• Each of the resulting solutions was then added to a separate launderometer canister.
• Into these canisters were added a quantity of 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., 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 (rp s) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
• FIG. 10 which shows that compositions containing CBH I, i.e., whole cellulase (GC010) and CBH II deleted cellulase, possessed the most strength loss whereas, the compositions containing no CBH I possessed significantly reduced strength loss as compared to whole cellulase and CBH II deleted cellulase. From these results, it is seen that the presence of CBH I in a cellulase composition imparts increased strength loss to the composition as compared to a similar composition not containing CBH I.
• CBH I whole cellulase (GC010) and CBH II deleted cellulase
• such compositions should be free of all CBH I type cellulase components and preferably, all CBH type cellulase components.
• acidic EG enriched cellulase compositions will result in even lower strength loss at pH > 7 than those results observed at pH 5 shown in FIG. 10.
• the first experiment measures the ability of a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma reesei to remove surface fibers from a cotton-containing fabric over various pHs. This cellulase was tested for its ability to remove surface fibers in a launderometer. 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.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• Samples were prepared and titrated so as to provide for samples at pH 5, pH 6, pH 7 and pH 7.5. Each of the resulting solution was then added to a separate launderometer canister. Into these canisters were added a quantity of marbles to facilitate fiber removal as well as a 7 inch x 5 inch cotton fabric (100% woven cotton, available as Style No. 439W from Test Fabrics, Inc., 200 Blackford Ave., Middlesex, NJ 08846). The canister was then closed and the canister lowered into the launderometer bath which was maintained at 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.
• rpms revolutions per minute
• 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 as follows: Rating Standard 2 not treated with cellulase treated 3 with 8 ppm cellulase treated with 16 ppm cellulase treated with 20 ppm cellulase treated with 40 ppm cellulase treated with 50 ppm cellulase treated with 100 ppm cellulase
• the fabric to be rated was provided a rating which most closely matched one of the standards.
• FIG. 11 illustrates that at the same pH, a dose dependent response is seen in the amount of fibers removed. That is to say that at the same pH, the fabrics treated with more cellulase provided for higher levels of fiber removal as compared to fabrics treated with less cellulase. Moreover, the results of this figure demonstrate that at higher pHs, fiber removal can still be effected merely by using higher concentrations of cellulase.
• the first cellulase composition analyzed was a complete cellulase system (CYTOLASE 123 cellulase, commercially available from Genencor International, South San Francisco, CA) produced by wild type Trichoderma reesei and is identified as GC010.
• CYTOLASE 123 cellulase commercially available from Genencor International, South San Francisco, CA
• the second cellulase composition analyzed was a CBH I and CBH II deleted cellulase composition prepared from Trichoderma reesei genetically modified in a manner similar to that described above so as to be incapable of producing CBH I and CBH II and is identified as CBHI/lid.
• 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 cellulase compositions were first normalized so that similar amounts of EG components were used. These compositions were tested for their ability to remove surface fibers in a launderometer. An appropriate amount of cellulase to provide for the requisite concentrations of EG components in the final compositions were added to separate solutions of 400 ml of a 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.
• the canister was then closed and the canister lowered into the launderometer bath which was maintained at 43°C.
• the canister was then rotated in the bath at a speed of at least about 40 revolutions per minute (rpms) for about 1 hour. Afterwards, the cloth is removed, rinsed well and dried in a standard drier.
• FIG. 12 illustrates that both GC010 and CBH I/IId cellulase compositions gave substantially identical fiber removal results.
• the results of this figure suggest that it is the EG components which provide for fiber removal.
• Example 17 Tergotometer Color Restoration This example is further to Example 17 and substantiates that CBH type components are not necessary for color restoration.
• the purpose of this example is to examine the ability of cellulase compositions deficient in CBH type components to restore color to cotton-containing fabrics. Because the activity of most of the EG components derived from Trichoderma reesei is greatest at or near pH 5, color restoration results will be most evident when the assay is conducted at about this pH. However, the results achieved at pH 5 are believed to correlate with color restoration results which would occur at other pHs and accordingly, are indicative of the relative color restoration capacity of the cellulase components analyzed.
• the cellulase composition employed in this example was a CBH I and CBH II deleted cellulase composition prepared from
• CBH I and CBH II comprises up to about 70 percent of the cellulase composition, deletion of this component results in enriched levels of all of the EG components.
• the assay was conducted by adding a sufficient concentration of this cellulase composition to a 50 mM citrate/phosphate buffer to provide 500 ppm of cellulase.
• the solution was titrated to pH 5 and contained 0.1 weight percent of nonionic surfactant (Grescoterg GL100 - commercially available from Gresco Mfg. , Thomasville, NC 27360) .
• a 10 inch x 10 inch faded cotton containing fabric was 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: Cotton Containing Material Result
• This example demonstrates that the presence of CBH type components are not essential for imparting improved softness to cotton-containing fabrics.
• this example employs a cellulase composition derived from Trichoderma reesei genetically engineered in the manner described above so as to be incapable of producing 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 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 ask to rate them with respect to softness based on softness criteria such as the pliability of the whole fabric. Swatches obtained from treatment with the five different enzyme concentrations and the blank were placed behind a 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.
• cellulase compositions enriched in acidic EG type components derived from microorganisms other than Trichoderma reesei could be used in place of the cellulase compositions described in these examples.
• the source of the cellulase composition enriched in acidic EG type components is not important to this invention and any fungal cellulase composition enriched in acidic EG type components can be used.
• fungal cellulases for use in preparing the fungal cellulase compositions used in this invention can be obtained from Trichoderma koningii. Pencilium sp.
• Example 20 Cellulases can be used, i.e., CELLUCLAST (available from Novo Industry, Copenhagen, Denmark) , RAPIDASE (available from Gist Brocades, N.V. , Delft, Holland), and the like.
• CELLUCLAST available from Novo Industry, Copenhagen, Denmark
• RAPIDASE available from Gist Brocades, N.V. , Delft, Holland
• a pyr4 defective derivative of T. reesei strain RutC30 (Sheir-Neiss and Montenecourt, (1984) , Appl. Microbiol. Biotechnol. 20:46-53) was obtained by the method outlined in Example 1. Protoplasts of this strain were transformed with undigested pEGIpyr4 and stable transformants were purified.
• EP4, EP5, EP6, EP11) , as well as untransformed RutC30 were inoculated into 50 ml of YEG medium (yeast extract, 5 g/1; glucose, 20 g/1) in 250 ml shake flasks and cultured with shaking for two days at 28°C.
• YEG medium yeast extract, 5 g/1; glucose, 20 g/1
• TSF medium 0.05M citrate-phosphate buffer, pH 5.0; Avicel microcrystalline cellulose, 10 g/1; KH 2 P0 4 , 2.0 g/1; (NH 4 ) 2 S0 4 , 1.4 g/1; proteose peptone, 1.0 g/1; Urea, 0.3 g/1; MgS0 4 .7H 2 0, 0.3 g/1; CaCl 2 , 0.3 g/1;
• FeS0 4 .7H 2 5.0 mg/1; MnS0 4 .H 2 0, 1.6 mg/1; ZnS0 4 , 1.4 mg/1; CoCl 2 , 2.0 mg/1; 0.1% Tween 80). These cultures were incubated with shaking for a further four days at 28°C. Samples of the supernatant were taken from these cultures and assays designed to measure the total amount of protein and of endoglucanase activity were performed as described below.
• the endoglucanase assay relied on the release of soluble, dyed oligosaccharides from Remazol Brilliant Blue-carboxymethylcellulose (RBB-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.
• pEGIpyr4 transformant culture supernatant or 0.1 M sodium acetate as a blank (10- 20 ⁇ l) were placed in tubes, 250 ⁇ l of substrate was added and the tubes were incubated for 30 minutes at 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 HCl, 76% ethanol) was then added. The tubes were vortexed and allowed to sit for five minutes before centrifuging for three minutes at approximately 13,000 x g. The optical density was measured spectrophotometrically at a wavelength of 590-600 nm.
• the protein assay used was the BCA (bicinchoninic acid) assay using reagents obtained from Pierce, Rockford, Illinois, USA.
• the standard was bovine serum albumin (BSA) .
• BCA reagent was made by mixing 1 part of reagent B with 50 parts of reagent A. One ml of the BCA reagent was mixed with 50 ⁇ l of appropriately diluted BSA or test culture supernatant. Incubation was for 30 minutes at 37°C and the optical density was finally measured spectrophotometrically at a wavelength of 562 nm.
• the transformants described in this Example were obtained using intact pEGIpyr4 and will contain DNA sequences integrated in the genome which were derived from the pUC plasmid. Prior to transformation it would be possible to digest pEGlpyr4 with Hindlll and isolate the larger DNA fragment containing only T. reesei DNA. Transformation of T. reesei with this isolated fragment of DNA would allow isolation of transformants which overproduced EGI and contained no heterologous DNA sequences except for the two short pieces of synthetic DNA shown in FIG. 8. It would also be possible to use pEGIpyr4 to transform a strain which was deleted for either the cbhl gene, or the cbh2 gene, or for both genes. In this way a strain could be constructed which would over-produce EGI and produce either a limited range of, or no, exo-cellobiohydrolases.
• Example 20 The methods of Example 20 could be used to produce T. reesei strains which would over-produce any of the other cellulase components, xylanase components or other proteins normally produced by T. reesei.
• pCEPCl A plasmid, pCEPCl, was constructed in which the coding sequence for EGI was functionally fused to the promoter from the cbhl gene. This was achieved using in vitro, site-specific mutagenesis to alter the DNA sequence of the cbhl and egll genes in order to create convenient restriction endonuclease cleavage sites just 5' (upstream) of their respective translation initiation sites. DNA sequence analysis was performed to verify the expected sequence at the junction between the two DNA segments. The specific alterations made are shown in FIG. 14.
• DNA fragments which were combined to form pCEPCl were inserted between the EcoRI sites of pUC4K and were as follows (see FIG. 15) : A) A 2.1 kb fragment from the 5' flanking region of the cbhl locus. This includes the promoter region and extends to the engineered Bell site and so contains no cbhl coding sequence.
• BamHI site and extending through the coding region and including approximately 0.5 kb beyond the translation stop codon.
• At the 3' end of the fragment is 18 bp derived from the pUC218 multiple cloning site and a 15 bp synthetic oligonucleotide used to link this fragment with the fragment below.
• the plasmid, pCEPCl was designed so that the EGI coding sequence would be integrated at the cbhl locus, replacing the coding sequence for CBHI without introducing any foreign DNA into the host strain. Digestion of this plasmid with EcoRI liberates a fragment which includes the cbhl promoter region, the egll coding sequence and transcription termination region, the __ reesei pyr4 gene and a segment of DNA from the 3' (downstream) flanking region of the cbhl locus (see Fig. 15) .
• Example 1 (Sheir-Neiss, supra) was obtained by the method outlined in Example 1. This strain was transformed with pCEPCl which had been digested with EcoRI. Stable transformants were selected and subsequently cultured in shaker flasks for cellulase production as described in Example 20. In order to visualize the cellulase proteins, isoelectric focusing gel electrophoresis was performed on samples from these cultures using the method described in Example 7. Of a total of 23 transformants analysed in this manner 12 were found to produce no CBHI protein, which is the expected result of integration of the CEPC1 DNA at the cbhl locus.
• Southern blot analysis was used to confirm that integration had indeed occurred at the cbhl locus in some of these transformants and that no sequences derived from the bacterial plasmid vector (pUC4K) were present (see Fig. 16) .
• the DNA from the transformants was digested with Pstl before being subjected to electrophoresis and blotting to a membrane filter.
• the resulting Southern blot was probed with radiolabelled plasmid pUC4K::cbhl (see Example 2) .
• the probe hybridised to the cbhl gene on a 6.5 kb fragment of DNA from the untransformed control culture (FIG. 16, lane A).
• Endoglucanase activity assays were performed on samples of culture supernatant from the untransformed culture and the transformants exactly as described in Example 20 except that the samples were diluted 50 fold prior to the assay so that the protein concentration in the samples was between approximately 0.03 and 0.07 mg/ml.
• the results of assays performed with the untransformed control culture and four different transformants are shown in Table 2.
• Transformants CEPC1-103 and CEPC1-112 are examples in which integration of the CEPCl fragment had led to loss of CBHI production.
• the egl3 gene encoding EGII (previously referred to as EGIII by others) , has been cloned from T. reesei and the DNA sequence published (Saloheimo et al., 1988, Gene 63:11-21).
• the latter vector, pUC219 is derived from pUC119 (described in Wilson et al., 1989, Gene 77:69-78) by expanding the multiple cloning site to include restriction sites for Bglll. Clal and Xhol.
• T ⁇ . reesei pyr4 gene present on a 2.7 kb Sail fragment of genomic DNA, was inserted into a Sail site within the EGII coding sequence to create plasmid pEGII::P-l (FIG. 17) .
• the plasmid, pEGII::P-l can be digested with Hindlll and BamHI to yield a linear fragment of DNA derived exclusively from T ⁇ . reesei except for 5 bp on one end and 16 bp on the other end, both of which are derived from the multiple cloning site of pUC219.
• T. reesei strain GC69 will be transformed with pEGII::P-l which had been previously digested with Hindlll and BamHI and stable transformants will be selected. Total DNA will be isolated from the transformants and Southern blot analysis used to identify those transformants in which the fragment of DNA containing the pyr4 and eg!3 genes had integrated at the egl3 locus and consequently disrupted the EGII coding sequence. The transformants will be unable to produce EGII. It would also be possible to use pEGII::P-l to transform a strain which was deleted for either or all of the cbhl. cbh2 f or egll genes. In this way a strain could be constructed which would only produce certain cellulase components and no EGII component.
• a pyr4 deficient derivative of strain P37P ⁇ CBH67 was obtained by the method outlined in Example 1.
• This strain P37P ⁇ 67P " 1 was transformed with pEGII::P-l which had been previously digested with Hindlll and BamHI and stable transformants were selected.
• Total DNA was isolated from transformants and Southern blot analysis used to identify strains in which the fragment of DNA containing the pyr4 and eg!3 genes had integrated at the egl3 locus and consequently disrupted the EGII coding sequence.
• the size of the band corresponding to the egl3 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37P ⁇ 67P " 1 strain (Lanes A and C) and the transformant disrupted for egl3 (FIG. 18, Lanes B and D) .
• the transformant containing the disrupted egl3 gene illustrated in FIG. 18 was named A22.
• the transformant identified in FIG. 18 is unable to produce CBHI, CBHII or EGII.
• the egll gene of T. reesei strain RL-P37 was obtained, as described in Example 12, as a 4.2 kb Hindlll fragment of genomic DNA. This fragment was inserted at the Hindlll site of pUClOO (a derivative of pUC18; Yanisch-Perron et al., 1985, Gene 33:103- 119, with an oligonucleotide inserted into the multiple cloning site adding restriction sites for Bglll. Clal and Xhol) .
• the plasmid pP ⁇ EGI-1 can be digested with Hindlll to release a DNA fragment comprising only T_ reesei genomic DNA having a segment of the egll gene at either end and the pyr4 gene replacing part of the EGI coding sequence, in the center.
• Transformation of a suitable __ reesei pyr4 deficient strain with the pP ⁇ EGI-1 digested with Hindlll will lead to integration of this DNA fragment at the egll locus in some proportion of the transformants. In this manner a strain unable to produce EGI will be obtained.
• Example 26 The expectation that the EGI gene could be inactivated using the method outlined in Example 26 is strengthened by this experiment.
• a plasmid, p ⁇ EGlpyr-3 was constructed which was similar to pP ⁇ EGI-1 except that the Aspergillus niqer pyr4 gene replaced the T_ reesei pyr4 gene as selectable marker.
• the egll gene was again present as a 4.2 kb Hindlll fragment inserted at the Hindlll site of pUClOO.
• Example 25 (from Example 25) will be obtained by the method outlined in Example 1.
• This strain will be transformed with pP ⁇ EGI-1 which had been previously digested with Hindlll to release a DNA fragment comprising only £_j_ reesei genomic DNA having a segment of the egll gene at either end with part of the EGI coding sequence replaced by the pyr4 gene.
• Stable pyr4+ transformants will be selected and total DNA isolated from the transformants.
• the DNA will be probed with 32 P labelled pP ⁇ EGI-1 after Southern blot analysis in order to identify transformants in which the fragment of DNA containing the pyr4 gene and egll sequences has integrated at the egll locus and consequently disrupted the EGI coding sequence.
• the transformants identified will be unable to produce CBHI, CBHII, EGI and EGII.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Zoology (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Mycology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Medicinal Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Textile Engineering (AREA)
• Detergent Compositions (AREA)
• Enzymes And Modification Thereof (AREA)

Applications Claiming Priority (4)

Publications (2)

Family

ID=27081827

Family Applications (1)

Country Status (5)

Families Citing this family (21)

Citations (3)

Family Cites Families (13)

• 1991
  • 1991-10-04 WO PCT/US1991/007273 patent/WO1992006210A1/en not_active Application Discontinuation
  • 1991-10-04 EP EP91919464A patent/EP0552276A4/en not_active Withdrawn
  • 1991-10-04 CA CA 2093425 patent/CA2093425A1/en not_active Abandoned
  • 1991-10-04 JP JP3517578A patent/JPH06502205A/ja active Pending
• 1993
  • 1993-04-01 FI FI931490A patent/FI931490A/fi not_active Application Discontinuation

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events