Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • 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
  • 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
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
  • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
  • D06P5/15—Locally discharging the dyes
  • D06P5/158—Locally discharging the dyes with other compounds

Definitions

• the present invention is related to a modified cellulase composition which results from treating cellulase with protease.
• the present invention is related to a modified cellulase composition in which specific components of the cellulase are differentially stable to proteolysis such that protease treatment preferentially degrades certain protein and cellulase components while leaving other cellulase and protein components relatively intact.
• the present invention contemplates use of the protease treated cellulase composition in textile treatment, pulp and paper production, grain and animal feed processing and detergents.
• Cellulases are enzymes which break down cellulose by hydrolyzing the ⁇ -1 ,4-glucan linkages to form glucose, cellobiose and various cello-oligosaccharides.
• Cellulases expressed in certain fungi and bacteria have been extensively characterized.
• cellulase produced by the fungal species Trichoderma sp. (especially Trichoderma longibrachiatum), has been subject to a high level of attention due to the capability of the species to produce in large quantities a complete cellulase system ("whole cellulase") which is effective in degrading crystalline forms of cellulose.
• cellulases produced by fungal species such as Neurospora sp., Penicillium sp., Fusarium sp., Humicola sp. and Aspergillus sp. and bacterial cellulases, for example those derived from Bacillus, have also been the subject of much study.
• Complete fungal cellulase systems comprise several different enzyme classifications 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”)(see e.g., Jrin, "Methods in Enzymology", 160, 25, pages 234 et seq. (1988)).
• CBH exo-cellobiohydrolases
• EG endoglucanases
• BG ⁇ -glucosidases
• Each of the fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification.
• cellulase compositions containing multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources.
• the complete cellulase system comprising CBH, EG and BG components is required to efficiently convert crystalline cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose to glucose. Moreover, a synergistic relationship is observed between the cellulase components, particulariy if they are of different classifications. For example, combinations of specific EG components and specific CBH components may be more or less efficient in breaking down cellulose. Similarly, various cellulase components or subcombinations thereof have been found to be more useful in industrial applications than co ⁇ esponding complete cellulase systems.
• CBHI, CBHII, EGI and EGII components derived from Trichoderma longibrachiatum problems have been observed when using certain cellulase components, e.g., CBHI, CBHII, EGI and EGII components derived from Trichoderma longibrachiatum, in detergents and textile treatment compositions due to fabric strength loss and dye redeposition, also known as backstaining.
• U.S. Patent No. 5,120,463 discloses that CBHI enriched cellulase compositions provide excellent cleaning characteristics while reducing fiber degradation effects.
• U.S. Patent No. 5,246,853 discloses that the presence of exo-cellobiohydrolase type components in combination with endoglucanase type components in a stonewashing compositions results in detrimental strength loss.
• U.S. Patent No. 5,290,474 discloses that the EGIII component of Trichoderma spp. provides for superior and unexpected advantages in detergent compositions as compared to the EGI and EGII components of Trichoderma reesei and that certain exo- cellobiohydrolase type components are responsible for undesirable characteristics such as increased strength loss.
• the various components of complete cellulase systems are also known to have differing optimal pH and pH activity profiles which can both adversely and advantageously effect their industrial application.
• isolated endoglucanase type components are of value in the textile industry due to the desirable characteristics they confer to fabrics
• many fungal endoglucanase components have their maximal activity at acidic pHs whereas most laundry detergent compositions are formulated for use at from neutral to alkaline (pH >7 to about 10) conditions.
• EGIII derived from Trichoderma longibrachiatum is known to have a relatively neutral pH activity profile and significant alkaline pH activity.
• substantially pure EGIII cellulase component can be used in a pre-washing step at an intermediate pH where sufficient activity exists to provide desired improvements in color retention and restoration, softening and feel as disclosed in U.S. Patent No. 5,290,474.
• EGIII cellulase component has also been described for home use in a stand alone composition suitable for restoring color to faded fabrics, see e.g., U.S. Patent No. 4,738,682.
• the high activity under neutral to alkaline conditions of EGIII is also beneficial in textile processes for treating cotton- containing fabrics (see U.S. Serial Nos. 07/954,113 and 08/210) as well as in silage and/or composting processes.
• U.S. Patent No. 5,328,841 discloses a method for purifying EGIII and xylanase from an extracellular culture media including a mixture of cellulase and xylanase by adding polyethylene glycol having a molecular weight of between about 5,000 and 10,000. Genetic engineering techniques have also been used to ease preparation of specific cellulase components from whole cellulase. For example, PCT Publication No.
• WO 92/06209 discloses preparation and cultivation of Trichoderma reesei strains which are deleted for one or more cellulase genes, thus producing enriched concentrations of, e.g., EGI, EGII, EGIII, CBHI or CBHII compared to the complete cellulase systems.
• a typical fermentation broth may include peptide compounds which deleteriously effect certain cellulase uses. These peptide components are believed to cause adverse effects in cellulase applications, for example, backstaining in stonewashing processes.
• the present invention is directed to the discovery that certain cellulase components and other peptides are differentially degraded by protease. More importantly, however, the present invention is directed to the discovery that cellulase modified according to the present invention will have desirable properties over unmodified cellulase.
• This discovery allows the use of protease modification of cellulase to produce a composition which has desirable attributes, e.g., decreased backstaining and/or decreased strength loss in textile applications.
• the present invention achieves the above stated objects through the use of a cellulase composition which has been modified with protease either simultaneously with, or prior to, the use of the cellulase composition in an intended application.
• a method of preparing a modified cellulase comprising the steps of (a) adding protease to a cellulase composition comprising at least one proteolytically stable cellulase; and (b) incubating said cellulase and said protease in an aqueous solution for a time and under conditions sufficient to degrade non- proteolytically stable cellulases.
• a modified cellulase composition is prepared according the above method.
• the modified cellulase composition so prepared is present in a detergent composition or a stonewashing composition for the treatment of textiles.
• Figure 1 shows an isoelectric focusing gel of an enzyme mixture after protease treatment on specific components of Trichoderma longibrachiatum whole cellulase.
• Cellulase proteins refers to enzymes including exo-cellobiohydrolase (CBH) proteins, endoglucanase (EG) proteins and ⁇ -glucosidase (BG) proteins derived from wild- type fungal sources or microorganisms genetically modified so as to incorporate and express all or part of the cellulase genes obtained from a wild-type fungal sources.
• CBH exo-cellobiohydrolase
• EG endoglucanase
• BG ⁇ -glucosidase
• cellulase proteins do not include other proteins expressed by cellulase producing organisms such as, for example xylanases, proteases, amylases.
• Complete cellulase or “whole cellulase” refers to a composition comprising a cellulase profile representative of that produced by a naturally occurring cellulase producing microorganism.
• Trichoderma longibrachiatum whole cellulase prepared under standard fermentation conditions will show a cellulase profile which contains approximately the following amount of the following components: 45-55% exo-cellobiohydrolase I ("CBHI:), 13-15% exo-cellobiohydrolase II (“CBHII”), 11-13% endoglucanase I (“EGI”), 8- 10% endoglucanase II (“EGII”), 1-4% endoglucanase III (“EGIII”) and 0.5-1% ⁇ -glucanase ("BG”), by weight of cellulase.
• CBHI exo-cellobiohydrolase I
• CBHII 13-15% exo-cellobiohydrolase II
• EGI endoglucanase
• Modified cellulase refers to a cellulase composition, for example a whole cellulase or portion thereof, which has been treated with protease to modify the relative activities therein.
• the resulting modified cellulase will comprise a relative increase in activity of EGIII and relative decrease in the activity of CBHI, CBHII, EGI and EGII compared to the complete cellulase.
• Cellulose binding domain or “binding domain” refers to a distinct structural attribute of many cellulases which is responsible for attaching a cellulase to cellulose.
• Binding domain as referred to herein means the structure known to exist in many cellulases comprised of a distinct sequence of amino acids which is attached via a linker region to a catalytic core subunit.
• Cellulose binding domain does not refer to a "binding region” which is structurally integral to the catalytic core.
• Endoglucanases refers generally to a cellulase enzyme which hydrolyzes the internal ⁇ (1-4) glycosidic linkages of a cellulose polymer. Endoglucanases are generally characterized by their ability to (a) hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (“CMC”), thereby reducing the viscosity of CMC containing solutions, and (b)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.).
• CMC carboxymethylcellulose
• Microbial sources often contain more than one type of endoglucanase.
• the different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities and different enzymatic action patterns.
• EGI, EGII and EGIII from Trichoderma longibrachiatum are all characterized by different biochemical characteristics.
• EGIII cellulase refers to the endoglucanase component derived from Trichoderma spp. or any microorganism producing a protein equivalent to EGIII produced by Trichoderma spp. characterized by a pH optimum of about 5.5 to 6.0, an isoelectric point (pl) of from about 7.2 to 8.0, and a molecular weight of about 23 to 28 Kdaltons.
• EGIII cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride.
• EGIII cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.5 to 6.0, an isoelectric point (pl) of about 7.4 and a molecular weight of about 25 to 28 Kdaltons.
• EGIII cellulase derived from Trichoderma viride has a pH optimum of about 5.5, an isoelectric point (pl) of about 7.7 and a molecular weight of about 23.5 Kdaltons.
• the amino acid sequence of the EGIII cellulase may be altered. Alteration of the active sites on this enzyme may lead to a variety of changes such as altered pH optima, altered temperature optima or altered affinities for the substrate.
• EGII has been previously referred to by the nomenclature “EGIII” by some authors but current nomenclature uses the term “EGII”.
• the EGII protein is recognized to be substantially different from the EGIII protein in its molecular weight, pl, and pH optimum as evidenced by Table 2 of Example 2 presented below
• Exo-cellobiohydrolase or “CBH” refers to a cellulase enzyme properly known as cellulose ⁇ (1-4)cellob ⁇ osidase.
• CBH components are generally believed to cleave glucose and cellobiose units from the end of the cellulose polymer
• CBH components are generally characterized by the fact that (a) they are competitively inhibited by cellobiose (K, approximately 1mM); (b) they are unable to significantly hydrolyze substituted celluloses such as carboxymethylcellulose and (c) they hydrolyze phosphonc acid swollen cellulase and to a lesser degree highly crystalline cellulose.
• K cellobiose
• a single microbial source can produce a vanety of CBH components
• Tnchoderma longibrachiatum is known to produce at least two distinct CBH components, CBHI and CBHII.
• ⁇ -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 cellooligosacchandes (“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 1mM).
• 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 charactenzed 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
• vanous cellulase components are further charactenzed in the characteristics they confer to products which inco ⁇ orate them
• stonewashing compositions are known to be adversely effected by CBH components which cause an increased level of backstaining
• This backstaining results in a less desirable finished textile product
• both EG and CBH components have been proposed for use in detergents alone
• methods, such as the present invention, to selectively remove one or the other of these components are desirable
• “Proteolytically stable cellulase” refers to a cellulase which is differentially resistant to proteolytic attack compared to other cellulases in the same incubation mixture.
• complete cellulase derived from Trichoderma longibrachiatum contains a component mixture of at least the following cellulases: CBHI, CBHII, EGI, EGII and EGIII.
• CBHI CBHII
• EGI EGII
• EGIII EGIII
• cellulases having a level of homology of amino acid sequence to the amino acid sequence of EGIII from T longibrachiatum of greater than 60%, preferably greater than 70% and most preferably greater than 90% will confer similar benefits in a protease/cellulase solution wherein it is desired to retain only significant activity from one such component (see PCT Publication No. WO 94/21801).
• cellulases lacking binding domains, such as EGIII from 7. longibrachiatum and many cellulases derived from bacterial species such as Bacillus will possess similar proteolytic resistance and will be valuable in protease/cellulase compositions.
• the composition will retain a significant amount of activity corresponding to the proteolytically stable cellulase while the activity of non-proteolytically stable cellulases will be significantly diminished.
• the remaining activity after protease treatment for the proteolytically stable cellulase will be greater than about 10 percent, more preferably greater than about 15 percent; and most preferably greater than about 30 percent relative to unmodified proteolytically stable cellulase.
• the remaining activity after protease treatment of non-proteolytically stable cellulase will preferably be less than about 10 percent relative to unmodified non-proteolytically stable cellulase.
• a cellulase mixture containing a desired proteolytically stable cellulase is incubated with protease for an appropriate time and under appropriate conditions to substantially hydrolyze or degrade non-proteolytically stable cellulases and background proteins in the solution.
• the resulting modified cellulase solution is then optionally subjected to well known separation techniques to remove the protease and/or the hydrolyzed contaminating proteins. In such a way, it is possible to reduce or eliminate the activity of certain cellulases while increasing the activity of proteolytically stable cellulases.
• the cellulase composition useful in the present invention may be derived from any source, e.g., a fungal or bacterial source.
• Preferred cellulases which may contain mixtures of proteolytically stable cellulases and non-proteolytically stable cellulases include those derived from fungal sources such as Trichoderma, Humicola, Aspergillus, Streptomyces, Erwinia, Neurospora, Penicillium, Thermomonospora, Microtetraspora or Fusarium or from bacterial sources such as Bacillus.
• the cellulases of Trichoderma longibrachiatum are particularly preferred.
• Methods for preparing cellulase solutions from sources such as those Iisted above are well known in the art.
• the cellulase mixture according to the present invention may be either an aqueous solution or a dry solid, e.g., a granular composition comprising cellulase.
• the protease according to the invention may be derived from either exogenous or endogenous sources.
• Added exogenous protease refers to proteases that differ from those naturally present in the enzyme complement of the microorganism from which the cellulase composition to be treated is derived.
• the added protease may be endogenous.
• added endogenous proteases refers to an amount of protease in a redepositing cellulase composition that is over and above what is naturally produced by the microorganism or produced via overexpression of the gene encoding the naturally occurring protease.
• Proteases are available from several sources including microbial, plant and animal sources.
• proteases are derived from Bacillus licheniformis, Bacillus subtilis and Aspergillus oryzae.
• Proteases suitable for the invention include, for example, serine, metallo and acid proteases, as well as endo- and exo-proteases.
• Subtilisins are serine proteases which generally act to cleave internal peptide bonds of proteins and peptides.
• Metallo proteases are exo- or endo-proteases which require a metal cofactor for activity.
• One of the preferred serine proteases is subtilisin.
• Particularly preferred proteases useful in the present invention are proteases obtained from a microorganism genetically modified as described in US Patents Nos. 5,182,204, 5,185,258 and 5,316,941, and U.S. Patent No. Re. 34,606 the disclosures of which are inco ⁇ orated herein by reference.
• the amount of added protease is determined by many factors such as the purity, concentration, activity and specificity of the added protease, as well as the length and temperature of the incubation with the cellulase composition. Therefore, the amount of added protease will vary with the type of protease added to the cellulase composition.
• the amount of protease added is an amount of protease in excess of the amount which is produced naturally by a microorganism.
• the added protease will proteolytically cleave the contaminating proteins, thus selectively retaining the desired proteolytically stable cellulase.
• Conditions appropriate to hydrolyze the contaminating proteins upon incubation of the cellulase and protease will be dependent on the type of protease used. Generally, conditions sufficient for proteolytic activity of a specific protease used will be suitable for the present pu ⁇ oses. Of course, the closer the incubation conditions are to the optimal conditions for the protease, the more efficient the hydrolysis of contaminating proteins will be.
• the temperature of the incubation should preferably be between 20 and 70°C, more preferably between 30 and 35°C.
• the time of incubation should preferably be shorter than 24 hours, more preferably between 1 and 120 minutes, and most preferably between 10 and 60 minutes.
• the pH of the incubation is preferably between about 4 and 10, more preferably between about 6 and about 8, depending on the pH optimum of the protease. However, the pH should not be so high as to oxidize or denature the proteolytically stable cellulase.
• the protease or hydrolyzed proteins may be optionally removed from the solution. Removal of the protease or hydrolyzed proteins from the proteolytically stable cellulase may be achieved through any art-recognized means. For example, the protease may be separated by precipitation, centrifugation, ultrafiltration, gel filtration, membrane filtration, ultramembrane filtration, affinity chromatography, pH or temperature inactivation. Similarly, the hydrolyzed cellulases and background proteins may be removed from solution according to such art-recognized means. Alternatively, the hydrolyzed proteins may be removed from the solution in conjunction with standard washing procedures.
• the protease/cellulase composition when used in a detergent to clean dirty fabrics, the protease/cellulase composition will generally be added during either the pre-wash or regular wash cycles. Upon being added to solution, the protease will degrade the background proteins and preferentially degrade non- proteolytically stable cellulases leaving a substantial portion of the proteolytically stable cellulases in solution.
• the proteolytically stable cellulases will tend to adhere to the fabrics to a greater degree than the degraded protein, upon removal of the wash liquor from the laundry chamber and replacement with either a subsequent wash cycle liquor or a rinse liquor, the degraded cellulases and background proteins will be removed leaving the intact cellulase in solution without the presence of the degraded background proteins and non-proteolytically stable cellulases.
• composition according to the present invention may be used for any purpose in which cellulase finds application.
• the present invention contemplates the use of the compositions disclosed herein in detergents, stonewashing applications, animal feed additives, and the treatment of lignocellulosic material and waste water.
• U.S. Patent No. 5,290,474, which disclosure is inco ⁇ orated by reference discloses how to make and use detergent compositions comprising EGIII.
• U.S. Patent No. 5,246,853 which disclosure is herein inco ⁇ orated by reference, discloses how to make and use compositions for treating cotton containing textiles, e.g., stonewashing, utilizing EGIII.
• the present invention provides a significant advance in the preparation of cellulase components.
• protease treatment of complete cellulase from Trichoderma longibrachiatum preferentially hydrolyzes proteins and cellulases other than EGIII.
• cellulases which are known to cause problems in industrial applications, for example CBH-type cellulases, are eliminated from solution leaving a relatively purified EGIII sample in terms of remaining activity.
• background proteins are hydrolyzed preferentially to EGIII. Applicants believe that these background proteins are responsible for numerous undesirable effects in industrial enzyme mixtures. For example, background proteins may result in allergenicity which is inappropriate for specific uses, e.g., laundry detergents. Additionally, background proteins may cause poor results in applications in which the cellulase is used, for example, in stonewashing where they may cause backstaining.
• a concentrated subtilisin protease solution derived from Bacillus subtilis was added to one aliquot, to give a final protease concentration of 0.4 mg/ml, and cellulase concentration of 4 mg/ml. The same volume of buffer was added to the other enzyme aliquot.

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• 1996
  • 1996-09-18 WO PCT/US1996/014976 patent/WO1997011165A2/en not_active Application Discontinuation
  • 1996-09-18 AU AU73637/96A patent/AU7363796A/en not_active Abandoned
  • 1996-09-18 NZ NZ320821A patent/NZ320821A/xx unknown
  • 1996-09-18 MX MX9801889A patent/MX9801889A/es unknown
  • 1996-09-18 EP EP96935853A patent/EP0851915A2/de not_active Withdrawn
  • 1996-09-18 CA CA002229780A patent/CA2229780A1/en not_active Abandoned

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