WO2012027374A2 - Glucides-hydrolases fongiques inédites - Google Patents

Glucides-hydrolases fongiques inédites Download PDF

Info

Publication number
WO2012027374A2
WO2012027374A2 PCT/US2011/048817 US2011048817W WO2012027374A2 WO 2012027374 A2 WO2012027374 A2 WO 2012027374A2 US 2011048817 W US2011048817 W US 2011048817W WO 2012027374 A2 WO2012027374 A2 WO 2012027374A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
acid sequence
protein
nucleic acid
amino acid
Prior art date
Application number
PCT/US2011/048817
Other languages
English (en)
Other versions
WO2012027374A3 (fr
Inventor
Johannes Visser
Sandra Hinz
Jan Werij
Jacob Visser
Vivi Joosten
Martijn Koetsier
Mark Emalfarb
Original Assignee
Dyadic International (Usa) Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dyadic International (Usa) Inc. filed Critical Dyadic International (Usa) Inc.
Publication of WO2012027374A2 publication Critical patent/WO2012027374A2/fr
Publication of WO2012027374A3 publication Critical patent/WO2012027374A3/fr

Links

Classifications

    • 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
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • 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
    • 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)
    • 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
    • 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)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This invention relates to novel enzymes and novel methods for producing the same. More specifically this invention relates to enzymes produced by fungi.
  • the invention also relates to a method to convert lignocellulosic biomass or cellulosic substrates to fermentable sugars with enzymes that degrade lignocellulosic, cellulosic, and even more complex plant cell wall material and to novel combinations of enzymes, including those that provide a combined or synergistic release of sugars from plant biomass.
  • the invention also relates to a method to release cellular contents by effecting degradation of the cell walls.
  • the invention also relates to methods of using the novel enzymes and compositions of such enzymes in a variety of other processes, such as washing or treating of clothing or fabrics, detergent processes, animal feed, food, baking, biofuel, starch preparation, liquification, beverage, biorefining, deinking and biobleaching of paper and pulp, oil and waste dispersing, and treatment of waste streams.
  • distillers' dried grains are lignocellulosic byproducts of the corn dry milling process. Milled whole corn kernels are treated with amylases to liquefy the starch within the kernels and hydrolyze it to glucose. The glucose so produced is then fermented in a second step to ethanol. The residual solids after the ethanol fermentation and distillation are centrifuged and dried, and the resulting product is DDG, which is used as an animal feed stock.
  • DDG composition can vary, a typical composition for DDG is: about 32% hemicellulose, 22%) cellulose, 30%> protein, 10%> lipids, 4% residual starch, and 4% inorganics.
  • the cellulose and hemicellulose fractions comprising about 54% of the weight of the DDG, can be efficiently hydrolyzed to fermentable sugars by enzymes; however, it has been found that the carbohydrates comprising lignocellulosic materials in DDG are more difficult to digest.
  • the efficiency of hydrolysis of these (hemi) cellulosic polymers by enzymes is much lower than the hydrolytic efficiency of starch, due to the more complex and recalcitrant nature of these substrates. Accordingly, the cost of producing the requisite enzymes is higher than the cost of producing amylases for starch hydrolysis.
  • Major polysaccharides comprising lignocellulosic materials include cellulose and hemicelluloses.
  • the enzymatic hydrolysis of these polysaccharides to soluble sugars (and finally to monomers such as glucose, xylose and other hexoses and pentoses) is catalyzed by several enzymes acting in concert.
  • endo- 1,4-P"glucanases (EGs) and exo-cellobiohydrolases (CBHs) catalyze the hydrolysis of insoluble cellulose to cellooligosachharides (with cellobiose as the main product), while ⁇ -glucosidases (BGLs) convert the oligosaccharides to glucose.
  • EGs endo- 1,4-P"glucanases
  • CBHs exo-cellobiohydrolases
  • BGLs ⁇ -glucosidases
  • xylanases together with other enzymes such as D-L- arabinofuranosidases, ferulic and acetylxylan esterases and ⁇ -xylosidases, catalyze the hydrolysis of hemicelluloses.
  • Enzymes useful for the hydrolysis of complex polysaccharides are also highly useful in a variety of industrial textile applications, as well as industrial paper and pulp applications, and in the treatment of waste streams.
  • methods for treating cellulose-containing fabrics for clothing with hydrolytic enzymes, such as cellulases are known to improve the softness or feel of such fabrics.
  • Cellulases are also used in detergent compositions, either for the purpose of enhancing the cleaning ability of the composition or as a softening agent.
  • Cellulases are also used in combination with polymeric agents in processes for providing a localized variation in the color density of fibers.
  • Such enzymes can also be used for the saccharification of lignocellulosic biomass in waste streams, such as municipal solid waste, for biobleaching of wood pulp, and for deinking of recycled print paper.
  • waste streams such as municipal solid waste
  • biobleaching of wood pulp and for deinking of recycled print paper.
  • hydrolysis of these polysaccharides in lignocellulosic materials for use as feedstocks described above the cost and hydrolytic efficiency of the enzymes are major factors that control the use of enzymes in these processes.
  • Enzymes are also used in the food and animal feed industry.
  • esterases can be utilized to degum vegetable oils; improving the production of various food products as well as enhancing the flavor of food products.
  • Esterases can be used in the feed to reduce the amount of phosphate in feed.
  • Carbohydrases can be used to increase the yield of fruit juice and oils; stimulate fermentation in the brewing industry; produce gelling agents; and modify starches, to name a few.
  • Carbohydrases in the feed industry include, but are not limited to, improving feed conversion, reducing the viscosity, and producing oligosaccharides.
  • Filamentous fungi such as Aspergillus sp. and Trichoderma sp. are sources of cellulases and hemicellulases, as well as other enzymes useful in the enzymatic hydrolysis of major polysaccharides.
  • strains of Trichoderma sp. such as T. viride, T. reesei and T. longibrachiatum, and Penicillium sp., and enzymes derived from these strains, have previously been used to hydro lyze crystalline cellulose.
  • the costs associated with producing enzymes from these fungi but foremost the narrow window of operating of these enzymes in terms of pH and temperature, remains a drawback. It is therefore desirable to produce inexpensive enzymes and enzyme mixtures that efficiently degrade cellulose and hemicellulose for use in a variety of agricultural and industrial applications.
  • the present invention comprises an isolated nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to an amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.
  • the nucleic acid sequence encodes an amino acid sequence that is at least about 90%, at least about 95%, at least about 97% or at least about 99% identical to the amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.
  • the nucleic acid sequence encodes a protein comprising an amino acid sequence selected from Sequences SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46, SEQ ID No: 48, SEQ ID No: 50, SEQ ID No: 52, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No: 58, SEQ ID No: 60, SEQ ID No: 62, SEQ ID No: 64, SEQ ID No: 66, SEQ ID No:
  • the nucleic acid sequence comprises a nucleic acid sequence selected from Sequences SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 31, SEQ ID No: 33, SEQ ID No: 35, SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 41, SEQ ID No: 43, SEQ ID No: 45, SEQ ID No: 47, SEQ ID No: 49, SEQ ID No: 51, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 56, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61, SEQ ID No: 63, SEQ ID No: 65
  • the present invention comprises nucleic acid sequences that are fully complementary to any of the nucleic acid sequences described above.
  • the present invention comprises an isolated protein comprising an amino acid sequence encoded by any of the nucleic acid molecules described above.
  • the present invention comprises an isolated fusion protein comprising an isolated protein of the present invention fused to a protein comprising an amino acid sequence that is heterologous to the isolated protein.
  • the present invention comprises a kit for degrading a lignocellulosic material to fermentable sugars comprising at least one isolated protein of the present invention.
  • the present invention comprises a detergent comprising at least one isolated protein of the present invention.
  • the present invention comprises a composition for the degradation of a lignocellulosic material comprising at least one isolated protein of the present invention.
  • the present invention comprises a recombinant nucleic acid molecule comprising an isolated nucleic acid molecule of the present invention, operatively linked to at least one expression control sequence.
  • the recombinant nucleic acid molecule comprises an expression vector.
  • the recombinant nucleic acid molecule comprises a targeting vector.
  • the present invention comprises an isolated host cell trans fected with a nucleic acid molecule of the present invention.
  • the host cell is a fungus.
  • the host cell is a filamentous fungus.
  • the filamentous fungus is from a genus selected from the group consisting of: Chrysosporium, Thielavia, Talaromyces, Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus, Cryplococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Talaromyces, Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and Trichoderma, and anamorphs and teleomorphs thereof.
  • the filamentous fungus is selected from the group consisting of: Trichoderma reesei, Trichoderma harzanium, Chrysosporium lucknowense, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Talaromyces flavus, Talaromyces emersonii and Myceliophthora thermophila.
  • the host cell is a bacterium.
  • the present invention comprises an oligonucleotide consisting essentially of at least 12 consecutive nucleotides of a nucleic acid sequence selected from Sequences SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 31, SEQ ID No: 33, SEQ ID No: 35, SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 41, SEQ ID No: 43, SEQ ID No: 45, SEQ ID No: 47, SEQ ID No: 49, SEQ ID No: 51, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 56, SEQ ID No: 57, SEQ ID No:
  • the present invention comprises a kit comprising at least one oligonucleotide of the present invention.
  • the present invention comprises methods for producing a protein of the present invention, comprising culturing a cell that has been transfected with a nucleic acid molecule comprising a nucleic acid sequence encoding the protein, and expressing the protein with the transfected cell. In some embodiments, the present invention further comprises recovering the protein from the cell or from a culture comprising the cell.
  • the present invention comprises a genetically modified organism comprising components suitable for degrading a lignocellulosic material to fermentable sugars, wherein the organism has been genetically modified to express at least one protein of the present invention.
  • the genetically modified organism is a plant, alga, fungus or bacterium.
  • the fungus is yeast, mushroom or filamentous fungus.
  • the filamentous fungus is from a genus selected from the group consisting of: Chrysosporium, Thielavia, Talaromyces, Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus, Cryplococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Talaromyces, Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and Trichoderma.
  • the filamentous fungus is selected from the group consisting of: Trichoderma reesei, Trichoderma harzanium, Chrysosporium lucknowense, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Talaromyces flavus, Talaromyces emersonii and Myceliophthora thermophila.
  • the genetically modified organism has been genetically modified to express at least one additional enzyme.
  • the additional enzyme is an accessory enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanases, exo- -D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.
  • the genetically modified organism is a plant.
  • the present invention comprises a recombinant enzyme isolated from a genetically modified microorganism of the present invention.
  • the recombinant enzyme has been subjected to a purification step.
  • the present invention comprises a crude fermentation product produced by culturing the cells from the genetically modified organism of the present invention, wherein the crude fermentation product contains at least one protein of the present invention.
  • the present invention comprises a multi-enzyme composition comprising enzymes produced by a genetically modified organism of the present invention , and recovered therefrom.
  • the present invention comprises a multi-enzyme composition comprising at least one protein of the present inventions, and at least one additional protein for degrading a lignocellulosic material or a fragment thereof that has biological activity.
  • the multi-enzyme composition comprises at least one cellobiohydrolase, at least one xylanase, at least one endoglucanase, at least one ⁇ -glucosidase, at least one ⁇ -xylosidase, and at least one accessory enzyme.
  • between about 50% and about 70% of the enzymes in the multi-enzyme composition are cellobiohydrolases.
  • between about 10%o and about 30%> of the enzymes in the composition are xylanases.
  • between about 5%> and about 15% of the enzymes in the composition are endoglucanases.
  • between about 1% and about 5%> of the enzymes in the composition are ⁇ -glucosidases. In some embodiments, between about 1% and about 3%> of the enzymes in the composition are ⁇ -xylosidases.
  • the multi-enzyme composition comprises about 60%> cellobiohydrolases, about 20%> xylanases, about 10% endoglucanases, about 3%> ⁇ -glucosidases, about 2%> ⁇ - xylosidases, and about 5%> accessory enzymes.
  • the xylanases are selected from the group consisting of: endoxylanases, exoxylanases, and ⁇ -xylosidases.
  • the accessory enzymes include an enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanase, exo ⁇ -D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.
  • the multi-enzyme composition comprises at least one hemicellulase.
  • the hemicellulase is selected from the group consisting of a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, and endo-galactanase, a mannanase, an endo arabinase, an exo arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xylogluconase, and mixtures thereof.
  • the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and ⁇ - xylosidase.
  • the multi-enzyme composition comprises at least one cellulase.
  • the composition is a crude fermentation product. In some embodiments, the composition is a crude fermentation product that has been subjected to a purification step.
  • the multi-enzyme composition further comprises one or more accessory enzymes.
  • the accessory enzymes include at least one enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanase, exo-P-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.
  • the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase. In some embodiments, the accessory enzyme is added as a crude or a semi-purified enzyme mixture. In some embodiments, the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.
  • the multi-enzyme composition comprises at least one protein of the present invention, and at least one additional protein for degrading an arabinoxylan-containing material or a fragment thereof that has biological activity.
  • the composition comprises at least one endoxylanase, at least one ⁇ -xylosidase, and at least one arabinofuranosidase.
  • the arabinofuranosidase comprises an arabinofuranosidase with specificity towards single substituted xylose residues, an arabinofuranosidase with specificity towards double substituted xylose residues, or a combination thereof.
  • the present invention comprises methods for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with at least one isolated protein of the present invention.
  • the methods of the present invention further comprise contacting the lignocellulosic material with at least one additional isolated protein comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from Sequences SEQ ID NO: 2, SEQ ID No: 4, SEQ ID NO:
  • the additional isolated protein is part of a multi-enzyme composition.
  • the present invention comprises methods for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with at least one multi-enzyme composition of the present invention.
  • the present invention comprises a method for producing an organic substance, comprising:
  • the steps of saccharifying and fermenting are performed simultaneously.
  • the organic substance is an alcohol, organic acid, ketone, amino acid, or gas.
  • the alcohol is ethanol.
  • the lignocellulosic material is selected from the group consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.
  • the lignocellulosic material is distiller's dried grains (DDG) or DDG with solubles.
  • DDG distiller's dried grains
  • the DDG or DDG with solubles is derived from corn.
  • the present invention comprises a method for degrading a lignocellulosic material consisting of DDG or DDG with solubles to sugars, the method comprising contacting the DDG or DDG with solubles with a multi- enzyme composition of the present invention, whereby at least about 10% of the fermentable sugars are liberated. In some embodiments, at least about 15%, at least 20%), or at least about 23% of the sugars are liberated.
  • the present invention further comprises a pretreatment process for pretreating the lignocellulosic material.
  • the pretreatment process is selected from the group consisting of physical treatment, metal ion, ultraviolet light, ozone, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment.
  • the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.
  • the heat treatment comprises heating the lignocellulosic material to 121°C for 15 minutes.
  • the present invention further comprises detoxifying the lignocellulosic material.
  • the present invention further comprises recovering the fermentable sugar.
  • the sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.
  • the present invention further comprises recovering the contacted lignocellulosic material after the fermentable sugars are degraded.
  • the present invention comprises a feed additive comprising the recovered lignocellulosic material of the present invention.
  • the protein content of the recovered lignocellulosic material is higher than that of the starting lignocellulosic material.
  • the present invention comprises methods of improving the performance of an animal which comprises administering to the animal the feed additive of the present invention.
  • the present invention comprises methods for improving the nutritional quality of an animal feed comprising adding the feed additive of the present invention to an animal feed.
  • the present invention comprises methods for stonewashing a fabric, comprising contacting the fabric with at least one isolated protein of the present invention.
  • the present invention comprises methods for stonewashing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of the present invention.
  • the fabric is denim.
  • the present invention comprises methods for enhancing the softness or feel of a fabric or depilling a fabric, comprising contacting the fabric with at least one isolated protein of the present invention, or a fragment thereof comprising a carbohydrate binding module (CBM) of the protein.
  • CBM carbohydrate binding module
  • the present invention comprises methods for enhancing the softness or feel of a fabric or depilling a fabric, comprising contacting the fabric with at least one multi-enzyme composition of the present invention.
  • the present invention comprises methods for restoring color to or brightening a fabric, comprising contacting the fabric with at least one isolated protein of the present invention.
  • the present invention comprises methods for restoring color to or brightening a fabric, comprising contacting the fabric with at least one multi- enzyme composition of the present invention.
  • the present invention comprises methods of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one isolated protein of the present invention.
  • the present invention comprises methods of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of the present invention.
  • the present invention comprises methods of biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one isolated protein of the present invention.
  • the present invention comprises methods of biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one multi-enzyme composition of the present invention
  • the present invention comprises methods for enhancing the cleaning ability of a detergent composition, comprising adding at least one isolated protein of the present invention to the detergent composition.
  • the present invention comprises methods for enhancing the cleaning ability of a detergent composition, comprising adding at least one multi- enzyme composition of the present invention to the detergent composition.
  • the present invention comprises a detergent composition, comprising at least one isolated protein of the present invention and at least one surfactant.
  • the present invention comprises a detergent composition, comprising at least one multi-enzyme composition of the present invention and at least one surfactant.
  • the present invention comprises methods for releasing cellular contents comprising contacting a cell with at least one protein of the present invention.
  • the cell may be a bacterium, an algal cell, a fungal cell or a plant cell. In preferred embodiments, the cell is an algal cell.
  • contacting the cell with at least one protein of the present invention degrades the cell wall.
  • the cellular contents are selected from the group consisting of: alcohols and oils.
  • the present invention comprises compositions for degrading cell walls comprising at least one protein of the present invention.
  • the present invention comprises methods for improving the nutritional quality of food comprising adding to the food at least one protein of the present invention.
  • the present invention comprises methods for improving the nutritional quality of food comprising pretreating the food with at least one protein of the present invention.
  • the present invention comprises methods for improving the nutritional quality of animal feed comprising adding to the animal feed at least one protein of the present invention.
  • the present invention comprises methods for improving the nutritional quality of animal feed comprising pretreating the feed with at least one isolated protein of the present invention.
  • the present invention comprises a genetically modified organism comprising at least one nucleic acid molecule encoding a protein of the present invention, in which the activity of one or more of the proteins is upregulated, the activity of one or more of the proteins downregulated, or the activity of one or more of the proteins is upregulated and the activity of one or more of the proteins is downregulated.
  • the present invention relates generally to proteins that play a role in the degradation of cellulose and hemicellulose and nucleic acids encoding the same.
  • the present invention relates to enzymes isolated from a filamentous fungal strain denoted herein as CI (Accession No. VKM F-3500-D), nucleic acids encoding the enzymes, and methods of producing and using the enzymes.
  • the invention also provides compositions that include at least one of the enzymes described herein for uses including, but not limited to, the hydrolysis of lignocellulose.
  • the invention stems, in part, from the discovery of a variety of novel cellulases and hemicellulases produced by the CI fungus that exhibit high activity toward cellulose and other components of biomass.
  • the present invention also provides methods and compositions for the conversion of plant biomass to fermentable sugars that can, in turn, be converted to useful products.
  • Such products may include, without limitation, metabolites, bioplastics, biopolymers and biofuels.
  • the methods include methods for degrading lignocellulosic material using enzyme mixtures to liberate sugars.
  • the compositions of the invention include enzyme combinations that break down lignocellulose.
  • biomass or "lignocellulosic material” includes materials containing cellulose and/or hemicellulose. Generally, these materials also contain pectin, lignin, protein, carbohydrates (such as starch and sugar) and ash. Lignocellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • Fermentable sugars refers to simple sugars, such as glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.
  • Biomass can include virgin biomass and/or non- virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper and yard waste.
  • Common forms of biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane bagasse, sugar beet, soybean, corn, corn husks, corn kernel including fiber from kernels, products and byproducts from milling of grains such as corn, tobacco, wheat and barley (including wet milling and dry milling) as well as municipal solid waste, waste paper and yard waste.
  • the biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues.
  • Agricultural biomass includes branches, bushes, canes, corn and corn husks, energy crops, algae, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, peat moss, mushroom compost and hard and soft woods (not including woods with deleterious materials).
  • agricultural biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. Agricultural biomass may be any of the aforestated singularly or in any combination or mixture thereof.
  • Energy crops are fast-growing crops that are grown for the specific purpose of producing energy, including without limitation, biofuels, from all or part of the plant.
  • Energy crops can include crops that are grown (or are designed to grow) for their increased cellulose, xylose and sugar contents. Examples of such plants include, without limitation, switchgrass, willow and poplar.
  • Energy crops may also include algae, for example, designer algae that are genetically engineered for enhanced production of hydrogen, alcohols, and oils, which can be further processed into diesel and jet fuels, as well as other bio-based products.
  • biomass high in starch, sugar, or protein such as corn, grains, fruits and vegetables are usually consumed as food.
  • biomass high in cellulose, hemicellulose and lignin are not readily digestible and are primarily utilized for wood and paper products, animal feed, fuel, or are typically disposed.
  • the substrate is of high lignocellulose content, including distillers' dried grains corn stover, corn cobs, rice straw, wheat straw, hay, sugarcane bagasse, sugar cane pulp, citrus peels and other agricultural biomass, switchgrass, forestry wastes, poplar wood chips, pine wood chips, sawdust, yard waste, and the like, including any combination thereof.
  • the lignocellulosic material is distillers' dried grains (DDG).
  • DDG also known as dried distiller's grain, or distiller's spent grain
  • the lignocellulosic material can also be distiller's dried grain with soluble material recycled back (DDGS). While reference will be made herein to DDG for convenience and simplicity, it should be understood that both DDG and DDGS are contemplated as desired lignocellulosic materials.
  • DDG is derived from corn.
  • distiller's grains do not necessarily have to be dried.
  • the grains normally are currently dried, water and enzymes are added to the DDG substrate in the present invention. If the saccharification were done on site, the drying step could be eliminated and enzymes could be added to the distiller's grains without drying.
  • the present invention includes enzymes or compositions thereof with, for example, cellobiohydrolase, endoglucanase, xylanase, ⁇ -glucosidase, and hemicellulase activities.
  • Fermentable sugars can be converted to useful value-added fermentation products, non-limiting examples of which include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol.
  • Specific value-added products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol); lactic acid; plastics; specialty chemicals; organic acids, including citric acid, succinic acid, itaconic acid and maleic acid; solvents; animal feed supplements; pharmaceuticals; vitamins; amino acids, such as lysine, methionine, tryptophan, threonine, and aspartic acid; industrial enzymes, such as proteases, cellulases, amylases, glucanases, xylanases, arabinanases, lactases, lipases, esterases, lyases, oxidoreductases, transferases ; and chemical feedstocks.
  • biofuels including ethanol and butanol
  • lactic acid plastics
  • specialty chemicals organic acids, including citric acid, succinic acid, itaconic acid and maleic acid
  • solvents including citric acid, succinic acid, itaconic acid
  • the enzymes of the present invention may also be used for stone washing cellulosic fabrics such as cotton (e.g., denim), linen, hemp, ramie, cupro, lyocell, newcell, rayon and the like. See, for example, U.S. Patent No. 6,015,707.
  • the enzymes and compositions of the present invention are suitable for industrial textile applications in addition to the stone washing process.
  • cellulases are used in detergent compositions, either for the purpose of enhancing the cleaning ability of the composition or as a softening agent. When so used, the cellulase will degrade a portion of the cellulosic material, e.g., cotton fabric, in the wash, which facilitates the cleaning and/or softening of the fabric.
  • endoglucanase components of fungal cellulases have also been used for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, and for use in improving the feel of cotton fabrics, and the like.
  • Enzymes and compositions of the present invention may also be used in the treatment of paper pulp (e.g., for improving the drainage or for de-inking of recycled paper) or for the treatment of wastewater streams (e.g., to hydrolyze waste material containing cellulose, hemicellulose and pectins to soluble lower molecular weight polymers).
  • the enzymes of the present invention may also be used to release the contents of a cell.
  • contacting or mixing the cells with the enzymes of the present invention will degrade the cell walls, resulting in cell lysis and release of the cellular contents.
  • Such cells can include bacteria, plant cells, fungi including yeasts, and algae.
  • the enzymes of the present invention may be used to degrade the cell walls of algal cells in order to release the materials contained within the algal cells.
  • such materials may include, without limitation, alcohols and oils. The alcohols and oils so released can be further processed to produce diesel, jet fuels, as well as other economically important bio- products.
  • the enzymes of the present invention may be used alone, or in combination with other enzymes, chemicals or biological materials.
  • the enzymes of the present invention may be used for in vitro applications in which the enzymes or mixtures thereof are added to or mixed with the appropriate substrates to catalyze the desired reactions. Additionally, the enzymes of the present invention may be used for in vivo applications in which nucleic acid molecules encoding the enzymes are introduced into cells and are expressed therein to produce the enzymes and catalyze the desired reactions within the cells.
  • enzymes capable of promoting cell wall degradation may be added to algal cells suspended in solutions to degrade the algal cell walls and release their content, whereas in some embodiments, nucleic acid molecules encoding such enzymes may be introduced into the algal cells to express the enzymes therein, so that these enzymes can degrade the algal cell walls from within.
  • Some embodiments may combine the in vitro applications with the in vivo applications.
  • nucleic acids encoding enzymes capable of catalyzing cell wall degradation may be introduced into algal cells to express the enzymes in those cells and to degrade their cell walls, while enzymes may also added to or mixed with the cells to further promote the cell wall degradation.
  • the enzymes used for in vitro applications may be different from the enzymes used for in vivo applications.
  • an enzyme with the laminarinase activity may be mixed with the cells, while an enzyme with the xyloglucanase activity is expressed within the cells.
  • the present invention includes proteins isolated from, or derived from the knowledge of enzymes from, a fungus such as Myceliophthora thermophila or a mutant or other derivative thereof, and more particularly, from the fungal strain denoted herein as CI (Accession No. VKM F-3500-D).
  • M. thermophila has previously appeared in patent applications and in the literature as Chrysosporium lucknowense or Sporotrichum thermophile.
  • the proteins of the invention possess enzymatic activity.
  • U.S. Patent No. 6,015,707 or U.S. Patent No. 6,573,086 a strain called CI (Accession No.
  • VKM F-3500-D was isolated from samples of forest alkaline soil from Sola Lake, Far East of the Russian Federation. This strain was deposited at the All-Russian Collection of Microorganisms of Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow, Russia, 113184, under the terms of the Budapest Treaty on the International Regulation of the Deposit of Microorganisms for the Purposes of Patent Procedure on August 29, 1996, as Chrysosporium lucknowense Garg 27K, VKM-F 3500 D. Various mutant strains of M. thermophila (C. lucknowense) CI have been constructed and these strains have also been deposited at the All- Russian Collection of Microorganisms of Russian Academy of Sciences (VKM), Bakhurhina St.
  • VKM All- Russian Collection of Microorganisms of Russian Academy of Sciences
  • Strain CI was mutagenised by subjecting it to ultraviolet light to generate strain UV13-6 (Accession No. VKM F-3632 D). This strain was subsequently further mutated with N-methyl-N'-nitro-N-nitrosoguanidine to generate strain NG7C-19 (Accession No. VKM F-3633 D).
  • strain UV18-25 (Accession No. VKM F-3631 D).
  • strain WIL (Accession No. CBS 122189), which was subsequently subjected to mutation by ultraviolet light, resulting in strain W1L#100L (Accession No. CBS122190).
  • Strain CI was previously classified as a Chrysosporium lucknowense based on morphological and growth characteristics of the microorganism, as discussed in detail in U.S. Patent No. 6,015,707, U.S. Patent No. 6,573,086 and patent PCT/NL2010/000045.
  • a protein of the invention comprises, consists essentially of, or consists of an amino acid sequence selected from Sequences SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46, SEQ ID No: 48, SEQ ID No: 50, SEQ ID No: 52, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No: 58, SEQ ID No: 60, SEQ ID No: 62, SEQ ID No: 64, SEQ ID No: 66,
  • the present invention also includes homologues or variants of any of the above sequences, including fragments and sequences having a given identity to any of the above sequences, wherein the homologue, variant, or fragment has at least one biological activity of the wild- type protein, as described herein.
  • the proteins disclosed herein possess carbohydrase enzymatic activity, or the ability to degrade carbohydrate-containing materials.
  • carbohydrase enzymatic activity or the ability to degrade carbohydrate-containing materials.
  • a review of enzymes involved in the degradation of polysaccharides can be found in de Vries et al, Microbiol. Mol. Biol. Rev. 65:497-522 (2001).
  • the proteins may possess cellulase activity such as endoglucanase activity (e.g., l,4-P-D-glucan-4- glucanohydrolases), exoglucanase activity (e.g., l,4-P-D-glucan cellobiohydrolases), and ⁇ -glucosidase activity.
  • the proteins may possess hemicellulase activity such as endoxylanase activity, exoxylanase activity, or ⁇ - xylosidase activity.
  • the proteins may possess laminarinase, xyloglucanase, galactanase, glucoamylase, pectate lyase, chitosanase, exo-P-D-glucosaminidase, cellobiose dehydrogenase, acetylxylan esterase, ligninase, amylase, glucuronidase, ferulic acid esterase, arabinofuranosidase, pectin methyl esterase, arabinase, lipase, glucosidase, ⁇ -hexosaminidase, rhamnogalacturonan acetylesterase, exo- rhamnogalacturonase, rhamnogalacturon
  • carbohydrase refers to any protein that catalyzes the hydrolysis of carbohydrates.
  • glycoside hydrolase refers to a protein that catalyzes the hydrolysis of the glycosidic bonds between carbohydrates or between a carbohydrate and a non- carbohydrate residue.
  • Endoglucanases cellobiohydrolases, ⁇ -glucosidases, a- glucosidases, xylanases, ⁇ -xylosidases, alpha- xylosidases, galactanases, a- galactosidases, ⁇ -galactosidases, a-amylases, glucoamylases, endo-arabinases, arabinofuranosidases, mannanases, ⁇ -mannosidases, pectinases, acetyl xylan esterases, acetyl mannan esterases, ferulic acid esterases, coumaric acid esterases, pectin methyl esterases, and chitosanases are examples of glycosidases.
  • Cellulase refers to a protein that catalyzes the hydrolysis of l,4 ⁇ -D-glycosidic linkages in cellulose (such as bacterial cellulose, cotton, filter paper, phosphoric acid swollen cellulose, Avicel ® ); cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose); plant lignocellulosic materials, beta-D-glucans or xyloglucans.
  • Cellulose is a linear beta-(l-4) glucan consisting of anhydrocellobiose units. Endoglucanases, cellobiohydrolases, and ⁇ - glucosidases are examples of cellulases.
  • Endoglucanase refers to a protein that catalyzes the hydrolysis of cellulose to oligosaccharide chains at random locations by means of an endoglucanase activity.
  • Cellobio hydrolase refers to a protein that catalyzes the hydrolysis of cellulose to cellobiose via an exoglucanase activity, sequentially releasing molecules of cellobiose from the reducing or non-reducing ends of cellulose or cello- oligo saccharides.
  • ⁇ -glucosidase refers to an enzyme that catalyzes the conversion of cellobiose and oligosaccharides to glucose.
  • Hemicellulase refers to a protein that catalyzes the hydrolysis of hemicellulose, such as that found in lignocellulosic materials. Hemicelluloses are complex polymers, and their composition often varies widely from organism to organism, and from one tissue type to another. Hemicelluloses include a variety of compounds, such as xylans, arabinoxylans, xyloglucans, mannans, glucomannans. Hemicellulose can also contain glucan, which is a general term for beta-linked glucose residues. In general, a main component of hemicellulose is beta-l,4-linked xylose, a five carbon sugar.
  • this xylose is often branched as beta- 1,3 linkages or beta- 1,2 linkages, and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, or by esterification to acetic acid.
  • the composition, nature of substitution, and degree of branching of hemicellulose is very different in dicotyledonous plants (dicots, i.e., plant whose seeds have two cotyledons or seed leaves such as lima beans, peanuts, almonds, peas, kidney beans) as compared to monocotyledonous plants (monocots; i.e., plants having a single cotyledon or seed leaf such as corn, wheat, rice, grasses, barley).
  • hemicellulose is comprised mainly of xyloglucans that are 1 ,4-beta- linked glucose chains with 1,6-alpha- linked xylosyl side chains.
  • xyloglucans 1 ,4-beta- linked glucose chains with 1,6-alpha- linked xylosyl side chains.
  • hetero xylans are primarily comprised of 1,4-beta- linked xylose backbone polymers with 1,2- or 1,3- alpha linkages to arabinose, linkage of galactose and mannose to arabinose or xylose in side chains, as well as xylose modified by ester-linked acetic acids.
  • beta glucans comprised of 1 ,3- and 1,4-beta- linked glucosyl chains.
  • cellulose, heteroxylans and beta glucans are present in roughly equal amounts, each comprising about 15-25% of the dry matter of cell walls.
  • Hemicellulo lytic enzymes i.e. hemicellulases, include both endo-acting and exo-acting enzymes, such as xylanases, ⁇ -xylosidases.
  • alpha-xylosidases galactanases, a-galactosidases, ⁇ -galactosidases, endo-arabinases, arabinofuranosidases, mannanases, ⁇ -mannosidases.
  • Hemicellulases also include the accessory enzymes, such as acetylesterases, ferulic acid esterases, and coumaric acid esterases.
  • xylanases and acetyl xylan esterases cleave the xylan and acetyl side chains of xylan and the remaining xylo-oligomers are unsubstituted and can thus be hydrolysed with ⁇ -xylosidase only.
  • xylanases, acetylesterases and ⁇ - xylosidases are examples of hemicellulases.
  • Xylanase specifically refers to an enzyme that hydro lyzes the ⁇ -1,4 bond in the xylan backbone, producing short xylooligosaccharides.
  • ⁇ -Mannanase or "endo-l,4 ⁇ -mannosidase” refers to a protein that hydrolyzes mannan-based hemicelluloses (mannan, glucomannan, galacto(gluco)mannan) and produces short P-l,4-marmooligosaccharides.
  • Mannan endo-l,6- D-mannosidase refers to a protein that hydrolyzes 1,6- D - mannosidic linkages in unbranched 1,6-mannans.
  • ⁇ -Mannosidase ⁇ -l,4-mannoside mannohydrolase; EC 3.2.1.25) refers to a protein that catalyzes the removal of ⁇ -D-mannose residues from the nonreducing ends of oligosaccharides.
  • Galactanase refers to a protein that catalyzes the hydrolysis of ⁇ -1,4- ⁇ - ⁇ - galactosidic linkages in arabinogalactans.
  • Glucoamylase refers to a protein that catalyzes the hydrolysis of terminal 1,4- linked a-D-glucose residues successively from non-reducing ends of the glycosyl chains in starch with the release of ⁇ -D-glucose.
  • ⁇ -hexosaminidase or " ⁇ - ⁇ -acetylglucosaminidase” refers to a protein that catalyzes the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl- ⁇ -D-hexosamines.
  • ⁇ -L-arabinofuranosidase refers to a protein that hydrolyzes arabinofuranosyl- containing hemicelluloses or pectins.
  • Some of these enzymes remove arabinofuranoside residues from 0-2 or 0-3 single substituted xylose residues, as well as from 0-2 and/or 0-3 double substituted xylose residues. Some of these enzymes remove arabinose residues from arabinan oligomers.
  • Endo-arabinase refers to a protein that catalyzes the hydrolysis of 1,5-a- arabinofuranosidic linkages in 1,5-arabinans.
  • Exo-arabinase refers to a protein that catalyzes the hydrolysis of 1,5-a- linkages in 1,5-arabinans or 1,5-a-L arabino-oligosaccharides, releasing mainly arabinobiose, although a small amount of arabinotriose can also be liberated.
  • ⁇ -xylosidase refers to a protein that hydrolyzes short l,4-P-D-xylooligomers into xylose.
  • Cellobiose dehydrogenase refers to a protein that oxidizes cellobiose to cellobiono lactone.
  • Chitosanase refers to a protein that catalyzes the endohydro lysis of ⁇ -1,4- linkages between D-glucosamine residues in acetylated chitosan (i.e., deacetylated chitin).
  • Exo-polygalacturonase refers to a protein that catalyzes the hydrolysis of terminal alpha 1,4-linked galacturonic acid residues from non-reducing ends thus converting polygalacturonides to galacturonic acid.
  • Alcohol xylan esterase refers to a protein that catalyzes the removal of the acetyl groups from xylose residues.
  • Alcohol mannan esterase refers to a protein that catalyzes the removal of the acetyl groups from mannose residues
  • ferulic esterase or "ferulic acid esterase” refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and ferulic acid.
  • Coumaric acid esterase refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and coumaric acid.
  • Acetyl xylan esterases, ferulic acid esterases and pectin methyl esterases are examples of carbohydrate esterases.
  • Pectate lyase and pectin lyases refer to proteins that catalyze the cleavage of 1 ,4-a-D-galacturonan by beta-elimination acting on polymeric and/or oligosaccharide substrates (pectates and pectins, respectively).
  • Endo-l,3-P-glucanase or “laminarinase” refers to a protein that catalyzes the cleavage of 1,3-linkages in ⁇ -D-glucans such as laminarin or lichenin. Laminarin is a linear polysaccharide made up of P-l,3-glucan with P-l,6-linkages.
  • lichenan refers to a protein that catalyzes the hydrolysis of lichenan, a linear, 1,3-1,4- ⁇ - ⁇ glucan.
  • Rhamnogalacturonan is composed of alternating a -1,4-rhamnose and a-l,2-linked galacturonic acid, with side chains linked 1,4 to rhamnose.
  • the side chains include Type I galactan, which is P-l,4-linked galactose with P-l,3-linked arabinose substituents; Type II galactan, which is P-l,3-l,6-linked galactoses (very branched) with arabinose substituents; and arabinan, which is P-l,5-linked arabinose with a- 1,3-linked arabinose branches.
  • the galacturonic acid substituents may be acetylated and/or methylated.
  • Exo-rhamnogalacturonanase refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin from the nonreducing end.
  • Rhamnogalacturonan acetylesterase refers to a protein that catalyzes the removal of the acetyl groups ester-linked to the highly branched rhamnogalacturonan (hairy) regions of pectin.
  • Rhamnogalacturonan lyase refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin via a ⁇ -elimination mechanism (see, e.g., Pages et al, J. Bacteriol. 185:4727-4733 (2003)).
  • Alpha-rhamnosidase refers to a protein that catalyzes the hydrolysis of terminal non-reducing a-L-rhamnose residues in a-L-rhamnosides.
  • Glycosidases glycoside hydrolases; GH
  • GH glycoside hydrolases
  • Glycosidases such as the proteins of the present invention may be assigned to families on the basis of sequence similarities, and there are now over 100 different such families defined (see the CAZy (Carbohydrate Active EnZymes database) website, maintained by the Architecture of Fonction de Macromolecules Bi Anlagens of the Centre National de lalich Scientifique, which describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds; Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated database approach. In “Recent Advances in Carbohydrate Bioengineering", H.J. Gilbert, G. Davies, B.
  • sequence homology may be used to identify particular domains within proteins, such as carbohydrate binding modules (CBMs; also known as carbohydrate binding domains (CBDs), sometimes called cellulose binding domains).
  • CBMs carbohydrate binding modules
  • CBDs carbohydrate binding domains
  • the CAZy homologies of proteins of the present invention are disclosed below.
  • An enzyme assigned to a particular CAZy family may exhibit one or more of the enzymatic activities or substrate specificities associated with the CAZy family. In other embodiments, the enzymes of the present invention may exhibit one or more of the enzyme activities discussed above.
  • Certain proteins of the present invention may be classified as "Family 61 glycosidases" based on homology of the polypeptides to CAZy Family GH61.
  • Family 61 glycosidases may exhibit cellulo lytic enhancing activity or endoglucanase activity. Additional information on the properties of Family 61 glycosidases may be found in U.S. Patent Application Publication Nos. 2005/0191736, 2006/0005279, 2007/0077630, and in PCT Publication No. WO 2004/031378.
  • cellulolytic enhancing activity refers to a biological activity that enhances the hydrolysis of a cellulosic material by proteins having cellulolytic activity.
  • saccharifying a cellulosic material with a cellulolytic protein in the presence of a Family 61 glycosidases may increase the degradation of cellulosic material compared to the presence of only the cellulolytic protein.
  • the cellulosic material can be any material containing cellulose.
  • the cellulolytic activity is a biological activity that hydrolyzes a cellulosic material.
  • Cellulolytic enhancing activity can be determined by measuring the increase in sugars from the hydrolysis of a cellulosic material by cellulolytic protein.
  • Proteins of the present invention may also include homologues, variants, and fragments of the proteins disclosed herein.
  • the protein fragments include, but are not limited to, fragments comprising a catalytic domain (CD) and/or a carbohydrate binding module (CBM) (also known as a cellulose-binding domain; both can be referred to herein as CBM).
  • CD catalytic domain
  • CBM carbohydrate binding module
  • the identity and location of domains within proteins of the present invention are disclosed in detail below.
  • the present invention encompasses all combinations of the disclosed domains.
  • a protein fragment may comprise a CD of a protein but not a CBM of the protein or a CBM of a protein but not a CD.
  • domains from different proteins may be combined.
  • Protein fragments comprising a CD, CBM or combinations thereof for each protein disclosed herein can be readily produced using standard techniques known in the art.
  • a protein fragment comprises a domain of a protein that has at least one biological activity of the full-length protein. Homologues or variants of proteins of the invention that have at least one biological activity of the full-length protein are described in detail below.
  • biological activity of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form of the protein as measured or observed in vitro or in vivo.
  • a protein fragment comprises a domain of a protein that has the catalytic activity of the full- length enzyme. Specific biological activities of the proteins of the invention, and structures within the proteins that are responsible for the activities, are described below.
  • Carbohydrases represent a category of various enzymes and polypeptides including amylases, cellulases, hemicellulases, pectinases, and chitinases that catalyze and/or enhance the hydrolysis or synthesis of a carbohydrate.
  • Applications of carbohydrases in the food industry include, but are not limited to, increasing the yield of fruit juice production in total liquefaction; increasing the pressing yield of oils e.g. from olives, cleaning filters, reduction of viscosity, hydro lyzing starch, and stimulating fermentation in the brewing industry; increasing the loaf volume and improving crust color in the baking industry; preventing/reducing the staling of bread; removing lactose from milk products; clarifying, filtrating, and extracting aroma and color (e.g., the wine industry); debittering or detoxifying plant glycosidic compounds; processing coffee; aiding in digestion; producing starch (e.g., separating starch from gluten); producing oligosaccharides (e.g., nutraceuticals); producing gelling agents; modifying viscosity; saccharification of starch and other biopolymers; and modifying starch.
  • oils e.g. from olives, cleaning filters, reduction of viscosity, hydro lyzing starch, and stimulating fermentation in the brewing industry
  • carbohydrases in the bio fuel industry include, but are not limited to, releasing fermentable sugars, and the saccharification of lignocelluloses and starch. Saccharification can also be used to produce fermentable sugars for the production of high value metabolites and chemical building blocks for producing derivatives thereof and fine chemical.
  • SEQ ID NO: 1 encodes a mannase (Sequence CH 1) enzyme having the amino acid sequence of SEQ ID NO: 2;
  • SEQ ID NO: 165 encodes a mannase (Sequence CH 86) having the amino acid sequence of SEQ ID NO: 166.
  • SEQ ID NO: 3 encodes a mannosidase (Sequence CH 2) having the amino acid sequence of SEQ ID NO: 4;
  • SEQ ID NO: 53 encodes a mannosidase (Sequence CH 27) having the amino acid sequence of SEQ ID NO: 54;
  • SEQ ID NO: 79 encodes a mannosidase (Sequence CH 41) having the amino acid sequence of SEQ ID NO: 80;
  • SEQ ID NO: 107 encodes a mannosidase (Sequence CH 55) having the amino acid sequence of SEQ ID NO: 108;
  • SEQ ID NO: 115 encodes a mannosidase (Sequence CH 60) having the amino acid sequence of SEQ ID NO: 116;
  • SEQ ID NO: 123 encodes a mannosidase (Sequence CH 64) having the amino acid sequence of SEQ ID NO: 124;
  • SEQ ID NO: 141 encodes a
  • Mannosidase (Sequence CH 73) having the amino acid sequence of SEQ ID NO: 142;
  • SEQ ID NO: 151 encodes a mannosidase (Sequence CH 79) having the amino acid sequence of SEQ ID NO: 152;
  • SEQ ID NO: 173 encodes a
  • Mannosidase (Sequence CH 90) having the amino acid sequence of SEQ ID NO: 174;
  • SEQ ID NO: 235 encodes a mannosidase (Sequence CH 121) having the amino acid sequence of SEQ ID NO: 236;
  • SEQ ID NO: 257 encodes a
  • SEQ ID NO: 132 having the amino acid sequence of SEQ ID NO: 258;
  • SEQ ID NO: 311 encodes a mannosidase (Sequence CH 159) having the amino acid sequence of SEQ ID NO: 312;
  • SEQ ID NO: 321 encodes a
  • SEQ ID NO: 164 having the amino acid sequence of SEQ ID NO: 322;
  • SEQ ID NO: 325 encodes a mannosidase (Sequence CH 166) having the amino acid sequence of SEQ ID NO: 326.
  • SEQ ID NO: 5 encodes a alpha-galactosidase (Sequence CH 3) having the amino acid sequence of SEQ ID NO: 6.
  • SEQ ID NO: 7 encodes a gycosylhydrolase (Sequence CH 3) having the amino acid sequence of SEQ ID NO: 8;
  • SEQ ID NO: 11 encodes a gyclosyl hydrolase (Sequence CH 6) having the amino acid sequence of SEQ ID NO: 12;
  • SEQ ID NO: 13 encodes a gyclosyl hydrolase (Sequence CH 7) having the amino acid sequence of SEQ ID NO: 14;
  • SEQ ID NO: 27 encodes a glycosylhydrolase (Sequence CH 14) having the amino acid sequence of SEQ ID NO: 28;
  • SEQ ID NO: 29 encodes a glycosylhydrolase (Sequence CH 15) having the amino acid sequence of SEQ ID NO: 30;
  • SEQ ID NO: 37 encodes a glycosylhydrolase (Sequence CH 19) having the amino acid sequence of SEQ ID NO: 38;
  • SEQ ID NO: 39 encodes
  • glycosylhydrolase (Sequence CH 20) having the amino acid sequence of SEQ ID NO: 40;
  • SEQ ID NO: 41 encodes a glycosylhydrolase (Sequence CH 21) having the amino acid sequence of SEQ ID NO: 42;
  • SEQ ID NO: 43 encodes a
  • glycosylhydrolase (Sequence CH 22) having the amino acid sequence of SEQ ID NO: 44;
  • SEQ ID NO: 45 encodes a glycosylhydrolase (Sequence CH 23) having the amino acid sequence of SEQ ID NO: 46;
  • SEQ ID NO: 49 encodes a
  • glycosylhydrolase (Sequence CH 25) having the amino acid sequence of SEQ ID NO: 50;
  • SEQ ID NO: 51 encodes a glycosylhydrolase (Sequence CH 26) having the amino acid sequence of SEQ ID NO: 52;
  • SEQ ID NO: 59 encodes a
  • glycosylhydrolase (Sequence CH 30) having the amino acid sequence of SEQ ID NO: 60;
  • SEQ ID NO: 63 encodes a glycosylhydrolase (Sequence CH 31) having the amino acid sequence of SEQ ID NO: 64;
  • SEQ ID NO: 75 encodes a
  • glycosylhydrolase (Sequence CH 39) having the amino acid sequence of SEQ ID NO: 76
  • SEQ ID NO: 83 encodes a glycosylhydrolase (Sequence CH 43) having the amino acid sequence of SEQ ID NO: 84
  • SEQ ID NO: 97 encodes a
  • glycosylhydrolase (Sequence CH 50) having the amino acid sequence of SEQ ID NO: 98; SEQ ID NO: 103 encodes a glycosylhydrolase (Sequence CH 53) having the amino acid sequence of SEQ ID NO: 104; SEQ ID NO: 157 encodes a glycosylhydrolase (Sequence CH 82) having the amino acid sequence of SEQ ID NO: 158; SEQ ID NO: 167 encodes a glycosylhydrolase (Sequence CH 87) having the amino acid sequence of SEQ ID NO: 168; SEQ ID NO: 171 encodes a glycosylhydrolase (Sequence CH 89) having the amino acid sequence of SEQ ID NO: 172; SEQ ID NO: 207 encodes a glycosylhydrolase (Sequence CH 107) having the amino acid sequence of SEQ ID NO: 208; SEQ ID NO: 211 encodes a glycosylhydrolase (Seque
  • SEQ ID NO: 9 encodes a xylanase (Sequence CH 5) having the amino acid sequence of SEQ ID NO: 10;
  • SEQ ID NO: 57 encodes a xylanase (Sequence CH 29) having the amino acid sequence of SEQ ID NO: 58;
  • SEQ ID NO: 87 encodes a xylanase (Sequence CH 45) having the amino acid sequence of SEQ ID NO: 88;
  • SEQ ID NO: 93 encodes a xylanase (Sequence CH 48) having the amino acid sequence of SEQ ID NO: 94;
  • SEQ ID NO: 129 encodes a xylanase (Sequence CH 67) having the amino acid sequence of SEQ ID NO: 130;
  • SEQ ID NO: 159 encodes a xylanase (Sequence CH 83) having the amino acid sequence of SEQ ID NO: 160;
  • SEQ ID NO: 15 encodes a arabinofuranosidase (Sequence CH 8) having the amino acid sequence of SEQ ID NO: 16;
  • SEQ ID NO: 47 encodes a arabinofuranosidase (Sequence CH 24) having the amino acid sequence of SEQ ID NO: 48;
  • SEQ ID NO: 69 encodes a arabinofuranosidase (Sequence CH 35) having the amino acid sequence of SEQ ID NO: 70;
  • SEQ ID NO: 119 encodes a arabinofuranosidase (Sequence CH 62) having the amino acid sequence of SEQ ID NO: 120;
  • SEQ ID NO: 191 encodes a arabinofuranosidase (Sequence CH 100) having the amino acid sequence of SEQ ID NO: 192;
  • SEQ ID NO: 289 encodes a arabinofuranosidase (Sequence CH 148) having the amino acid sequence of SEQ ID NO: 290.
  • SEQ ID NO: 17 encodes a al ha-glucosidase (Sequence CH 9) having the amino acid sequence of SEQ ID NO: 18;
  • SEQ ID NO: 23 encodes a alpha-glucosidase (Sequence CH 12) having the amino acid sequence of SEQ ID NO: 24;
  • SEQ ID NO: 25 encodes a alpha-glucosidase (Sequence CH 13) having the amino acid sequence of SEQ ID NO: 26.
  • SEQ ID NO: 19 encodes a cellobio hydrolase (Sequence CH 10) having the amino acid sequence of SEQ ID NO: 20;
  • SEQ ID NO: 179 encodes a cellobiohydrolase (Sequence CH 93) having the amino acid sequence of SEQ ID NO: 180;
  • SEQ ID NO: 253 encodes a cellobiohydrolase (Sequence CH 130) having the amino acid sequence of SEQ ID NO: 254.
  • SEQ ID NO: 21 encodes a galacturonidase (Sequence CH 11) having the amino acid sequence of SEQ ID NO: 22.
  • SEQ ID NO: 31 encodes a Glycosidase (Sequence CH 16) having the amino acid sequence of SEQ ID NO: 32.
  • SEQ ID NO: 33 encodes a Glycosidase (Sequence CH 17) having the amino acid sequence of SEQ ID NO: 34.
  • SEQ ID NO: 35 encodes a deacetylase (Sequence CH 18) having the amino acid sequence of SEQ ID NO: 36;
  • SEQ ID NO: 67 encodes a deacetylase (Sequence CH 34) having the amino acid sequence of SEQ ID NO: 68;
  • SEQ ID NO: 277 encodes a deacetylase (Sequence CH 142) having the amino acid sequence of SEQ ID NO: 278;
  • SEQ ID NO: 333 encodes a deacetylase (Sequence CH 170) having the amino acid sequence of SEQ ID NO: 334;
  • SEQ ID NO: 335 encodes a deacetylase (Sequence CH 171) having the amino acid sequence of SEQ ID NO: 336.
  • SEQ ID NO: 55 encodes a phytase (Sequence CH 28) having the amino acid sequence of SEQ ID NO: 56;
  • SEQ ID NO: 183 encodes a phytase (Sequence CH 95) having the amino acid sequence of SEQ ID NO: 184.
  • SEQ ID NO: 61 encodes a chitinase (Sequence CH 31) having the amino acid sequence of SEQ ID NO: 62.; SEQ ID NO: 65 encodes a chitinase (Sequence CH 33) having the amino acid sequence of SEQ ID NO: 66; SEQ ID NO: 105 encodes a chitinase (Sequence CH 54) having the amino acid sequence of SEQ ID NO: 106; SEQ ID NO: 113 encodes a chitinase (Sequence CH 58) having the amino acid sequence of SEQ ID NO: 114 check sequence; SEQ ID NO: 163 encodes a chitinase (Sequence CH 85) having the amino acid sequence of SEQ ID NO: 164; SEQ ID NO: 193 encodes a chitinase (Sequence CH 99) having the amino acid sequence of SEQ ID NO: 194; SEQ ID NO: 243 encodes a
  • SEQ ID NO: 71 encodes a galacturonidase (Sequence CH 37) having the amino acid sequence of SEQ ID NO: 72.
  • SEQ ID NO: 73 encodes a glucosidase (Sequence CH 38) having the amino acid sequence of SEQ ID NO: 74;
  • SEQ ID NO: 95 encodes a glucosidase (Sequence CH 49) having the amino acid sequence of SEQ ID NO: 96;
  • SEQ ID NO: 101 encodes a glucosidase (Sequence CH 52) having the amino acid sequence of SEQ ID NO: 102;
  • SEQ ID NO: 121 encodes a glucosidase (Sequence CH 63) having the amino acid sequence of SEQ ID NO: 122;
  • SEQ ID NO: 135 encodes a glucosidase (Sequence CH 70) having the amino acid sequence of SEQ ID NO: 136;
  • SEQ ID NO: 137 encodes a glucosidase (Sequence CH 71) having the amino acid sequence of SEQ ID NO: 138;
  • SEQ ID NO: 77 encodes a carbohydrate hydrolase (Sequence CH 40) having the amino acid sequence of SEQ ID NO: 78;
  • SEQ ID NO: 341 encodes a carbohydrate hydrolase (Sequence CH 174) having the amino acid sequence of SEQ ID NO: 342;
  • SEQ ID NO: 343 encodes a carbohydrate hydrolase (Sequence CH 175) having the amino acid sequence of SEQ ID NO: 344.
  • SEQ ID NO: 81 encodes a hydrolase (Sequence CH 42) having the amino acid sequence of SEQ ID NO: 82;
  • SEQ ID NO: 347 encodes a hydrolase (Sequence CH 177) having the amino acid sequence of SEQ ID NO: 348;
  • SEQ ID NO: 349 encodes a hydrolase (Sequence CH 178) having the amino acid sequence of SEQ ID NO: 350.
  • SEQ ID NO: 85 encodes a glucanase (Sequence CH 44) having the amino acid sequence of SEQ ID NO: 86;
  • SEQ ID NO: 99 encodes a glucanase (Sequence CH 51) having the amino acid sequence of SEQ ID NO: 100;
  • SEQ ID NO: 249 encodes a glucanase (Sequence CH 128) having the amino acid sequence of SEQ ID NO: 250;
  • SEQ ID NO: 265 encodes a glucanase (Sequence CH 136) having the amino acid sequence of SEQ ID NO: 266;
  • SEQ ID NO: 307 encodes a glucanase (Sequence CH 157) having the amino acid sequence of SEQ ID NO: 308;
  • SEQ ID NO: 309 encodes a glucanase (Sequence CH 158) having the amino acid sequence of SEQ ID NO: 310.
  • SEQ ID NO: 89 encodes a glyco hydrolase (Sequence CH 46) having the amino acid sequence of SEQ ID NO: 90;
  • SEQ ID NO: 91 encodes a glycohydrolase (Sequence CH 47) having the amino acid sequence of SEQ ID NO: 92.
  • SEQ ID NO: 109 encodes a trehalase (Sequence CH 56) having the amino acid sequence of SEQ ID NO: 110;
  • SEQ ID NO: 223 encodes a trehalase (Sequence CH 115) having the amino acid sequence of SEQ ID NO: 224.
  • SEQ ID NO: 111 encodes a cellulase (Sequence CH 57) having the amino acid sequence of SEQ ID NO: 112;
  • SEQ ID NO: 117 encodes a cellulase (Sequence CH 61) having the amino acid sequence of SEQ ID NO: 118;
  • SEQ ID NO: 143 encodes a cellulase (Sequence CH 74) having the amino acid sequence of SEQ ID NO: 144;
  • SEQ ID NO: 201 encodes a cellulase (Sequence CH 104) having the amino acid sequence of SEQ ID NO: 202;
  • SEQ ID NO: 203 encodes a cellulase (Sequence CH 105) having the amino acid sequence of SEQ ID NO: 204;
  • SEQ ID NO: 217 encodes a cellulase (Sequence CH 112) having the amino acid sequence of SEQ ID NO: 218;
  • SEQ ID NO: 237 encodes a
  • SEQ ID NO: 363 encodes a glucanase/glucosidase (Sequence CH 59) having the amino acid sequence of SEQ ID NO:364.
  • SEQ ID NO: 125 encodes a galactosidase (Sequence CH 65) having the amino acid sequence of SEQ ID NO: 126;
  • SEQ ID NO: 127 encodes a galactosidase (Sequence CH 66) having the amino acid sequence of SEQ ID NO: 128;
  • SEQ ID NO: 139 encodes a galactosidase (Sequence CH 72) having the amino acid sequence of SEQ ID NO: 140;
  • SEQ ID NO: 155 encodes a galactosidase (Sequence CH 81) having the amino acid sequence of SEQ ID NO: 156;
  • SEQ ID NO: 181 encodes a galactosidase (Sequence CH 94) having the amino acid sequence of SEQ ID NO: 182;
  • SEQ ID NO: 233 encodes a galactosidase
  • sequence CH 120 having the amino acid sequence of SEQ ID NO: 234;
  • SEQ ID NO: 255 encodes a galactosidase (Sequence CH 131) having the amino acid sequence of SEQ ID NO: 256;
  • SEQ ID NO: 267 encodes a galactosidase
  • SEQ ID NO: 131 encodes a endoglucanase (Sequence CH 68) having the amino acid sequence of SEQ ID NO: 132;
  • SEQ ID NO: 145 encodes a endoglucanase (Sequence CH 76) having the amino acid sequence of SEQ ID NO: 146;
  • SEQ ID NO: 161 encodes a endoglucanase (Sequence CH 84) having the amino acid sequence of SEQ ID NO: 162.
  • SEQ ID NO: 133 encodes a glycoside hydrolase (Sequence CH 69) having the amino acid sequence of SEQ ID NO: 134.
  • SEQ ID NO: 365 encodes a glucanase/glucosidase (Sequence CH 75) having the amino acid sequence of SEQ ID NO: 366.
  • SEQ ID NO: 147 encodes an amylase (Sequence CH 77) having the amino acid sequence of SEQ ID NO: 148;
  • SEQ ID NO: 279 encodes an amylase (Sequence CH 143) having the amino acid sequence of SEQ ID NO: 280;
  • SEQ ID NO: 283 encodes an amylase (Sequence CH 145) having the amino acid sequence of SEQ ID NO: 284.
  • SEQ ID NO: 149 encodes a galactanase (Sequence CH 78) having the amino acid sequence of SEQ ID NO: 150.
  • SEQ ID NO: 169 encodes a ferulic acid esterase (Sequence CH 88) having the amino acid sequence of SEQ ID NO: 170.
  • SEQ ID NO: 177 encodes a betaglucanase (Sequence CH 92) having the amino acid sequence of SEQ ID NO: 178SEQ ID NO: 185 encodes a
  • xylosidase/arabinofuranosidase (Sequence CH 96) having the amino acid sequence of SEQ ID NO: 186.
  • SEQ ID NO: 187 encodes a xylosidase (Sequence CH 97) having the amino acid sequence of SEQ ID NO: 188;
  • SEQ ID NO: 197 encodes a xylosidase (Sequence CH 102) having the amino acid sequence of SEQ ID NO: 198.
  • SEQ ID NO: 209 encodes a glucanase/glucosidase (Sequence CH 108) having the amino acid sequence of SEQ ID NO: 210;
  • SEQ ID NO: 215 encodes a
  • glucanase/glucosidase (Sequence CH 111) having the amino acid sequence of SEQ ID NO: 216.
  • SEQ ID NO: 213 encodes a beta-glucanase (Sequence CH 110) having the amino acid sequence of SEQ ID NO: 214.
  • SEQ ID NO: 221 encodes a mannanase (Sequence CH 114) having the amino acid sequence of SEQ ID NO: 222;
  • SEQ ID NO: 229 encodes a mannanase (Sequence CH 118) having the amino acid sequence of SEQ ID NO: 230;
  • SEQ ID NO: 287 encodes a mannanase (Sequence CH 147) having the amino acid sequence of SEQ ID NO: 288.
  • SEQ ID NO: 263 encodes a scaffold00142.pathl .genel90 cellulase (Sequence CH 135) having the amino acid sequence of SEQ ID NO: 264
  • SEQ ID NO: 285 encodes a glucan synthase (Sequence CH 146) having the amino acid sequence of SEQ ID NO: 286.
  • SEQ ID NO: 291 encodes an arabinase (Sequence CH 149) having the amino acid sequence of SEQ ID NO: 292;
  • SEQ ID NO: 305 encodes an arabinase (Sequence CH 156) having the amino acid sequence of SEQ ID NO: 306.
  • SEQ ID NO: 295 encodes a xylosidase (Sequence CH 151) having the amino acid sequence of SEQ ID NO: 296;
  • SEQ ID NO: 297 encodes a xylosidase (Sequence CH 82)152 having the amino acid sequence of SEQ ID NO: 298.
  • SEQ ID NO: 323 encodes a glucanosyltransferase (Sequence CH 165) having the amino acid sequence of SEQ ID NO: 324.
  • SEQ ID NO: 337 encodes a chitosanase (Sequence CH 172) having the amino acid sequence of SEQ ID NO: 338;
  • SEQ ID NO: 339 encodes a chitosanase (Sequence CH 173) having the amino acid sequence of SEQ ID NO: 340.
  • SEQ ID NO: 345 encodes a carbo hydro lytic enhancer (Sequence CH 176) having the amino acid sequence of SEQ ID NO: 346.
  • Enzyme C105111 (Sequence CH 177) is a glycosidehydrolase and/or polypeptide having cellulo lytic enhancing activity in family GH61 encoded by SEQ ID NO: 351, having the 278 amino acid sequence of SEQ ID NO: 352. An exon is present from nucleotides 329 to 1165.
  • the cDNA of SEQ ID NO 17 encodes amino acid sequence SEQ ID NO: 352. A singal sequence is believed to span amino acids 1- 19 of SEQ ID NO: 352 with the mature protein being encoded by amino acids 20- 278 of SEQ ID NO: 352.
  • Enzyme CL03830 (Sequence CH 178) is a glycosidehydrolase and/or polypeptide having cellulo lytic enhancing activity in family GH61 encoded by the nucleotides of SEQ ID NO: 353, having the 405 amino acid sequence of SEQ ID NO: 354. An exon is present from nucleotides 530 to 1747.
  • the cDNA of SEQ ID NO: 368 encodes the sequence of SEQ ID NO: 354. A singal sequence is believed to span amino acids 1-21 of SEQ ID NO: 354 with the mature protein being encoded by amino acids 22-405 of SEQ ID NO: 354.
  • Enzyme CL9131 (Sequence CH 179) is a glycosidehydrolase and/or polypeptide having cellulo lytic enhancing activity in family GH61 having the 418 amino acid sequence of SEQ ID NO: 356 and encoded by nucleotide SEQ ID NO: 355. Exons are present in SEQ ID NO: 356 from nucleotides 512 to 1398 and from nucleotides 1480 to 1849. An intron spans nucleotides 1399 to 1479. The cDNA of SEQ ID NO: 369 also encodes amino acid SEQ ID NO: 356. A singal sequence is believed to span amino acids 1-22 of SEQ ID NO: 354 with the mature protein being encoded by amino acids 23-418 of SEQ ID NO: 354.
  • Enzyme CL00632 (Sequence CH 180) is a glycosidehydrolase and/or polypeptide having cellulo lytic enhancing activity in family GH61 having the amino acid sequence of SEQ ID NO: 358 and encoded by the nucleotides of SEQ ID NO: 357. Exons are present from nucleotides 146 to 381; 521 to 775; 864 to 932; 1036 to 1186; and 1246 to 1848. Introns are present from nucleotides 382 to 520; 776 to 863; 933 to 1035; and 1187 to 1245. A singal sequence is believed to span amino acids 1-23 of SEQ ID NO: 356 with the mature protein being encoded by amino acids 24-437 of SEQ ID NO: 356.
  • an isolated protein or polypeptide in the present invention includes full-length proteins and their glycosylated or otherwise modified forms forms, fusion proteins, or any fragment or homologue or variant of such a protein.
  • an isolated protein such as an enzyme according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, synthetically produced proteins, proteins complexed with lipids, soluble proteins, and isolated proteins associated with other proteins, for example.
  • a "Myceliophthora thermophila or M. thermophila protein” or “ Myceliophthora thermophila or M. thermophila enzyme” refers to a protein (generally including a homologue or variant of a naturally occurring protein) from Myceliophthora thermophila or to a protein that has been otherwise produced from the knowledge of the structure (e.g., sequence) and perhaps the function of a naturally occurring protein from Myceliophthora thermophila. In other words, a M.
  • thermophila protein includes any protein that has substantially similar structure and function of a naturally occurring M. thermophila protein or that is a biologically active (i.e., has biological activity) homologue or variant of a naturally occurring protein from thermophila as described in detail herein.
  • a thermophila protein can include purified, partially purified, recombinant, mutated/modified and synthetic proteins.
  • modifications can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of a M. thermophila protein (or nucleic acid sequences) described herein.
  • An isolated protein according to the present invention can be isolated from its natural source, produced recombinantly or produced synthetically.
  • modification and “mutation” can be used interchangeably, particularly with regard to the modifications/mutations to the primary amino acid sequences of a protein or peptide (or nucleic acid sequences) described herein.
  • modification can also be used to describe post- translational modifications to a protein or peptide including, but not limited to, methylation, farnesylation, carboxymethylation, geranyl geranylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, and/or amidation.
  • Modification can also included the cleavage of a signal peptide, or methionine, or other portions of the peptide that require cleavage to generate the mature peptide.
  • the terms "homologue” or “variants” are used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the "prototype” or “wild-type” protein) by minor modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form.
  • Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes one or a few amino acids, including deletions (e.g., a truncated version of the protein or peptide), insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation.
  • a homologue or variant can have either enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide.
  • Homologues or variants can be the result of natural allelic variation or natural mutation.
  • a naturally occurring allelic variant of a nucleic acid encoding a protein is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes such protein, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence.
  • Homologous can also be the result of a gene duplication and rearrangement, resulting in a different location.
  • Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
  • allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art.
  • Homologues or variants can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • Modifications of a protein, such as in a homologue or variant may result in proteins having the same biological activity as the naturally occurring protein, or in proteins having decreased or increased biological activity as compared to the naturally occurring protein. Modifications which result in a decrease in protein expression or a decrease in the activity of the protein, can be referred to as inactivation (complete or partial), down-regulation, or decreased action of a protein. Similarly, modifications which result in an increase in protein expression or an increase in the activity of the protein, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein.
  • an isolated protein including a biologically active homologue, variant, or fragment thereof, has at least one characteristic of biological activity of a wild-type, or naturally occurring, protein.
  • the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form of the protein as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions).
  • the biological activity of a protein of the present invention can include an enzyme activity (catalytic activity and/or substrate binding activity), such as cellulase activity, hemicellulase activity, ⁇ -glucanase activity, ⁇ - glucosidase activity, D-galactosidase activity, ⁇ -galactosidase activity, xylanase activity or any other activity disclosed herein.
  • an enzyme activity catalytic activity and/or substrate binding activity
  • cellulase activity hemicellulase activity
  • ⁇ -glucanase activity ⁇ - glucosidase activity
  • D-galactosidase activity ⁇ -galactosidase activity
  • xylanase activity xylanase activity
  • Such assays include, but are not limited to, measurement of enzyme activity ⁇ e.g., catalytic activity), measurement of substrate binding, and the like. It is noted that an isolated protein of the present invention (including homologues or variants) is not required to have a biological activity such as catalytic activity.
  • a protein can be a truncated, mutated or inactive protein, or lack at least one activity of the wild-type enzyme, for example. Inactive proteins may be useful in some screening assays, for example, or for other purposes such as antibody production.
  • Methods to measure protein expression levels of a protein according to the invention include, but are not limited to: western blotting, immuno cytochemistry, flow cytometry or other immuno logic-based assays; assays based on a property of the protein including but not limited to, ligand binding or interaction with other protein partners.
  • Homologues or variants of a protein encompassed by the present invention can comprise, consist essentially of, or consist of, in one embodiment, an amino acid sequence that is at least about 35% identical, and more preferably at least about 40% identical, and more preferably at least about 45% identical, and more preferably at least about 50% identical, and more preferably at least about 55% identical, and more preferably at least about 60% identical, and more preferably at least about 65% identical, and more preferably at least about 70% identical, and more preferably at least about 75% identical, and more preferably at least about 80% identical, and more preferably at least about 85% identical, and more preferably at least about 90% identical, and more preferably at least about 95% identical, and more preferably at least about 96% identical, and more preferably at least about 97% identical, and more preferably at least about 98% identical, and more preferably at least about 99% identical, or any percent identity between 35% and 99%, in whole integers (i.e., 36%o, 37%), etc.), to an amino acid sequence
  • the amino acid sequence of the homologue or variant has a biological activity of the wild-type or reference protein or of a biologically active domain thereof (e.g., a catalytic domain).
  • a biologically active domain thereof e.g., a catalytic domain.
  • the amino acid position of the wild-type is typically used.
  • the wild-type can also be referred to as the "parent.” Additionally, any generation before the variant at issue can be a parent.
  • a protein of the present invention comprises, consists essentially of, or consists of an amino acid sequence that, alone or in combination with other characteristics of such proteins disclosed herein, is less than 100% identical to an amino acid sequence selected from SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46, SEQ ID No: 48, SEQ ID No: 50, SEQ ID No: 52, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No: 58, SEQ ID No: 60, SEQ ID No
  • a protein of the present invention can be less than 100% identical, in combination with being at least about 35% identical, to a given disclosed sequence.
  • a homologue or variant according to the present invention has an amino acid sequence that is less than about 99% identical to any of such amino acid sequences, and in another embodiment, is less than about 98%> identical to any of such amino acid sequences, and in another embodiment, is less than about 97% identical to any of such amino acid sequences, and in another embodiment, is less than about 96% identical to any of such amino acid sequences, and in another embodiment, is less than about 95% identical to any of such amino acid sequences, and in another embodiment, is less than about 94% identical to any of such amino acid sequences, and in another embodiment, is less than about 93% identical to any of such amino acid sequences, and in another embodiment, is less than about 92% identical to any of such amino acid sequences, and in another embodiment, is less than about 91% identical to any of such amino acid sequences, and in another embodiment, is less than about 90%>
  • reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res.
  • PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologues or variants.
  • the program first performs a gapped BLAST database search.
  • the PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.
  • BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment.
  • BLAST 2.0 Gapped BLAST search
  • a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
  • a protein of the present invention can also include proteins having an amino acid sequence comprising at least 10 contiguous amino acid residues of any of the sequences described herein (i.e., 10 contiguous amino acid residues having 100% identity with 10 contiguous amino acids of the amino acid sequences of Sequences CH 1- CH 183).
  • a homologue or variant of a protein amino acid sequence includes amino acid sequences comprising at least 20, or at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 125, or at least 150, or at least 175, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350 contiguous amino acid residues of any of the amino acid sequence represented disclosed herein.
  • fragments of proteins without biological activity are useful in the present invention, for example, in the preparation of antibodies against the full-length protein or in a screening assay (e.g., a binding assay). Fragments can also be used to construct fusion proteins, for example, where the fusion protein comprises functional domains from two or more different proteins (e.g., a CBM from one protein linked to a CD from another protein). In one embodiment, a homologue or variant has a measurable or detectable biological activity associated with the wild-type protein (e.g., enzymatic activity).
  • the term "contiguous” or “consecutive”, with regard to nucleic acid or amino acid sequences described herein, means to be connected in an unbroken sequence.
  • a first sequence to comprise 30 contiguous (or consecutive) amino acids of a second sequence means that the first sequence includes an unbroken sequence of 30 amino acid residues that is 100% identical to an unbroken sequence of 30 amino acid residues in the second sequence.
  • a first sequence to have "100% identity" with a second sequence means that the first sequence exactly matches the second sequence with no gaps between nucleotides or amino acids.
  • a protein of the present invention includes a protein having an amino acid sequence that is sufficiently similar to a natural amino acid sequence that a nucleic acid sequence encoding the homologue or variant is capable of hybridizing under moderate, high or very high stringency conditions (described below) to (i.e., with) a nucleic acid molecule encoding the natural protein (i.e., to the complement of the nucleic acid strand encoding the natural amino acid sequence).
  • a homologue or variant of a protein of the present invention is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under low, moderate, or high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising, consisting essentially of, or consisting of, an amino acid sequence represented by any of Sequences CH 1- CH 183.
  • hybridization conditions are described in detail below.
  • a nucleic acid sequence complement of nucleic acid sequence encoding a protein of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to the strand which encodes the protein. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand having a sequence that is a complement to the single strand DNA.
  • nucleic acid molecules of the present invention can be either double-stranded or single- stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes an amino acid sequence such as the amino acid sequences of SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46, SEQ ID No: 48, SEQ ID No: 50, SEQ ID No: 52, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No
  • hybridization conditions refers to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al, ibid., (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al, 1984, Anal. Biochem. 138, 267-284; Meinkoth et al, ibid.
  • moderate stringency hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides).
  • High stringency hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides).
  • Very high stringency hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 90%> nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10%> or less mismatch of nucleotides).
  • conditions permitting about 10%> or less mismatch of nucleotides i.e., one of skill in the art can use the formulae in Meinkoth et al, ibid, to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:R A or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10°C less than for DNA:R A hybrids.
  • stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 20°C and about 35°C (lower stringency), more preferably, between about 28°C and about 40°C (more stringent), and even more preferably, between about 35°C and about 45°C (even more stringent), with appropriate wash conditions.
  • 6X SSC 0.9 M Na +
  • stringent hybridization conditions for DNA:R A hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, T m can be calculated empirically as set forth in Sambrook et al, supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions.
  • hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25 °C below the calculated T m of a particular hybrid
  • wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated T m of the particular hybrid.
  • hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X-0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC).
  • the minimum size of a protein and/or homologue or variant of the present invention is a size sufficient to have biological activity or, when the protein is not required to have such activity, sufficient to be useful for another purpose associated with a protein of the present invention, such as for the production of antibodies that bind to a naturally occurring protein.
  • the protein of the present invention is at least 20 amino acids in length, or at least about 25 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, or at least about 60 amino acids in length, or at least about 70 amino acids in length, or at least about 80 amino acids in length, or at least about 90 amino acids in length, or at least about 100 amino acids in length, or at least about 125 amino acids in length, or at least about 150 amino acids in length, or at least about 175 amino acids in length, or at least about 200 amino acids in length, or at least about 250 amino acids in length, and so on up to a full length of each protein, and including any size in between in increments of one whole integer (one amino acid).
  • the protein can include a portion of a protein or a full-length protein, plus additional sequence (e.g., a fusion protein sequence), if desired.
  • the present invention also includes a fusion protein that includes a domain of a protein of the present invention (including a homologue or variant) attached to one or more fusion segments, which are typically heterologous in sequence to the protein sequence (i.e., different than protein sequence).
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; provide other desirable biological activity; and/or assist with the purification of the protein (e.g., by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, solubility, action or biological activity; and/or simplifies purification of a protein).
  • Fusion segments can be joined to amino and/or carboxyl termini of the domain of a protein of the present invention and can be susceptible to cleavage in order to enable straight-forward recovery of the protein.
  • Fusion proteins are preferably produced by culturing a recombinant cell transfected with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a domain of a protein of the present invention.
  • proteins of the present invention also include expression products of gene fusions (for example, used to overexpress soluble, active forms of the recombinant protein), of mutagenized genes (such as genes having codon modifications to enhance gene transcription and translation), and of truncated genes (such as genes having membrane binding modules removed to generate soluble forms of a membrane protein, or genes having signal sequences removed which are poorly tolerated in a particular recombinant host).
  • gene fusions for example, used to overexpress soluble, active forms of the recombinant protein
  • mutagenized genes such as genes having codon modifications to enhance gene transcription and translation
  • truncated genes such as genes having membrane binding modules removed to generate soluble forms of a membrane protein, or genes having signal sequences removed which are poorly tolerated in a particular recombinant host.
  • any of the amino acid sequences described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence.
  • the resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence.
  • the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived.
  • the present invention also provides enzyme combinations that break down lignocellulose material.
  • Such enzyme combinations or mixtures can include a multi-enzyme composition that contains at least one protein of the present invention in combination with one or more additional proteins of the present invention or one or more enzymes or other proteins from other microorganisms, plants, or similar organisms.
  • Synergistic enzyme combinations and related methods are contemplated.
  • the invention includes methods to identify the optimum ratios and compositions of enzymes with which to degrade each lignocellulosic material. These methods entail tests to identify the optimum enzyme composition and ratios for efficient conversion of any lignocellulosic substrate to its constituent sugars.
  • the Examples below include assays that may be used to identify optimum ratios and compositions of enzymes with which to degrade lignocellulosic materials.
  • any combination of the proteins disclosed herein is suitable for use in the multi- enzyme compositions of the present invention. Due to the complex nature of most biomass sources, which can contain cellulose, hemicellulose, pectin, lignin, protein, and ash, among other components, preferred enzyme combinations may contain enzymes with a range of substrate specificities that work together to degrade biomass into fermentable sugars in the most efficient manner.
  • a multi-enzyme complex for lignocellulose saccharification is a mixture of cellobiohydrolase(s), xylanase(s), endoglucanase(s), P-glucosidase(s), P-xylosidase(s), and accessory enzymes.
  • any of the enzymes described specifically herein can be combined with any one or more of the enzymes described herein or with any other available and suitable enzymes, to produce a multi-enzyme composition.
  • the invention is not restricted or limited to the specific exemplary combinations listed below.
  • the cellobiohydrolase(s) comprise between about 30% and about 90% or between about 40% and about 70% of the enzymes in the composition, and more preferably, between about 55% and 65%, and more preferably, about 60% of the enzymes in the composition (including any percentage between 40% and 70% in 0.5% increments (e.g., 40%, 40.5%, 41%, etc.).
  • the xylanase(s) comprise between about 10%> and about 30%> of the enzymes in the composition, and more preferably, between about 15% and about 25%, and more preferably, about 20% of the enzymes in the composition (including any percentage between 10%> and 30%> in 0.5%> increments).
  • the endoglucanase(s) comprise between about 5% and about 15% of the enzymes in the composition, and more preferably, between about 7% and about 13%, and more preferably, about 10% of the enzymes in the composition (including any percentage between 5% and 15% in 0.5% increments).
  • the P-glucosidase(s) comprise between about 1% and about 15% of the enzymes in the composition, and preferably between about 2% and 10%, and more preferably, about 3% of the enzymes in the composition (including any percentage between 1% and 15% in 0.5% increments).
  • the P-xylosidase(s) comprise between about 1% and about 3% of the enzymes in the composition, and preferably, between about 1.5% and about 2.5%, and more preferably, about 2% of the enzymes in the composition (including any percentage between 1% and 3% in 0.5% increments.
  • the accessory enzymes comprise between about 2% and about 8%) of the enzymes in the composition, and preferably, between about 3% and about 7%, and more preferably, about 5% of the enzymes in the composition (including any percentage between 2% and 8% in 0.5% increments.
  • One particularly preferred example of a multi-enzyme complex for lignocellulose saccharification is a mixture of about 60% cellobiohydrolase(s), about 20% xylanase(s), about 10% endoglucanase(s), about 3% P-glucosidase(s), about 2% ⁇ - xylosidase(s) and about 5% accessory enzyme(s).
  • Enzymes and multi-enzyme compositions of the present invention may also be used to break down arabinoxylan or arabinoxylan-containing substrates.
  • Arabino xylan is a polysaccharide composed of xylose and arabinose, wherein D-L- arabinofuranose residues are attached as branch-points to a P-(l,4)-linked xylose polymeric backbone.
  • the xylose residues may be mono-substituted at the C2 or C3 position, or di-substituted at both positions.
  • Ferulic acid or coumaric acid may also be ester-linked to the C5 position of arabinosyl residues. Further details on the hydrolysis of arabinoxylan can be found in International Publication No. WO 2006/114095.
  • the substitutions on the xylan backbone can inhibit the enzymatic activity of xylanases, and the complete hydrolysis of arabinoxylan typically requires the action of several different enzymes.
  • a multi-enzyme complex for arabinoxylan hydrolysis is a mixture of endoxylanase(s), P-xylosidase(s), and arabinofuranosidase(s), including those with specificity towards single and double substituted xylose residues.
  • the multi-enzyme complex may further comprise one or more carbohydrate esterases, such as acetyl xylan esterases, ferulic acid esterases, coumaric acid esterases or pectin methyl esterases. Any combination of two or more of the above-mentioned enzymes is suitable for use in the multi-enzyme complexes. However, it is to be understood that the invention is not restricted or limited to the specific exemplary combinations listed herein.
  • the endoxylanase(s) comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 40%>, at least about 50%>, at least about 70%> of the enzymes in the composition (including any percentage between 5% and 70%> in 0.5%> increments (e.g., 5.0%, 5.5%, 6.0%>, etc.).
  • Endoxylanase(s) either alone or as part of a multi-enzyme complex, may be used in amounts of 0.001 to 2.0 g/kg, 0.005 to 1.0 g/kg, or 0.05 to 0.2 g/kg of substrate.
  • the P-xylosidase(s) comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 40%>, at least about 50%>, at least about 70%> of the enzymes in the composition (including any percentage between 5% and 70% in 0.5% increments (e.g., 5.0%>, 5.5%, 6.0%>, etc.).
  • P-xylosidase(s) either alone or as part of a multi-enzyme complex, may be used in amounts of 0.001 to 2.0 g/kg, 0.005 to 1.0 g/kg, or 0.05 to 0.2 g/kg of substrate.
  • the arabinofuranosidase(s) comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 40%, at least about 50%, at least about 70% of the enzymes in the composition (including any percentage between 5% and 70% in 0.5%) increments (e.g., 5.0%>, 5.5%, 6.0%>, etc.).
  • the total percentage of arabinofuranosidase(s) present in the composition may include arabinofuranosidase(s) with specificity towards single substituted xylose residues, arabinofuranosidase(s) with specificity towards double substituted xylose residues, or any combination thereof.
  • Arabinofuranosidase(s) either alone or as part of a multi-enzyme complex, may be used in amounts of 0.001 to 2.0 g/kg, 0.005 to 1.0 g/kg, or 0.05 to 0.2 g/kg of substrate.
  • One or more components of a multi-enzyme composition can be obtained from or derived from a microbial, plant, or other source or combination thereof, and will contain enzymes capable of degrading lignocellulosic material.
  • Examples of enzymes included in the multi- enzyme compositions of the invention include cellulases, hemicellulases (such as xylanases, including endoxylanases, exoxylanases, and ⁇ -xylosidases; mannanases, including endomannanases, exomannanases, and ⁇ -mannosidases), ligninases, amylases, glucuronidases, proteases, esterases (including ferulic acid esterase), lipases, glucosidases (such as ⁇ -glucosidase), and xyloglucanases.
  • cellulases such as xylanases, including endoxylanases, exoxylanases, and ⁇ -xylosidases
  • mannanases including endomannanases, exomannanases, and ⁇ -mannosidases
  • ligninases amylases
  • the multi-enzyme composition may contain many types of enzymes, mixtures comprising enzymes that increase or enhance sugar release from biomass are preferred, including hemicellulases.
  • the hemicellulase is selected from a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, an endo-galactanase, a mannanase, an endo-arabinase, an exo- arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xyloglucanase, or mixtures of any of these.
  • the enzymes can include glucoamylase, ⁇ -xylosidase and/or ⁇ -glucosidase.
  • mixtures comprising enzymes that are capable of degrading cell walls and releasing
  • the enzymes of the multi-enzyme composition can be provided by a variety of sources.
  • the enzymes can be produced by growing organisms such as bacteria, algae, fungi, and plants which produce the enzymes naturally or by virtue of being genetically modified to express the enzyme or enzymes.
  • at least one enzyme of the multi-enzyme composition is a commercially available enzyme.
  • the multi-enzyme compositions comprise an accessory enzyme.
  • An accessory enzyme is any additional enzyme capable of hydrolyzing lignocellulose or enhancing or promoting the hydrolysis of lignocellulose, wherein the accessory enzyme is typically provided in addition to a core enzyme or core set of enzymes.
  • An accessory enzyme can have the same or similar function or a different function as an enzyme or enzymes in the core set of enzymes. These enzymes have been described elsewhere herein, and can generally include cellulases, xylanases, ligninases, amylases, lipidases, or glucuronidases, for example.
  • Accessory enzymes can include enzymes that when contacted with biomass in a reaction, allow for an increase in the activity of enzymes (e.g., hemicellulases) in the multi-enzyme composition.
  • An accessory enzyme or enzyme mix may be composed of enzymes from (1) commercial suppliers; (2) cloned genes expressing enzymes; (3) complex broth (such as that resulting from growth of a microbial strain in media, wherein the strains secrete proteins and enzymes into the media); (4) cell lysates of strains grown as in (3); and, (5) plant material expressing enzymes capable of degrading lignocellulose.
  • the accessory enzyme is a glucoamylase, a pectinase, or a ligninase.
  • a ligninase is an enzyme that can hydrolyze or break down the structure of lignin polymers, including lignin peroxidases, manganese peroxidases, laccases, and other enzymes described in the art known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydro lyzing bonds formed between hemicellulosic sugars (notably arabinose) and lignin.
  • the multi-enzyme compositions comprise a biomass comprising microorganisms or a crude fermentation product of microorganisms.
  • a crude fermentation product refers to the fermentation broth which has been separated from the microorganism biomass (by filtration, for example).
  • the microorganisms are grown in fermentors, optionally centrifuged or filtered to remove biomass, and optionally concentrated, formulated, and dried to produce an enzyme(s) or a multi-enzyme composition that is a crude fermentation product.
  • enzyme(s) or multi-enzyme compositions produced by the microorganism are subjected to one or more purification steps, such as ammonium sulfate precipitation, chromatography, and/or ultrafiltration, which result in a partially purified or purified enzyme(s).
  • the enzyme(s) will include recombinant enzymes.
  • the enzyme(s) may include both naturally occurring and recombinant enzymes.
  • compositions comprising at least about 500 ng, and preferably at least about 1 ⁇ g, and more preferably at least about 5 ⁇ g, and more preferably at least about 10 ⁇ g, and more preferably at least about 25 ⁇ g, and more preferably at least about 50 ⁇ g, and more preferably at least about 75 ⁇ g, and more preferably at least about 100 ⁇ g, and more preferably at least about 250 ⁇ g, and more preferably at least about 500 ⁇ g, and more preferably at least about 750 ⁇ g, and more preferably at least about 1 mg, and more preferably at least about 5 mg, of an isolated protein comprising any of the proteins or homologues, variants, or fragments thereof discussed herein.
  • composition of the present invention may include any carrier with which the protein is associated by virtue of the protein preparation method, a protein purification method, or a preparation of the protein for use in any method according to the present invention.
  • a carrier can include any suitable buffer, extract, or medium that is suitable for combining with the protein of the present invention so that the protein can be used in any method described herein according to the present invention.
  • an immobilized enzyme includes immobilized isolated enzymes, immobilized microbial cells which contain one or more enzymes of the invention, other stabilized intact cells that produce one or more enzymes of the invention, and stabilized cell/membrane homogenates.
  • Stabilized intact cells and stabilized cell/membrane homogenates include cells and homogenates from naturally occurring microorganisms expressing the enzymes of the invention and preferably, from genetically modified microorganisms as disclosed elsewhere herein.
  • nucleic acid molecules that encode a protein of the present invention, as well as homologues, variants, or fragments of such nucleic acid molecules.
  • a nucleic acid molecule of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence encoding any of the isolated proteins disclosed herein, including a fragment or a homologue or variant of such proteins, described above.
  • Nucleic acid molecules can include a nucleic acid sequence that encodes a fragment of a protein that does not have biological activity, and can also include portions of a gene or polynucleotide encoding the protein that are not part of the coding region for the protein (e.g., introns or regulatory regions of a gene encoding the protein). Nucleic acid molecules can include a nucleic acid sequence that is useful as a probe or primer (oligonucleotide sequences).
  • a nucleic acid molecule of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence represented in SEQ ID No: 1 , SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 1 1 , SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21 , SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 31 , SEQ ID No: 33, SEQ ID No: 35, SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 41 , SEQ ID No: 43, SEQ ID No: 45, SEQ ID No: 47, SEQ ID No: 49, SEQ ID No: 51 , SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 56
  • a nucleic acid sequence encodes a protein (including fragments and homologues or variatns thereof) useful in the invention, or encompasses useful oligonucleotides or complementary nucleic acid sequences.
  • a nucleic molecule of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence encoding an amino acid sequence represented in SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46
  • nucleic acid molecules include isolated nucleic acid molecules that hybridize under moderate stringency conditions, and more preferably under high stringency conditions, and even more preferably under very high stringency conditions, as described above, with the complement of a nucleic acid sequence encoding a protein of the present invention (i.e., including naturally occurring allelic variants encoding a protein of the present invention).
  • an isolated nucleic acid molecule encoding a protein of the present invention comprises a nucleic acid sequence that hybridizes under moderate, high, or very high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising an amino acid sequence represented in SEQ ID NO: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 32, SEQ ID No: 34, SEQ ID No: 36, SEQ ID No: 38, SEQ ID No: 40, SEQ ID No: 42, SEQ ID No: 44, SEQ ID No: 46, SEQ ID No: 48, SEQ ID No: 50, SEQ ID No: 52, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No
  • an isolated nucleic acid molecule is a nucleic acid molecule (polynucleotide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA, including cDNA.
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule
  • nucleic acid sequence primarily refers to the sequence of nucleotides on the nucleic acid molecule
  • the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein.
  • An isolated nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates.
  • An isolated nucleic acid molecule can include degeneracies.
  • nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons.
  • nucleic acid sequence of a nucleic acid molecule that encodes a protein of the present invention can vary due to degeneracies. It is noted that a nucleic acid molecule of the present invention is not required to encode a protein having protein activity. A nucleic acid molecule can encode a truncated, mutated or inactive protein, for example. In addition, nucleic acid molecules of the invention are useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules.
  • the nucleic acid molecule is an oligonucleotide, such as a probe or primer
  • the oligonucleotide preferably ranges from about 5 to about 50 or about 500 nucleotides, more preferably from about 10 to about 40 nucleotides, and most preferably from about 15 to about 40 nucleotides in length.
  • reference to a gene includes all nucleic acid sequences related to a natural (i.e. wild-type) gene, such as regulatory regions that control production of the protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • a gene in another embodiment, can be a naturally occurring allelic variant that includes a similar but not identical sequence to the nucleic acid sequence encoding a given protein. Allelic variants have been previously described above. Genes can include or exclude one or more introns or any portions thereof or any other sequences or which are not included in the cDNA for that protein.
  • the phrases "nucleic acid molecule” and “gene” can be used interchangeably when the nucleic acid molecule comprises a gene as described above.
  • an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis.
  • Isolated nucleic acid molecules include any nucleic acid molecules and homologues or variants thereof that are part of a gene described herein and/or that encode a protein described herein, including, but not limited to, natural allelic variants and modified nucleic acid molecules (homologues or variants) in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications provide the desired effect on protein biological activity or on the activity of the nucleic acid molecule.
  • Allelic variants and protein homologues or variants e.g., proteins encoded by nucleic acid homologues or variants
  • a nucleic acid molecule homologue or variant (i.e., encoding a homologue or variant of a protein of the present invention) can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al.).
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis and recombinant DNA techniques e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification
  • nucleic acid molecule homologues or variants can be selected by hybridization with a gene or polynucleotide, or by screening for the function of a protein encoded by a nucleic acid molecule ⁇ i.e., biological activity).
  • the minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein (including a fragment, homologue, or variant of a full-length protein) having biological activity, sufficient to encode a protein comprising at least one epitope which binds to an antibody, or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding a natural protein (e.g., under moderate, high, or high stringency conditions).
  • the size of the nucleic acid molecule encoding such a protein can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration).
  • the minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT -rich.
  • nucleic acid molecule of the present invention can include a portion of a protein encoding sequence, a nucleic acid sequence encoding a full-length protein (including a gene), including any length fragment between about 20 nucleotides and the number of nucleotides that make up the full length cDNA encoding a protein, in whole integers (e.g., 20, 21, 22, 23, 24, 25 nucleotides), or multiple genes, or portions thereof.
  • the heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
  • the polynucleotide probes or primers of the invention are conjugated to detectable markers.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H,
  • the polynucleotide probes are immobilized on a substrate such as: artificial membranes, organic supports, biopolymer supports and inorganic supports.
  • One embodiment of the present invention relates to a recombinant nucleic acid molecule which comprises the isolated nucleic acid molecule described above which is operatively linked to at least one expression control sequence. More particularly, according to the present invention, a recombinant nucleic acid molecule typically comprises a recombinant vector and any one or more of the isolated nucleic acid molecules as described herein. According to the present invention, a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and/or for introducing such a nucleic acid sequence into a host cell.
  • the recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell.
  • a vector typically contains nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid sequences of the present invention or which are useful for expression of the nucleic acid molecules of the present invention (discussed in detail below).
  • the vector can be either R A or DNA, either prokaryotic or eukaryotic, and typically is a plasmid.
  • the vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant host cell, although it is preferred if the vector remains separate from the genome for most applications of the invention.
  • the entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention.
  • An integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome.
  • a recombinant vector of the present invention can contain at least one selectable marker.
  • a recombinant vector used in a recombinant nucleic acid molecule of the present invention is an expression vector.
  • expression vector is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest, such as an enzyme of the present invention).
  • a nucleic acid sequence encoding the product to be produced e.g., the protein or homologue or variant thereof is inserted into the recombinant vector to produce a recombinant nucleic acid molecule.
  • the nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell.
  • a recombinant nucleic acid molecule includes at least one nucleic acid molecule of the present invention operatively linked to one or more expression control sequences (e.g., transcription control sequences or translation control sequences).
  • expression control sequences e.g., transcription control sequences or translation control sequences.
  • the phrase "recombinant molecule” or “recombinant nucleic acid molecule” primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to a transcription control sequence, but can be used interchangeably with the phrase “nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein.
  • the phrase "operatively linked” refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conducted) into a host cell.
  • Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced. Transcription control sequences may also include any combination of one or more of any of the foregoing.
  • Recombinant nucleic acid molecules of the present invention can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell.
  • a recombinant molecule of the present invention including those which are integrated into the host cell chromosome, also contains secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein.
  • Suitable signal segments include a signal segment that is naturally associated with the protein to be expressed or any heterologous signal segment capable of directing the secretion of the protein according to the present invention.
  • a recombinant molecule of the present invention comprises a leader sequence to enable an expressed protein to be delivered to and inserted into the membrane of a host cell.
  • Suitable leader sequences include a leader sequence that is naturally associated with the protein, or any heterologous leader sequence capable of directing the delivery and insertion of the protein to the membrane of a cell.
  • the term "transfection” is generally used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell.
  • transformation can be used interchangeably with the term “transfection” when such term is used to refer to the introduction of nucleic acid molecules into microbial cells or plants and describes an inherited change due to the acquisition of exogenous nucleic acids by the microorganism that is essentially synonymous with the term “transfection.”
  • Transfection techniques include, but are not limited to, transformation, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.
  • One or more recombinant molecules of the present invention can be used to produce an encoded product (e.g., a protein) of the present invention.
  • an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein.
  • a preferred method to produce an encoded protein is by transfecting a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transfect include, but are not limited to, any bacterial, fungal (e.g., filamentous fungi or yeast or mushrooms), algal, plant, insect, or animal cell that can be transfected. Host cells can be either untransfected cells or cells that are already transfected with at least one other recombinant nucleic acid molecule.
  • Suitable cells may include any microorganism (e.g., a bacterium, a protist, an alga, a fungus, or other microbe), and is preferably a bacterium, a yeast or a filamentous fungus.
  • Suitable bacterial genera include, but are not limited to, Escherichia, Bacillus, Lactobacillus, Pseudomonas and Streptomyces .
  • Suitable bacterial species include, but are not limited to, Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus Stearothermophilus, Lactobacillus brevis, Pseudomonas aeruginosa and Streptomyces lividans.
  • Suitable genera of yeast include, but are not limited to, Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, and Phaffia.
  • Suitable yeast species include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, P. canadensis, Kluyveromyces marxianus and Phaffia rhodozyma.
  • Suitable fungal genera include, but are not limited to: Chrysosporium, Thielavia, , Talaromyces, Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus, Cryptococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and Trichoderma, and anamorphs and teleomorphs thereof.
  • Suitable fungal species include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Aspergillus japonicus, Absidia coerulea, Rhizopus oryzae, Chrysosporium lucknowense, Neurospora crassa, Neurospora intermedia, Trichoderma reesei, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Talaromyces emersonii and Talaromyces flavus.
  • the present invention includes proteins isolated from, or derived from the knowledge of enzymes from, a fungus such as Myceliophthora thermophila or a mutant or other derivative thereof, and more particularly, from the fungal strain denoted herein as CI (Accession No. VKM F-3500-D).
  • M. thermophila has previously appeared in patent applications and in the literature as Chrysosporium lucknowense or Sporotrichum thermophile.
  • the proteins of the invention possess enzymatic activity.
  • U.S. Patent No. 6,015,707 or U.S. Patent No. 6,573,086 a strain called CI (Accession No.
  • VKM F-3500-D was isolated from samples of forest alkaline soil from Sola Lake, Far East of the Russian Federation. This strain was deposited at the All-Russian Collection of Microorganisms of Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow, Russia, 113184, under the terms of the Budapest Treaty on the International Regulation of the Deposit of Microorganisms for the Purposes of Patent Procedure on August 29, 1996, as Chrysosporium lucknowense Garg 27K, VKM-F 3500 D. Various mutant strains of M. thermophila (C. lucknowense) CI have been produced and these strains have also been deposited at the All-USDn Collection of Microorganisms of Russian Academy of Sciences (VKM), Bakhurhina St.
  • VKM All-Russian Collection of Microorganisms of Russian Academy of Sciences
  • Strain CI was mutagenised by subjecting it to ultraviolet light to generate strain UV13-6 (Accession No. VKM F-3632 D). This strain was subsequently further mutated with N-methyl-N'-nitro-N-nitrosoguanidine to generate strain NG7C-19 (Accession No. VKM F-3633 D).
  • strain UV18-25 accesion No. VKM F-3631 D
  • strain W1L accesion No. CBS 122189
  • strain W1L#100L accesion No. CBS122190
  • Strain CI was previously classified as a Chrysosporium lucknowense based on morphological and growth characteristics of the microorganism, as discussed in detail in U.S. Patent No. 6,015,707, U.S. Patent No. 6,573,086 and patent PCT/NL2010/000045.
  • suitable host cells include insect cells (most particularly Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells), nematode cells (particularly C. elegans cells), avian cells, amphibian cells (particularly Xenopus laevis cells), reptilian cells, and mammalian cells (most particularly human, simian, canine, rodent, bovine, or sheep cells, e.g. NIH3T3, CHO (Chinese hamster ovary cell), COS, VERO, BHK, HEK, and other rodent or human cells).
  • insect cells most particularly Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells
  • nematode cells particularly C. elegans cells
  • avian cells particularly amphibian cells (particularly Xenopus laevis cells)
  • one or more protein(s) expressed by an isolated nucleic acid molecule of the present invention are produced by culturing a cell that expresses the protein (i.e., a recombinant cell or recombinant host cell) under conditions effective to produce the protein.
  • the protein may be recovered, and in others, the cell may be harvested in whole, either of which can be used in a composition.
  • Microorganisms used in the present invention are cultured in an appropriate fermentation medium.
  • An appropriate, or effective, fermentation medium refers to any medium in which a cell of the present invention, including a genetically modified microorganism (described below), when cultured, is capable of expressing enzymes useful in the present invention and/or of catalyzing the production of sugars from lignocellulosic biomass.
  • the microorganisms can be cultured by any fermentation process which includes, but is not limited to, batch, fed-batch, cell recycle, and continuous fermentation.
  • the fungal strains are grown in fermentors, optionally centrifuged or filtered to remove biomass, and optionally concentrated, formulated, and dried to produce an enzyme(s) or a multi-enzyme composition that is a crude fermentation product.
  • fermentors optionally centrifuged or filtered to remove biomass, and optionally concentrated, formulated, and dried to produce an enzyme(s) or a multi-enzyme composition that is a crude fermentation product.
  • Particularly suitable conditions for culturing filamentous fungi are described, for example, in U.S. Patent No. 6,015,707 and U.S. Patent No. 6,573,086, supra.
  • resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the culture medium; be secreted into a space between two cellular membranes; or be retained on the outer surface of a cell membrane.
  • the phrase "recovering the protein” refers to collecting the whole culture medium containing the protein and need not imply additional steps of separation or purification.
  • Proteins produced according to the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential precipitation or solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential precipitation or solubilization.
  • Proteins of the present invention are preferably retrieved, obtained, and/or used in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the protein in any method according to the present invention.
  • a protein to be useful in any of the methods described herein or in any method utilizing enzymes of the types described herein according to the present invention it is substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in a method disclosed by the present invention ⁇ e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein of the present invention (including homologues and variants) when it is used in a method disclosed by the present invention (described in detail below).
  • a "substantially pure" protein is a protein that can be produced by any method (i.e., by direct purification from a natural source, recombinantly, or synthetically), and that has been purified from other protein components such that the protein comprises at least about 80% weight/weight of the total protein in a given composition (e.g., the protein of interest is about 80% of the protein in a solution/composition/buffer), and more preferably, at least about 85%, and more preferably at least about 90%, and more preferably at least about 91%, and more preferably at least about 92%, and more preferably at least about 93%, and more preferably at least about 94%, and more preferably at least about 95%, and more preferably at least about 96%, and more preferably at least about 97%, and more preferably at least about 98%>, and more preferably at least about 99%, weight/weight of the total protein in a given composition.
  • the protein of interest is about 80% of the protein in a solution/composition/buffer
  • Recombinant techniques useful for controlling the expression of nucleic acid molecules include, but are not limited to, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites), modification of nucleic acid molecules to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, operators, enhancers
  • substitutions or modifications of translational control signals e.g., ribosome binding sites
  • a genetically modified microorganism that has been transfected with one or more nucleic acid molecules of the present invention.
  • a genetically modified microorganism can include a genetically modified bacterium, alga, yeast, filamentous fungus, or other microbe.
  • Such a genetically modified microorganism has a genome which is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (i.e., increased or modified activity and/or production of at least one enzyme or a multi-enzyme composition for the conversion of lignocellulosic material to fermentable sugars).
  • Genetic modification of a microorganism can be accomplished using classical strain development and/or molecular genetic techniques. Such techniques known in the art and are generally disclosed for microorganisms, for example, in Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press or Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as "Sambrook”).
  • a genetically modified microorganism can include a microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect within the microorganism.
  • a genetically modified microorganism can endogenously contain and express an enzyme or a multi-enzyme composition for the conversion of lignocellulosic material to fermentable sugars, and the genetic modification can be a genetic modification of one or more of such endogenous enzymes, whereby the modification has some effect on the ability of the microorganism to convert lignocellulosic material to fermentable sugars (e.g., increased expression of the protein by introduction of promoters or other expression control sequences, or modification of the coding region by homologous recombination to increase the activity of the encoded protein).
  • a genetically modified microorganism can endogenously contain and express an enzyme for the conversion of lignocellulosic material to fermentable sugars, and the genetic modification can be an introduction of at least one exogenous nucleic acid sequence (e.g., a recombinant nucleic acid molecule), wherein the exogenous nucleic acid sequence encodes at least one additional enzyme useful for the conversion of lignocellulosic material to fermentable sugars and/or a protein that improves the efficiency of the enzyme for the conversion of lignocellulosic material to fermentable sugars.
  • the microorganism can also have at least one modification to a gene or genes comprising its endogenous enzyme(s) for the conversion of lignocellulosic material to fermentable sugars.
  • the genetically modified microorganism does not necessarily endogenously (naturally) contain an enzyme for the conversion of lignocellulosic material to fermentable sugars, but is genetically modified to introduce at least one recombinant nucleic acid molecule encoding at least one enzyme or a multiplicity of enzymes for the conversion of lignocellulosic material to fermentable sugars.
  • a microorganism can be used in a method of the invention, or as a production microorganism for crude fermentation products, partially purified recombinant enzymes, and/or purified recombinant enzymes, any of which can then be used in a method of the present invention.
  • a cell extract that contains the activity to test can be generated. For example, a lysate from the host cell is produced, and the supernatant containing the activity is harvested and/or the activity can be isolated from the lysate. In the case of cells that secrete enzymes into the culture medium, the culture medium containing them can be harvested, and/or the activity can be purified from the culture medium.
  • the extracts/activities prepared in this way can be tested using assays known in the art. Accordingly, methods to identify mutli-enzyme compositions capable of degrading lignocellulosic biomass are provided.
  • DDG dinitro salicylic acid assay
  • the present invention is not limited to fungi and also contemplates genetically modified organisms such as algae, bacterial, and plants transformed with one or more nucleic acid molecules of the invention.
  • the plants may be used for production of the enzymes, and/or as the lignocellulosic material used as a substrate in the methods of the invention.
  • Methods to generate recombinant plants are known in the art. For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al, "Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E. Eds.
  • Some embodiments of the present invention include genetically modified organisms comprising at least one nucleic acid molecule encoding at least one enzyme of the present invention, in which the activity of the enzyme is downregulated by manipulating the efficiency with which those nucleic acid molecules are transcribed, translated, or post-translationally modified, or by "knocking out" the endogenous copy of the gene so that the expression of the gene is substantially reduced or eliminated.
  • the activity of the enzyme may be upregulated.
  • the present invention also contemplates downregulating activity of one or more enzymes while simultaneously upregulating activity of one or more enzymes to achieve the desired outcome.
  • Proteins of the present invention at least one protein of the present invention, compositions comprising such protein(s) of the present invention, and multi- enzyme compositions (examples of which are described above) may be used in any method where it is desirable to hydrolyze glycosidic linkages in lignocellulosic material, or any other method wherein enzymes of the same or similar function are useful.
  • the present invention includes the use of at least one protein of the present invention, compositions comprising at least one protein of the present invention, or multi-enzyme compositions in methods for hydrolyzing lignocellulose and the generation of fermentable sugars therefrom.
  • the method comprises contacting the lignocellulosic material with an effective amount of one or more proteins of the present invention, composition comprising at least one protein of the present invention, or a multi-enzyme composition, whereby at least one fermentable sugar is produced (liberated).
  • the lignocellulosic material may be partially or completely degraded to fermentable sugars. Economical levels of degradation at commercially viable costs are contemplated.
  • the amount of enzyme or enzyme composition contacted with the lignocellulose will depend upon the amount of glucan present in the lignocellulose.
  • the amount of enzyme or enzyme composition contacted with the lignocellulose may be from about 0.1 to about 200 mg enzyme or enzyme composition per gram of glucan; in other embodiments, from about 3 to about 20 mg enzyme or enzyme composition per gram of glucan.
  • the invention encompasses the use of any suitable or sufficient amount of enzyme or enzyme composition between about 0.1 mg and about 200 mg enzyme per gram glucan, in increments of 0.05 mg (i.e., 0.1 mg, 0.15 mg, 0.2 mg... 199.9 mg, 199.95 mg, 200 mg).
  • the invention provides a method for degrading DDG, preferably, but not limited to, DDG derived from corn, to sugars.
  • the method comprises contacting the DDG with a protein of the present invention, a composition comprising at least one protein of the present invention, or a multi- enzyme composition. In certain embodiments, at least 10% of fermentable sugars are liberated.
  • the at least 15% of the sugars are liberated, or at least 20%> of the sugars are liberated, or at least 23% of the sugars are liberated, or at least 24% of the sugars are liberated, or at least 25% of the sugars are liberated, or at least 26% of the sugars are liberated, or at least 27% of the sugars are liberated, or at least 28% of the sugars are liberated.
  • the invention provides a method for producing fermentable sugars comprising cultivating a genetically modified microorganism of the present invention in a nutrient medium comprising a lignocellulosic material, whereby fermentable sugars are produced.
  • Accessory enzymes have been described elsewhere herein.
  • the accessory enzyme or enzymes may be added at the same time, prior to, or following the addition of a protein of the present invention, a composition comprising at least one protein of the present invention, or a multi-enzyme composition, or can be expressed (endogenously or overexpressed) in a genetically modified microorganism used in a method of the invention.
  • the protein of the present invention, a composition comprising at least one protein of the present invention, or a multi- enzyme composition will be compatible with the accessory enzymes selected.
  • a composition comprising at least one protein of the present invention, or a multi-enzyme composition
  • the conditions such as temperature and pH
  • the accessory enzyme may also be present in the lignocellulosic material itself as a result of genetically modifying the plant.
  • the nutrient medium used in a fermentation can also comprise one or more accessory enzymes.
  • the method comprises a pretreatment process.
  • a pretreatment process will result in components of the lignocellulose being more accessible for downstream applications or so that it is more digestible by enzymes following treatment in the absence of hydrolysis.
  • the pretreatment can be a chemical, physical or biological pretreatment.
  • the lignocellulose may have been previously treated to release some or all of the sugars, as in the case of DDG.
  • Physical treatments such as grinding, boiling, freezing, milling, vacuum infiltration, and the like may also be used with the methods of the invention.
  • the heat treatment comprises heating the lignocellulosic material to 121°C for 15 minutes.
  • a physical treatment such as milling can allow a higher concentration of lignocellulose to be used in the methods of the invention.
  • a higher concentration refers to about 20%, up to about 25%, up to about 30%>, up to about 35%), up to about 40%>, up to about 45%, or up to about 50%> lignocellulose.
  • the lignocellulose may also be contacted with a metal ion, ultraviolet light, ozone, and the like.
  • Additional pretreatment processes are known to those skilled in the art, and can include, for example, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment, including ammonia fiber explosion (AFEX) technology. Details on pretreatment technologies and processes can be found in Wyman et al, Bioresource Tech.
  • the method comprises detoxifying the lignocellulosic material.
  • Dextoxification may be desirable in the event that inhibitors are present in the lignocellulosic material.
  • Such inhibitors can be generated by a pretreatment process, deriving from sugar degradation or are direct released from the lignocellulose polymer.
  • Detoxifying can include the reduction of their formation by adjusting sugar extraction conditions; the use of inhibitor-tolerant or inhibitor- degrading strains of microorganisms. Detoxifying can also be accomplished by the addition of ion exchange resins, active charcoal, enzymatic detoxification using, e.g., laccase, and the like. In some embodiments, the proteins, compositions or products of the present invention further comprises detoxifying agents.
  • the methods may be performed one or more times in whole or in part. That is, one may perform one or more pretreatments, followed by one or more reactions with a protein of the present invention, composition or product of the present invention and/or accessory enzyme.
  • the enzymes may be added in a single dose, or may be added in a series of small doses. Further, the entire process may be repeated one or more times as necessary. Therefore, one or more additional treatments with heat and enzymes are contemplated.
  • the fermentable sugars may be recovered and/or purified by any method known in the art.
  • the sugars can be subjected to further processing; e.g., they can also be sterilized, for example, by filtration.
  • the invention provides means for improving quality of lignocellulosic material, including DDG for animal nutrition.
  • the treated lignocellulosic material e.g., a lignocellulosic material which has been saccharified
  • the recovered material can be used as an animal feed additive. It is believed that the recovered material will have beneficial properties for animal nutrition, possibly due to a higher protein content.
  • the amount of enzyme or enzyme composition contacted with the lignocellulosic material may be from about 0.0001 % to about 1.0 % of the weight of the lignocellulosic material; in other embodiments, from about 0.005 % to about 0.1 % of the weight of the lignocellulosic material.
  • the invention includes the use of any amount of enzyme or enzyme composition between about 0.0001 % and about 1.0 %, in increments of 0.0001 (i.e., 0.0001, 0.0002, 0.0003...etc.).
  • the invention provides a method for producing an organic substance, comprising saccharifying a lignocellulosic material with an effective amount of a protein of the present invention or a composition comprising at least one protein of the present invention, fermenting the saccharified lignocellulosic material obtained with one or more fermentating microorganisms, and recovering the organic substance from the fermentation.
  • Sugars released from biomass can be converted to useful fermentation products including but not limited to amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol.
  • Specific products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol); lactic acid; plastics; specialty chemicals; organic acids, including citric acid, succinic acid, itaconic acid and maleic acid; solvents; animal feed supplements; pharmaceuticals; vitamins; amino acids, such as lysine, methionine, tryptophan, threonine, and aspartic acid; industrial enzymes, such as proteases, cellulases, amylases, glucanases, lactases, lipases, lyases, oxidoreductases, and transferases; and chemical feedstocks.
  • the methods of the invention are also useful to generate feedstocks for fermentation by fermenting microorganisms.
  • the method further comprises the addition of at least one fermenting organism.
  • fertilizing organism refers to an organism capable of fermentation, such as bacteria and fungi, including yeast. Such feedstocks have additional nutritive value above the nutritive value provided by the liberated sugars.
  • the present invention provides methods for improving the nutritional quality of food (or animal feed) comprising adding to the food (or the animal feed) at least one protein of the present invention.
  • the present invention provides methods for improving the nutritional quality of the food (or animal feed) comprising pretreating the food (or the animal feed) with at least one isolated protein of the present invention.
  • use of the enzymes xylanases and arabinofuranosidases in bread making has been known to improve the nutritional quality of the dough by degrading the arabinoxylans in the dough.
  • Improving the nutritional quality can mean making the food (or the animal feed) more digestible and/or less allergenic, and encompasses changes in the caloric value, taste and/or texture of the food.
  • the proteins of the present invention may be used as part of nutritional supplements.
  • the proteins of the present invention may be used as part of digestive aids, and may help in providing relief from digestive disorders such as acid reflux and celiac disease.
  • Proteins of the present invention and compositions comprising at least one protein of the present invention are also useful in a variety of other applications involving the hydrolysis of glycosidic linkages in lignocellulosic material, such as stone washing, color brightening, depilling and fabric softening, as well as other applications well known in the art. Proteins of the present invention and compositions comprising at least one protein of the present invention are also readily amenable to use as additives in detergent and other media used for such applications. These and other methods of use will readily suggest themselves to those of skill in the art based on the invention described herein.
  • proteins and compositions of the present invention can be used in stone washing procedures for fabrics or other textiles.
  • the proteins and compositions can be used in stone washing procedures for denim jeans. Examples of these uses can be found in U.S. Patent Application Publication No. 2003/0157595.
  • the cellulase compositions of this invention can be used to reduce or eliminate the harshness associated with a fabric or textile by contacting the fabric or textile with a protein or composition of the present invention.
  • the fabric or textile may be made from cellulose or cotton.
  • a preferred range for reducing or eliminating the harshness associated with a fabric or textile is between about pH 8 to about 12, or between about pH 10 to about 11.
  • the proteins or compositions of the subject invention can be used in detergent compositions.
  • the detergent composition may comprise at least one protein or composition of the present invention and one or more surfactants.
  • the detergent compositions may also include any additional detergent ingredient known in the art.
  • the detergent compositions of this invention preferably employ a surface active agent, i.e., surfactant, including anionic, non- ionic, and amp ho lytic surfactants well known for their use in detergent compositions.
  • a surface active agent i.e., surfactant, including anionic, non- ionic, and amp ho lytic surfactants well known for their use in detergent compositions.
  • the detergent compositions of this invention can additionally contain one or more componentsExamples of detergent compositions employing cellulases are exemplified in U.S. Pat. Nos. 4,435,307; 4,443,355; 4,661,289; 4,479,881; 5,120,463.
  • 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 method including a spray- drying method and/or a granulation method.
  • the granulation method are the most preferred because of the non-dusting nature of granules compared to spray dry products.
  • the detergent base is a liquid, it may be either a homogenous solution or an inhomogeneous solution.
  • compositions of the present invention include, but are not limited to, garment dyeing applications such as enzymatic mercerizing of viscose, bio-polishing applications, enzymatic surface polishing; bio wash (washing or washing down treatment of textile materials), enzymatic micro fibrillation, enzymatic "cottonization” of linen, ramie and hemp; and treatment of Lyocel® or Newcell® (i.e., "TENCEL®” from Courtauld's), Cupro® and other cellulosic fibers or garments, dye removal from dyed cellulosic substrates such as dyed cotton (Leisola & Linko ⁇ (1976) Analytical Biochemistry, v.
  • garment dyeing applications such as enzymatic mercerizing of viscose, bio-polishing applications, enzymatic surface polishing
  • bio wash washing or washing down treatment of textile materials
  • enzymatic micro fibrillation enzymatic "cottonization” of linen, ramie and hemp
  • the amount of enzyme or enzyme composition contacted with a textile may vary with the particular application. Typically, for biofinishing and denim washing applications, from about 0.02 wt. % to about 5 wt. % of an enzyme or enzyme composition may be contacted with the textile. In some embodiments, from about 0.5 wt. % to about 2 wt. % of an enzyme or enzyme composition may be contacted with the textile. For bioscouring, from about 0.1 to about 10, or from about 0.1 to about 1.0 grams of an enzyme or enzyme composition per kilogram of textile may be used, including any amount between about 0.1 grams and about 10 grams, in increments of 0.1 grams.
  • the proteins or compositions of the present invention can be used in the saccharification of lignocellulose biomass from agriculture, forest products, municipal solid waste, and other sources, for biobleaching of wood pulp, and for de-inking of recycled print paper all by methods known to one skilled in the art.
  • the amount of enzyme or enzyme composition used for pulp and paper modification typically varies depending upon the stock that is used, the pH and temperature of the system, and the retention time.
  • the amount of enzyme or enzyme composition contacted with the paper or pulp may be from about 0.01 to about 50 U; from about 0.1 to about 15 U; or from about 0.1 to about 5 U of enzyme or enzyme composition per dry gram of fiber, including any amount between about 0.01 and about 50 U, in 0.01 U increments.
  • the amount of enzyme or enzyme composition contacted with the paper or pulp may be from about 1 to about 2000 grams or from about 100 to about 500 grams enzyme or enzyme composition per dry ton of pulp, including any amount between about 1 and about 2000 grams, in 1 gram increments.
  • Proteins or compositions of the present invention can added to wastewater to reduce the amount of solids such as sludge or to increase total biochemical oxygen demand (BOD) and chemical oxygen demand (COD) removal.
  • BOD biochemical oxygen demand
  • COD chemical oxygen demand
  • proteins or compositions of the present invention can be used to transform particulate COD to soluble COD in wastewater produced from grain/fruit/cellulose industrial processes or to increase the BOD/COD ratio by increasing waste biodegradability (soluble lower molecular weight polymers in cellulosic/hemicellulosic wastes are typically more readily biodegradable than non- soluble material).
  • proteins or compositions of the present invention can also be used to increase waste digestion by aerobic and/or anaerobic bacteria.
  • Chitosanases of the present invention can hydro lyze the P-l,4-linkages between D- glucosamine residues in acetylated chitosan (i.e., deacetylated chitin) and thus may be used to degrade chitin- or chitosan-containing materials.
  • acetylated chitosan i.e., deacetylated chitin
  • Examples of chitin- containing materials include fungal cell walls, insect exoskeletons, the eggs of parasitic worms, and crustacean shells.
  • Chitosanases may be used to inhibit or reduce fungal growth, including the treatment of fungal infections. Chitosanases may also by used as lysing enzymes for the generation of protoplasts from fungi (see, e.g., Yano et al, Biosci Biotechnol Biochem. 70: 1754 (2006).
  • Chitosanases or compositions containing chitosanases may be used as a biological control agent such as an insecticide (see, e.g., Kramer et al, Insect Biochem Mol Biol. 27:887 (1997).
  • Chitin-degrading enzymes such as chitinases and chitosanases have been shown to be effective for controlling white-fly larvae in laboratory tests.
  • chitosanases may be applied to crops, plants and the like to control insect infestations.
  • This assay measures the release of /?-nitrophenol by the action of a- arabinofuranosidase on /?-nitrophenyl a-L-arabinofuranoside (PNPA).
  • Acetate buffer (0.1 M, pH 5.0) is prepared as follows: 8.2 g of anhydrous sodium acetate or 13.6 g of sodium acetate * 3H 2 0 is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • PNPA (Fluka, Switzerland, cat. # 73616) is used as the assay substrate. 8.25 mg of PNPA is dissolved in 5 mL of distilled water and 5 mL 0.1 M acetate buffer using a magnetic stirrer to obtain a 1 mM stock solution. The solution is stable for 2 days with storage at 4°C.
  • the stop reagent (0.25 M sodium carbonate solution) is prepared as follows: 26.5 g of anhydrous sodium carbonate is dissolved in 800 ml of distilled water, and the solution volume is adjusted to 1000 ml. This reagent is used to terminate the enzymatic reaction.
  • ⁇ 405 A s - A SB
  • DF the enzyme dilution factor
  • 21 the dilution of 10 ⁇ enzyme solution in 210 ⁇ reaction volume
  • 1.33 is the conversion factor of microtiter plates to cuvettes
  • 13.700 is the extinction coefficient 13700 M "1 cm "1 of
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH43, GH51, GH54, and GH62 enzymes.
  • arabinoxylans For the complete degradation of arabinoxylans to arabinose and xylose, several enzyme activities are needed, including endo-xylanases and arabinofuranosidases.
  • the arabinoxylan molecule from wheat is highly substituted with arabinosyl residues. These can be substituted either to the C 2 or the C 3 position of the xylosyl residue (single substitution), or both to the C 2 and C 3 position of the xylose (double substitution).
  • Single and double substituted oligosaccharides are prepared by incubating wheat arabinoxylan (WAX; 10 mg/rnL; Megazyme, Bray, Ireland) in 50 mM acetate buffer pH 5 with 0.3 mg Pentopan Mono (mono component endo-l,4- -xylanase, an enzyme from Thermomyces lanuginosus produced in Aspergillus oryzae; Sigma, St. Louis, USA) for 16 hours at 30°C. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 3100 x g. The supernatant is used for further experiments. Degradation of the arabinoxylan is followed by analysis of the formed reducing sugars and High Performance Anion Exchange Chromatography (HPAEC).
  • HPAEC High Performance Anion Exchange Chromatography
  • Double substituted arabinoxylan oligosaccharides are prepared by incubation of 800 ⁇ of the supernatant described above with 0.18 mg of the arabinofuranosidase Abfl (Abfl is arabinofuranosidase from M. thermophila with activity towards single arabinose substituted xylose residues and is disclosed in U.S. Application No. 11/833,133, filed August 2, 2007, the contents of which are incorporated herein by reference) in 50 mM acetate buffer pH 5 for 20 hours at 30°C. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10,000 x g, and the supernatant is used for further experiments. Degradation of the arabinoxylan is followed by analysis of the formed reducing sugars and HPAEC.
  • the enzyme (25 ⁇ g total protein) is incubated with single and double substituted arabinoxylan oligosaccharides (100 supernatant of Pentopan Mono treated WAX) in 50 mM acetate buffer at 30°C during 20 hours. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10,000 x g. Degradation of the arabinoxylan is followed by HPAEC analysis.
  • the enzyme (25 ⁇ g total protein) from B. adolescentis ( ⁇ , 0.02 U; Megazyme, Bray, Ireland) is incubated with double substituted arabinoxylan oligosaccharides (125 ⁇ supernatant of Pentopan Mono and Abfl treated WAX) in 50 mM acetate buffer at 35°C during 24 hours. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10,000 x g. Degradation of the arabinoxylan is followed by HPAEC analysis.
  • the amount of reducing sugars is measured with help of the DNS (3,5- dinitro salicylic acid) assay.
  • DNS 3,5- dinitro salicylic acid
  • 0.5 mL of DNS reagent (3,5-dinitrosalicylic acid and sodium potassium tartrate dissolved in dilute sodium hydroxide) is added to the sample (50 ⁇ ), containing 0 - 5 mg/ml reducing sugar.
  • the reaction mixture is heated at 100°C for 5 minutes and rapidly cooled in ice to room temperature.
  • the absorbance at 570 nm is measured. Glucose is used as a standard.
  • oligosaccharides are also present after xylanase treatment.
  • the single substituted oligosaccharides is removed from the xylanase-treated WAX mixture by the enzyme Abfl as described above.
  • the double substituted oligosaccharides are removed from the xylanase-treated WAX mixture by the enzyme AXHd3 as described above.
  • Samples containing only single substituted oligosaccharides or double substituted oligosaccharides are treated with the target enzyme or AXHd 3 from B. adolescentis as a reference enzyme as described above.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH43, GH51, GH54, and GH62 enzymes.
  • the cellulase standard solution is prepared, which contains 2 units of cellulase per ml of 0.2 M HAc/NaOH, pH 5. Subsequently, a standard series of 0 to 2 U/ml is prepared (12 samples).
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is conducted in micro titer plate format. Each well contains 50 ⁇ of xyloglucan substrate (0,25%(w/v) tamarind xyloglucan in water), 30 ⁇ of 0,2 M HAc/NaOH pH 5, 20 ⁇ xyloglucanase sample or cellulase standard sample. These are incubated at 37°C for 2 hours. After incubation 25 ⁇ of each well are mixed with 125 ⁇ working reagent. These solutions are heated at 95°C for 5 minutes. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A410) as As (enzyme sample). The standard curve is determined and from that the enzyme activities are determined.
  • Activity is calculated as follows: xyloglucanase activity is determined by reference to a standard curve of the cellulase standard solution.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC the slope of the standard curve
  • DF the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH12, GH16, GH44, and GH74 enzymes.
  • the following example illustrates the assay to measure the a-glucuronidase activity towards arabinoxylan oligosaccharides from Eucalyptus wood. This assay measures the release of glucuronic acid by the action of the ⁇ -glucuronidase on the arabinoxylan oligosaccharides.
  • Sodium acetate buffer (0.01 M, pH 5.0) is prepared as follows. 0.82 g of anhydrous sodium acetate or 1.36 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 0.6 g (0.572 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.01 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • Acetylated, 4-O-MeGlcA substituted xylo -oligosaccharides with 2-4 xylose residues or 4-10 xylose residues from Eucalyptus wood are prepared using the method described in Lucas et al, 2002. 1 mg of xylo-oligosaccharides is dissolved in 1 mL distilled water using magnetic stirrer. 4-O-MeGlcA is purified by using the method described in Lucas et al. 2002. Aldo-biuronic acid (XiG), aldo-triuronic acid (X 2 G), and aldo-tetrauronic acid (X3G) are obtained from Megazyme.
  • XiG aldo-triuronic acid
  • X3G aldo-tetrauronic acid
  • the analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (1 mm ID x 25 mm) and a Dionex EDetl PAD-detector (Dionex Co., Sunnyvale).
  • a flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-50 min, 0-500 mM.
  • Each elution is followed by a washing step of 5 min using 1 M sodium acetate in 0.1 M NaOH and an equilibration step of 15 min using 0.1 M NaOH.
  • CE-LIF Capillary Electrophoresis-Laser induced fluorescence detector
  • CE-LIF is performed using ProteomeLab PA800 Protein Characterization System (Beckman Coulter), controlled by 32 Karat Software.
  • the capillary column used is polyvinyl alcohol coated capillary (N-CHO capillary, Beckman Coulter), with 50 ⁇ ID, 50.2 cm length, 40 cm to detector window.
  • 25 mM sodium acetate buffer pH 4.75 containing 0.4% polyethyleneoxide (Carbohydrate separation buffer, Beckman Coulter) is used as running buffer.
  • the sample (about 3.5 nL) is injected to the capillary by a pressure of 0.5 psi for 3 seconds.
  • the separation is done for 20 minutes at 30 kV separating voltage, with reversed polarity.
  • the samples are stored at 10°C.
  • the labeled XOS are detected using LIF detector at 488 nm excitation and 520 nm emission wavelengths.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH67 and GH115 enzymes.
  • This assay measures the release of /?-nitrophenol by the action of ⁇ -xylosidase on /?-nitrophenyl ⁇ -D-xylopyranoside (PNPX).
  • ⁇ -xylosidase unit of activity is the amount of enzyme that liberates 1 micromole of /?-nitrophenol in one minute.
  • Sodium acetate buffer (0.1 M, pH 5.0) is prepared as follows. 8.2 g of anhydrous sodium acetate or 13.6 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 6.0 g (5.72 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.1 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • PNPX from Extrasynthese (France, cat. # 4244) is used as the assay substrate. 16.5 mg of PNPX is dissolved in 5 mL of distilled water and 5 mL 0.1 M sodium acetate buffer using magnetic stirrer to obtain 2 mM stock solution. The solution is stable for 2 days on storage at 4 °C.
  • the stop reagent (0.25 M sodium carbonate solution) is prepared as follows. 26.5 g of anhydrous sodium carbonate is dissolved in 800 mL of distilled water, and the solution volume is adjusted to 1000 mL. This reagent is used to terminate the enzymatic reaction.
  • ⁇ 405 A s (enzyme sample) - A SB (substrate blank)
  • DF the enzyme dilution factor
  • 21 the dilution of 10 ⁇ enzyme solution in 210 ⁇ reaction volume
  • 1.33 is the conversion factor of microtiter plates to cuvettes
  • 13.700 is the extinction coefficient 13700 M ⁇ 1 cm "1 of /?-nitrophenol released corrected for mol/L to ⁇ / ⁇
  • RT is the reaction time in minutes.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.
  • the following example illustrates the assay used to measure ⁇ -galactosidase activity.
  • This assay measures the action of ⁇ -galactosidase on 5-Bromo-4-chloro-3- indolyl ⁇ -D-galactoside (X-Gal) to yield galactose and 5-bromo-4-chloro-3- hydroxyindole.
  • 5-bromo-4-chloro-3-hydroxyindole is oxidized into 5,5'-dibromo- 4,4'-dichloro-indigo, which is an insoluble blue product.
  • Sodium acetate buffer (0.05 M, pH 5.0) is prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 5.0.
  • X-Gal from Fermentas (St. Leon Rot, Germany) is used as the assay substrate. 1.0 mg of X-Gal is dissolved in 10 mL 0.05 M sodium acetate buffer using magnetic stirrer.
  • ⁇ 590 A s (enzyme sample) - A SB (substrate blank) and DF is the enzyme dilution factor.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH2 and GH42 enzymes.
  • the following example illustrates the assay used to measure arabinofuranosidase/arabinase activity. This assay measures the release of arabinose by the action of the a-arabinofuranosidase on linear and branched arabinan.
  • Sodium acetate buffer (0.05 M, pH 5.0) is prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • Linear and branched arabinan is purchased from British Sugar.
  • the enzyme sample (40-55 ⁇ g total protein) is incubated with 5 mg/mL of linear or branched arabinan in 50 mM sodium acetate buffer at 40°C during 24 hours. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10,000 x g. Degradation of the arabinan is followed by HPAEC analysis.
  • Substrate Blank (40-55 ⁇ g total protein) is incubated with 5 mg/mL of linear or branched arabinan in 50 mM sodium acetate buffer at 40°C during 24 hours. The reaction is stopped by heating the samples at 100°C for 10 minutes. The samples are centrifuged for 5 minutes at 10,000 x g. Degradation of the arabinan is followed by HPAEC analysis.
  • the analysis of the samples is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (1 mm ID x 25 mm) and a Dionex EDetl PAD- detector (Dionex Co., Sunnyvale).
  • a flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.
  • Each elution is followed by a washing step of 5 min 1,000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH43, GH51, GH54, GH62, and GH93 enzymes.
  • This example illustrates the assay used to determine the ability of a protein to bind chitin.
  • a plastic column (6.8x150 mm) is filled with the mixture and it is washed with water overnight at 4°C.
  • the method is repeated with the unbound material and fresh chitin.
  • the unbound material is analyzed by SDS-gel electrophoresis.
  • the bound proteins, including the matrix are heated for 10 minutes at 95°C in sample buffer and separated by SDS-gel electrophoresis. Specific bands from this gel are analyzed by MS/MS.
  • This example illustrates the assay to determine the ability of a protein to bind
  • This example illustrates the assay to measure the endo-xylanase activity towards AZO-wheat arabinoxylan.
  • This substrate is insoluble in buffered solutions, but rapidly hydrates to form gel particles which are readily and rapidly hydrolysed by specific endo-xylanases releasing soluble dye-labeled fragments.
  • Sodium acetate buffer (0.2 M, pH 5.0) is prepared as follows. 16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 12.0 g (11.44 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.2 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • AZO-wheat arabinoxylan from Megazyme (Bray, Ireland, Cat. # I- AWAXP) is used as the assay substrate. 1 g of AZO-WAX is suspended in 3 mL ethanol and adjusted to 100 mL with 0.2 M sodium acetate buffer pH 5.0 using magnetic stirrer.
  • the assay is performed as detailed below.
  • 0.2 mL of 10 mg/ml AZO-WAX stock solution is preheated at 40° C for 10 minutes. This preheated stock solution is mixed with 0.2 mL of the enzyme sample
  • 0.2 mL of 10 mg/ml AZO-WAX stock solution is preheated at 40° C for 10 minutes. This preheated stock solution is mixed with 200 ⁇ of 0.2 M sodium acetate buffer pH 5.0 (preheat at 40° C for 10 min) and incubated at 40 °C for 10 minutes. After exactly 10 minutes of incubation, 1.0 mL of 96% ethanol is added and then the absorbance at 590 nm (A590) is measured as A SB (substrate blank).
  • Activity is calculated as follows: endo-xylanase activity is determined by reference to a standard curve, produced from an endo-xylanase with known activity towards AZO-WAX.
  • ⁇ 590 A s (enzyme sample) - A SB (substrate blank)
  • SC the slope of the standard curve
  • DF the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH8, GH10, and GH11.
  • This example illustrates the assay to measure xylanase activity.
  • This assay measures the release of xylose and xylo -oligosaccharides by the action of xylanases on wheat arabinoxylan oligosaccharides (WAX).
  • Sodium acetate buffer (0.05 M, pH 5.0) is prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 5.0. [0437] Wheat arabinoxylan is purchased from Megazyme (Bray Ireland, Cat. # P- WAXYI).
  • the analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (1 mm ID x 25 mm) and a Dionex EDetl PAD-detector (Dionex Co., Sunnyvale).
  • a flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-50 min, 0-500 mM.
  • Each elution is followed by a washing step of 5 min 1,000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH5, GH8, GH10, and GH11.
  • This example illustrates the assay to determine the ability of a protein to bind
  • the bound proteins including the matrix, are heated for 10 minutes at 95°C in sample buffer and separated by SDS-gel electrophoresis.
  • This assay measures the amount of reducing sugars released from polygalacturonic acid (PGA) by the action of a polygalacturonase.
  • One unit of activity is defined as 1 ⁇ of reducing sugars liberated per minute under the specified reaction conditions.
  • Sodium acetate buffer (0.2 M, pH 5.0) is prepared as follows. 16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 12.0 g (11.44 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.2 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • PGA Polygalacturonic acid
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is performed as detailed below.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC is the slope of the standard curve
  • DF is the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH28.
  • This assay measures the release of /?-nitrophenol by the action of ⁇ -galactosidase on /?-nitrophenyl-P-D-galactopyranoside (PNPGa).
  • PNPGa ⁇ -galactosidase unit of activity is the amount of enzyme that liberates 1 micromole of /?-nitrophenol in one minute.
  • Mcllvain buffer (pH 4.0) is prepared as follows. 21.01 g of citric acid monohydrate (CeHsC ⁇ ⁇ O) is dissolved in Millipore water so that the final volume of the solution to be 1000 mL (Solution A). In a separate flask, 53.62 g of Na 2 HP0 4 *7H 2 0 is dissolved in Millipore water to make the total volume of 1000 mL (Solution B). The final Mcllvain buffer, pH 4.0, is prepared by mixing 614.5 mL Solution A with 385.5 mL Solution B. The pH of the resulting solution is equal to 7.0
  • PNPGa from Fluka (Switzerland, cat. # 46021) is used as the assay substrate. 2.7 mg of PNPGa is dissolved in 10 mL of Mcllvain buffer using magnetic stirrer to obtain 1.5 mM stock solution. The solution is stable for 2 days on storage at 4 °C.
  • the stop reagent (0.25 M sodium carbonate solution) is prepared as follows. 26.5 g of anhydrous sodium carbonate is dissolved in 800 mL of distilled water, and the solution volume is adjusted to 1000 mL. This reagent is used to terminate the enzymatic reaction.
  • the assay is performed as detailed below.
  • ⁇ 410 A S (enzyme sample) - A SB (substrate blank)
  • DF the enzyme dilution factor
  • 20 the dilution of 50 ⁇ enzyme solution in 1000 ⁇ reaction volume
  • 1.33 is the conversion factor of microtiter plates to cuvettes
  • 13.700 is the extinction coefficient 13700 M "1 cm "1 of /?-nitrophenol released corrected for mol/L to ⁇ ⁇ / ⁇
  • RT is the reaction time in minutes.
  • This example illustrates the assay to measure ⁇ -glucosidase activity.
  • This assay measures the release of /?-nitrophenol by the action of ⁇ -glucosidase on p- nitrophenyl ⁇ -D-glucopyranoside (PNPG).
  • PNPG p- nitrophenyl ⁇ -D-glucopyranoside
  • One ⁇ -glucosidase unit of activity is the amount of enzyme that liberates 1 micromole of /?-nitrophenol in one minute.
  • Sodium acetate buffer (0.2 M, pH 5.0) is prepared as follows. 16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 12 g (11.44 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.1 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • PNPG from Sigma (St.Louis, USA) is used as the assay substrate. 20 mg of PNPG is dissolved in 5 mL of sodium acetate buffer using magnetic stirrer. The solution is stable for 2 days on storage at 4 °C.
  • the stop reagent (0.25 M Tris-HCl, pH 8.8) is prepared as follows. 30.29 g of Tris is dissolved in 900 mL of distilled water (Solution A). The final 0.25 M Tris-HCl pH 8.5 is prepared by mixing solution A with 37% HC1 until the pH of the resulting solution is equal to 8.8. The solution volume is adjusted to 1000 mL. This reagent is used to terminate the enzymatic reaction.
  • the assay is performed as detailed below.
  • PNPG stock solution 0.025 mL of PNPG stock solution is mixed with 1 of the enzyme sample, 0.075 mL buffer and 0.099 mL Millipore water and incubated at 37 °C for 4 minutes. Every minute during 4 minutes a 0.04 mL sample is taken and added to 0.06 mL stop reagent. The absorbance at 410 nm (A410) is measured in microtiter plates as A s (enzyme sample).
  • Substrate Blank 0.025 mL of PNPG stock solution is mixed with 0.075 mL buffer and 0.1 mL Millipore water and incubated at 37 °C for 4 minutes. Every minute during 4 minutes a 0.04 mL sample is taken and added to 0.06 mL stop reagent. The absorbance at 410 nm (A 410 ) is measured in microtiter plates as A SB (substrate blank sample).
  • a 410 values are plotted against time in minutes (X-axis). The slope of the graph is calculated (dA). Enzyme activity is calculated by using the following formula:
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH1, GH3, GH9, and GH30 enzymes.
  • This example illustrates the assay to measure ⁇ -xylosidase activity. This assay measures the release of xylose by the action of ⁇ -xylosidase on xylobiose.
  • Sodium acetate buffer (0.05 M, pH 5.0) is prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • Xylobiose is purchased from Megazyme (Bray Ireland, Cat. # P-WAXYI). 25 mg is dissolved in 5 mL sodium acetate buffer pH 5.0. [0497] The assay is performed as detailed below.
  • mL of 5.0 mg/mL substrate solution is mixed with 0.02 mL of the enzyme sample at 50 °C and pH 5.0 for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100°C. The release of xylose and arabinoxylan oligosaccharides are analyzed by High Performance Anion Exchange Chromatography.
  • mL of 5.0 mg/mL substrate solution is mixed with 0.02 mL of the buffer at 50 °C and pH 5.0 for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100°C. The release of xylose and arabinoxylan oligosaccharides are analyzed by High Performance Anion Exchange Chromatography.
  • the analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID x 250 mm) column in combination with a CarboPac PA guard column (1 mm ID x 25 mm) and a Dionex EDetl PAD-detector (Dionex Co., Sunnyvale).
  • a flow rate of 0.25 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-15 min, 0-150 mM.
  • Each elution is followed by a washing step of 5 min using 1 M sodium acetate in 0.1 M NaOH and an equilibration step of 15 min using 0.1 M NaOH.
  • This assay can be used to test the activity of enzymes such as, but not limited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is conducted in micro titer plate format. Each well contains 50 ⁇ of ⁇ - glucan substrate (1 % (w/v) Barley ⁇ -glucan, laminarin, lichenan or curdlan in water), 30 ⁇ of 0,2 M HAc/NaOH pH 5, 20 ⁇ P-l,3-glucanase sample. These are incubated at 37°C for 2 hours. After incubation 25 ⁇ of each well are mixed with 125 ⁇ working reagent. These solutions are heated at 95°C for 5 minutes. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A 410 ) as As (enzyme sample). The standard curve is determined and from that the enzyme activities are determined.
  • Activity is calculated as follows: P-l,3-glucanase activity is determined by reference to a standard curve of the cellulase standard solution.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC is the slope of the standard curve
  • DF is the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as the GH5, GH12,
  • the following examples illustrates the assay to measure a-1, 6-Mannanase activity. Such activity is demonstrated by using a-l,6-linked mannobiose as the substrate and a D-mannose detection kit (Megazyme International) as the detection method, using a four enzyme coupled assay, using ATP and NADP+.
  • D-mannose detection kit Megazyme International
  • Reactions are conducted at 37°C in 100 mM MOPS (pH 7.0), containing 0.1 mM ZnS04, 1 mg mL-1 BSA, and 20 ⁇ of a-1, 6-Mannanase sample.
  • Mannose liberated by a-1, 6-Mannanase is phosphorylated to mannose-6-phosphate by hexokinase (HK).
  • Mannose-6-phosphate is subsequently converted to fructose-6- phosphate by phosphomannose isomerase (PMI) which is then isomerized to glucose-6-phosphate by phosphoglucose isomerase (PGI) .
  • PMI phosphomannose isomerase
  • glucose-6- phosphate is oxidized to gluconate-6-phosphate by glucose-6-phosphate dehydrogenase (G6P-DH)
  • G6P-DH glucose-6-phosphate dehydrogenase
  • the concurrent reduction of the NADP+ cofactor to NADPH is monitored at 340 nm using an extinction coefficient of 6223 (M-l -cm-l).
  • the enzymes are individually obtained from Sigma.
  • dA slope in A/min
  • Va reaction volume in 1
  • d dilution factor of assay mix
  • ⁇ extinction coefficient for NAD(P)H of 0.006223 ⁇ "1 cm "1
  • 1 length of cell in cm
  • [protein] protein stock concentration in mg/ml
  • Vp volume of protein stock added to assay in ml.
  • This assay can be used to test the activity of enzymes such as, but not limited to,
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is conducted in micro titer plate format. Each well contains 50 ⁇ of rhamnogalacturonan substrate (1 %(w/v) in water), 30 ⁇ of 0,2 M HAc/NaOH pH 5, 20 ⁇ rhamnogalacturonyl hydrolase sample. These are incubated at 37°C for 2 hours. After incubation 25 ⁇ of each well are mixed with 125 ⁇ working reagent. These solutions are heated at 95 °C for 5 minutes. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A 410 ) as As (enzyme sample). The standard curve is determined and from that the enzyme activities are determined.
  • Activity is calculated as follows: rhamnogalacturonyl hydrolase activity is determined by reference to a standard curve of the cellulase standard solution.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC the slope of the standard curve
  • DF the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as, but not limited to,
  • the following examples illustrates the assay to measure a-Amylase activity. Such activity is demonstrated by using amylose as a substrate and a reducing sugars assay (PAHBAH) as the detection method.
  • PAHBAH reducing sugars assay
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is conducted in micro titer plate format. Each well contains 50 ⁇ of amylose substrate (0.15 % (w/v) in water), 30 ⁇ of 0,2 M HAc/NaOH pH 5, 20 ⁇ ⁇ -amylase sample. These are incubated at 37°C for 15 minutes. After incubation 25 ⁇ of each well are mixed with 125 ⁇ working reagent. These solutions are heated at 95 °C for 5 minutes. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A 410 ) as As (enzyme sample).
  • Activity is calculated as follows: a-amylase activity is determined by reference to a standard curve of the cellulase standard solution.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC is the slope of the standard curve
  • DF is the enzyme dilution factor.
  • This example illustrates the assay to measure a-glucosidase activity.
  • This assay measures the release of /?-nitrophenol by the action of ⁇ -glucosidase on p- nitrophenyl a-D-glucopyranoside.
  • One ⁇ -glucosidase unit of activity is the amount of enzyme that liberates 1 micromole of /?-nitrophenol in one minute.
  • Sodium acetate buffer (0.2 M, pH 5.0) is prepared as follows. 16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate * 3H 2 0 is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 12 g (11.44 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.1 M sodium acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • the stop reagent (0.25 M Tris-HCl, pH 8.8) is prepared as follows. 30.29 g of Tris is dissolved in 900 mL of distilled water (Solution A). The final 0.25 M Tris-HCl pH 8.5 is prepared by mixing solution A with 37% HC1 until the pH of the resulting solution is equal to 8.8. The solution volume is adjusted to 1000 mL. This reagent is used to terminate the enzymatic reaction.
  • the assay is performed as detailed below.
  • Substrate Blank 0.025 mL of /?-nitrophenyl a-D-glucopyranoside stock solution is mixed with 0.075 mL buffer and 0.1 mL Millipore water and incubated at 37 °C for 4 minutes. Every minute during 4 minutes a 0.04 mL sample is taken and added to 0.06 mL stop reagent. The absorbance at 410 nm (A 410 ) is measured in microtiter plates as A SB (substrate blank sample).
  • a 410 values are plotted against time in minutes (X-axis). The slope of the graph is calculated (dA). Enzyme activity is calculated by using the following formula:
  • ⁇ extinction coefficient of /?-nitrophenol (0.0137 ⁇ "1 cm “1 )
  • [0573] protein stock concentration in mg/ml
  • Vp volume of protein stock added to assay in ml
  • This assay can be used to test the activity of enzymes such as, but not limited to, the GH4, GH13, GH31 and GH63 enzymes.
  • This assay measures the release of /?-nitrophenol by the action of glucoamylase on p-nitrophenyl-P-maltoside (PNPM).
  • PNPM p-nitrophenyl-P-maltoside
  • Acetate buffer (0.1 M, pH 5.0) is prepared as follows: 8.2 g of anhydrous sodium acetate or 13.6 g of sodium acetate * 3H 2 0 is dissolved in distilled water so that the final volume of the solution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 5.0, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 5.0.
  • PNPM Sigma- Aldrich, cat. # N1884.
  • PNPM is dissolved in 5 mL of distilled water and 5 mL 0.1 M acetate buffer using a magnetic stirrer to obtain a 4 mM stock solution.
  • the stop reagent (0.1 M sodium tetraborate solution) is prepared as follows: 20.1 g of anhydrous sodium tetraborate is dissolved in 800 ml of distilled water, and the solution volume is adjusted to 1000 ml. This reagent is used to terminate the enzymatic reaction.
  • ⁇ 405 A S - A SB
  • DF the enzyme dilution factor
  • 21 the dilution of 10 ⁇ enzyme solution in 210 ⁇ reaction volume
  • 1.33 is the conversion factor of microtiter plates to cuvettes
  • 13.700 is the extinction coefficient 13700 M "1 cm "1 of /?-nitrophenol released corrected for mol/L to umol/mL
  • 360 minutes is the reaction time.
  • This assay can be used to test the activity of enzymes such as, but not limited to, the GH15.
  • enzymes such as, but not limited to, the GH15.
  • glucanase activity is demonstrated by using a glucan (e.g. dextran, glycogen, pullulan, amylose, amylopectin, cellulose, curdlan, laminarin, chrysolaminarin, lentinan, lichenin, pleuran, zymosan, etc.) as the substrate and a reducing sugars assay (PAHBAH) as the detection method.
  • glucan e.g. dextran, glycogen, pullulan, amylose, amylopectin, cellulose, curdlan, laminarin, chrysolaminarin, lentinan, lichenin, pleuran, zymosan, etc.
  • PAHBAH reducing sugars assay
  • Reagent A 10 g of / ⁇ -Hydroxy benzoic acid hydrazide (PAHBAH) is suspended in 60 mL water. 10 mL of concentrated hydrochloric acid is added and the volume is adjusted to 200 ml.
  • Reagent B 24.9 g of trisodium citrate is dissolved in 500 ml of water. To this solution 2.2 g of calcium chloride and 40 g sodium hydroxide are added. The volume is adjusted to 2 L with water. Both reagents are stored at room temperature.
  • Working Reagent 10 ml of Reagent A is added to 90 ml of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses.
  • the assay is conducted in micro titer plate format. Each well contains 50 ⁇ of glucan substrate (1 % (w/v) glucan in water), 30 ⁇ of 0,2 M HAc/NaOH pH 5, 20 ⁇ endo-glucanase sample. These are incubated at 37°C for 2 hours. After incubation 25 ⁇ of each well are mixed with 125 ⁇ working reagent. These solutions are heated at 95 °C for 5 minutes. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A 410 ) as As (enzyme sample). The standard curve is determined and from that the enzyme activities are determined.
  • glucan substrate (1 %(w/v) glucan in water) is mixed with 50 ⁇ , 0.2 M sodium acetate buffer pH 5.0 and incubated at 37 °C for 2 hours.
  • 50 ⁇ , 0.2 M sodium acetate buffer pH 5.0 is mixed with 50 ⁇ , 0.2 M sodium acetate buffer pH 5.0 and incubated at 37 °C for 2 hours.
  • 125 ⁇ ⁇ of working solution is added to 25 ⁇ ⁇ of this reaction mixture.
  • the samples are heated for 5 minutes at 95°C. After cooling down, the samples are analyzed by measuring the absorbance at 410 nm (A 410 ) as A SB (substrate blank sample).
  • Activity is calculated as follows: endo-glucanase activity is determined by reference to a standard curve of the cellulase standard solution.
  • ⁇ 410 A s (enzyme sample) - A SB (substrate blank)
  • SC the slope of the standard curve
  • DF the enzyme dilution factor
  • This assay can be used to test the activity of enzymes such as, but not limited to, the GH5, GH6, GH7, GH8, GH9, GH12, GH13, GH14, GH15, GH16, GH17,

Abstract

La présente invention concerne des enzymes inédites et leurs procédés, tout aussi inédits, de production. L'invention concerne, plus précisément, diverses enzymes fongiques, les molécules d'acides nucléiques codant pour lesdites enzymes, des compositions en contenant, des cellules hôtes recombinantes et génétiquement modifiées en produisant et leurs procédés d'utilisation. L'invention concerne également un procédé de conversion de biomasse lignocellulosique en sucres fermentescibles au moyen d'enzymes capables de dégrader les matériaux lignocellulosiques, ainsi que des combinaisons inédites d'enzymes, notamment des combinaisons d'enzymes assurant une libération synergique de sucres à partir de biomasse végétale. L'invention concerne également un procédé de libération du contenu cellulaire par dégradation des parois cellulaires. L'invention concerne, en outre, des procédés d'utilisation desdites enzymes inédites et des compositions en contenant pour améliorer la digestibilité des aliments pour animaux, ainsi que dans divers autres procédés, notamment la transformation des aliments et des boissons, la boulangerie, le lavage des vêtements, les processus faisant intervenir un détergent, le bioraffinage, le désencrage et le bioblanchiment du papier et de la pâte à papier ou, encore, le traitement des flux de déchets.
PCT/US2011/048817 2010-08-23 2011-08-23 Glucides-hydrolases fongiques inédites WO2012027374A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37609210P 2010-08-23 2010-08-23
US61/376,092 2010-08-23

Publications (2)

Publication Number Publication Date
WO2012027374A2 true WO2012027374A2 (fr) 2012-03-01
WO2012027374A3 WO2012027374A3 (fr) 2012-08-16

Family

ID=45724021

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/048817 WO2012027374A2 (fr) 2010-08-23 2011-08-23 Glucides-hydrolases fongiques inédites

Country Status (1)

Country Link
WO (1) WO2012027374A2 (fr)

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2606131A1 (fr) * 2010-08-20 2013-06-26 Codexis, Inc. Utilisation de protéines de la famille 61 des glycoside hydrolases dans le traitement de la cellulose
WO2013096369A1 (fr) 2011-12-19 2013-06-27 Novozymes A/S Procédés et compositions permettant d'augmenter la digestibilité de matières cellulosiques
EP2609195A2 (fr) * 2010-08-23 2013-07-03 Codexis, Inc. Enzymes recombinantes capables de dégrader la lignocellulose utilisables en vue de la production de sucres solubles à partir de biomasse cellulosique
WO2014005499A1 (fr) * 2012-07-02 2014-01-09 Novozymes A/S Polypeptides présentant une activité de xylanase et polynucléotides codant pour ceux-ci
WO2013182669A3 (fr) * 2012-06-08 2014-01-30 Dsm Ip Assets B.V. Nouvelles enzymes de myriococcum thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2013182670A3 (fr) * 2012-06-08 2014-03-20 Dsm Ip Assets B.V. Nouvelles enzymes de scytalidium thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2013188313A3 (fr) * 2012-06-11 2014-04-10 Codexis, Inc. Xylanases et xylosidases fongiques
EP2772545A1 (fr) * 2013-03-01 2014-09-03 Nederlandse Organisatie voor toegepast -natuurwetenschappelijk onderzoek TNO Protéines d'amidon actives
WO2015081139A1 (fr) 2013-11-26 2015-06-04 Novozymes A/S Compositions enzymatiques et leurs utilisations
KR101535526B1 (ko) * 2012-06-26 2015-07-09 한국생명공학연구원 야생형보다 분비능이 증가된 gh61 융합 단백질
EP2780450A4 (fr) * 2011-11-15 2015-09-09 Novozymes Inc Polypeptides présentant une activité de cellobiohydrolase et polynucléotides codant pour ceux-ci
WO2015187935A1 (fr) 2014-06-06 2015-12-10 Novozymes A/S Compositions enzymatiques et leurs utilisations
EP2861739A4 (fr) * 2012-06-08 2016-08-03 Univ Concordia Nouvelles enzymes de déconstruction de la paroi cellulaire de scytalidium thermophilum, myriococcum thermophilum et aureobasidium pullulans, et leurs utilisations
WO2016145350A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique à étages multiple d'une biomasse lignocellulosique
WO2016145363A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique multi-étape d'une biomasse lignocellulosique faisant appel à une oxydoréductase et à un polypeptide aa9
WO2016145358A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique avec des enzymes hémicellulolytiques
WO2016205127A1 (fr) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides ayant une activité tréhalase et leur utilisation dans un procédé de production de produits de fermentation
CN108004153A (zh) * 2017-12-07 2018-05-08 青岛蔚蓝生物集团有限公司 一株高产果胶裂解酶的里氏木霉菌株及其应用
CN108265092A (zh) * 2017-12-26 2018-07-10 天津大学 一种具有优良抗氧化活性的香菇寡糖及制备方法
WO2019005755A1 (fr) 2017-06-28 2019-01-03 Novozymes A/S Polypeptides présentant une activité tréhalase et polynucléotides codant pour ceux-ci
WO2019030165A1 (fr) 2017-08-08 2019-02-14 Novozymes A/S Polypeptides ayant une activité tréhalase et leur utilisation dans un procédé de production de produits de fermentation
EP3467171A1 (fr) * 2017-10-06 2019-04-10 Lenzing Aktiengesellschaft Denim à filaments lyocellulaires
CN110072996A (zh) * 2016-12-21 2019-07-30 诺维信公司 具有溶菌酶活性的多肽、编码其的多核苷酸及其用途和组合物
WO2019231944A2 (fr) 2018-05-31 2019-12-05 Novozymes A/S Procédés d'amélioration de la croissance et de la productivité de levures
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
JP2020513771A (ja) * 2017-01-04 2020-05-21 ノボザイムス アクティーゼルスカブ 過敏性腸症候群または炎症性腸疾患の処置での使用のための微生物リゾチーム
CN112272671A (zh) * 2018-06-01 2021-01-26 诺维信公司 多肽
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
WO2024003143A1 (fr) * 2022-06-30 2024-01-04 Novozymes A/S Mutanases et compositions d'hygiène buccale les comprenant
US11896033B2 (en) 2016-12-21 2024-02-13 Novozymes A/S Polypeptides having lysozyme activity, polynucleotides encoding same and uses and compositions thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060185036A1 (en) * 2000-10-20 2006-08-17 Sticklen Masomeh B Transgenic plants containing ligninase and cellulase which degrade lignin and cellulose to fermentable sugars
WO2008151079A2 (fr) * 2007-05-31 2008-12-11 Novozymes, Inc. Compositions pour dégrader de la matière cellulosique
US20100003234A1 (en) * 2005-03-15 2010-01-07 Verenium Corporation Cellulases, Nucleic Acids Encoding Them and Methods for Making and Using Them

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060185036A1 (en) * 2000-10-20 2006-08-17 Sticklen Masomeh B Transgenic plants containing ligninase and cellulase which degrade lignin and cellulose to fermentable sugars
US20100003234A1 (en) * 2005-03-15 2010-01-07 Verenium Corporation Cellulases, Nucleic Acids Encoding Them and Methods for Making and Using Them
WO2008151079A2 (fr) * 2007-05-31 2008-12-11 Novozymes, Inc. Compositions pour dégrader de la matière cellulosique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CLAUDIA GROPOSO ET AL.: 'Contribution to the lignocellulolytic fungi (Basidiomycetes) of the Atlantic Rain Forest in Southern Brazil.' MYCOTAXON. vol. 92, 2005, pages 103 - 113 *
DATABASE GENBANK 09 April 2008 Database accession no. XM_001228634.1 *
DATABASE GENBANK 09 April 2008 Database accession no. XP_001228635.1 *

Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2606131A4 (fr) * 2010-08-20 2014-07-23 Codexis Inc Utilisation de protéines de la famille 61 des glycoside hydrolases dans le traitement de la cellulose
EP2606131A1 (fr) * 2010-08-20 2013-06-26 Codexis, Inc. Utilisation de protéines de la famille 61 des glycoside hydrolases dans le traitement de la cellulose
US9493802B2 (en) 2010-08-20 2016-11-15 Codexis, Inc. Use of glycohydrolase 61 protein variants with improved thermostability for processing cellulose
EP2609195A2 (fr) * 2010-08-23 2013-07-03 Codexis, Inc. Enzymes recombinantes capables de dégrader la lignocellulose utilisables en vue de la production de sucres solubles à partir de biomasse cellulosique
EP2609195A4 (fr) * 2010-08-23 2014-03-05 Codexis Inc Enzymes recombinantes capables de dégrader la lignocellulose utilisables en vue de la production de sucres solubles à partir de biomasse cellulosique
EP2780450A4 (fr) * 2011-11-15 2015-09-09 Novozymes Inc Polypeptides présentant une activité de cellobiohydrolase et polynucléotides codant pour ceux-ci
WO2013096369A1 (fr) 2011-12-19 2013-06-27 Novozymes A/S Procédés et compositions permettant d'augmenter la digestibilité de matières cellulosiques
EP2861739A4 (fr) * 2012-06-08 2016-08-03 Univ Concordia Nouvelles enzymes de déconstruction de la paroi cellulaire de scytalidium thermophilum, myriococcum thermophilum et aureobasidium pullulans, et leurs utilisations
WO2013182670A3 (fr) * 2012-06-08 2014-03-20 Dsm Ip Assets B.V. Nouvelles enzymes de scytalidium thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
WO2013182669A3 (fr) * 2012-06-08 2014-01-30 Dsm Ip Assets B.V. Nouvelles enzymes de myriococcum thermophilum de déconstruction de la paroi cellulaire et leurs utilisations
US9476077B2 (en) 2012-06-11 2016-10-25 Codexis, Inc. Fungal beta-xylosidase variants
WO2013188313A3 (fr) * 2012-06-11 2014-04-10 Codexis, Inc. Xylanases et xylosidases fongiques
US8956844B2 (en) 2012-06-11 2015-02-17 Codexis, Inc. Fungal xylanases and xylosidases
US8980578B2 (en) 2012-06-11 2015-03-17 Codexis, Inc. Fungal beta-xylosidase variants
WO2013188305A3 (fr) * 2012-06-11 2014-04-24 Codexis, Inc. Variants de la beta-xylosidase fongique
EP2859096A4 (fr) * 2012-06-11 2016-05-11 Codexis Inc Xylanases et xylosidases fongiques
KR101535526B1 (ko) * 2012-06-26 2015-07-09 한국생명공학연구원 야생형보다 분비능이 증가된 gh61 융합 단백질
WO2014005499A1 (fr) * 2012-07-02 2014-01-09 Novozymes A/S Polypeptides présentant une activité de xylanase et polynucléotides codant pour ceux-ci
EP2772545A1 (fr) * 2013-03-01 2014-09-03 Nederlandse Organisatie voor toegepast -natuurwetenschappelijk onderzoek TNO Protéines d'amidon actives
WO2015081139A1 (fr) 2013-11-26 2015-06-04 Novozymes A/S Compositions enzymatiques et leurs utilisations
WO2015187935A1 (fr) 2014-06-06 2015-12-10 Novozymes A/S Compositions enzymatiques et leurs utilisations
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
WO2016145358A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique avec des enzymes hémicellulolytiques
WO2016145363A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique multi-étape d'une biomasse lignocellulosique faisant appel à une oxydoréductase et à un polypeptide aa9
WO2016145350A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique à étages multiple d'une biomasse lignocellulosique
WO2016205127A1 (fr) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides ayant une activité tréhalase et leur utilisation dans un procédé de production de produits de fermentation
CN108350442A (zh) * 2015-06-18 2018-07-31 诺维信公司 具有海藻糖酶活性的多肽及其在产生发酵产物的方法中的用途
US11685910B2 (en) 2015-06-18 2023-06-27 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
US10676727B2 (en) 2015-06-18 2020-06-09 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
US11896033B2 (en) 2016-12-21 2024-02-13 Novozymes A/S Polypeptides having lysozyme activity, polynucleotides encoding same and uses and compositions thereof
US11896034B2 (en) 2016-12-21 2024-02-13 Novozymes A/S Polypeptides having lysozyme activity, polynucleotides encoding same and uses and compositions thereof
US10986850B2 (en) 2016-12-21 2021-04-27 Novozymes A/S Polypeptides having lysozyme activity, polynucleotides encoding same and uses and compositions thereof
CN110072996A (zh) * 2016-12-21 2019-07-30 诺维信公司 具有溶菌酶活性的多肽、编码其的多核苷酸及其用途和组合物
EP3559224A4 (fr) * 2016-12-21 2021-01-20 Novozymes A/S Polypeptides ayant une activité lysozyme, polynucléotides codant pour ceux-ci et utilisations et compositions de ceux-ci
JP2020513771A (ja) * 2017-01-04 2020-05-21 ノボザイムス アクティーゼルスカブ 過敏性腸症候群または炎症性腸疾患の処置での使用のための微生物リゾチーム
US11584783B2 (en) 2017-06-28 2023-02-21 Novozymes A/S Processes for producing a fermentation product
WO2019005755A1 (fr) 2017-06-28 2019-01-03 Novozymes A/S Polypeptides présentant une activité tréhalase et polynucléotides codant pour ceux-ci
EP4015524A1 (fr) 2017-06-28 2022-06-22 Novozymes A/S Polypeptides présentant une activité tréhalase et polynucléotides codant pour ceux-ci
US11220679B2 (en) 2017-08-08 2022-01-11 Novozymes A/S Polypeptides having trehalase activity
WO2019030165A1 (fr) 2017-08-08 2019-02-14 Novozymes A/S Polypeptides ayant une activité tréhalase et leur utilisation dans un procédé de production de produits de fermentation
WO2019068476A1 (fr) * 2017-10-06 2019-04-11 Lenzing Aktiengesellschaft Denim à filaments lyocellulaires
RU2753288C1 (ru) * 2017-10-06 2021-08-12 Ленцинг Актиенгеселльшафт Деним из лиоцелловых волокон
EP3467171A1 (fr) * 2017-10-06 2019-04-10 Lenzing Aktiengesellschaft Denim à filaments lyocellulaires
CN108004153A (zh) * 2017-12-07 2018-05-08 青岛蔚蓝生物集团有限公司 一株高产果胶裂解酶的里氏木霉菌株及其应用
CN108265092A (zh) * 2017-12-26 2018-07-10 天津大学 一种具有优良抗氧化活性的香菇寡糖及制备方法
WO2019231944A2 (fr) 2018-05-31 2019-12-05 Novozymes A/S Procédés d'amélioration de la croissance et de la productivité de levures
EP3802565A4 (fr) * 2018-06-01 2022-09-14 Novozymes A/S Polypeptides
CN112272671A (zh) * 2018-06-01 2021-01-26 诺维信公司 多肽
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
WO2024003143A1 (fr) * 2022-06-30 2024-01-04 Novozymes A/S Mutanases et compositions d'hygiène buccale les comprenant

Also Published As

Publication number Publication date
WO2012027374A3 (fr) 2012-08-16

Similar Documents

Publication Publication Date Title
EP2197893B1 (fr) Enzymes fongiques inédites
WO2012027374A2 (fr) Glucides-hydrolases fongiques inédites
US7923236B2 (en) Fungal enzymes
CA2657684C (fr) Construction de compositions de cellulase hautement efficaces pour une hydrolyse enzymatique de la cellulose
US9133448B2 (en) Polypeptide having cellobiohydrolase activity and uses thereof
EP3318574B1 (fr) Polypeptide doté d'une activité bêta-glucosidase et utilisations associées
EP2588603B1 (fr) Polypeptide améliorant la cellulase et utilisations correspondantes
US20130280764A1 (en) Method of improving the activity of cellulase enzyme mixtures in the saccharification (ligno)cellulosic material
WO2012018691A2 (fr) Nouvelles enzymes fongiques
US8790894B2 (en) Mutant cellobiohydrolase
EA023558B1 (ru) ТЕРМОСТАБИЛЬНЫЙ ПОЛИПЕПТИД С АКТИВНОСТЬЮ β-ЦЕЛЛОБИОГИДРОЛАЗЫ И ЕГО ПРИМЕНЕНИЕ ДЛЯ ПРОИЗВОДСТВА САХАРОВ ИЗ ЛИГНОЦЕЛЛЮЛОЗНОГО МАТЕРИАЛА
WO2012021883A2 (fr) Nouvelles enzymes fongiques
US20160168581A1 (en) Enzymes and uses thereof
WO2012078741A2 (fr) Nouvelles estérases fongiques
MX2012014990A (es) Polipeptido que tiene actividad suolenina y sus usos.
WO2013176205A1 (fr) Xylanase et procédé de production de sucre à l'aide de celle-ci
JP2013243954A (ja) キシラナーゼおよびそれを用いた糖の製造方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11820526

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11820526

Country of ref document: EP

Kind code of ref document: A2