Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/248—Xylanases
  • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
  • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
  • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
• • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • 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)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
• • 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
• • 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
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
  • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
  • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

• the present invention relates to polypeptides having xylanase activity, and polynucleotides encoding the polypeptides.
• the invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
• the invention further relates to a process of using the polypeptides having xylanase activity in a process for improving the nutritional quality of distillers dried grains (DGS) or distillers dried grains with solubles (DDGS) produced as a co product of a fermentation product production process, a process for producing fermentation products, as well as the use of enzyme blends comprising the polypeptides having xylanase activity in the processes.
• DGS distillers dried grains
• DDGS distillers dried grains with solubles
• Processes for producing fermentation products, such as ethanol, from a starch or lignocellulose containing material are well known in the art.
• the preparation of the starch containing material such as corn for utilization in such fermentation processes typically begins with grinding the corn in a dry-grind or wet-milling process.
• Wet-milling processes involve fractionating the corn into different components where only the starch fraction enters the fermentation process.
• Dry-grind processes involve grinding the corn kernels into meal and mixing the meal with water and enzymes. Generally, two different kinds of dry-grind processes are used.
• the most commonly used process includes grinding the starch-containing material and then liquefying gelatinized starch at a high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) carried out in the presence of a glucoamylase and a fermentation organism.
• SSF simultaneous saccharification and fermentation
• Another well-known process often referred to as a “raw starch hydrolysis” process (RSH process) includes grinding the starch-containing material and then simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.
• the liquid fermentation products are recovered from the fermented mash (often referred to as “beer mash”), e.g., by distillation, which separates the desired fermentation product, e.g. ethanol, from other liquids and/or solids.
• the remaining fraction is referred to as“whole stillage”.
• Whole stillage typically contains about 10 to 20% solids.
• the whole stillage is separated into a solid and a liquid fraction, e.g., by centrifugation.
• the separated solid fraction is referred to as“wet cake” (or“wet grains”) and the separated liquid fraction is referred to as“thin stillage”.
• Wet cake and thin stillage contain about 35 and 7% solids, respectively.
• Wet cake, with optional additional dewatering is used as a component in animal feed or is dried to provide“Distillers Dried Grains” (DDG) used as a component in animal feed.
• DDG “Distillers Dried Grains”
• Thin stillage is typically evaporated to provide evaporator condensate and syrup or may alternatively be recycled to the slurry tank as“backset”. Evaporator condensate may either be forwarded to a methanator before being discharged and/or may be recycled to the slurry tank as“cook water”.
• the syrup may be blended into DDG or added to the wet cake before or during the drying process, which can comprise one or more dryers in sequence, to produce DDGS (Distillers Dried Grain with Solubles).
• Syrup typically contains about 25% to 35% solids. Oil can also be extracted from the thin stillage and/or syrup as a by-product for use in biodiesel production, as a feed or food additive or product, or other biorenewable products.
• Distiller’s grain with solubles (DGS) and distiller’s dried grain with solubles (DDGS) are co-products of the grain to ethanol industry, which are used for animal feed.
• DGS and DDGS are rich in fiber, and therefore the highest feasible inclusion rate for monogastric animals, such as e.g. poultry and swine, is lower than for ruminants such as e.g. cattle.
• Glycohydrolase enzymes such as e.g. endoxylanase, are added to feed blends to increase the digestibility of fiber rich feed blends.
• enzymes added to feed blends e.g. homogeneous mixing of the enzymes into the feed blend, heat stability of the enzyme protein during pelletization of the feed, stability of the enzyme protein during the low pH gastric passage, and relatively short residence time in the guts of some animal species.
• the present invention overcomes the above challenges by adding a presently disclosed polypeptide having xylanase activity or an enzyme blend comprising the polypeptide having xylanase activity and/or cellulolytic composition upstream during the fermentation product production process, for example during the simultaneous saccharification and fermentation (SSF) step, where there is continuous mixing of a free flowing slurry, the temperature is stable (e.g., between 30 to 35°C), the pH is stable (e.g., between about pH4 and pH5), and the residence time is typically in the range of 54 to 80 hours.
• SSF simultaneous saccharification and fermentation
• the present invention more particularly relates to the addition of a presently disclosed polypeptide having xylanase activity or enzyme blend comprising the polypeptide having xylanase activity during the SSF process to produce a DDGS product, or DGS product, with higher digestibility for animals (e.g., monogastric animals).
• a presently disclosed polypeptide having xylanase activity or enzyme blend comprising the polypeptide having xylanase activity during the SSF process to produce a DDGS product, or DGS product, with higher digestibility for animals (e.g., monogastric animals).
• the fiber e.g., corn
• solubilized fiber may be fermented by the gut microbiome to metabolizable products such as fatty acids.
• the solubilized fiber has a positive effect on gut health by acting as a substrate for beneficial gut flora.
• GH98 xylanases are known to produce larger oligosaccharides compared to GH30 xylanases (Rogowski et al. 2015). It is believed that the larger oligosaccharide profile may have a positive effect on gut health, feed value, and the color of DDGS (e.g., due to decreased formation of carmelization and Maillard products during drying).
• polypeptide having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 5 or 7;
• polypeptide encoded by a polynucleotide that hybridizes under very-high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 2, 6 or 8, or (ii) the full-length complement of (i) or (ii);
• polypeptide encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2, 6 or 8;
• the present invention also relates to polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells comprising the polynucleotides; and methods of producing the polypeptides.
• the present invention relates to a GH98 xylanase or enzyme blend comprising a GH98 xylanase.
• the enzyme blend further comprises a cellulolytic composition.
• the cellulolytic composition is present in the blend the ratio of the xylanase and cellulolytic composition is from about 5:95 to about 95:5.
• the ratio of the xylanase and the cellulolytic composition in the blend is about 10:90. In an embodiment, the ratio of the xylanase and the cellulolytic composition in the blend is about 20:80. In an embodiment, the ratio of the xylanase and the cellulolytic composition in the blend is about 50:50.
• the enzyme blend comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase.
• the enzyme blend comprises at least 5%, at least 10% xylanase, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% cellulolytic composition.
• the xylanase is from the genus Microbacterium or Paenibacillus. In an embodiment, the xylanase is a GH98 xylanase selected from the group consisting of:
• the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an
• Aspergillus fumigatus cellobiohydrolase I Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the cellulolytic composition comprises: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 16;
• the cellulolytic composition comprises: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; and (ii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 16 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 863 of SEQ ID NO: 15
• the cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain selected from the group consisting of Aspergillus, Penicilium, Talaromyces, and Trichoderma, optionally wherein: (i) the Aspergillus strain is selected from the group consisting of Aspergillus aurantiacus, Aspergillus niger and Aspergillus oryzae ; (ii) the Penicilium strain is selected from the group consisting of Penicilium emersonii and Penicilium oxalicum ; (iii) the
• the Talaromyces strain is selected from the group consisting of Talaromyces aurantiacus and Talaromyces emersonii ; and (iv) the Trichoderma strain is Trichoderma reesei.
• the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition.
• the present invention relates to a process of producing a fermentation product, comprising the following steps: (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature with an alpha-amylase, a glucoamylase, and a GH98 xylanase or an enzyme blend or composition comprising the GH98 xylanase; and (b) fermenting using a fermentation organism.
• the present invention relates to a process for producing a fermentation product from starch-containing material comprising the steps of: (a) liquefying a starch-containing material with an alpha-amylase; (b) saccharifying the liquefied material obtained in step (a) with a glucoamylase and a GH98 xylanase of the present invention or an enzyme blend or composition comprising the GH98 xylanase; and (c) fermenting using a fermenting organism.
• saccharification and fermentation is performed simultaneously.
• the starch-containing material comprises maize, corn, wheat, rye, barley, triticale, sorghum, switchgrass, millet, pearl millet, foxtail millet.
• the fermentation product is alcohol, particularly ethanol.
• the fermenting organism is yeast, particularly Saccharomyces sp., more particularly Saccharomyces cerevisiae.
• the present invention relates to a process for improving the nutritional quality of distillers dried grains (DGS) or distillers dried grains with solubles (DDGS) produced as a co-product of a fermentation product production process, the process comprising performing a process for producing a fermentation product of the present invention, and recovering the fermentation product to produce DGS or DDGS as a co product, wherein the DGS or DDGS produced have improved nutritional quality.
• DGS distillers dried grains
• DDGS distillers dried grains with solubles
• the true metabolizable energy of the DGS or DDGS is increased by at least 5%, at least 10%, at least 15%, or at least 20%, as compared to the TME of DGS or DDGS produced when a GH98 xylanase of the present invention or an enzyme blend or composition comprising the GH98 xylanase is not present during the saccharification step, fermentation step, and/or simultaneous saccharification and fermentation step of a process for producing a fermentation product of the present invention.
• the animal is a monogastric animal.
• the DGS or DDGS produced are not darkened after drying as compared to DGS or DDGS produced when a GH98 xylanase of the present invention or an enzyme blend or composition comprising the GH98 xylanase is not present during the saccharification step, fermentation step, and/or simultaneous saccharification and fermentation step of a process of the present invention.
• the present invention relates to the use of a GH98 xylanase of the present invention or an enzyme blend or composition comprising the GH98 xylanase for improving the nutritional quality of DGS or DDGS produced as a co-product of a fermentation product production process, preferably without resulting in a darkening the DDG or DDGS.
• the present invention relates to the use of a GH98 xylanase of the present invention or an enzyme blend or composition comprising the GH98 xylanase for solubilizing fiber, preferably for solubilizing xylose and arabinose.
• FIG. 1 shows the average DP4+ yield (g/L) for each of the GH98 enzyme blends as compared to the control cellulolytic composition not supplemented with xylanase.
• FIG. 2 shows the average glucose yield (g/L) for each of the GH98 enzyme blends as compared to control cellulolytic composition not supplemented with xylanase.
• SEQ ID NO: 1 is the amino acid sequence of a GH98 xylanase from Microbacterium oxydans.
• SEQ ID NO: 2 is the polynucleotide sequence encoding the GH98 xylanase of SEQ ID NO: 1.
• SEQ ID NO: 3 is the amino acid sequence of a GH98 xylanase from Microbacterium hydrocarbonoxydans.
• SEQ ID NO: 4 is the amino acid sequence of a GH98 xylanase from Microbacterium sp. SA39.
• SEQ ID NO: 5 is the amino acid sequence of a GH98 xylanase from Paenibacillus glycanilyticus.
• SEQ ID NO: 6 is the polynucleotide sequence encoding the GH98 xylanase of SEQ
• SEQ ID NO: 7 is the amino acid sequence of the mature GH98 xylanase from Paenibacillus terrigena.
• SEQ ID NO: 8 is the polynucleotide sequence encoding the GH98 xylanase of SEQ ID NO: 7.
• SEQ ID NO: 9 is the amino acid sequence of a GH98 xylanase from Paenibacillus terrigena.
• SEQ ID NO: 10 is the amino acid sequence of the mature GH98 xylanase from Paenibacillus sp. DMB20.
• SEQ ID NO: 11 is the amino acid sequence of the glucoamylase from Talaromyces emersonii.
• SEQ ID NO: 12 is the amino acid sequence of the glucoamylase from Gloeophyllum sepiarium.
• SEQ ID NO: 13 is the amino acid sequence of the glucoamylase from Gloeophyllum trabeum.
• SEQ ID NO: 14 is the amino acid sequence of the Rhizomucor pusillus alpha- amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) having the following substitutions G128D+D143N.
• SEQ ID NO: 15 is the amino acid sequence of the full-length cellobiohydrolase I from Aspergillus fumigatus.
• SEQ ID NO: 16 is the amino acid sequence of the full-length cellobiohydrolase II from Aspergillus fumigatus.
• SEQ ID NO: 17 is the amino acid sequence of the full-length beta-glucosidase from Aspergillus fumigatus.
• SEQ ID NO: 18 is the amino acid sequence of the full-length GH61 polypeptide from Penicillium emersonii.
• SEQ ID NO: 19 is the amino acid sequence of the full-length alpha-amylase from Bacillus stearothermophilus.
• SEQ ID NO: 20 is the amino acid sequence of the full-length GH 10 xylanase from Dictyogllomus thermophilum.
• SEQ ID NO: 21 is the amino acid sequence of the full-length GH11 xylanase from Dictyogllomus thermophilum.
• SEQ ID NO: 22 is the amino acid sequence of the full-length GH10 xylanase from Rasomsonia byssochlamydoides.
• SEQ ID NO: 23 is the amino acid sequence of the full-length GH10 xylanase from Talaromyces leycettanus.
• SEQ ID NO: 24 is the amino acid sequence of the full-length GH10 xylanase from Aspergillus fumigatus.
• SEQ ID NO: 25 is the amino acid sequence of the full-length endoglucanase from Talaromyces leycettanus.
• SEQ ID NO: 26 is the amino acid sequence of the full-length endoglucanase from Penicillium capsulatum.
• SEQ ID NO: 27 is the amino acid sequence of the full-length endoglucanase from Trichophaea saccata.
• SEQ ID NO: 28 is the amino acid sequence of the full-length GH45 endoglucanase from Sordaria fimicola.
• SEQ ID NO: 29 is the amino acid sequence of the full-length GH45 endoglucanase from Thielavia terrestris.
• SEQ ID NO: 30 is the amino acid sequence of the full-length glucoamylase from Penicillium oxalicum.
• SEQ ID NO: 31 is the amino acid sequence of the full-length protease from
• SEQ ID NO: 32 is the amino acid sequence of the full-length protease from
• Thermoascus aurantiacus.
• SEQ ID NO: 33 is the codon-optimized polynucleotide sequence of the GH98 xylanase from Microbacterium oxydans.
• SEQ ID NO: 34 is the amino acid sequence of the Bacillus clausii secretion signal.
• SEQ ID NO: 35 is the amino acid sequence of the polyhistidine tag.
• allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
• An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
• Alpha-Amylases (alpha-1 , 4-glucan-4-glucanohydrolases, EC 3.2.1.1) are a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched
• Animal refers to all animals except humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, cattle, e.g., beef cattle, cows, and young calves, deer, yank, camel, llama and kangaroo.
• Non-ruminant animals include mono-gastric animals, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), young calves; fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike,
• Animal feed refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal.
• Animal feed for a mono-gastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).
• Beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose.
• beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var.
• beta-glucosidase is defined as 1.0 pmole of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01% TWEEN® 20 (polyoxyethylene sorbitan monolaurate).
• Body Weight Gain means an increase in live weight of an animal during a given period of time, e.g., the increase in weight from day 1 to day 21.
• cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
• the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
• Cellobiohydrolase means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei
• Cellobiohydrolase activity is determined according to the procedures described by Lever et ai, 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et ai, 1982, FEBS Letters,
• the Tomme et al. method can be used to determine cellobiohydrolase activity.
• Cellulolytic enzyme, cellulolytic composition, or cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
• the two basic enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
• approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases,
• Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
• the most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose,
• Cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in Pretreated Corn Stover (“PCS”) (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, compared to a control hydrolysis without addition of cellulolytic enzyme protein.
• PCS Pretreated Corn Stover
• Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSCU, 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
• Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a variant.
• the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA.
• the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
• control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the present invention.
• Each control sequence may be native (/.e., from the same gene) or foreign (/.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other.
• control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
• the control sequences include a promoter, and transcriptional and translational stop signals.
• the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
• Endoglucanase means an endo-1 ,4-(1 ,3;1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
• Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention,
• CMC carboxymethyl cellulose
• expression includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
• Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.
• Family 61 glycoside hydrolase The term“Family 61 glycoside hydrolase” or “Family GH61” or“GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J.
• the enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1,4-beta-D-glucanase activity in one family member.
• the structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
• Feed Conversion Ratio The term“feed conversion ratio” the amount of feed fed to an animal to increase the weight of the animal by a specified amount.
• An improved feed conversion ratio means a lower feed conversion ratio.
• lower feed conversion ratio or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition.
• Feed efficiency means the amount of weight gain per unit of feed when the animal is fed ad-libitum or a specified amount of food during a period of time.
• increase feed efficiency it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present.
• fragment means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature
• a fragment contains at least 85%, e.g., at least 90% or at least 95% of the amino acid residues of the mature polypeptide of an enzyme.
• Glucoamylases are a group of enzymes, which catalyze the hydrolysis of terminal (1 4)-linked a-D-glucose residues successively from non-reducing ends of the chains with release of beta-D-glucose.
• Hemicellulolytic enzyme or hemicellulase means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases, Current Opinion In Microbiology 6(3): 219-228. Hemicellulases are key components in the degradation of plant biomass.
• hemicellulases include, but are not limited to, an acetylmannan esterase, an acetyxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
• the substrates of these enzymes are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation.
• the catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups.
• GHs glycoside hydrolases
• CEs carbohydrate esterases
• catalytic modules based on homology of their primary sequence, can be assigned into GH and CE families marked by numbers. Some families, with overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A).
• An informative and updated classification of these and other carbohydrate active enzymes is available on the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chern. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C.
• Host cell means any cell type that is susceptible to
• host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
• Isolated means a substance in a form or environment which does not occur in nature.
• isolated substances include (1) any non- naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
• An isolated substance may be present in a fermentation broth sample.
• Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
• the mature polypeptide of a Microbacterium oxydans xylanase is amino acids 33 to 884 of SEQ ID NO: 1 and amino acids 1 to 32 of SEQ ID NO: 1 are a signal peptide. In one aspect, the mature polypeptide of a Micro bacterium
• hydrocarbonoxydans xylanase is amino acids 35 to 890 of SEQ ID NO: 3 and amino acids 1 to 34 of SEQ ID NO: 3 are a signal peptide.
• the mature polypeptide of a Microbacterium sp. SA39 xylanase is amino acids 35 to 890 of SEQ ID NO: 4 and amino acids 1 to 34 of SEQ ID NO: 4 are a signal peptide.
• the mature polypeptide of a Paenibacillus glycanilyticus xylanase is amino acids 31 to 826 of SEQ ID NO: 5 and amino acids 1 to 30 of SEQ ID NO: 5 are a signal peptide.
• the mature polypeptide of a Paenibacillus terrigena xylanase is amino acids 35 to 831 of SEQ ID NO: 7 and amino acids 1 to 34 of SEQ ID NO: 7 are a signal peptide.
• the mature polypeptide of a Paenibacillus terrigena xylanase is amino acids 35 to 831 of SEQ ID NO: 9 and amino acids 1 to 34 of SEQ ID NO: 9 are a signal peptide.
• the mature polypeptide of a Paenibacillus sp. DMB20 xylanase is amino acids 1 to 794 of SEQ ID NO: 10.
• the mature polypeptide of an A is amino acids 35 to 831 of SEQ ID NO: 7 and amino acids 1 to 34 of SEQ ID NO: 7 are a signal peptide.
• the mature polypeptide of a Paenibacillus terrigena xylanase is amino acids 35 to 831 of SEQ ID NO:
• fumigatus cellobiohydrolase I is amino acids 27 to 532 of SEQ ID NO: 15 and amino acids 1 to 26 of SEQ ID NO: 15 are a signal peptide.
• the mature polypeptide of an A. fumigates cellobiohydrolase II is amino acids 20 to 454 of SEQ ID NO: 16 and amino acids 1 to 19 of SEQ ID NO: 16 are a signal peptide.
• the mature polypeptide of an A. fumigatus beta-glucosidase is amino acids 20 to 863 of SEQ ID NO: 17 and amino acids 1 to 19 of SEQ ID NO: 17 are a signal peptide.
• the mature polypeptide of a Penicillium sp. GH61 polypeptide is amino acids 26 to 253 of SEQ ID NO: 18 and amino acids 1 to 25 of SEQ ID NO: 18 are a signal peptide.
• a host cell may produce a mixture of two of more different mature polypeptides (/.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
• Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide.
• nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
• Nutrient Digestibility means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro intestinal tract, e.g., the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g., the ileum.
• Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro-intestinal tract or a segment of the gastro-intestinal tract.
• Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash.
• Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed.
• Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.
• Energy digestibility means the gross energy of the feed consumed minus the gross energy of the faeces or the gross energy of the feed consumed minus the gross energy of the remaining digest a on a specified segment of the gastro-intestinal tract of the animal, e.g., the ileum.
• Metabolizable energy refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the faeces or the digest a present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed.
• operbly linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
• Percentage solubilized xylan means the amount of xylose measured in the supernatant after incubation with an enzyme compared to the total amount of xylose present in the substrate before the incubation with the enzyme.
• the percentage solubilized xylan may be calculated using defatted destarched maize (DFDSM) as substrate.
• DFDSM is prepared according to ‘Preparation of Defatted Destarched Maize (DFDSM)’ in the experimental section.
• the percentage solubilized xylan from defatted destarched maize may be determined using the reaction conditions 20 pg enzyme / g DFDSM and incubation at 40°C, pH 5 for 2.5 hours as described in the‘Xylose solubilization assay’ herein.
• the term‘is performed under the reaction conditions 20 pg xylanase variant per gram defatted destarched maize (DFDSM) and incubation at 40°C, pH 5 for 2.5 hours’ is to be understood that the percentage solubilised xylan is calculated as described in the‘Xylose solubilization assay’ herein.
• 2% (w/w) DFDSM suspension was prepared in 100 mM sodium acetate, 5 mM CaC , pH 5 and allowed to hydrate for 30 min at room
• enzyme/substrate mixtures were left for hydrolysis in 2.5 h at 40°C under gently agitation (500 RPM) in a plate incubator. After enzymatic hydrolysis, the enzyme/substrate plates were centrifuged for 10 min at 3000 RPM and 50 pi supernatant was mixed with 100 pi 1.6 M HCI and transferred to 300 pi PCR tubes and left for acid hydrolysis for 40 min at 90°C in a PCR machine. Samples were neutralized with 125 pi 1.4 M NaOH after acid hydrolysis and loaded on the HPAE-PAD for mono-saccharide analysis.
• Polypeptide having cellulolytic enhancing activity means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity.
• cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS).
• suitable temperature e.g., 50°C, 55°C, or 60°C
• pH e.g., 5.0 or 5.5
• a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvasrd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.
• the GH61 polypeptide having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20- fold.
• Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter“sequence identity”.
• sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et ai,
• the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
• the output of Needle labeled“longest identity” is used as the percent identity and is calculated as follows:
• deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), e.g., version 5.0.0 or later.
• the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
• the output of Needle labeled“longest identity” (obtained using the - nobrief option) is used as the percent identity and is calculated as follows:
• variant means a polypeptide having enzyme or enzyme enhancing activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
• a substitution means replacement of the amino acid occupying a position with a different amino acid;
• a deletion means removal of the amino acid occupying a position; and
• an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
• Wild-type xylanase The term“wild-type” xylanase means a xylanase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
• xylanase means an endo-1 ,4-beta-xylanase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans.
• xylanase activity can be determined with 0.2 percent AZCL-arabinoxylan as substrate in 0.01 percent TRITON(R) X-100 and 200 mM sodium phosphate buffer pH 6 at 37 degrees centigrade or 0.2 percent AZCL-xylan as substrate in 0.01 percent TRITON(R) X-100 and 20 mM sodium acetate buffer pH 5.0 at 50 degrees centigrade (see Example 17).
• xylanase activity is defined as 1.0 micro mole of azurine produced per minute at 37 degrees centigrade, pH 6 from 0.2 percent AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 or at 50 degrees centigrade, pH 5 from 0.2 percent AZCL-xylan as substrate in 20 mM sodium acetate pH 5.
• xylanase activity can be determined according to the assay described in the Materials & Methods section.
• any of SEQ ID NOs: 1 , 3-5, 7, or 9-10 can be used to determine the corresponding amino acid residue in another xylanase.
• the amino acid sequence of another xylanase is aligned with any of SEQ ID NOs: 1 , 3-5, 7, or 9-10, and based on the alignment, the amino acid position number corresponding to any amino acid residue in SEQ ID NOs: 1 , 3-5, 7, or 9-10 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al.
• the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
• Identification of the corresponding amino acid residue in another xylanase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log- expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1794), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059- 3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et ai, 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.
• MUSCLE multiple sequence
• Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
• algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
• substitutions For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as“Thr226Ala” or“T226A”. Multiple mutations are separated by addition marks (“+”), e.g.,“Gly205Arg + Ser411 Phe” or“G205R + S411 F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively. Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *.
• deletion of glycine at position 195 is designated as “Gly195*” or“G195*”.
• Multiple deletions are separated by addition marks (“+”), e.g.,“Gly195* + Ser411*” or“G195* + S411*”.
• Insertions For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated“Gly195Glyl_ys” or“G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as“Gly195Glyl_ysAla” or“G195GKA”.
• the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s).
• the sequence would thus be:
• variants comprising multiple alterations are separated by a plus sign (“+”), e.g.,“Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
• + e.g.,“Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
• the present invention relates to novel GH98 xylanases, enzyme blends or compositions comprising the novel GH98 xylanases, and the use thereof in a process for improving the nutritional quality of distillers dried grains (DDG) or distillers dried grains with solubles (DDGS) produced as a co-product of a fermentation product production process, a process for producing fermentation products, and for solubilizing fiber, preferably for solubilizing xylose and arabinose.
• DDG distillers dried grains
• DDGS distillers dried grains with solubles
• DDGS is typically fed to cattle because the high fiber content limits the nutritional value for monogastric animals (e.g., poultry and swine).
• monogastric animals e.g., poultry and swine.
• DDGS monogastric animals
• One way to solubilize fiber is by adding enzymes to the feed blend, however, the shorter residence time and less than ideal conditions in vivo limits the efficacy of enzymes added to feed.
• the work described herein demonstrates that the addition of a presently disclosed GH98 xylanase or an enzyme blend comprising the GH98 xylanase upstream during the fermentation product production process (e.g., during simultaneous saccharification and fermentation) significantly increases the degree of fiber solubilization.
• the presently disclosed GH98 xylanases and enzyme blends or compositions comprising the GH98 xylanase significantly increase the degree of fiber solubilization without resulting in the darkening of DDGS during the drying process.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 1 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 1.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 1.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 3 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 3.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 3.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 3 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 4.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 4.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 1 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 3 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 3 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO: 3.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• polynucleotides of SEQ ID NO: 2, or subsequences thereof, as well as the polypeptides of SEQ ID NO: 2 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having protease activity from strains of different genera or species according to methods well known in the art.
• probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
• Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
• the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
• Both DNA and RNA probes can be used.
• the probes are typically labelled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
• a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity.
• Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
• DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
• the carrier material is used in a Southern blot.
• hybridization indicates that the
• polynucleotide hybridizes to a labelled nucleic acid probe corresponding to (i) SEQ ID NO: 2; (ii) the mature polypeptide coding sequence of SEQ ID NO: 2; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions.
• Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
• the nucleic acid probe is nucleotides 1 to 2655 of SEQ ID NO: 2. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 1 ; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 2. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 3; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 4; the mature polypeptide thereof; or a fragment thereof.
• the present invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2 of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
• the polypeptide has been isolated.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 1 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 3 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ I D NO: 4 is up to 10, e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 5 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 5.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 5.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 5 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• polynucleotides of SEQ ID NO: 6, or subsequences thereof, as well as the polypeptides of SEQ ID NO: 5 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having protease activity from strains of different genera or species according to methods well known in the art.
• probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
• Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
• the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
• Both DNA and RNA probes can be used.
• the probes are typically labelled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
• a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity.
• Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
• DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
• the carrier material is used in a Southern blot.
• hybridization indicates that the
• polynucleotide hybridizes to a labelled nucleic acid probe corresponding to (i) SEQ ID NO: 6; (ii) the mature polypeptide coding sequence of SEQ ID NO: 6; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions.
• Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
• the nucleic acid probe is nucleotides 1 to 2481 of SEQ ID NO: 6. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 5; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 6.
• the present invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 6 of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
• the polypeptide has been isolated.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 5 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 5 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 5 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 5 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 7 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 7.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 7.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 9 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 9.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 9.
• the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 10.
• the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 10.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 7 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO: 7.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 9 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO: 7.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 9 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO: 7.
• the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the xylanase activity of the mature polypeptide of SEQ ID NO:
• the polynucleotides of SEQ ID NO: 8, or subsequences thereof, as well as the polypeptides of SEQ ID NO: 8 or a fragments thereof may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having protease activity from strains of different genera or species according to methods well known in the art.
• probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
• Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
• the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
• Both DNA and RNA probes can be used.
• the probes are typically labelled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
• a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity.
• Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
• DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
• the carrier material is used in a Southern blot.
• hybridization indicates that the
• polynucleotide hybridizes to a labelled nucleic acid probe corresponding to (i) SEQ ID NO: 8; (ii) the mature polypeptide coding sequence of SEQ ID NO: 2; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions.
• Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
• the nucleic acid probe is nucleotides 1 to 2496 of SEQ ID NO: 8. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 7; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 8. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 9; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 10; the mature polypeptide thereof; or a fragment thereof.
• the present invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 8 of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
• the polypeptide has been isolated.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 7 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 7 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 9 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 9 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• the present invention relates to variants of the mature polypeptide of SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
• the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 10 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20- 25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
• conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
• Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
• Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
• the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance,
• Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152- 2156; WO 95/17413; or WO 95/22625.
• Other methods that can be used include error-prone PCR, phage display ⁇ e.g., Lowman et ai., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et ai., 1986, Gene 46: 145; Ner et ai., 1988, DNA 7: 127).
• Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et ai., 1999, Nature Biotechnology 17: 893-896).
• Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
• the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
• the polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
• a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
• Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
• Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post- translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et ai., 1994, Science 266: 776-779).
• a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et ai, 2003, J. Ind. Microbiol. Bioiechnol. 3: 568-576; Svetina et ai, 2000, J.
• the signal peptide of the polypeptide having xylanase activity is replaced with another signal peptide to enhance expression of the polypeptide.
• the signal peptide is from the genus Bacillus, for instance the Bacillus clausii signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 1 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 3 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 4 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 5 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 7 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 9 is replaced with the signal peptide of SEQ ID NO: 34.
• the signal peptide of the polypeptide of SEQ ID NO: 10 is replaced with the signal peptide of SEQ ID NO: 34.
• the polypeptide having xylanase activity comprises a tag, for instance, to facilitate purification of the polypeptide.
• Purification tags are well known in the art.
• the polypeptide can include a poly-histidine tag.
• the polypeptide having xylanase activity has an poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 1 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 3 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 4 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 5 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 7 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 9 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• the mature polypeptide of SEQ ID NO: 10 or variant thereof has a poly-histidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35.
• a polypeptide having xylanase activity of the present invention may be obtained from microorganisms of the genus Microbacterium.
• the polypeptide having xylanase activity is a Microbacterium oxydans polypeptide, for instance, the Microbacterium oxydans xylanase of SEQ ID NO: 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto.
• the variant is the Microbacterium hydrocarbonoxydans polypeptide of SEQ ID NO: 3.
• the variant is the Microbacterium sp. SA39
• a polypeptide having xylanase activity of the present invention may be obtained from microorganisms of the genus Paenibacillus.
• the polypeptide having xylanase activity is a Paenibacillus glycanilyticus polypeptide, for instance, the Paenibacillus glycanilyticus of SEQ ID NO: 5 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto.
• the polypeptide having xylanase activity is a Paenibacillus terrigena polypeptide, for instance, the Paenibacillus terrigena polypeptide of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto.
• the variant is the Paenibacillus terrigena polypeptide of SEQ ID NO: 9.
• the variant is the Paenibacillus sp. DMB20 polypeptide of SEQ ID NO: 10.
• ATCC American Type Culture Collection
• DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• CBS Centraalbureau Voor Schimmelcultures
• NRRL Northern Regional Research Center
• the polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
• the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
• the present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein.
• the polynucleotide encoding the polypeptide the present invention has been isolated.
• the techniques used to isolate or clone a polynucleotide include isolation from genomic DNA or cDNA, or a combination thereof.
• the cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et ai, 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
• the polynucleotides may also be cloned from a strain of Microbacterium, particularly from Microbacterium oxydans, Microbacterium hydrocarbonoxydans, Microbacterium sp. SA39, or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
• the polynucleotides may be cloned from a strain of Paenibacillus, such as from Paenibacillus glycanilyticus, Paenibacillus terrigena,
• Paenibacillus sp. DMB20 or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
• the present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
• at least one control sequence is heterologous to the polynucleotide encoding a variant of the present invention.
• the nucleic acid construct would not be found in nature.
• the polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
• the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
• the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
• the promoter may be any polynucleotide that shows transcriptional activity in the host cell including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
• suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene ( amyQ ), Bacillus licheniformis alpha- amylase gene ( amyL ), Bacillus licheniformis penicillinase gene ( penP ), Bacillus
• amyM Bacillus subtilis levansucrase gene
• sacB Bacillus subtilis xylA and xylB genes
• Bacillus thuringiensis crylllA gene Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107)
• E. coli lac operon E. coli trc promoter
• streptomyces coelicolor agarase gene dagA
• prokaryotic beta-lactamase gene Villa-Kamaroff et ai, 1978, Proc. Natl. Acad.
• tandem promoters are disclosed in WO 99/43835.
• the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
• the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
• Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ( aprH ), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA ( rrnB ).
• the control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
• mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et ai,
• the control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell.
• the leader is operably linked to the
• the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell’s secretory pathway.
• the 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
• the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
• a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
• a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide.
• any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
• Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ( nprT , nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
• the signal peptide comprises or consists of the Bacillus clausii signal peptide of SEQ ID NO: 34.
• the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
• the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
• a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
• the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease ( aprE ), Bacillus subtilis neutral protease ( nprT ), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
• the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
• the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
• the polynucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
• at least one control sequence is heterologous to the polynucleotide of the present invention.
• the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
• the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
• the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
• the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
• the vector may be a linear or closed circular plasmid.
• the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
• the vector may contain any means for assuring self-replication.
• the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
• a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
• the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
• a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
• Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin,
• chloramphenicol kanamycin, neomycin, spectinomycin, or tetracycline resistance.
• the selectable marker may be a dual selectable marker system as described in WO 2010/039889.
• the dual selectable marker is an hph-tk dua ⁇ selectable marker system.
• the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
• the vector may rely on the
• polynucleotide s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
• the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
• the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
• the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
• the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
• the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
• the term “origin of replication” or“plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
• bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and rAMb1 permitting replication in Bacillus.
• More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide.
• An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
• the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.
• the one or more control sequences are heterologous to the polynucleotide of the present invention.
• a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
• the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
• the host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
• the prokaryotic host cell may be any Gram-positive.
• Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
• Gram negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and
• the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
• the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823- 829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278).
• protoplast transformation see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115
• competent cell transformation see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81
• the introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower ei al., 1988, Nucleic Acids Res. 16: 6127- 6145).
• the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong ei al., 2004, Folia Microbiol. (Praha) 49: 399- 405), conjugation (see, e.g., Mazodier ei al., 1989, J. Bacteriol.
• the introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et ai, 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51-57).
• electroporation see, e.g., Choi et ai, 2006, J. Microbiol. Methods 64: 391-397
• conjugation see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51-57.
• Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et ai., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409- 436).
• any method known in the art for introducing DNA into a host cell can be used.
• the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
• the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
• the host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
• the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
• the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published
• compositions e.g., in catalogues of the American Type Culture Collection.
• the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
• the polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.
• the polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
• chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
• electrophoretic procedures e.g., preparative isoelectric focusing
• differential solubility e.g., ammonium sulfate precipitation
• SDS-PAGE or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989)
• polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide is used as a source of the polypeptide.
• the present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention.
• the fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products.
• the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
• fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
• fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
• the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
• the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation.
• the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
• the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof.
• the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
• the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
• the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
• the fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
• a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
• the cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
• the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis.
• the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
• the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
• a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
• the whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
• the present invention also relates to compositions comprising a polypeptide of the present invention.
• the compositions may comprise a GH98 xylanase of the present invention as the major enzymatic component, e.g., a mono-component composition.
• compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta- xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, a a
• the composition comprises a xylanase of the invention and a glucoamylase.
• the composition comprises a xylanase of the invention and a glucoamylase derived from Talaromyces emersonii (e.g., SEQ ID NO: 11).
• the composition comprises a trehalase of the invention and a glucoamylase derived from Gloeophyllum, such as G. serpiarium (e.g., SEQ ID NO: 12) or G. trabeum (e.g., SEQ ID NO: 13).
• the composition comprises a trehalase of the invention, a glucoamylase and an alpha-amylase.
• the composition comprises a trehalase of the invention, a glucoamylase and an alpha-amylase derived from Rhizomucor, preferably a strain the Rhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase hybrid having an linker (e.g., from Aspergillus niger) and starch-bonding domain (e.g., from Aspergillus niger).
• the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase and a cellulolytic enzyme composition.
• the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase and a cellulolytic enzyme composition, wherein the cellulolytic composition is derived from Trichoderma reesei.
• the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase and a protease.
• the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase and a protease.
• the protease may be derived from
• the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, a cellulolytic enzyme composition and a protease.
• the composition comprises a trehalase of the invention, a glucoamylase, e.g., derived from Talaromyces emersonii, Gloeophyllum serpiarium or Gloephyllum trabeum, an alpha-amylase, e.g., derived from Rhizomucor pusillus, in particular one having a linker and starch-binding domain, in particular derived from
• Aspergillus niger in particular one having the following substitutions: G128D+D143N (using SEQ ID NO: 14 for numbering); a cellulolytic enzyme composition derived from Trichoderma reesei, and a protease, e.g., derived from Thermoascus aurantiacus or Meripilus giganteus.
• Examples of specifically contemplated secondary enzymes e.g., a glucoamylase from Talaromyces emersonii shown in SEQ ID NO: 11 herein or a glucoamylase having, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 11 herein can be found in the“Enzymes” section below.
• compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
• the compositions may be stabilized in accordance with methods known in the art.
• compositions of the present invention are given below of preferred uses of the compositions of the present invention.
• dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.
• the present invention relates to enzyme blends comprising a xylanase and/or a cellulolytic composition.
• the enzyme blends can be used for solubilization of fiber (e.g., corn fiber, e.g., arabinose, xylose, etc.), for example, during the SSF step (or pre saccharification step) of a fermentation product production process (e.g., ethanol), preferably without resulting in darkening of DDG or DDGS produced as a co-product of the
• the ratio of the xylanase and the cellulolytic composition can be optimized to increase fiber solubilization of any particular substrate (e.g., corn fiber) and minimize or prevent darkening of downstream DDG or DDGS.
• the present invention relates to an enzyme blend comprising a xylanase.
• the present invention relates to an enzyme blend comprising a xylanase and a cellulolytic composition, wherein the ratio of the xylanase and cellulolytic composition in the blend is from about 5:95 to about 95:5.
• the ratio of the xylanase and cellulolytic composition is 10:90.
• the ratio of the xylanase and cellulolytic composition is 15:85.
• the ratio of the xylanase and cellulolytic composition is 20:80.
• the ratio of the xylanase and cellulolytic composition is 25:75.
• composition is 30:70.
• composition is 35:65.
• composition is 40:60.
• composition is 45:55. In an embodiment, the ratio of the xylanase and cellulolytic composition is 50:50. In an embodiment, the ratio of the xylanase and cellulolytic
• composition is 55:45.
• composition is 60:40.
• composition is 65:35.
• composition is 70:30. In an embodiment, the ratio of the xylanase and cellulolytic composition is 75:25. In an embodiment, the ratio of the xylanase and cellulolytic composition is 80:20. In an embodiment, the ratio of the xylanase and cellulolytic
• composition is 85:15.
• composition is 90:10.
• the present invention contemplates using any xylanase that, when optionally blended together with a cellulolytic composition in various ratios, is capable of solubilizing fiber (e.g., arabinose, xylose, etc.) in a fermentation product production process, such as especially ethanol, preferably without resulting in a darkening of the DDGS after drying.
• solubilizing fiber e.g., arabinose, xylose, etc.
• the xylanase is from the taxonomic order Micrococcales or Bacillales, or preferably the taxonomic family Microbacteriaceae, Bacillaceae or
• Paenibacillaceae or more preferably from the taxonomic genus Microbacterium, Bacillus, or Paenibacillus, or even more preferably from the taxonomic species Microbacterium oxydans, Microbacterium hydrocarbonoxydans, or Microbacterium sp. SA39, Bacillus
• the xylanase has at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 4 and is obtained or obtainable from the taxonomic order Micrococcales, or preferably the taxonomic family Microbacteriaceae, or more preferably from the taxonomic genus
• the xylanase has at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10 and is obtainable from the taxonomic order Bacillales, or preferably the taxonomic family Bacillaceae or Paenibacillaceae, or more preferably from the taxonomic genus Bacillus or Paenibacillus, or even more preferably from the taxonomic species Bacillus subtilis, Bacillus amyloliquefaciens, Bacill
• the xylanase is a GH98 family xylanase (herein referred to as GH98 xylanases).
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 , e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 1.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 1 , is a fragment of SEQ ID NO: 1 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 1 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 3, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 3.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 3, is a fragment of SEQ ID NO: 2 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 3 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 4, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 4.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 4, is a fragment of SEQ ID NO: 4 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 4 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 5, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 5.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 5, is a fragment of SEQ ID NO: 5 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 5 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 7, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 7.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 7, is a fragment of SEQ ID NO: 7 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 7 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 9, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 9.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 9, is a fragment of SEQ ID NO: 9 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 9 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase may be (a) a polypeptide having at least 70% sequence identity to SEQ ID NO: 10, e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xylanase activity.
• the amino acid sequence of the xylanase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 10.
• the xylanase comprises or consists of the amino acid sequence of SEQ ID NO: 10, is a fragment of SEQ ID NO: 10 wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO: 10 and an N- and/or C-terminal extension or deletion of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
• the xylanase comprises a variant xylanase having one or more substitutions described in EP Application No. 17177304.7 (incorporated herein by reference in its entirety).
• the xylanase comprises a variant xylanase having one or more substitutions described in International Patent Application No. PCT/EP2017/065336
• the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
• the xylanase may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
• a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
• Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
• Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post- translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
• a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
• cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.
• the xylanase may be obtained from microorganisms of any genus.
• the term“obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.
• the polypeptide may be a bacterial polypeptide.
• the polypeptide may be a bacterial polypeptide.
• polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or preferably the taxonomic family Bacillaceae or Paenibacillaceae, or more preferably from the taxonomic genus Bacillus or Paenibacillus, or even more preferably from the taxonomic species Bacillus subtilis, Bacillus amyloliquefaciens, Paenibacillus glycanilyticus, Bacillus licheniformis, Paenibacillus pabuli or Paenibacillus terrigena.
• the polypeptide is from a bacterium of the class Actinobacteria, such as from the order Micrococcales, or preferably the taxonomic family Microbacteriaceae or Micrococcaceae, or more preferably from the taxonomic genus Microbacterium,
• Micrococcus or Micrococoides or even more preferably from the taxonomic species
• Microbacterium oxydans Microbacterium hydrocarbonoxydans, or Microbacterium sp. SA39.
• the xylanase is a Paenibacillus glycanilyticus, Paenibacillus pabuli, or Paenibacillus terrigena xylanse.
• the xylanase is a Microbacterium oxydans, Microbacterium hydrocarbonoxydans, or Microbacterium sp. SA39 xylanase.
• the xylanase is a Microbacterium oxydans xylanase, e.g., the xylanase having the amino acid sequence of SEQ ID NO: 1.
• the xylanase is a Paenibacillus glycanilyticus xylanase, e.g., the xylanase having the amino acid sequence of SEQ ID: 5.
• the xylanase is a Paenibacillus terrigena xylanase, e.g., the xylanase having the amino acid sequence of SEQ ID: 7.
• ATCC American Type Culture Collection
• DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• CBS Centraalbureau Voor Schimmelcultures
• NRRL Northern Regional Research Center
• the xylanase may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
• the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et ai, 1989, supra).
• the xylanase is a Paenibacillus GH98 xylanase. In an embodiment, the xylanase is a Microbacterium GH98 xylanase.
• Exemplary GH98 xylanases of use in the enzyme blends and processes of the present invention include those from the taxonomic genera of Bacteroides, Cellvibrio, Clostridium, Cystobacter, Bacillus, Dickeya, Fibrobacter, Geobacillus, Meloidogyne, Microbacterium, Micromonospora, Mucilaginibacter, Paenibacillus, Paludibacter, Radopholus, Ruminococcus, Serratia, Streptomyces,
• Verrucosispora Verrucosispora, and Xanthomonas.
• the xylanase is a GH98 xylanase selected from the group consisting of: (i) the Microbacterium oxydans xylanase of SEQ ID NO: 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (ii) the Microbacterium oxydans xylanase of SEQ ID NO: 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 8
• the xylanase e.g., GH98 xylanase, for example of SEQ NOs: 1 , 3-5, 7, 8-10, or variants thereof, is dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in a concentration of between 0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as 0.001-0.1 mg EP/g DS.
• EP Enzyme Protein
• the present invention contemplates using any cellulolytic composition that, when blended with a xylanase in various ratios, is capable of solubilizing fiber (e.g., arabinose, xylose, etc.) in a fermentation product production process, such as especially ethanol, without resulting in a darkening of the DDGS after drying.
• the cellulolytic composition used in an enzyme blend or process of the invention for producing fermentation products may be derived from any microorganism.
• “derived from any microorganism” means that the cellulolytic composition comprises one or more enzymes that were expressed in the microorganism.
• a cellulolytic composition derived from a strain of Trichoderma reesei means that the cellulolytic composition comprises one or more enzymes that were expressed in Trichoderma reesei.
• the cellulolytic composition is derived from a strain of
• Aspergillus such as a strain of Aspergillus aurantiacus, Aspergillus niger or Aspergillus oryzae.
• the cellulolytic composition is derived from a strain of
• Chrysosporium such as a strain of Chrysosporium lucknowense.
• the cellulolytic composition is derived from a strain of Humicola, such as a strain of Humicola insolens.
• the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum. In an embodiment, the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum. In an embodiment, the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum. In an embodiment, the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum. In an embodiment, the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum. In an embodiment, the cellulo
• Talaromyces such as a strain of Talaromyces aurantiacus or Talaromyces emersonii.
• the cellulolytic composition is derived from a strain of
• Trichoderma such as a strain of Trichoderma reesei.
• the cellulolytic composition is derived from a strain of Trichoderma reesei.
• the Trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I or a cellobiohydrolase II; (ii) a beta- glucosidase; and (iii) an endoglucanase.
• the Trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus beta-glucosidase; and (iii) an endoglucanase.
• composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 8
• cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
• the Trichoderma reesei cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Aspergillus aurantiacus.
• the Aspergillus aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Aspergillus aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Aspergillus aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at
• cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
• the Aspergillus aurantiacus cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Aspergillus niger.
• the Aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta- glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%
• the Aspergillus niger cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Aspergillus oryzae.
• composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Aspergillus oryzae cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Aspergillus oryzae cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least a cellobiohydr
• the Aspergillus oryzae cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Penicilium emersonii.
• the Penicilium emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Penicilium emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Penicilium emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least
• the Penicilium emersonii cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Penicilium oxalicum.
• the Penicilium oxalicum cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Penicilium oxalicum cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 8
• cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
• the Penicilium oxalicum cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Talaromyces aurantiacus.
• the Talaromyces aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a
• the Talaromyces aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus
• cellobiohydrolase I an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Talaromyces aurantiacus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least
• cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
• the Talaromyces aurantiacus cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain of Talaromyces emersonii.
• the Talaromyces emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I; (ii) a cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
• the Talaromyces emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) an Aspergillus fumigatus cellobiohydrolase I; (ii) an Aspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
• the Talaromyces emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15; (ii) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least
• cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16; (iii) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
• the Talaromyces emersonii cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta- galactosidase, beta-glucanase, beta-glucosidase, glucan 1 ,4-a-glucosidase, glucan 1 ,4- alpha-maltohydrolase, glucan 1 ,4-a-glucosidase, glucan 1 ,4-alpha-maltohydrolase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosi
• the cellulolytic composition comprises one or more formulating agents as disclosed herein, preferably one or more of the compounds selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose.
• formulating agents as disclosed herein, preferably one or more of the compounds selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate
• the cellulolytic composition e.g., derived from a strain of Aspergillus, Penicilium, Talaromyces, or Trichoderma, such as a Trichoderma reesei cellulolytic composition, is dosed in pre-saccharification, saccharification, and/or
• the invention also relates to processes for producing a fermentation product from starch-containing material using a fermenting organism, wherein a polypeptide having xylanase activity (GH98 xylanase) or an enzyme blend or composition comprising a xylanase and a cellulolytic composition (e.g., from Trichoderma reesei) is added before and/or during fermentation.
• a polypeptide having xylanase activity GH98 xylanase activity
• an enzyme blend or composition comprising a xylanase and a cellulolytic composition (e.g., from Trichoderma reesei) is added before and/or during fermentation.
• a cellulolytic composition e.g., from Trichoderma reesei
• the invention relates to processes for producing fermentation products from starch-containing material without gelatinization (i.e., without cooking) of the starch- containing material (often referred to as a“raw starch hydrolysis” process), wherein a presently disclosed GH98 xylanase, or enzyme blend or composition comprising the xylanase and a cellulolytic composition is added.
• the fermentation product such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water.
• a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s) to produce sugars that can be fermented into the fermentation product by a suitable fermenting organism.
• the desired fermentation product e.g., ethanol
• un-gelatinized i.e., uncooked
• cereal grains such as corn.
• the invention relates to processes for producing a fermentation product from starch-containing material comprising simultaneously
• step (i) and/or (ii) is carried out using at least a glucoamylase and a GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase.
• the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during saccharifying step (i). In an embodiment, the a GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during fermenting step (ii).
• an alpha amylase in particular a fungal alpha-amylase, is also added in step (i). Steps (i) and (ii) may be performed simultaneously.
• the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during simultaneous saccharification and fermentation (SSF).
• the fermenting organism is yeast and the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during yeast propagation.
• the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to a process for producing a fermentation product from starch-containing material comprising the steps of:
• GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added before or during step (c).
• the slurry is heated to above the gelatinization temperature and an alpha-amylase variant may be added to initiate liquefaction (thinning).
• the slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a).
• Liquefaction may in an embodiment be carried out as a three-step hot slurry process.
• the slurry is heated to between 60-95°C, preferably between 70-90°C, such as preferably between 80-85°C at a pH of 4-6, in particular at a pH of 4.5-5.5, and alpha-amylase variant, optionally together with a protease, a carbohydrate-source generating enzyme, such as a glucoamylase, a phospholipase, a phytase, and/or pullulanase, are added to initiate liquefaction (thinning).
• the liquefaction process is usually carried out at a pH of 4-6, in particular at a pH from 4.5 to 5.5. Saccharification step (b) may be carried out using conditions well known in the art.
• a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature from 20-75°C, in particular 40-70°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5.
• SSF process simultaneous saccharification and fermentation process
• SSF simultaneous saccharification and fermentation
• a fermenting organism such as yeast, and enzyme(s)
• SSF may typically be carried out at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around about 32°C.
• fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
• the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during saccharifying step (b).
• the a GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during fermenting step (c). Steps (b) and (c) may be performed simultaneously.
• the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during simultaneous saccharification and fermentation (SSF).
• the fermenting organism is yeast and the GH98 xylanase of the present invention or enzyme blend or composition comprising the xylanase is added during yeast propagation.
• Any suitable starch-containing starting material may be used in a process of the present invention.
• the starting material is generally selected based on the desired fermentation product.
• starch-containing starting materials suitable for use in the processes of the present invention, include barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof.
• the starch-containing material may also be a waxy or non-waxy type of corn and barley.
• the starch-containing material is corn.
• the starch-containing material is wheat.
• Fermentation product means a product produced by a method or process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes;
• alcohols e.g., ethanol, methanol, butanol
• organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid
• ketones e.g., acetone
• amino acids e.g., glutamic acid
• gases e.g., H2 and CO2
• antibiotics e
• the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry.
• Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
• the fermentation product is ethanol.
• fermenting organism refers to any organism, including bacterial and fungal organisms, such as yeast and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermenting organisms are able to ferment, i.e., convert, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose, or xylose, directly or indirectly into the desired fermentation product.
• fermentable sugars such as arabinose, fructose, glucose, maltose, mannose, or xylose
• fermenting organisms examples include fungal organisms such as yeast.
• Preferred yeast include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarunr, strains of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris ; strains of Candida, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis.
• Other fermenting organisms include strains of Hansenula, in particular Hansenula anomala or Hansenula polymorpha ; strains of Kluyveromyces, in particular Kluyveromyces fragilis or Kluyveromyces marxianus ; and strains of
• Schizosaccharomyces in particular Schizosaccharomyces pombe.
• the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
• the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
• the fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product.
• One skilled in the art can easily determine suitable fermentation conditions.
• the fermentation may be carried out at conventionally used conditions.
• Preferred fermentation processes are anaerobic processes.
• fermentations may be carried out at temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
• temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
• bacteria with a significantly lower temperature optimum down to around room temperature (around 20°C) are also known. Examples of suitable fermenting organisms can be found in the“Fermenting
• the fermentation may go on for 24 to 96 hours, in particular for 35 to 60 hours.
• the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C.
• the pH is from pH 3 to 6, preferably around pH 4 to 5.
• the fermentation product may be separated from the fermentation medium.
• the slurry may be distilled to extract the desired fermentation product (e.g., ethanol).
• the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques.
• the fermentation product may also be recovered by stripping or other method well known in the art.
• the fermentation product e.g., ethanol, with a purity of up to, e.g., about 96 vol. percent ethanol is obtained.
• the method of the invention further comprises distillation to obtain the fermentation product, e.g., ethanol.
• the fermentation and the distillation may be carried out simultaneously and/or separately/sequentially; optionally followed by one or more process steps for further refinement of the fermentation product.
• the material remaining is considered the whole stillage.
• whole stillage includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.
• the fermentation product can optionally be recovered by any method known in the art.
• the whole stillage is separated or partitioned into a solid and liquid phase by one or more methods for separating the thin stillage from the wet cake.
• Separating whole stillage into thin stillage and wet cake in order to remove a significant portion of the liquid/water may be done using any suitable separation technique, including centrifugation, pressing and filtration.
• the separation/dewatering is carried out by centrifugation.
• Preferred centrifuges in industry are decanter type centrifuges, preferably high speed decanter type centrifuges.
• An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval which is a high-performance decanter.
• the separation is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment.
• Thin stillage is the term used for the supernatant of the centrifugation of the whole stillage.
• the thin stillage contains 4-6 percent dry solids (DS) (mainly proteins, soluble fiber, fine fibers, and cell wall components) and has a temperature of about 60-90 degrees centigrade.
• the thin stillage stream may be condensed by evaporation to provide two process streams including: (i) an evaporator condensate stream comprising condensed water removed from the thin stillage during evaporation, and (ii) a syrup stream, comprising a more concentrated stream of the non-volatile dissolved and non-dissolved solids, such as non-fermentable sugars and oil, remaining present from the thin stillage as the result of removing the evaporated water.
• oil can be removed from the thin stillage or can be removed as an intermediate step to the evaporation process, which is typically carried out using a series of several evaporation stages.
• Syrup and/or de-oiled syrup may be introduced into a dryer together with the wet grains (from the whole stillage separation step) to provide a product referred to as distillers dried grain with solubles, which also can be used as animal feed.
• syrup and/or de-oiled syrup is sprayed into one or more dryers to combine the syrup and/or de-oiled syrup with the whole stillage to produce distillers dried grain with solubles.
• the recycled thin stillage may constitute from about 1-70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% of the slurry formed in step (a).
• the process further comprises recycling at least a portion of the thin stillage stream to the slurry, optionally after oil has been extracted from the thin stillage stream.
• the wet cake containing about 25-40 wt-%, preferably 30-38 wt-% dry solids, has been separated from the thin stillage (e.g., dewatered) it may be dried in a drum dryer, spray dryer, ring drier, fluid bed drier or the like in order to produce“Distillers Dried Grains” (DDG).
• DDG is a valuable feed ingredient for animals, such as livestock, poultry and fish. It is preferred to provide DDG with a content of less than about 10-12 wt.-% moisture to avoid mold and microbial breakdown and increase the shelf life. Further, high moisture content also makes it more expensive to transport DDG.
• the wet cake is preferably dried under conditions that do not denature proteins in the wet cake.
• the wet cake may be blended with syrup separated from the thin stillage and dried into DDG with Solubles (DDGS).
• DDG DDG with Solubles
• Partially dried intermediate products such as are sometimes referred to as modified wet distillers grains, may be produced by partially drying wet cake, optionally with the addition of syrup before, during or after the drying process.
• the present invention relates to a process for improving the nutritional quality of distillers dried grains (DGS) or distillers dried grains with solubles (DDGS) produced as a co-product of a fermentation product production process.
• DGS distillers dried grains
• DDGS distillers dried grains with solubles
• a process for improving the nutritional quality of DGS or DDGS produced as a co-product of a fermentation production process comprises performing a process for producing a fermentation product described above in Section XI (e.g., a RSH process or a conventional cook process including a liquefaction step) or Examples herein, and recovering the fermentation product to produce DGS or DDGS as a co-product, wherein the DGS or DDGS produced have improved nutritional quality.
• Section XI e.g., a RSH process or a conventional cook process including a liquefaction step
• the step of recovering the fermentation product to produce DGS or DDGS as the co-product may include any one or combination of the above described steps of recovery of fermentation product(s), for example by distillation, to produce whole stillage, separating whole stillage into wet cake and thin stillage, processing of thin stillage, drying of wet cake and producing DDG or DDGS, etc.
• “improved nutritional quality” means an increase in the true metabolizable energy (TME) of the DDG or DDGS by at least 5% as compared to DDG or DDGS produced in a fermentation product production process (e.g., an RSH process or conventional cook process including a liquefaction step as set forth in Section XI) in which a presently disclosed GH98 xylanase or enzyme blend or composition comprising the xylanase was not added during pre-saccharification, saccharification, fermentation, and/or
• TME true metabolizable energy
• the GH98 xylanase or enzyme blends and compositions comprising the xylanase and processes of the present invention increase the TME of the DDG or DDGS by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% as compared to DDG or DDGS produced in a fermentation product production process (e.g., an RSH process or
• the GH98 xylanase or enzyme blends or compositions comprising the xylanase, and processes of the present invention improve the nutritional quality of the DDG or DDGS without resulting in a darkening of the DDG or DDGS after drying.
• a GH98 xylanase of the present invention, or an enzyme blend or composition comprising a xylanase during a fermentation product production process can be readily assessed, for example, by measuring the DDG or DDGS color using the Hunter Color scale (see Examples herein).
• the cellulolytic composition used in a process of the invention for producing fermentation products may be derived from any microorganism.
• “derived from any microorganism” means that the cellulolytic composition comprises one or more enzymes that were expressed in the microorganism.
• a cellulolytic composition derived from a strain of Trichoderma reesei means that the cellulolytic composition comprises one or more enzymes that were expressed in Trichoderma reesei.
• the cellulolytic composition is derived from a strain of
• Aspergillus such as a strain of Aspergillus aurantiacus, Aspergillus niger or Aspergillus oryzae.
• the cellulolytic composition is derived from a strain of
• Chrysosporium such as a strain of Chrysosporium lucknowense.
• the cellulolytic composition is derived from a strain of Humicola, such as a strain of Humicola insolens.
• the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum.
• the cellulolytic composition is derived from a strain of
• Talaromyces such as a strain of Talaromyces aurantiacus or Talaromyces emersonii.
• the cellulolytic composition is derived from a strain of
• Trichoderma such as a strain of Trichoderma reesei.
• the cellulolytic composition is derived from a strain of Trichoderma reesei.
• the cellulolytic composition may comprise one or more of the following
• polypeptides including enzymes: GH61 polypeptide having cellulolytic enhancing activity, beta-glucosidase, CBHI and CBHII, or a mixture of two, three, or four thereof.
• the cellulolytic composition comprising a beta- glucosidase having a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
• the cellulolytic composition may comprise some hemicellulase, such as, e.g., xylanase and/or beta-xylosidase.
• the hemicellulase may come from the cellulolytic composition producing organism or from other sources, e.g., the hemicellulase may be foreign to the cellulolytic composition producing organism, such as, e.g., Trichoderma reesei.
• the hemicellulase content in the cellulolytic composition constitutes less than 10 wt.% such as less than 5 wt. % of the cellulolytic composition.
• the cellulolytic composition comprises a beta-glucosidase.
• the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.
• the cellulolytic composition comprises a beta-glucosidase and a CBH.
• the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and a CBHI.
• the cellulolytic composition comprises a beta-glucosidase and a CBHI.
• the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a CBHI, and a CBHII.
• the cellulolytic composition comprises a beta-glucosidase, a CBHI, and a CBHII.
• the cellulolytic composition may further comprise one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
• the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
• endoglucanase is an endoglucanase I.
• endoglucanase is an endoglucanase II.
• the cellulolytic composition used according to the invention may in one
• the cellulolytic composition comprises one or more CBH I (cellobiohydrolase I).
• the cellulolytic composition comprises a cellobiohydrolase I (CBHI), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the Cel7A CBHI disclosed in SEQ ID NO: 6 in WO 2011/057140 or SEQ ID NO: 15 herein, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
• CBHI cellobiohydrolase I
• a cellobiohydrolase I comprising an amino acid sequence having at least 70%, e.g. , 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 15 herein;
• a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%,
• a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 in WO 2013/148993 or the full-length complement thereof.
• the cellulolytic composition used according to the invention may in one
• CBH II cellobiohydrolase II
• the cellobiohydrolase II such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one in SEQ ID NO: 16 herein or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
• CBHII cellobiohydrolase II
• a cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 16 herein;
• a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%,
• the cellulolytic composition used according to the invention may in one
• the beta-glucosidase may in one embodiment be one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta- glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as such as one disclosed in WO 2005/047499 or SEQ ID NO: 17 herein or an Aspergillus fumigatus beta-glucosidase variant, such as one disclosed in WO 2012/044915 or co-pending PCT application PCT/US11/054185 (or US provisional application # 61/388,997), such as one with the following substitutions: F100D, S283G, N456E, F512Y.
• Aspergillus oryzae such as the one disclosed in WO 2002/095014 or the fusion protein having beta- glucosidase activity disclosed in WO 2008/0576
• beta-glucosidase is derived from a strain of the genus Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
• a beta-glucosidase comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 17 herein;
• a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993; and
• the beta-glucosidase is a variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 17 herein, wherein the variant has beta-glucosidase activity.
• the parent beta-glucosidase of the variant is (a) a polypeptide comprising the mature polypeptide of SEQ ID NO: 17 herein; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 17 herein; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5 in WO
• the beta-glucosidase variant has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent beta-glucosidase.
• the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 17 herein.
• the beta-glucosidase is from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 17 herein), which comprises one or more substitutions selected from the group consisting of L89M, G91 L, F100D, 1140V, 1186V, S283G, N456E, and F512Y; such as a variant thereof with the following substitutions:
• the number of substitutions is between 1 and 4, such as 1 , 2, 3, or 4 substitutions.
• the variant comprises a substitution at a position corresponding to position 100, a substitution at a position corresponding to position 283, a substitution at a position corresponding to position 456, and/or a substitution at a position corresponding to position 512.
• the beta-glucosidase variant comprises the following substitutions: Phe100Asp, Ser283Gly, Asn456Glu, Phe512Tyr in SEQ ID NO: 17 herein.
• the beta-glucosidase has a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
• the cellulolytic composition used according to the invention may in one
• the enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity, such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as the one described in WO 2010/138754 as SEQ ID NO: 2; or one derived from a strain derived from Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed in WO
• Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of:
• a GH61 polypeptide having cellulolytic enhancing activity comprising the mature polypeptide of SEQ ID NO: 18 herein;
• a GH61 polypeptide having cellulolytic enhancing activity comprising an amino acid seguence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 18 herein;
• polynucleotide comprising a nucleotide seguence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding seguence of SEQ ID NO: 7 in WO 2013/148993; and
• the cellulolytic composition may comprise a number of difference polypeptides, such as enzymes.
• the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (WO 2005/074656) and Aspergillus oryzae beta- glucosidase fusion protein (WO 2008/057637).
• the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499).
• the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Penicillium emersonii GH61 A polypeptide having cellulolytic enhancing activity disclosed in WO 2011/041397, Aspergillus fumigatus beta- glucosidase (SEQ ID NO: 2 of WO 2005/047499) or a variant thereof with the following substitutions: F100D, S283G, N456E, F512Y.
• the enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, e.g., Trichoderma host cell, as a source of the enzymes.
• the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
• Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
• the cellulolytic composition comprising a beta- glucosidase having a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
• cellulolytic enzyme composition is dosed (i.e. during
• step ii) and/or fermentation in step iii) or SSF) from 0.0001-3 mg EP/g DS, preferably 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS, more preferred from 0.005- 0.5 mg EP/g DS, even more preferred 0.01-0.1 mg EP/g DS.
• an alpha-amylase is present and/or added in liquefaction optionally together with a hemicellulase, an endoglucanase, a protease, a carbohydrate- source generating enzyme, such as a glucoamylase, a phospholipase, a phytase, and/or pullulanase.
• the alpha-amylase added during liquefaction step i) may be any alpha-amylase.
• bacterial alpha-amylase means any bacterial alpha-amylase classified under EC 3.2.1.1.
• a bacterial alpha-amylase used according to the invention may, e.g., be derived from a strain of the genus Bacillus, which is sometimes also referred to as the genus Geobacillus.
• Bacillus alpha-amylase is derived from a strain of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus sp. TS-23, or Bacillus subtilis, but may also be derived from other Bacillus sp.
• the bacterial alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS:
• the alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 19 herein.
• the Bacillus stearothermophilus alpha-amylase may be a mature wild- type or a mature variant thereof.
• the mature Bacillus stearothermophilus alpha-amylases, or variant thereof, may be naturally truncated during recombinant production.
• the mature Bacillus stearothermophilus alpha-amylase may be truncated at the C-terminal so it is around 491 amino acids long (compared to SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 19 herein), such as from 480-495 amino acids long.
• the Bacillus alpha-amylase may also be a variant and/or hybrid. Examples of such a variant can be found in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, WO 02/10355 and W02009/061380 (all documents are hereby incorporated by reference). Specific alpha-amylase variants are disclosed in U.S. Patent Nos.
• BSG alpha-amylase Bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having a deletion of one or two amino acids at any of positions R179, G180, 1181 and/or G182, preferably the double deletion disclosed in WO 96/23873 - see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to deletion of positions 1181 and G182 compared to the amino acid sequence of Bacillus
• BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
• stearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 19 herein or the deletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 19 herein.
• Bacillus alpha-amylases especially Bacillus stearothermophilus (BSG) alpha-amylases, which have at one or two amino acid deletions corresponding to positions R179, G180, 1181 and G182, preferably which have a double deletion corresponding to R179 and G180, or preferably a deletion of positions 181 and 182 (denoted 1181* + G182*), and optionally further comprises a N193F substitution (denoted 1181* + G182* + N193F) compared to the wild-type BSG alpha- amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 19 herein.
• BSG Bacillus stearothermophilus
• the bacterial alpha-amylase may also have a substitution in a position corresponding to S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or a S242 variant in the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 19 herein.
• the variant is a S242A, E or Q variant, preferably a S242Q or A variant, of the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 19 herein for numbering).
• the variant is a position E188 variant, preferably E188P variant of the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 19 herein for numbering).
• the bacterial alpha-amylase may also be a hybrid bacterial alpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467).
• this hybrid has one or more, especially all, of the following substitutions:
• variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylases): H154Y, A181T, N190F, A209V and Q264S and/or the deletion of two residues between positions 176 and 179, preferably the deletion of E178 and G179 (using SEQ ID NO: 5 of WO 99/19467 for position numbering).
• the bacterial alpha-amylase is the mature part of the chimeric alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29):. 267501-26507, referred to as BD5088 or a variant thereof.
• This alpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO 2007134207.
• the mature enzyme sequence starts after the initial“Met” amino acid in position 1.
• the alpha-amylase is optionally used in combination with a hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C.
• a hemicellulase preferably xylanase
• an endoglucanase having a Melting Point (DSC) above 70°C, such as above 75°C, in particular above 80°C may be included.
• the thermostable alpha-amylase such as a bacterial an alpha-amylase, is preferably derived from Bacillus stearothermophilus or Bacillus sp. TS-23.
• the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb of at least 10.
• the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaC , of at least 15. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 20. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 25. In an embodiment the alpha-amylase has a T 1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 30.
• the alpha-amylase has a T 1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 40. In an embodiment the alpha- amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 50. In an embodiment the alpha-amylase has a T 1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, of at least 60. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCb, between 10-70.
• the alpha-amylase has a T 1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 15-70. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5,
• the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 25-70. In an embodiment the alpha-amylase has a T 1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 30-70. In an embodiment the alpha- amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 40-70.
• the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 50-70. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 60-70.
• the alpha-amylase is a bacterial alpha-amylase, preferably derived from the genus Bacillus, especially a strain of Bacillus stearothermophilus, in particular the Bacillus stearothermophilus as disclosed in WO 99/19467 as SEQ ID NO: 3 or SEQ ID NO: 19 herein with one or two amino acids deleted at positions R179, G180, 1181 and/or G182, in particular with R179 and G180 deleted, or with 1181 and G182 deleted, with mutations in below list of mutations.
• the Bacillus stearothermophilus alpha- amylases have double deletion 1181 + G182, and optional substitution N193F, optionally further comprising mutations selected from below list:
• the bacterial alpha-amylase such as Bacillus alpha-amylase, such as Bacillus stearothermophilus alpha-amylase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 19 herein.
• the bacterial alpha-amylase variant such as Bacillus alpha- amylase variant, such as Bacillus stearothermophilus alpha-amylase variant has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO: 19 herein.
• Bacillus stearothermophilus alpha- amylase and variants thereof are normally produced naturally in truncated form.
• the truncation may be so that the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 19 herein, or variants thereof, are truncated in the C-terminal and are typically around 491 amino acids long, such as from 480- 495 amino acids long.
• an optional hemicellulase preferably xylanase, having a Melting Point (DSC) above 80°C is present and/or added to liquefaction step i) in
• alpha-amylase such as a bacterial alpha-amylase (described above).
• thermostability of a hemicellulase may be determined as described in the“Materials & Methods’-section under“Determination of T d by Differential Scanning Calorimetry for Endoglucanases and Hemicellulases”.
• the hemicellulase, in particular xylanase, especially GH10 or GH11 xylanase has a Melting Point (DSC) above 82°C, such as above 84°C, such as above 86°C, such as above 88°C, such as above 88°C, such as above 90°C, such as above 92°C, such as above 94°C, such as above 96°C, such as above 98°C, such as above 100°C, such as between 80°C and 110°C, such as between 82°C and 110°C, such as between 84°C and 110°C.
• DSC Melting Point
• the hemicellulase, in particular xylanase, especially GH10 xylanase has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 20 herein, preferably derived from a strain of the genus Dictyoglomus, such as a strain of Dictyogllomus thermophilum.
• the hemicellulase, in particular xylanase, especially GH11 xylanase has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 21 herein, preferably derived from a strain of the genus Dictyoglomus, such as a strain of Dictyogllomus thermophilum.
• the hemicellulase, in particular xylanase, especially GH10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 22 herein, preferably derived from a strain of the genus Rasamsonia, such as a strain of Rasomsonia byssochlamydoides.
• the hemicellulase, in particular xylanase, especially GH10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 23 herein, preferably derived from a strain of the genus Talaromyces, such as a strain of Talaromyces leycettanus.
• the hemicellulase, in particular xylanase, especially GH 10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 24 herein, preferably derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus.
• an optional endoglucanase (“E”) having a Melting Point (DSC) above 70°C, such as between 70°C and 95°C may be present and/or added in liquefaction step i) in combination with an alpha-amylase, such as a thermostable bacterial alpha-amylase and an optional hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C.
• an alpha-amylase such as a thermostable bacterial alpha-amylase and an optional hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C.
• thermostability of an endoglucanase may be determined as described in the “Materials & Methods’-section of WO 2017/112540 (incorporated herein by reference in its entirety) under the heading“Determination of T d by Differential Scanning Calorimetry for Endoglucanases and Hemicellulases”.
• the endoglucanase has a Melting Point (DSC) above 72°C, such as above 74°C, such as above 76°C, such as above 78°C, such as above 80°C, such as above 82°C, such as above 84°C, such as above 86°C, such as above 88°C, such as between 70°C and 95°C, such as between 76°C and 94°C, such as between 78°C and 93°C, such as between 80°C and 92°C, such as between 82°C and 91 °C, such as between 84°C and 90°C.
• DSC Melting Point
• the endogluconase used in a process of the invention or comprised in a composition of the invention is a Glycoside Hydrolase Family 5 endoglucnase or GH5 endoglucanase (see the CAZy database on the“www.cazy.org” webpage.
• the GH5 endoglucanase is from family EG II, such as the
• the endoglucanase is a family GH45 endoglucanase.
• the GH45 endoglucanase is from family EG V, such as the Sordaria fimicola shown in SEQ ID NO: 28 herein or the Thielavia terrestris endoglucanase shown in SEQ ID NO: 29 herein.
• the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 25 herein.
• the endoglucanase is derived from a strain of the genus Talaromyces, such as a strain of Talaromyces leycettanus.
• the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 26 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium capsulatum.
• the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 27 herein, preferably derived from a strain of the genus Trichophaea, such as a strain of Trichophaea saccata.
• the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 28 herein, preferably derived from a strain of the genus Sordaria, such as a strain of Sordaria fimicola.
• the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 29 herein, preferably derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris.
• the endoglucanase is added in liquefaction step i) at a dose from 1-10,000 pg EP (Enzymes Protein) /g DS), such as 10-1 ,000 pg EP/g DS.
• EP Enzymes Protein
• an optional carbohydrate-source generating enzyme in particular a glucoamylase, preferably a thermostable glucoamylase, may be present and/or added in liquefaction together with an alpha-amylase and optional hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C, and an optional endoglucanase having a Melting Point (DSC) above 70°C, and an optional a pullulanase and/or optional phytase.
• a glucoamylase preferably a thermostable glucoamylase
• carbohydrate-source generating enzyme includes any enzymes generating fermentable sugars.
• a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol.
• the generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol.
• a mixture of carbohydrate-source generating enzymes may be used. Specific examples include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators).
• the carbohydrate-source generating enzyme is thermostable.
• the carbohydrate-source generating enzyme in particular thermostable glucoamylase, may be added together with or separately from the alpha-amylase and the thermostable protease.
• the carbohydrate-source generating enzyme is a thermostable glucoamylase, preferably of fungal origin, preferably a filamentous fungi, such as from a strain of the genus Penicillium, especially a strain of Penicillium oxalicum , in particular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO
• thermostable glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 30 herein.
• the carbohydrate-source generating enzyme in particular thermostable glucoamylase, is the Penicillium oxalicum glucoamylase shown in SEQ ID NO: 30 herein.
• the carbohydrate-source generating enzyme is a variant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 30 herein, having a K79V substitution (referred to as“PE001”) (using the mature sequence shown in SEQ ID NO: 14 therein for numbering).
• the K79V glucoamylase variant has reduced sensitivity to protease degradation relative to the parent as disclosed in WO 2013/036526 (which is hereby incorporated by reference).
• Penicillium oxalicum glucoamylase variants are disclosed in
• thermostability compared to the parent.
• the glucoamylase has a K79V
• substitution (using SEQ ID NO: 30 herein for numbering), corresponding to the PE001 variant, and further comprises at least one of the following substitutions or combination of substitutions:
• Penicillium oxalicum glucoamylase variant has a K79V substitution using SEQ ID NO: 23 herein for numbering (PE001 variant), and further comprises one of the following mutations:
• the glucoamylase variant such as Penicillium oxalicum glucoamylase variant has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature polypeptide of SEQ ID NO: 30 herein.
• the carbohydrate-source generating enzyme in particular glycoamylase, may be added in amounts from 0.1- 100 micrograms EP/g DS, such as 0.5-50 micrograms EP/g DS, such as 1-25 micrograms EP/g DS, such as 2-12 micrograms EP/g DS.
• Pullulanase Present and/or Added During Liquefaction may be added in amounts from 0.1- 100 micrograms EP/g DS, such as 0.5-50 micrograms EP/g DS, such as 1-25 micrograms EP/g DS, such as 2-12 micrograms EP/g DS.
• a pullulanase may be present and/or added during liquefaction step i) together with an alpha-amylase and an optional hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C.
• an alpha-amylase and an optional hemicellulase preferably xylanase, having a melting point (DSC) above 80°C.
• a protease a carbohydrate-source generating enzyme, preferably a thermostable glucoamylase, may also optionally be present and/or added during liquefaction step i).
• the pullulanase may be present and/or added during liquefaction step i) and/or saccharification step ii) or simultaneous saccharification and fermentation.
• Pullulanases (E.C. 3.2.1.41 , pullulan 6-glucano-hydrolase), are debranching enzymes characterized by their ability to hydrolyze the alpha-1 , 6-glycosidic bonds in, for example, amylopectin and pullulan.
• Contemplated pullulanases according to the present invention include the
• pullulanases from Bacillus amyloderamificans disclosed in U.S. Patent No. 4,560,651 (hereby incorporated by reference), the pullulanase disclosed as SEC ID NO: 2 in WO 01/151620 (hereby incorporated by reference), the Bacillus deramificans disclosed as SEC ID NO: 4 in WO 01/151620 (hereby incorporated by reference), and the pullulanase from Bacillus acidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (hereby incorporated by reference) and also described in FEMS Mic. Let. (1994) 115, 97-106.
• pullulanases contemplated according to the present invention included the pullulanases from Pyrococcus woesei, specifically from Pyrococcus woesei DSM No. 3773 disclosed in WO 92/02614.
• the pullulanase is a family GH57 pullulanase.
• the pullulanase includes an X47 domain as disclosed in WO 2011/087836 (which are hereby incorporated by reference). More specifically the pullulanase may be derived from a strain of the genus Thermococcus, including Thermococcus litoralis and Thermococcus
• hydrothermalis such as the Thermococcus hydrothermalis pullulanase shown WO
• the pullulanase may also be a hybrid of the Thermococcus litoralis and Thermococcus hydrothermalis pullulanases or a T hydrothermalis/T. litoralis hybrid enzyme with truncation site X4 disclosed in WO 2011/087836 (which is hereby incorporated by reference).
• the pullulanase is one comprising an X46 domain disclosed in WO 2011/076123 (Novozymes).
• the pullulanase may according to the invention be added in an effective amount which include the preferred amount of about 0.0001-10 mg enzyme protein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gram DS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.
• Pullulanase activity may be determined as NPUN.
• An Assay for determination of NPUN is described in the“Materials & Methods”-section below.
• Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYMETM D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int, USA), and AMANO 8 (Amano, Japan).
• a phytase may be present and/or added in liquefaction in combination with an alpha-amylase and optional hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C.
• an alpha-amylase and optional hemicellulase preferably xylanase, having a melting point (DSC) above 80°C.
• a phytase used according to the invention may be any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) or from any salt thereof (phytates).
• Phytases can be classified according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
• the phytase to be used in the invention may have any specificity, e.g., be a 3-phytase (EC 3.1.3.8), a 6-phytase (EC 3.1.3.26) or a 5-phytase (no EC number).
• the phytase has a temperature optimum above 50°C, such as in the range from 50-90°C.
• the phytase may be derived from plants or microorganisms, such as bacteria or fungi, e.g., yeast or filamentous fungi.
• a plant phytase may be from wheat-bran, maize, soy bean or lily pollen. Suitable plant phytases are described in Thomlinson et al, Biochemistry, 1 (1962), 166-171 ;
• a bacterial phytase may be from genus Bacillus, Citrobacter, Hafnia ,
• Citrobacter braakii Citrobacter freundii, Hafnia alvei, Buttiauxella gaviniae, Buttiauxella agrestis, Buttiauxella noackies and E. coli.
• Suitable bacterial phytases are described in Paver and Jagannathan, 1982, Journal of Bacteriology 151 : 1 102-1108; Cosgrove, 1970, Australian Journal of Biological Sciences 23: 1207-1220; Greiner et al, Arch. Biochem.
• a yeast phytase may be derived from genus Saccharomyces or Schwanniomyces, specifically species Saccharomyces cerevisiae or Schwanniomyces occidentalis.
• the former enzyme has been described as a Suitable yeast phytases are described in Nayini et al,
• Phytases from filamentous fungi may be derived from the fungal phylum of Ascomycota (ascomycetes) or the phylum Basidiomycota, e.g., the genus Aspergillus, Thermomyces (also called Humicola), Myceliophthora, Manascus, Penicillium, Peniophora, Agrocybe, Paxillus, or Trametes, specifically the species Aspergillus terreus, Aspergillus niger,
• Aspergillus niger var. awamori Aspergillus ficuum, Aspergillus fumigatus, Aspergillus oryzae, T. lanuginosus (also known as H. lanuginosa), Myceliophthora thermophila,
• Peniophora lycii Agrocybe pediades, Manascus anka, Paxillus involtus, or Trametes pubescens. Suitable fungal phytases are described in Yamada et al., 1986, Agric. Biol.
• the phytase is derived from Buttiauxella, such as Buttiauxella gaviniae, Buttiauxella agrestis, or Buttiauxella noackies, such as the ones disclosed as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, respectively, in WO
• the phytase is derived from Citrobacter, such as Citrobacter
• Citrobacter braakii such as one disclosed in WO 2006/037328 (hereby incorporated by reference).
• Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 897010; EP 897985; WO 99/49022; WO
• BIO-FEED BIO-FEED
• PHYTASETM, PHYTASE NOVOTM CT or L all from Novozymes
• LIQMAX DuPont
• RONOZYMETM NP RONOZYME® HiPhos
• RONOZYME® P5000 CT
• NATUPHOSTM NG 5000 from DSM.
• a carbohydrate-source generating enzyme preferably a glucoamylase, is present and/or added during saccharification and/or fermentation.
• the carbohydrate-source generating enzyme is a glucoamylase, of fungal origin, preferably from a stain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae ; or a strain of Trichoderma, preferably T. reesel ⁇ , or a strain of
• Talaromyces preferably T. emersonii
• glucoamylase According to the invention the glucoamylase present and/or added in saccharification and/or fermentation may be derived from any suitable source, e.g., derived from a
• glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921 , Aspergillus oryzae glucoamylase (Agric. Biol. Chem.
• variants or fragments thereof include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and
• glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see US patent no. 4,727,026 and (Nagasaka et al. (1998)“Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent no. Re. 32,153),
• the glucoamylase used during saccharification and/or fermentation is the Talaromyces emersonii glucoamylase disclosed in WO 99/28448.
• Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C.
• thermohydrosulfuricum (WO 86/01831).
• Contemplated fungal glucoamylases include Trametes cingulata, Pachykytospora papyracea ; and Leucopaxillus giganteus all disclosed in WO 2006/069289; and Peniophora rufomarginata disclosed in W02007/124285; or a mixture thereof.
• hybrid glucoamylase are contemplated according to the invention. Examples include the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).
• Pycnoporus in particular a strain of Pycnoporus as described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genus Gloephyllum, in particular a strain of
• glucoamylases which exhibit a high identity to any of the above- mentioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to any one of the mature parts of the enzyme sequences mentioned above.
• Glucoamylases may in an embodiment be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.
• compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U, SPIRIZYMETM ULTRA, SPIRIZYMETM EXCEL, SPIRIZYMETM ACHIEVE and AMGTM E (from Novozymes A/S); OPTIDEXTM 300, GC480, GC417 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Danisco US).
• the carbohydrate-source generating enzyme present and/or added during saccharification and/or fermentation may also be a maltogenic alpha-amylase.
• A“maltogenic alpha-amylase” (glucan 1 ,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration.
• a maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in US Patent nos. 4,598,048, 4,604,355 and 6, 162,628, which are hereby incorporated by reference.
• the maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.
• an optional protease such as a thermostable protease
• a thermostable protease may be present and/or added in liquefaction together with an alpha-amylase, such as a thermostable alpha-amylase, and a hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C, and optionally an endoglucanase, a carbohydrate-source generating enzyme, in particular a glucoamylase, optionally a pullulanase and/or optionally a phytase.
• an alpha-amylase such as a thermostable alpha-amylase
• a hemicellulase preferably xylanase, having a melting point (DSC) above 80°C
• DSC melting point
• an endoglucanase a carbohydrate-source generating enzyme, in particular a glucoamylase, optional
• Proteases are classified on the basis of their catalytic mechanism into the following groups: Serine proteases (S), Cysteine proteases (C), Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yet unclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J. Barrett, N.D. Rawlings, J.F.Woessner (eds), Academic Press (1998), in particular the general introduction part.
• S Serine proteases
• C Cysteine proteases
• A Aspartic proteases
• M Metallo proteases
• U Unknown, or as yet unclassified, proteases
• thermostable protease used according to the invention is a“metallo protease” defined as a protease belonging to EC 3.4.24
• metalloendopeptidases preferably EC 3.4.24.39 (acid metallo proteinases).
• protease is a metallo protease or not
• determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.
• Protease activity can be measured using any suitable assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question.
• Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples of assay- temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80°C.
• protease substrates examples include casein, such as Azurine-Crosslinked Casein (AZCL-casein).
• AZCL-casein Azurine-Crosslinked Casein
• Two protease assays are described below in the“Materials & Methods”- section of WO 2017/112540 (incorporated herein by reference), of which the so-called “AZCL-Casein Assay” is the preferred assay.
• thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the protease activity of the JTP196 variant (Example 2 from WO 2017/112540) or Protease Pfu (SEQ ID NO: 31 herein) determined by the AZCL-casein assay described in the “Materials & Methods”-section in WO 2017/112540.
• thermostable protease used in a process or composition of the invention as long as it fulfills the thermostability properties defined below.
• the protease is of fungal origin.
• thermostable protease is a variant of a metallo protease as defined above.
• thermostable protease used in a process or composition of the invention is of fungal origin, such as a fungal metallo protease, such as a fungal metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).
• thermostable protease is a variant of the mature part of the metallo protease shown in SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 and shown as SEQ ID NO: 32 herein further with mutations selected from below list:
• thermostable protease is a variant of the mature metallo protease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO
• the protease variant has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 32 herein.
• thermostable protease may also be derived from any bacterium as long as the protease has the thermostability properties defined according to the invention.
• thermostable protease is derived from a strain of the bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfu protease).
• protease is one shown as SEQ ID NO: 1 in US patent No. 6,358,726-B1 (Takara Shuzo Company) and SEQ ID NO: 31 herein.
• thermostable protease is one disclosed in SEQ ID NO: 31 herein or a protease having at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1 in US patent no. 6,358,726-B1 or SEQ ID NO: 31 herein.
• the Pyroccus furiosus protease can be purchased from Takara Bio,
• the Pyrococcus furiosus protease is a thermostable protease according to the invention.
• the commercial product Pyrococcus furiosus protease (Pfu S) was found (see Example 5 of ) to have a thermostability of 110% (80°C/70°C) and 103% (90°C/70°C) at pH 4.5 determined as described in Example 2 of WO 2017/112540.
• thermostable protease has a thermostability value of more than 20% determined as Relative Activity at 80°C/70°C determined as described in Example 2.
• the protease has a thermostability of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, such as more than 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C.
• protease has a thermostability of between 20 and 50%, such as between 20 and 40%, such as 20 and 30% determined as Relative Activity at 80°C/70°C.
• the protease has a thermostability between 50 and 115%, such as between 50 and 70%, such as between 50 and 60%, such as between 100 and 120%, such as between 105 and 115% determined as Relative Activity at 80°C/70°C.
• the protease has a thermostability value of more than 10% determined as Relative Activity at 85°C/70°C determined as described in Example 2 of WO 2017/112540.
• the protease has a thermostability of more than 10%, such as more than 12%, more than 14%, more than 16%, more than 18%, more than 20%, more than 30%, more than 40%, more that 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 110% determined as Relative Activity at 85°C/70°C.
• the protease has a thermostability of between 10 and 50%, such as between 10 and 30%, such as between 10 and 25% determined as Relative Activity at 85°C/70°C.
• the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%
• the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%
• the protease may have a themostability for above 90, such as above 100 at 85°C as determined using the Zein-BCA assay as disclosed in Example 3 of WO 2017/112540.
• the protease has a themostability above 60%, such as above 90%, such as above 100%, such as above 110% at 85°C as determined using the Zein-BCA assay.
• protease has a themostability between 60-120, such as between 70-120%, such as between 80-120%, such as between 90-120%, such as between 100- 120%, such as 110-120% at 85°C as determined using the Zein-BCA assay.
• thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the activity of the JTP196 protease variant or Protease Pfu determined by the AZCL-casein assay described in the“Materials & Methods”-section of WO 2017/112540.
• an enzyme blend of the present invention for improving the nutritional quality of distillers dried grains (DGS) or distillers dried grains with solubles (DDGS) produced as a co-product of a fermentation product production process of the present invention, preferably without resulting in a darkening the DDG or DDGS.
• DDG distillers dried grains
• DDGS distillers dried grains with solubles
• Any enzyme blend disclosed in Section I herein can be used in this manner.
• an additional enzyme such as an enzyme or enzyme composition described under the“Enzymes” section can be used in combination together with an enzyme blend of the present invention.
• the enzyme blend is used to improve the nutritional quality of DGS or DDGS by increasing the TME of the DDG or DDGS when administered to an animal (e.g., non-ruminant, e.g., monogastric, e.g., poultry or swine, etc.) by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13% at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to the TME of the DDG or DDGS produced as a co-product when an enzyme blend of the present invention is not present during the saccharification,
• an animal e.g., non-ruminant, e.g., monogastric, e.g., poultry or swine, etc.
• the enzyme blend is used to improve the nutritional quality of DGS or DDGS by increasing the TME of the DDG or DDGS in an animal (e.g., non-ruminant, e.g.,
• monogastric e.g., poultry or swine, etc.
• monogastric e.g., poultry or swine, etc.
• an enzyme blend of the present invention for increasing the solubilisation of fiber present in a fermentation mash during a fermentation product production process of the present invention, preferably without resulting in a darkening the DDG or DDGS.
• the enzyme blend is used to increase fiber solubilisation during the production of alcohol (e.g., ethanol) from a starch- containing material.
• the enzyme blend is used to increase the enzyme blend to increase the enzyme blend to increase the
• solubilisation of corn fiber in an ethanol production process such as a RSH process or convention cook including a liquefaction step.
• the enzyme blend is used to increase the solubilisation of arabinose.
• the enzyme blend is used to increase the solubilisation of xylose.
• Any enzyme blend disclosed in Section I herein can be used in this manner.
• an additional enzyme such as an enzyme or enzyme composition described under the“Enzymes” section can be used in combination together with an enzyme blend of the present invention.
• the enzyme blend is used to increase the solubilisation of fiber (e.g., arabinose, xylose, etc.) contacted with the enzyme blend by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13% at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to the solubilisation of fiber not contacted with an enzyme blend of the present invention.
• fiber e.g., arabinose, xylose, etc.
• the enzyme blend is used to increase the solubilisation of fiber (e.g., arabinose, xylose, etc.) contacted with the enzyme blend by at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33% at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% compared to fiber not contacted with the enzyme blend.
• fiber e.g., arabinose, xylose, etc.
• a polypeptide having xylanase activity selected from the group consisting of:
• polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 5, or 7;
• polypeptide encoded by a polynucleotide that hybridizes under very-high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 2, 6, or 8 or the full-length complement of any thereof;
• the mature polypeptide is amino acids 33 to 884 of SEQ ID NO: 1 ;
• the mature polypeptide is amino acids 31 to 826 of SEQ ID NO: 5;
• the mature polypeptide is amino acids 35 to 831 of SEQ ID NO: 7.
• a nucleic acid construct or recombinant expression vector comprising the polynucleotide of paragraph 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
• a recombinant host cell comprising the polynucleotide of paragraph 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide.
• a method of producing a polypeptide having xylanase activity comprising (a) cultivating the host cell of paragraph 8 under conditions conducive for production of the polypeptide and (b) optionally recovering the polypeptide.
• a process of producing a fermentation product comprising the following steps:
• a process for producing a fermentation product from starch-containing material comprising the steps of:
• step (b) saccharifying the liquefied material obtained in step (a) with a glucoamylase and a GH98 xylanase or enzyme blend comprising the GH98 xylanase;
• starch-containing material comprises maize, corn, wheat, rye, barley, triticale, sorghum, switchgrass, millet, pearl millet, foxtail millet.
• cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of:
• cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of:
• cellulolytic composition comprises:
• a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID NO: 15;
• a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 16 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 454 of SEQ ID NO: 16;
• a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 17 or a variant thereof having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 863 of SEQ ID NO: 17; and/or
• a GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID NO: 18 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 26 to 253 of SEQ ID NO: 18.
• cellulolytic composition further comprises an endoglucanase.
• the cellulolytic composition is derived from a strain selected from the group consisting of Aspergillus, Penicilium, Talaromyces, and Trichoderma, optionally wherein: (i) the Aspergillus strain is selected from the group consisting of Aspergillus aurantiacus, Aspergillus niger and Aspergillus oryzae (ii) the Penicilium strain is selected from the group consisting of Penicilium emersonii and Penicilium oxalicum ; (iii) the Talaromyces strain is selected from the group consisting of Talaromyces aurantiacus and Talaromyces emersonii ; and (iv) the Trichoderma strain is Trichoderma reesei. 29. The enzyme blend of any of paragraphs 3-5 or the process of any of paragraphs IQ- 28, wherein the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition.
• Alpha-Amylase 369 Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (SEQ ID NO: 19 herein) truncated to 491 amino acids.
• Cellulolytic composition Cellulolytic composition derived from Trichoderma reesei comprising: Aspergillus fumigatus Cel7A CBH1 disclosed as SEC ID NO: 6 in
• E-SEP Blend comprising transgenic GH10 xylanase expressing, and a GH62 arabinofuranosidase expressing Trichoderma reesei cellulose strain.
• Glucoamylase SA Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ ID NO: 34 in W099/28448, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) disclosed in SEQ ID NO: 14 herein having the following substitutions G128D+D143N (activity ratio in AGU:AGU:FAU-F is about 20:5:1).
• Protease Pfu Protease derived from Pyrococcus furiosus shown in SEQ ID NO: 31 herein.
• GH98#1 GH98 xylanase from Paenibacillus terrigena having the amino acid sequence of SEQ ID NO: 7.
• GH98#2 GH98 xylanase from Paenibacillus glycanilyticus having the amino acid sequence of SEQ ID NO: 5.
• GH98#3 GH98 xylanase from Microbacterium oxydans having the amino acid sequence of SEQ I D NO: 1.
• Yeast ETHANOL REDTM available from Red Star/Lesaffre, USA.
• the activity of a xylanase variant towards defatted destrached Maize can be measured by High-Performance Anion-Exchange Chromatography with Pulsed
• 2% (w/w) DFDSM suspension can be prepared in 100 mM sodium acetate, 5 mM CaCh, pH 5 and allowed to hydrate for 30 minutes at room temperature under gentle stirring. After hydration, 200 pi substrate suspension can be pipetted into a 96 well plate and mixed with 20 mI enzyme solution to obtain a final enzyme concentration of 20 PPM relative to substrate (20 mg enzyme / g substrate).
• enzyme/substrate mixtures can then be left for hydrolysis in 2.5 hours at 40°C under gentle agitation (500 RPM) in a plate incubator (Biosan PST-100 HL). After enzymatic hydrolysis, the enzyme/substrate plates can be centrifuged for 10 minutes at 3000 RPM and 50 mI supernatant (hydrolysate) is mixed with 100 mI 1.6 M HCI and transferred to 300 mI PCR tubes and left for acid hydrolysis for 40 minutes at 90°C in a PCR machine.
• the purpose of the acid hydrolysis is to convert soluble polysaccharides, released by the xylanase variant, into mono-saccharides, which can be quantified using HPAE-PAD.
• [xylose] denotes the concentration of xylose in the supernatant measured by HPAE- PAD, V the volume of the sample, MW, the molecular weight of internal xylose in arabino- xylan (132 g/mol), Xxyl, the fraction of xylose in DFDSM (0.102) and Msub, the mass of DFDSM in the sample.
• the GH98 endo-beta-1 ,4-xylanases were derived from bacterial strains isolated from environmental sample by standard microbiological isolation techniques. The isolated pure strains were identified, and taxonomy was assigned based on DNA sequencing of the 16S ribosomal genes (Table 1).
• Chromosomal DNA was isolated from pure cultures and subjected to full genome sequencing using ILLUMINA® technology. Genome sequencing, the subsequent assembly of reads and the gene discovery (i.e. annotation of gene functions) is known to the person skilled in the art and the service can be purchased commercially.
• the genome sequences were analyzed for putative endo-beta-1,4-xylanases from the CAZY database GH98 family (Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014). The Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490-D495.).
• This analysis identified 3 genes encoding putative GH98 endo- beta-1 ,4-xylanases, which were subsequently ordered as linear DNA fragments from GeneArt® and recombinantly expressed in Bacillus subtilis.
• the DNA encoding the putative GH98 endo-beta-1 ,4-xylanase from Micro bacterium oxydans was codon-optimized (SEQ ID NO: 33) for Bacillus subtilis before ordering.
• the integration constructs used for were plasmid-based products made by fusion of the gene of interest between two B. subtilis chromosomal regions along with strong promoters and a chloramphenicol resistance marker.
• the GH98 endo-beta-1 ,4-xylanase genes were expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including stabilizing sequence.
• the genes were fused with DNA encoding a Bacillus clausii secretion signal (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 34), replacing the native secretion signal. Furthermore, the expression construct results in the addition of an amino-terminal poly histidine tag consisting of the amino acid sequence HHHHHHPR (SEQ ID NO: 35) to the mature GH98 endo-beta-1 ,4-xylanases to facilitate easy purification by immobilized metal affinity chromatography.
• the resulting plasmid was transformed into Bacillus subtilis and integrated in the chromosome by homologous recombination into the pectate lyase locus. Subsequently, a recombinant Bacillus subtilis clone containing the integrated expression construct was grown in liquid culture. The culture broth was centrifuged (20000 x g, 20 min) and the supernatant was carefully decanted from the precipitate and used for purification of the enzyme or alternatively sterile filtered supernatant was used directly for assays.
• the pH of the cleared supernatant was adjusted to pH 8, filtrated through a 0.2mM filter, and the supernatant applied to a 5 ml HisTrapTM excel column.
• the column Prior to loading, the column had been equilibrated in 5 column volumes (CV) of 50 mM Tris/HCI pH 8.
• CV column volumes
• the column was washed with 8 CV of 50 mM Tris/HCI pH 8, and elution of the target was obtained with 50 mM HEPES pH 7 + 10mM imidazole.
• the eluted protein was desalted on a HiPrepTM 26/10 desalting column, equilibrated using 3 CV of 50 mM HEPES pH 7 + 100 mM NaCI. This buffer was also used for elution of the target, and the flow rate was 10 ml/min. Relevant fractions were selected and pooled based on the chromatogram and SDS-PAGE analysis.
• Example 2 demonstrates the effectiveness of various GH98 xylanases of the present invention and enzyme blends comprising 10:90 ratios of GH98 xylanases and the cellulolytic composition listed in the Materials & Methods section on corn fiber.
• Saccharification assays were done on high-solids corn fiber at 15% DS, pH5 and 32°C for 3 days. Each xylanase was tested as a 10% replacement (by protein) in the cellulolytic composition (“VD”) to a total protein loading of 0.25 mg EP/g Dry Solids (DS) (protein concentration for each of the xylanases in the assay was 0.00375 mg/ml). The incubations were supplemented with GSA (0.6 AGU/g DS).
• FIG. 1 shows the average DP4+ yield (g/L) for each of the GH98 enzyme blends as compared to the control cellulolytic composition not supplemented with xylanase.
• FIG. 2 shows the average glucose yield (g/L) for each of the GH98 enzyme blends as compared to control cellulolytic composition not supplemented with xylanase.
• FIG. 1 and FIG. 2 show that a GH98 xylanase from Microbacterium oxydans (#3; SEQ ID NO: 1) performed better than the GH98 xylanases from Paenibacillus.

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